U.S. patent application number 16/466193 was filed with the patent office on 2019-12-12 for method for producing trans-polyisoprenoid, vector, transgenic plant, method for producing pneumatic tire and method for producin.
This patent application is currently assigned to SUMITOMO RUBBER INDUSTRIES, LTD.. The applicant listed for this patent is KANAZAWA UNIVERSITY, SUMITOMO RUBBER INDUSTRIES, LTD., TOHOKU UNIVERSITY. Invention is credited to Kazuhisa FUSHIHARA, Yukino INOUE, Toru NAKAYAMA, Yuko SAKURAI, Seiji TAKAHASHI, Haruhiko YAMAGUCHI, Satoshi YAMASHITA.
Application Number | 20190376093 16/466193 |
Document ID | / |
Family ID | 62626349 |
Filed Date | 2019-12-12 |
United States Patent
Application |
20190376093 |
Kind Code |
A1 |
SAKURAI; Yuko ; et
al. |
December 12, 2019 |
METHOD FOR PRODUCING TRANS-POLYISOPRENOID, VECTOR, TRANSGENIC
PLANT, METHOD FOR PRODUCING PNEUMATIC TIRE AND METHOD FOR PRODUCING
RUBBER PRODUCT
Abstract
The present invention aims to provide a method for producing a
trans-polyisoprenoid which can increase trans rubber production.
The present invention is directed to a method for producing a
trans-polyisoprenoid in vitro, which involves the use of a gene
coding for a trans-prenyltransferase (tPT) family protein and
further involves the use of rubber particles bound to a protein
encoded by the gene, or a method for producing a
trans-polyisoprenoid, which includes introducing into a plant a
vector including a promoter having a promoter activity that drives
laticifer-specific gene expression and a gene coding for a tPT
family protein linked to the promoter to express a protein encoded
by the gene specifically in laticifers.
Inventors: |
SAKURAI; Yuko; (Kobe-shi,
Hyogo, JP) ; YAMAGUCHI; Haruhiko; (Kobe-shi, Hyogo,
JP) ; INOUE; Yukino; (Kobe-shi, Hyogo, JP) ;
FUSHIHARA; Kazuhisa; (Kobe-shi, Hyogo, JP) ;
TAKAHASHI; Seiji; (Sendai-shi, JP) ; YAMASHITA;
Satoshi; (Kanazawa-shi, JP) ; NAKAYAMA; Toru;
(Sendai-shi, JP) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
SUMITOMO RUBBER INDUSTRIES, LTD.
TOHOKU UNIVERSITY
KANAZAWA UNIVERSITY |
Kobe-shi, Hyogo
Sendai-shi, Miyagi
Kanazawa-shi, Ishikawa |
|
JP
JP
JP |
|
|
Assignee: |
SUMITOMO RUBBER INDUSTRIES,
LTD.
Kobe-shi, Hyogo
JP
TOHOKU UNIVERSITY
Sendai-shi, Miyagi
JP
KANAZAWA UNIVERSITY
Kanazawa-shi, Ishikawa
JP
|
Family ID: |
62626349 |
Appl. No.: |
16/466193 |
Filed: |
November 21, 2017 |
PCT Filed: |
November 21, 2017 |
PCT NO: |
PCT/JP2017/041732 |
371 Date: |
June 3, 2019 |
Current U.S.
Class: |
1/1 |
Current CPC
Class: |
C12N 9/1085 20130101;
C12N 5/10 20130101; C12P 5/007 20130101; B60C 1/00 20130101; C12N
15/8243 20130101; B29D 30/0601 20130101; C12P 5/02 20130101; A01H
6/00 20180501; C12N 15/09 20130101 |
International
Class: |
C12P 5/02 20060101
C12P005/02; A01H 6/00 20060101 A01H006/00; B60C 1/00 20060101
B60C001/00 |
Foreign Application Data
Date |
Code |
Application Number |
Dec 21, 2016 |
JP |
2016-248330 |
Claims
1. A method for producing a trans-polyisoprenoid, the method
comprising binding a protein expressed from a gene coding for a
trans-prenyltransferase (tPT) family protein to rubber particles in
vitro.
2. The method for producing a trans-polyisoprenoid according to
claim 1, wherein the trans-prenyltransferase (tPT) family protein
comprises, at positions 183 to 187 in the amino acid sequence of
HbSDS from Hevea brasiliensis represented by SEQ ID NO:2 or at
corresponding positions, the following amino acid sequence (A1):
DDX.sub.1X.sub.2D (A1) wherein X.sub.1 and X.sub.2 are the same as
or different from each other and each represent any amino acid
residue, or the following amino acid sequence (A2):
DDX.sub.1X.sub.2X.sub.3X.sub.4D (A2) wherein X.sub.1, X.sub.2,
X.sub.3, and X.sub.4 are the same as or different from each other
and each represent any amino acid residue, and the
trans-prenyltransferase (tPT) family protein comprises, at
positions 310 to 314 in the amino acid sequence of HbSDS from Hevea
brasiliensis represented by SEQ ID NO:2 or at corresponding
positions, the following amino acid sequence (B):
DDX.sub.11X.sub.12D (B) wherein X.sub.11 and X.sub.12 are the same
as or different from each other and each represent any amino acid
residue.
3. The method for producing a trans-polyisoprenoid according to
claim 1, wherein the gene coding for a trans-prenyltransferase
(tPT) family protein is derived from a plant.
4. The method for producing a trans-polyisoprenoid according to
claim 3, wherein the gene coding for a trans-prenyltransferase
(tPT) family protein is derived from a rubber-producing plant.
5. The method for producing a trans-polyisoprenoid according to
claim 4, wherein the gene coding for a trans-prenyltransferase
(tPT) family protein is derived from Hevea brasiliensis.
6. The method for producing a trans-polyisoprenoid according to
claim 1, wherein the binding comprises performing protein synthesis
in the presence of both rubber particles and a cell-free protein
synthesis solution containing an mRNA coding for a
trans-prenyltransferase (tPT) family protein to bind the tPT family
protein to the rubber particles.
7. The method for producing a trans-polyisoprenoid according to
claim 6, wherein the cell-free protein synthesis solution comprises
a germ extract.
8. The method for producing a trans-polyisoprenoid according to
claim 7, wherein the germ extract is derived from wheat.
9. The method for producing a trans-polyisoprenoid according to
claim 6, wherein the rubber particles are present in the cell-free
protein synthesis solution at a concentration of 5 to 50 g/L.
10. A method for producing a pneumatic tire, the method comprising:
kneading a trans-polyisoprenoid produced by the method for
producing a trans-polyisoprenoid according to claim 1 with an
additive to obtain a kneaded mixture; building a green tire from
the kneaded mixture; and vulcanizing the green tire.
11. A method for producing a rubber product, the method comprising:
kneading a trans-polyisoprenoid produced by the method for
producing a trans-polyisoprenoid according to claim 1 with an
additive to obtain a kneaded mixture; forming a raw rubber product
from the kneaded mixture; and vulcanizing the raw rubber
product.
12. A vector, comprising: a promoter having a promoter activity
that drives laticifer-specific gene expression; and a gene coding
for a trans-prenyltransferase (tPT) family protein functionally
linked to the promoter.
13. The vector according to claim 12, wherein the promoter having a
promoter activity that drives laticifer-specific gene expression is
at least one selected from the group consisting of a promoter of a
gene coding for rubber elongation factor (REF), a promoter of a
gene coding for small rubber particle protein (R PP), a promoter of
a gene coding for Hevein 2.1 (HEV2.1), and a promoter of a gene
coding for MYC1 transcription factor (MYC1).
14. A transgenic plant into which the vector according to claim 12
has been introduced.
15. A method for enhancing trans-polyisoprenoid production in a
plant by introducing the vector according to claim 12 into the
plant.
16. A method for producing a pneumatic tire, the method comprising:
kneading a trans-polyisoprenoid produced by a transgenic plant with
an additive to obtain a kneaded mixture, the transgenic plant being
produced by introducing the vector according to claim 12 into a
plant; building a green tire from the kneaded mixture; and
vulcanizing the green tire.
17. A method for producing a rubber product, the method comprising:
kneading a trans-polyisoprenoid produced by a transgenic plant with
an additive to obtain a kneaded mixture, the transgenic plant being
produced by introducing the vector according to claim 12 into a
plant; forming a raw rubber product from the kneaded mixture; and
vulcanizing the raw rubber product.
Description
TECHNICAL FIELD
[0001] The present invention relates to a method for producing a
trans-polyisoprenoid, a vector, a transgenic plant, a method for
producing a pneumatic tire, and a method for producing a rubber
product.
BACKGROUND ART
[0002] At present, natural rubber (one example of polyisoprenoids)
for use in industrial rubber products is produced by cultivating
rubber-producing plants, such as para rubber tree (Hevea
brasiliensis) of the family Euphorbiaceae or Indian rubber tree
(Ficus elastica) of the family Moraceae. Such natural rubber is a
polyisoprenoid (cis-natural rubber) in which isoprene units are
linked in a cis configuration. Other polyisoprenoids in which
isoprene units are trans-linked, trans-polyisoprenoids (trans
rubber), also exist in the nature.
[0003] A few plants in the nature, such as Eucommia ulmoides
belonging to the family Eucommiaceae of the order Eucommiales, a
high deciduous tree native to China, are known to produce
trans-polyisoprenoids (trans rubber) which can be extracted from
the seeds or pericarp tissue of Eucommia ulmoides. Trans rubber can
also be chemically synthesized. Such trans rubber has different
characteristics from cis-natural rubber and has been used in
crack-resistant golf balls or dental materials used to fill
cavities in teeth.
[0004] The trans-polyisoprenoid extracted and purified from
Eucommia ulmoides is a polyisoprenoid having a weight average
molecular weight of about 1.8.times.10.sup.6 in which at least 99%
of the units of the straight chain are trans-linked, and has been
used as eucommia lastomer. However, if Eucommia ulmoides, which is
used as a healthy food or herbal medicine, is industrially used to
extract and purify a trans-polyisoprenoid, this may potentially
compete with use as a food material.
[0005] Meanwhile, the chemically synthesized trans-polyisoprenoids
do not have a trans content of 100% but contain about 1.2 to 4% of
cis bonds. They also have a molecular weight of about 250,000, and
it is very difficult to synthesize a trans-polyisoprenoid having an
ultra-high molecular weight of 1,000,000 or higher. Furthermore,
their chemical synthesis requires a supply of raw materials,
including petroleum-derived materials, which is hardly an
eco-friendly (environmentally friendly) procurement process.
[0006] As described, both methods based on extraction and
purification from Eucommia ulmoides and on chemical synthesis may
cause a problem associated with energy or competition with
foodstuff. There is therefore a need for methods capable of stable
and large quantity procurement of trans-polyisoprenoids (trans
rubber).
[0007] Trans rubber has a trans-1,4-polyisoprene structure that is
biosynthesized by addition polymerization of isopentenyl
diphosphate (IPP) with a starting substrate such as dimethylallyl
diphosphate (DMAPP) or farnesyl diphosphate (FPP), and the nature
of this structure suggests that a trans-prenyltransferase (tPT) may
be involved in trans rubber biosynthesis.
[0008] For example, Patent Literature 1 describes that
trans-1,4-polyisoprene can be efficiently produced by transforming
a plant with an expression vector containing a gene coding for a
long-chain trans-prenyl diphosphate synthase
(trans-prenyltranspherase) to produce a plant containing an
increased amount of trans-1,4-polyisoprene, and cultivating the
plant.
CITATION LIST
Patent Literature
[0009] Patent Literature 1: JP 2016-93186 A
SUMMARY OF INVENTION
Technical Problem
[0010] As discussed above, a need has existed for methods capable
of stable and large quantity procurement of trans-polyisoprenoids
(trans rubber). Unfortunately, very little research and development
has been devoted to such methods, and there is still much room for
improvement in terms of methods for stable and large quantity
procurement of trans-polyisoprenoids (trans rubber).
[0011] In this context, one possible approach to solving these
problems is to stabilize and enhance the activity of tPT in trans
rubber biosynthesis in order to increase trans rubber
production.
[0012] The present invention aims to solve the problems and provide
a method for producing a trans-polyisoprenoid which can increase
trans rubber production in vitro.
[0013] The present invention also aims to solve the above problems
and provide a vector that can be introduced into a plant using
genetic transformation techniques to enhance trans-polyisoprenoid
production. Further objects are to provide a transgenic plant into
which the vector has been introduced and to provide a method for
enhancing production of a trans-isoprenoid or trans-polyisoprenoid
in a plant by introducing the vector into the plant.
Solution to Problem
[0014] The present invention relates to a method for producing a
trans-polyisoprenoid, the method including binding a protein
expressed from a gene coding for a trans-prenyltransferase (tPT)
family protein to rubber particles in vitro. This invention is
hereinafter called the first aspect of the present invention and
also referred to as the first invention.
[0015] Preferably, the trans-prenyltransferase (tPT) family protein
contains, at positions 183 to 187 in the amino acid sequence of
HbSDS from Hevea brasiliensis represented by SEQ ID NO:2 or at
corresponding positions,
[0016] the following amino acid sequence (A1):
DDX.sub.1X.sub.2D (A1)
wherein X.sub.1 and X.sub.2 are the same as or different from each
other and each represent any amino acid residue, or the following
amino acid sequence (A2):
DDX.sub.1X.sub.2X.sub.3X.sub.4D (A2)
wherein X.sub.1, X.sub.2, X.sub.3, and X.sub.4 are the same as or
different from each other and each represent any amino acid
residue, and
[0017] the trans-prenyltransferase (tPT) family protein contains,
at positions 310 to 314 in the amino acid sequence of HbSDS from
Hevea brasiliensis represented by SEQ ID NO:2 or at corresponding
positions,
[0018] the following amino acid sequence (B):
DDX.sub.11X.sub.12D (B)
wherein X.sub.11 and X.sub.12 are the same as or different from
each other and each represent any amino acid residue.
[0019] Preferably, the gene coding for a trans-prenyltransferase
(tPT) family protein is derived from a plant.
[0020] Preferably, the gene coding for a trans-prenyltransferase
(tPT) family protein is derived from a rubber-producing plant.
[0021] Preferably, the gene coding for a trans-prenyltransferase
(tPT) family protein is derived from Hevea brasiliensis.
[0022] Preferably, the binding includes performing protein
synthesis in the presence of both rubber particles and a cell-free
protein synthesis solution containing an mRNA coding for a
trans-prenyltransferase (tPT) family protein to bind the tPT family
protein to the rubber particles.
[0023] Preferably, the cell-free protein synthesis solution
contains a germ extract.
[0024] Preferably, the germ extract is derived from wheat.
[0025] Preferably, the rubber particles are present in the
cell-free protein synthesis solution at a concentration of 5 to 50
g/L.
[0026] The first invention is also directed to a method for
producing a pneumatic tire, the method including: kneading a
trans-polyisoprenoid produced by the method for producing a
trans-polyisoprenoid of the first invention with an additive to
obtain a kneaded mixture; building a green tire from the kneaded
mixture; and vulcanizing the green tire.
[0027] The first invention is also directed to a method for
producing a rubber product, the method including: kneading a
trans-polyisoprenoid produced by the method for producing a
trans-polyisoprenoid of the first invention with an additive to
obtain a kneaded mixture; forming a raw rubber product from the
kneaded mixture; and vulcanizing the raw rubber product.
[0028] The present invention also relates to a vector, including: a
promoter having a promoter activity that drives laticifer-specific
gene expression; and a gene coding for a trans-prenyltransferase
(tPT) family protein functionally linked to the promoter. This
invention is hereinafter called the second aspect of the present
invention and also referred to as the second invention.
[0029] Preferably, the promoter having a promoter activity that
drives laticifer-specific gene expression is at least one selected
from the group consisting of a promoter of a gene coding for rubber
elongation factor (REF), a promoter of a gene coding for small
rubber particle protein (SRPP), a promoter of a gene coding for
Hevein 2.1 (HEV2.1), and a promoter of a gene coding for MYC1
transcription factor (MYC1).
[0030] The second invention is also directed to a transgenic plant
into which any one of the above-described vectors has been
introduced.
[0031] The second invention is also directed to a method for
enhancing trans-isoprenoid production in a plant by introducing any
one of the above-described vectors into the plant.
[0032] The second invention is also directed to a method for
enhancing trans-polyisoprenoid production in a plant by introducing
any one of the above-described vectors into the plant.
[0033] The second invention is also directed to a method for
producing a pneumatic tire, the method including: kneading a
trans-polyisoprenoid produced by a transgenic plant with an
additive to obtain a kneaded mixture, the transgenic plant being
produced by introducing any one of the above-described vectors into
a plant; building a green tire from the kneaded mixture; and
vulcanizing the green tire.
[0034] The second invention is also directed to a method for
producing a rubber product, the method including: kneading a
trans-polyisoprenoid produced by a transgenic plant with an
additive to obtain a kneaded mixture, the transgenic plant being
produced by introducing any one of the above-described vectors into
a plant; forming a raw rubber product from the kneaded mixture; and
vulcanizing the raw rubber product.
Advantageous Effects of Invention
[0035] The method for producing a trans-polyisoprenoid of the first
invention includes binding a protein expressed from a gene coding
for a trans-prenyltransferase (tPT) family protein to rubber
particles in vitro. Thus, by binding a tPT family protein to rubber
particles, trans rubber can be synthesized in the rubber particles,
and therefore it is possible to efficiently produce trans rubber in
reaction vessels (e.g., test tubes, industrial plants).
[0036] The method for producing a pneumatic tire of the first
invention includes kneading a trans-polyisoprenoid produced by the
method for producing a trans-polyisoprenoid of the first invention
with an additive to obtain a kneaded mixture; building a green tire
from the kneaded mixture; and vulcanizing the green tire. With this
method, which produces a pneumatic tire from a trans-polyisoprenoid
obtained by a highly efficient polyisoprenoid production method, it
is possible to use plant resources effectively to produce
environmentally friendly pneumatic tires.
[0037] The method for producing a rubber product of the first
invention includes kneading a trans-polyisoprenoid produced by the
method for producing a trans-polyisoprenoid of the first invention
with an additive to obtain a kneaded mixture; forming a raw rubber
product from the kneaded mixture; and vulcanizing the raw rubber
product. With this method, which produces a rubber product from a
trans-polyisoprenoid obtained by a highly efficient polyisoprenoid
production method, it is possible to use plant resources
effectively to produce environmentally friendly rubber
products.
[0038] The vector of the second invention includes a promoter
having a promoter activity that drives laticifer-specific gene
expression and a gene coding for a trans-prenyltransferase (tPT)
family protein functionally linked to the promoter. By introducing
the vector into a plant, the gene coding for a protein involved in
trans-polyisoprenoid biosynthesis in the vector can be expressed
specifically in laticifers, thereby enhancing trans-isoprenoid or
trans-polyisoprenoid production in the plant.
[0039] The method for producing a pneumatic tire of the second
invention includes kneading a trans-polyisoprenoid produced by a
transgenic plant with an additive to obtain a kneaded mixture, the
transgenic plant being produced by introducing the vector of the
second invention into a plant; building a green tire from the
kneaded mixture; and vulcanizing the green tire. With this method,
which produces a pneumatic tire from a trans-polyisoprenoid
produced by a transgenic plant with an enhanced
trans-polyisoprenoid production, it is possible to use plant
resources effectively to produce environmentally friendly pneumatic
tires.
[0040] The method for producing a rubber product of the second
invention includes kneading a trans-polyisoprenoid produced by a
transgenic plant with an additive to obtain a kneaded mixture, the
transgenic plant being produced by introducing the vector of the
second invention into a plant; forming a raw rubber product from
the kneaded mixture; and vulcanizing the raw rubber product. With
this method, which produces a pneumatic tire from a
trans-polyisoprenoid produced by a transgenic plant with an
enhanced trans-polyisoprenoid production, it is possible to use
plant resources effectively to produce environmentally friendly
rubber products.
BRIEF DESCRIPTION OF DRAWINGS
[0041] FIG. 1 is a presumptive diagram illustrating rubber
synthesis by tPT on a rubber particle.
[0042] FIG. 2 is a schematic diagram illustrating part of a
trans-polyisoprenoid biosynthesis pathway.
[0043] FIG. 3 is an outline diagram illustrating the dialysis
process in Example.
[0044] FIG. 4 illustrates a graph of the measured molecular weight
distributions of the very long chain polyisoprenoids synthesized in
Example 1 and Comparative Example 2.
[0045] FIG. 5 is an outline diagram illustrating the results of
multiple sequence alignment of tPT family proteins derived from
various organisms.
DESCRIPTION OF EMBODIMENTS
[0046] Herein, the first invention and the second invention are
also referred to collectively as the present invention. The first
invention will be described first, and the second invention will be
described later.
First Invention
[0047] The method for producing a trans-polyisoprenoid of the first
invention includes binding a protein expressed from a gene coding
for a trans-prenyltransferase (tPT) family protein to rubber
particles in vitro.
[0048] The inventors were the first to discover that a
trans-polyisoprenoid (trans rubber) can be synthesized by binding a
tPT family protein to rubber particles in vitro. It is presumed
that tPT family proteins are disposed on rubber particles to
synthesize rubber as shown in FIG. 1. FIG. 1 schematically
illustrates an exemplary synthesis of trans rubber within a rubber
particle by polymerization of an isopentenyl diphosphate (IPP)
substrate by a tPT family protein depicted as tPT. Thus, by binding
a tPT family protein to rubber particles in vitro, for example in a
reaction vessel (e.g., a test tube or industrial plant) as in the
production method of the first invention, a trans-polyisoprenoid
(trans rubber) can be synthesized in the rubber particles, and
therefore it is possible to efficiently produce trans rubber in a
reaction vessel (e.g., a test tube or industrial plant).
[0049] The production method of the first invention may include any
other step as long as it involves the above binding step, and each
step may be performed once or repeated multiple times.
[0050] The amount of the tPT family protein to be bound to the
rubber particles is not particularly limited in the first
invention.
[0051] Herein, the expression "binding a tPT family protein to
rubber particles" means that, for example, the tPT family protein
is fully or partially incorporated into the rubber particles or
inserted into the membrane structure of the rubber particles. It is
not limited to these embodiments and also includes embodiments in
which, for example, the tPT family protein is localized on the
surface or inside of the rubber particles. Moreover, the concept of
binding to rubber particles also includes embodiments in which the
tPT family protein forms a complex with another protein bound to
the rubber particles to exist in the form of the complex on the
rubber particles.
[0052] A supplementary description of the present invention is
given below.
[0053] First, for example, even if it were known that cis-natural
rubber could be synthesized by binding rubber particles to a
cis-prenyltranspherase (CPT) family protein which is considered to
be deeply involved in the biosynthesis of cis-natural rubber, a
person skilled in the art, in light of the experimental results of
the CPT family protein, would not attempt to simply change the CPT
family protein to a tPT family protein because tPT family proteins
and CPT family proteins belong to very different protein families
and have very different protein structures.
[0054] Furthermore, since tPT family proteins are not present on
rubber particles in vivo, particularly in rubber-producing plants
capable of producing cis-natural rubber, a person skilled in the
art has no motivation to bind a tPT family protein to rubber
particles. If a skilled person were to consider biding a tPT family
protein to rubber particles, the skilled person, who knows the
above fact, could not predict at all that the binding of a tPT
family protein to rubber particles would lead to rubber
synthesis.
[0055] In such circumstances, it has been found that binding a tPT
family protein to rubber particles enables synthesis of a
trans-polyisoprenoid (trans rubber) in the rubber particles. Thus,
this is considered a surprising result which could not have been
predicted by one skilled in this art.
[0056] The origin of the rubber particles is not particularly
limited. For example, the rubber particles may be derived from the
latex of a rubber-producing plant such as Hevea brasiliensis,
Taraxacum kok-saghyz, Parthenium argentatum, Sonchus oleraceus, or
Ficus elastica.
[0057] The particle size of the rubber particles is also not
particularly limited. Rubber particles having a predetermined
particle size may be sorted out and used, or a mixture of rubber
particles having different particle sizes may be used. When rubber
particles having a predetermined particle size are sorted out and
used, the rubber particles may be either small rubber particles
(SRP) having a small particle size or large rubber particles (LRP)
having a large particle size.
[0058] In order to sort out the rubber particles having a
predetermined particle size, commonly used methods may be used,
including, for example, methods which involve centrifugation,
preferably multistage centrifugation. A specific method includes
centrifugation at 500-1500.times.g, centrifugation at
1700-2500.times.g, centrifugation at 7000-9000.times.g,
centrifugation at 15000-25000.times.g, and centrifugation at
40000-60000.times.g, carried out in that order. The duration of
each centrifugation treatment is preferably 20 minutes or longer,
more preferably 30 minutes or longer, still more preferably 40
minutes or longer, but is preferably 120 minutes or shorter, more
preferably 90 minutes or shorter. The temperature for each
centrifugation treatment is preferably 0 to 10.degree. C., more
preferably 2 to 8.degree. C., particularly preferably 4.degree.
C.
[0059] In the binding step, a protein expressed from a gene coding
for a trans-prenyltransferase (tPT) family protein is bound to
rubber particles in vitro.
[0060] The origin of the gene coding for a trans-prenyltransferase
(tPT) family protein is not particularly limited. The gene may be
derived from a microorganism, an animal, or a plant, preferably a
plant, more preferably a rubber-producing plant, still more
preferably at least one selected from the group consisting of
plants of the genera Hevea, Sonchus, Taraxacum, and Parthenium. In
particular, it is further preferably derived from at least one
species of plant selected from the group consisting of Hevea
brasiliensis, Sonchus oleraceus, Parthenium argentatum, and
Taraxacum kok-saghyz, particularly preferably from Hevea
brasiliensis.
[0061] The plant is not particularly limited, and examples include
Hevea species such as Hevea brasiliensis; Sonchus species such as
Sonchus oleraceus, Sonchus asper, and Sonchus brachyotus; Solidago
species such as Solidago altissima, Solidago virgaurea subsp.
asiatica, Solidago virgaurea subsp. leipcarpa, Solidago virgaurea
subsp. leipcarpa f. paludosa, Solidago virgaurea subsp. gigantea,
and Solidago gigantea Ait. var. leiophylla Fernald; Helianthus
species such as Helianthus annus, Helianthus argophyllus,
Helianthus atrorubens, Helianthus debilis, Helianthus decapetalus,
and Helianthus giganteus; Taraxacum species such as dandelion
(Taraxacum), Taraxacum venustum H. Koidz, Taraxacum hondoense
Nakai, Taraxacum platycarpum Dahlst, Taraxacum japonicum, Taraxacum
officinale Weber, Taraxacum kok-saghyz, and Taraxacum
brevicorniculatum; Ficus species such as Ficus carica, Ficus
elastica, Ficus pumila L., Ficus erecta Thumb., Ficus ampelas Burm.
f., Ficus benguetensis Merr., Ficus irisana Elm., Ficus microcarpa
L. f., Ficus septica Burm. f., and Ficus benghalensis; Parthenium
species such as Parthenium argentatum, Parthenium hysterophorus,
and Ambrosia artemisiifolia (Parthenium hysterophorus); lettuce
(Lactuca sativa); Ficus benghalensis; Arabidopsis thaliana; and
Eucommia ulmoides.
[0062] Herein, the term "trans-prenyltransferase (tPT) family
protein" refers to an enzyme that catalyzes a reaction of
trans-chain elongation of an isoprenoid compound. Specifically, for
example, in plants, trans-polyisoprenoids are biosynthesized via
trans-polyisoprenoid biosynthesis pathways as shown in FIG. 2, in
which tPT family proteins are considered to be enzymes that
catalyze the reactions enclosed by the dotted frame in FIG. 2. The
tPT family proteins are characterized by having an amino acid
sequence contained in the trans-IPPS HT domain (NCBI Accession No.
cd00685).
[0063] Herein, the term "isoprenoid compound" refers to a compound
containing an isoprene unit (C.sub.5H.sub.8). The term
"trans-isoprenoid" refers to a compound including an isoprenoid
compound in which isoprene units are trans-linked (in particular,
the content of trans bonds is preferably at least 90%, more
preferably at least 95%, still more preferably at least 97% of the
total bonds), and examples include trans-polyisoprenoids (trans
rubber) such as farnesyl diphosphate, geranylgeranyl diphosphate,
hexaprenyl diphosphate, heptaprenyl diphosphate, and
trans-1,4-polyisoprene.
[0064] FIG. 5 is an outline diagram illustrating the results of
multiple sequence alignment of tPT family proteins derived from
various organisms. According to literature, such as Andrew H.-J.
Wang et al., Eur. J. Biochem. 269, pp. 3339-3354 (2002), box A
(corresponding to positions 183 to 187 of HbSDS from Hevea
brasiliensis represented by SEQ ID NO:2) and box B (corresponding
to positions 310 to 314 of HbSDS from Hevea brasiliensis
represented by SEQ ID NO:2) in FIG. 5 are parts of highly conserved
regions of tPT family proteins derived from various organisms. The
term "conserved region" refers to a site having a similar sequence
(structure) which is presumed to have a similar protein function.
In particular, it is considered that an amino acid sequence at
positions corresponding to positions 183 to 187 of HbSDS from Hevea
brasiliensis represented by SEQ ID NO:2, and an amino acid sequence
at positions corresponding to positions 310 to 314 of HbSDS from
Hevea brasiliensis represented by SEQ ID NO:2 are conserved as
specific motifs, and proteins having these motifs at the respective
positions have the functions of tPT family proteins.
[0065] The multiple sequence alignment can be carried out as
described later in EXAMPLES.
[0066] Specifically, the trans-prenyltransferase (tPT) family
protein preferably contains, at positions 183 to 187 in the amino
acid sequence of HbSDS from Hevea brasiliensis represented by SEQ
ID NO:2 or at corresponding positions, the following amino acid
sequence (A1):
DDX.sub.1X.sub.2D (A1)
wherein X.sub.1 and X.sub.2 are the same as or different from each
other and each represent any amino acid residue, or the following
amino acid sequence (A2):
DDX.sub.1X.sub.2X.sub.3X.sub.4D (A2)
wherein X.sub.1, X.sub.2, X.sub.3, and X.sub.4 are the same as or
different from each other and each represent any amino acid
residue, and
[0067] the trans-prenyltransferase (tPT) family protein contains,
at positions 310 to 314 in the amino acid sequence of HbSDS from
Hevea brasiliensis represented by SEQ ID NO:2 or at corresponding
positions, the following amino acid sequence (B):
DDX.sub.11X.sub.12D (B)
wherein X.sub.11 and X.sub.12 are the same as or different from
each other and each represent any amino acid residue. As described
above, the tPT family protein having such a sequence is considered
to have the functions of tPT family proteins, including the
function as an enzyme that catalyzes a reaction of trans-chain
elongation of an isoprenoid compound. By binding this tPT family
protein to rubber particles, it is possible to synthesize trans
rubber in the rubber particles.
[0068] The tPT family protein preferably contains, at positions 183
to 187 in the amino acid sequence of HbSDS from Hevea brasiliensis
represented by SEQ ID NO:2 or at corresponding positions, the
following amino acid sequence (A1):
DDX.sub.1X.sub.2D (A1)
wherein X.sub.1 and X.sub.2 are the same as or different from each
other and each represent any amino acid residue, or the following
amino acid sequence (A2):
DDX.sub.1X.sub.2X.sub.3X.sub.4D (A2)
wherein X.sub.1, X.sub.2, X.sub.3, and X.sub.4 are the same as or
different from each other and each represent any amino acid
residue. More preferably, in the amino acid sequences (A1) and
(A2), X.sub.1 denotes M, I, or V, and X.sub.2 denotes M, I, or
L.
[0069] The tPT family protein contains, at positions 310 to 314 in
the amino acid sequence of HbSDS from Hevea brasiliensis
represented by SEQ ID NO:2 or at corresponding positions, the
following amino acid sequence (B):
DDX.sub.11X.sub.12D (B)
wherein X.sub.11 and X.sub.12 are the same as or different from
each other and each represent any amino acid residue. More
preferably, in the amino acid sequence (B), X.sub.11 denotes Y, M,
I, or V, and X.sub.12 denotes L.
[0070] Specifically, the conserved region corresponding to
positions 183 to 187 of HbSDS from Hevea brasiliensis represented
by SEQ ID NO:2 corresponds for example to:
[0071] positions 100 to 104 of FPPS from yeast represented by SEQ
ID NO:3;
[0072] positions 99 to 103 of EuFPPS from Eucommia ulmoides
represented by SEQ ID NO:4;
[0073] positions 93 to 97 of HbFPPS from Hevea brasiliensis
represented by SEQ ID NO:5;
[0074] positions 91 to 95 of TPT from yeast represented by SEQ ID
NO:6;
[0075] positions 171 to 175 of AtSDS1 from Arabidopsis thaliana
represented by SEQ ID NO:7;
[0076] positions 141 to 145 of HsTPT from human represented by SEQ
ID NO:8; or
[0077] positions 101 to 105 of MmTPT from mouse represented by SEQ
ID NO:9.
[0078] The conserved region corresponding to positions 310 to 314
of HbSDS from Hevea brasiliensis represented by SEQ ID NO: 2
corresponds for example to:
[0079] positions 240 to 244 of FPPS from yeast represented by SEQ
ID NO:3;
[0080] positions 238 to 242 of EuFPPS from Eucommia ulmoides
represented by SEQ ID NO:4;
[0081] positions 232 to 236 of HbFPPS from Hevea brasiliensis
represented by SEQ ID NO:5;
[0082] positions 262 to 266 of TPT from yeast represented by SEQ ID
NO:6;
[0083] positions 298 to 302 of AtSDS1 from Arabidopsis thaliana
represented by SEQ ID NO:7;
[0084] positions 268 to 272 of HsTPT from human represented by SEQ
ID NO:8; or
[0085] positions 228 to 232 of MmTPT from mouse represented by SEQ
ID NO:9.
[0086] Examples of the tPT family protein include: tPT derived from
yeast, such as FPPS (Erg20p [Saccharomyces cerevisiae R103]) and
TPT (Coq1p [Saccharomyces cerevisiae YJM1342]); tPT derived from
Eucommia ulmoides, such as EuFPPS (farnesyl pyrophosphate
synthetase [Eucommia ulmoides]); tPT derived from Hevea
brasiliensis, such as HbFPPS (farnesyl diphosphate synthase [Hevea
brasiliensis]) and HbSDS (solanesyl diphosphate synthase [Hevea
brasiliensis]); tPT derived from Arabidopsis thaliana, such as
AtSDS1 (solanesyl diphosphate synthase 1 [Arabidopsis thaliana]);
tPT derived from human, such as HsTPT (trans-prenyltransferase
[Homo sapiens]); and tPT derived from mouse, such as MmTPT
(trans-prenyltransferase [Mus musculus]).
[0087] In addition to rubber-producing plants which produce rubber,
other organisms such as plants, animals, and microorganisms have
genes coding for the tPT family proteins. Of course the tPT family
proteins from these organisms are naturally not involved in rubber
synthesis. In spite of this, according to the present invention,
trans rubber can be synthesized in rubber particles by binding any
tPT family protein, regardless of the origin, type, and other
factors of the protein, to the rubber particles. Thus, according to
the present invention, trans rubber can be synthesized in rubber
particles by using any tPT family protein, for example, regardless
of whether the gene coding for the tPT family protein is derived
from a rubber-producing plant or any other organism or whether it
is naturally involved in rubber synthesis. This is strongly
suggested by the mechanism (which indicates that the host to be
transfected, or in other words the environment in which the
cis-prenyltransferase (CPT) family protein is expressed is more
important for rubber synthesis activity than the origin or type of
the CPT family protein) already suggested in PCT/JP2016/069172 by
the present inventors.
[0088] The tPT family protein used in the present invention
desirably has a transmembrane domain on the N-terminal side to have
a higher affinity for rubber particles. In the case of a wild type
having no transmembrane domain, a transmembrane domain may be
artificially fused to the N-terminal side of the tPT family
protein. The transmembrane domain to be fused may have any amino
acid sequence, desirably an amino acid sequence of the
transmembrane domain of a protein inherently bound to rubber
particles in nature.
[0089] Specific examples of the tPT family protein include the
following protein [1]:
[1] a protein having the amino acid sequence represented by SEQ ID
NO:2.
[0090] Moreover, it is known that proteins having one or more amino
acid substitutions, deletions, insertions, or additions relative to
the original amino acid sequence can have the inherent function.
Thus, another specific example of the tPT family protein is the
following protein [2]:
[2] a protein having an amino acid sequence containing one or more
amino acid substitutions, deletions, insertions, and/or additions
relative to the amino acid sequence represented by SEQ ID NO:2, and
having an enzyme activity that catalyzes a reaction of trans-chain
elongation of an isoprenoid compound.
[0091] In order to maintain the function of the tPT family protein,
the protein preferably has an amino acid sequence containing one or
more, more preferably 1 to 83, still more preferably 1 to 62,
further preferably 1 to 41, particularly preferably 1 to 20, most
preferably 1 to 8, yet most preferably 1 to 4 amino acid
substitutions, deletions, insertions, and/or additions relative to
the amino acid sequence represented by SEQ ID NO:2.
[0092] Among other amino acid substitutions, conservative
substitutions are preferred. Specific examples include
substitutions within each of the following groups in the
parentheses: (glycine, alanine), (valine, isoleucine, leucine),
(aspartic acid, glutamic acid), (asparagine, glutamine), (serine,
threonine), (lysine, arginine), and (phenylalanine, tyrosine).
[0093] It is also known that proteins with amino acid sequences
having high sequence identity to the original amino acid sequence
can also have similar functions. Thus, another specific example of
the tPT family protein is the following protein [3]:
[3] a protein having an amino acid sequence with at least 80%
sequence identity to the amino acid sequence represented by SEQ ID
NO: 2, and having an enzyme activity that catalyzes a reaction of
trans-chain elongation of an isoprenoid compound.
[0094] In order to maintain the function of the tPT family protein,
the sequence identity to the amino acid sequence represented by SEQ
ID NO:2 is preferably at least 85%, more preferably at least 90%,
still more preferably at least 95%, particularly preferably at
least 98%, most preferably at least 99%.
[0095] Herein, the sequence identity between amino acid sequences
or nucleotide sequences may be determined using the algorithm BLAST
[Pro. Natl. Acad. Sci. USA, 90, 5873 (1993)] developed by Karlin
and Altschul or FASTA [Methods Enzymol., 183, 63 (1990)].
[0096] Whether it is a protein having the above enzyme activity or
not may be determined by conventional techniques, such as by
expressing a target protein in a transformant produced by
introducing a gene coding for the target protein into Escherichia
coli or other host organisms, and analyzing the presence or absence
of the function of the target protein by the corresponding activity
measuring method.
[0097] The gene coding for the tPT family protein is not
particularly limited as long as it codes for the tPT family protein
to express and produce the tPT family protein. Specific examples of
the gene include the following DNAs [1] and [2]:
[1] a DNA having the nucleotide sequence represented by SEQ ID
NO:1; and [2] a DNA which hybridizes under stringent conditions to
a DNA having a nucleotide sequence complementary to the nucleotide
sequence represented by SEQ ID NO:1, and which codes for a protein
having an enzyme activity that catalyzes a reaction of trans-chain
elongation of an isoprenoid compound.
[0098] Herein, the term "hybridize" means a process in which a DNA
hybridizes to a DNA having a specific nucleotide sequence or a part
of the DNA. Thus, the DNA having a specific nucleotide sequence or
part of the DNA may have a nucleotide sequence long enough to be
usable as a probe in Northern or Southern blot analysis or as an
oligonucleotide primer in polymerase chain reaction (PCR) analysis.
The DNA to be used as a probe may have a length of at least 100
bases, preferably at least 200 bases, more preferably at least 500
bases although it may be a DNA of at least 10 bases, preferably at
least 15 bases.
[0099] Techniques to perform DNA hybridization experiments are well
known. The hybridization conditions under which experiments are
carried out may be determined in accordance with, for example,
Molecular Cloning, 2nd ed. and 3rd ed. (2001), Methods for General
and Molecular Bacteriology, ASM Press (1994), Immunology methods
manual, Academic press (Molecular), and many other standard
textbooks.
[0100] The stringent conditions may include, for example, an
overnight incubation at 42.degree. C. of a DNA-immobilized filter
and a DNA probe in a solution containing 50% formamide, 5.times.SSC
(750 mM sodium chloride, 75 mM sodium citrate), 50 mM sodium
phosphate (pH 7.6), 5.times.Denhardt's solution, 10% dextran
sulfate, and 20 .mu.g/L denatured salmon sperm DNA, followed by
washing the filter for example in a 0.2.times.SSC solution at
approximately 65.degree. C. Less stringent conditions may also be
used. Changes in stringency may be accomplished through the
manipulation of formamide concentration (lower percentages of
formamide result in lower stringency), salt concentrations or
temperature. For example, low stringent conditions include an
overnight incubation at 37.degree. C. in a solution containing
6.times.SSCE (20.times.SSCE: 3 mol/L sodium chloride, 0.2 mol/L
sodium dihydrogen phosphate, 0.02 mol/L EDTA, pH 7.4), 0.5% SDS,
30% formamide, and 100 .mu.g/L denatured salmon sperm DNA, followed
by washing in a 1.times.SSC solution containing 0.1% SDS at
50.degree. C. In addition, to achieve even lower stringency, washes
performed following hybridization may be done at higher salt
concentrations (e.g. 5.times.SSC) in the above-mentioned low
stringent conditions.
[0101] Variations in the above conditions may be accomplished
through the inclusion or substitution of blocking reagents used to
suppress background in hybridization experiments. The inclusion of
blocking reagents may require modification of the hybridization
conditions for compatibility.
[0102] The DNA capable of hybridization under stringent conditions
as described above may have a nucleotide sequence with at least
80%, preferably at least 90%, more preferably at least 95%, still
more preferably at least 98%, particularly preferably at least 99%
sequence identity to the nucleotide sequence represented by SEQ ID
NO:1 as calculated using a program such as BLAST or FASTA with the
parameters mentioned above.
[0103] Whether the DNA which hybridizes to the aforementioned DNA
under stringent conditions is a DNA coding for a protein having a
predetermined enzyme activity or not may be determined by
conventional techniques, such as by expressing a target protein in
a transformant produced by introducing a gene coding for the target
protein into Escherichia coli or other host organisms, and
analyzing the presence or absence of the function of the target
protein by the corresponding activity measuring method.
[0104] Conventional techniques may be employed to identify the
amino acid sequence or nucleotide sequence of the protein. For
example, total RNA is extracted from a growing plant, the mRNA is
optionally purified, and a cDNA is synthesized by a reverse
transcription reaction. Subsequently, degenerate primers are
designed based on the amino acid sequence of a known protein
corresponding to the target protein, a DNA fragment is partially
amplified by RT-PCR, and the sequence is partially identified.
Then, the full-length nucleotide sequence or amino acid sequence is
identified, e.g. by the RACE method. The RACE method (rapid
amplification of cDNA ends method) refers to a method in which,
when the nucleotide sequence of a cDNA is partially known, PCR is
performed based on the nucleotide sequence data of such a known
region to clone an unknown region extending to the cDNA terminal.
This method is capable of cloning full-length cDNA by PCR without
preparing a cDNA library.
[0105] The degenerate primers may each preferably be prepared from
a plant-derived sequence having a highly similar sequence part to
the target protein.
[0106] If the nucleotide sequence coding for the protein is known,
the full-length nucleotide sequence or amino acid sequence can be
identified by designing a primer containing a start codon and a
primer containing a stop codon using the known nucleotide sequence,
followed by performing RT-PCR using a synthesized cDNA as a
template.
[0107] In the binding step, additional proteins may further be
bound to the rubber particles as long as the protein expressed from
a gene coding for a trans-prenyltransferase (tPT) family protein is
bound to the rubber particles in vitro.
[0108] The origin of the additional proteins is not particularly
limited, but preferably the additional proteins are derived from
any of the plants mentioned above, more preferably rubber-producing
plants, still more preferably at least one selected from the group
consisting of plants of the genera Hevea, Sonchus, Taraxacum, and
Parthenium. In particular, they are further preferably derived from
at least one species of plant selected from the group consisting of
Hevea brasiliensis, Sonchus oleraceus, Parthenium argentatum, and
Taraxacum kok-saghyz, particularly preferably from Hevea
brasiliensis.
[0109] The additional proteins are not limited and may each be any
protein, but in view of rubber synthesis activity of the rubber
particles, they are each preferably a protein that inherently
exists on rubber particles in a rubber-producing plant. The protein
that exists on rubber particles may be a protein bound to a large
part of the membrane surface of rubber particles, or a protein
inserted into and bound to the membrane of rubber particles, or a
protein that forms a complex with another protein bound to the
membrane to exist on the membrane surface.
[0110] Examples of the protein that inherently exists on rubber
particles in a rubber-producing plant include Nogo-B receptor
(NgBR), rubber elongation factor (REF), small rubber particle
protein (SRPP), .beta.-1,3-glucanase, and Hevein.
[0111] The binding step may be carried out by any method that can
bind a tPT family protein to rubber particles in vitro, such as,
for example, by performing protein synthesis in the presence of
both rubber particles and a cell-free protein synthesis solution
containing an mRNA coding for a tPT family protein to bind the tPT
family protein to the rubber particles.
[0112] The binding step preferably includes performing protein
synthesis in the presence of both rubber particles and a cell-free
protein synthesis solution containing an mRNA coding for a tPT
family protein to bind the tPT family protein to the rubber
particles, among other methods.
[0113] In other words, it is preferred to obtain rubber particles
bound to a tPT family protein by performing protein synthesis in
the presence of both rubber particles and a cell-free protein
synthesis solution containing an mRNA coding for the tPT family
protein (more specifically, using a mixture of rubber particles
with a cell-free protein synthesis solution containing an mRNA
coding for the tPT family protein).
[0114] Since liposomes are artificially produced as lipid bilayer
membranes formed of phospholipids, glyceroglycolipids, cholesterol,
or other components, no protein is bound to the surface of the
produced liposomes. In contrast, although rubber particles
collected from the latex of rubber-producing plants are also coated
with a lipid membrane, the membrane of the rubber particles is a
naturally derived membrane in which proteins that have been
synthesized in the plants are already bound to the surface of the
membrane. In view of this, it is expected to be more difficult to
bind an additional protein to rubber particles that are already
bound to and coated with proteins than to bind it to liposomes not
bound to any protein. There is also concern that the proteins
already bound to rubber particles could inhibit cell-free protein
synthesis. For these reasons, difficulties have been anticipated in
performing cell-free protein synthesis in the presence of rubber
particles. Under such circumstances, the present inventors have
conducted cell-free synthesis of a tPT family protein in the
presence of rubber particles, which had never been attempted in the
past, and it has been found that with this method, it is possible
to produce rubber particles bound to a tPT family protein.
[0115] The protein synthesis in the presence of both rubber
particles and a cell-free protein synthesis solution containing an
mRNA coding for a tPT family protein is namely the synthesis of a
tPT family protein by cell-free protein synthesis, and the
synthesized tPT family protein maintains its biological function
(native state). As the cell-free protein synthesis is performed in
the presence of rubber particles, the synthesized tPT family
protein in its native state can be bound to the rubber
particles.
[0116] The binding of a tPT family protein to rubber particles by
protein synthesis in the presence of both the cell-free protein
synthesis solution and the rubber particles means that, for
example, each tPT family protein synthesized by the protein
synthesis is fully or partially incorporated into the rubber
particles or inserted into the membrane structure of the rubber
particles. It is not limited to these embodiments and also
includes, for example, embodiments in which the protein is
localized on the surface or inside of the rubber particles.
Moreover, the concept of binding to rubber particles also includes
embodiments in which the protein forms a complex with another
protein bound to the rubber particles as described above to exist
in the form of the complex on the rubber particles.
[0117] Each mRNA coding for a tPT family protein serves as a
translation template that can be translated to synthesize the tPT
family protein.
[0118] The origin of the mRNA coding for a tPT family protein is
not particularly limited, and the mRNA may be derived from a
microorganism, an animal, or a plant, preferably a plant, more
preferably any of the plants mentioned above, still more preferably
a rubber-producing plant, further preferably at least one selected
from the group consisting of plants of the genera Hevea, Sonchus,
Taraxacum, and Parthenium. In particular, it is especially
preferably derived from at least one species of plant selected from
the group consisting of Hevea brasiliensis, Sonchus oleraceus,
Parthenium argentatum, and Taraxacum kok-saghyz, most preferably
from Hevea brasiliensis.
[0119] The mRNA coding for a tPT family protein may be prepared by
any method as long as the prepared mRNA serves as a translation
template that can be translated to synthesize the tPT family
protein. For example, the mRNA may be prepared by extracting total
RNA from the latex of a rubber-producing plant by, for example, the
hot phenol method, synthesizing cDNA from the total RNA, obtaining
a DNA fragment of a gene coding for a tPT family protein using
primers prepared based on the nucleotide sequence data of the gene
coding for a tPT family protein, and performing an ordinary in
vitro transcription reaction of the DNA fragment.
[0120] As long as the cell-free protein synthesis solution contains
the mRNA coding for a tPT family protein, it may contain mRNAs
coding for additional proteins.
[0121] The mRNAs coding for additional proteins may be ones that
can be translated to express the additional proteins. The
additional proteins may be as described above.
[0122] In the binding step in the first invention, cell-free
synthesis of a tPT family protein is preferably performed in the
presence of rubber particles. The cell-free protein synthesis may
be carried out using the cell-free protein synthesis solution in a
similar manner to conventional methods. The cell-free protein
synthesis system used may be a common cell-free protein synthesis
means, such as rapid translation system RTS500 (Roche Diagnostics);
or wheat germ extracts prepared in accordance with Proc. Natl.
Acad. Sci. USA, 97:559-564 (2000), JP 2000-236896 A, JP 2002-125693
A, and JP 2002-204689 A, or cell-free protein synthesis systems
using the wheat germ extracts (JP 2002-204689 A, Proc. Natl. Acad.
Sci. USA, 99:14652-14657 (2002)). Systems using germ extracts are
preferred among these. Thus, in another suitable embodiment of the
first invention, the cell-free protein synthesis solution contains
a germ extract.
[0123] The source of the germ extract is not particularly limited.
From the standpoint of translation efficiency, it is preferred to
use a plant-derived germ extract for cell-free protein synthesis of
a plant protein. It is particularly preferred to use a
wheat-derived germ extract. Thus, in another suitable embodiment of
the first invention, the germ extract is derived from wheat.
[0124] The method for preparing the germ extract is not
particularly limited, and may be carried out conventionally, as
described in, for example, JP 2005-218357 A.
[0125] The cell-free protein synthesis solution preferably further
contains a cyclic nucleoside monophosphate derivative or a salt
thereof (hereinafter, also referred to simply as "activity
enhancer"). Protein synthesis activity can be further enhanced by
the inclusion of the activity enhancer.
[0126] The cyclic nucleoside monophosphate derivative or salt
thereof is not particularly limited as long as it can enhance
cell-free protein synthesis activity. Examples include
adenosine-3',5'-cyclic monophosphoric acid and its salts;
adenosine-3',5'-cyclic monophosphorothioic acid (Sp-isomer) and its
salts; adenosine-3',5'-cyclic monophosphorothioic acid (Rp-isomer)
and its salts; guanosine-3',5'-cyclic monophosphoric acid and its
salts; guanosine-3',5'-cyclic monophosphorothioic acid (Sp-isomer)
and its salts; guanosine-3',5'-cyclic monophosphorothioic acid
(Rp-isomer) and its salts; 8-bromoadenosine-3',5'-cyclic
monophosphoric acid (bromo-cAMP) and its salts;
8-(4-chlorophenylthio)adenosine-3',5'-cyclic monophosphoric acid
(chlorophenylthio-cAMP) and its salts;
5,6-dichloro-1-.beta.-D-ribofuranosylbenzimidazole
adenosine-3',5'-cyclic monophosphoric acid
(dichlororibofuranosylbenzimidazole cAMP) and its salts;
adenosine-2',5'-cyclic monophosphoric acid and its salts;
adenosine-2',5'-cyclic monophosphorothioic acid (Sp-isomer) and its
salts; adenosine-2',5'-cyclic monophosphorothioic acid (Rp-isomer)
and its salts; guanosine-2',5'-cyclic monophosphoric acid and its
salts; guanosine-2',5'-cyclic monophosphorothioic acid (Sp-isomer)
and its salts; and guanosine-2',5'-cyclic monophosphorothioic acid
(Rp-isomer) and its salts.
[0127] The base that forms a salt with the cyclic nucleoside
monophosphate derivative is not particularly limited as long as it
is biochemically acceptable and forms a salt with the derivative.
Preferred are, for example, alkali metal atoms such as sodium or
potassium, and organic bases such as tris-hydroxyaminomethane,
among others.
[0128] Of these activity enhancers, adenosine-3',5'-cyclic
monophosphoric acid or adenosine-3',5'-cyclic monophosphate sodium
salt is particularly preferred. These activity enhancers may be
used alone or in combinations of two or more.
[0129] The activity enhancer may be added to the cell-free protein
synthesis solution in advance. If the activity enhancer is unstable
in the solution, it is preferably added during the protein
synthesis reaction performed in the presence of both the cell-free
protein synthesis solution and rubber particles.
[0130] The amount of the activity enhancer added is not
particularly limited as long as the activity enhancer is at a
concentration that can activate (increase) the protein synthesis
reaction in the cell-free protein synthesis solution. Specifically,
the final concentration in the reaction system may usually be at
least 0.1 millimoles/liter. The lower limit of the concentration is
preferably 0.2 millimoles/liter, more preferably 0.4
millimoles/liter, particularly preferably 0.8 millimoles/liter,
while the upper limit of the concentration is preferably 24
millimoles/liter, more preferably 6.4 millimoles/liter,
particularly preferably 3.2 millimoles/liter.
[0131] The temperature of the cell-free protein synthesis solution
to which the activity enhancer is added is not particularly
limited, but is preferably 0 to 30.degree. C., more preferably 10
to 26.degree. C.
[0132] In addition to the mRNA (translation template) coding for a
tPT family protein, the cell-free protein synthesis solution also
contains ATP, GTP, creatine phosphate, creatine kinase, L-amino
acids, potassium ions, magnesium ions, and other components
required for protein synthesis, and optionally an activity
enhancer. Such a cell-free protein synthesis solution can serve as
a cell-free protein synthesis reaction system.
[0133] Since the germ extract prepared as described in JP
2005-218357 A contains tRNA in an amount necessary for protein
synthesis reaction, addition of separately prepared tRNA is not
required when the germ extract prepared as above is used in the
cell-free protein synthesis solution. In other words, tRNA may be
added to the cell-free protein synthesis solution, if
necessary.
[0134] The binding step in the first invention preferably includes
performing protein synthesis in the presence of both rubber
particles and a cell-free protein synthesis solution containing an
mRNA coding for a tPT family protein. Specifically, this can be
accomplished by adding rubber particles to the cell-free protein
synthesis solution at a suitable point either before or after
protein synthesis, preferably before protein synthesis.
[0135] The rubber particles are preferably present in the cell-free
protein synthesis solution at a concentration of 5 to 50 g/L. In
other words, 5 to 50 g of rubber particles are preferably present
in 1 L of the cell-free protein synthesis solution. If the
concentration of rubber particles present in the cell-free protein
synthesis solution is less than 5 g/L, a rubber layer may not be
formed by separation treatment (e.g., ultracentrifugation) for
collecting the rubber particles bound to the synthesized tPT family
protein, and therefore it may be difficult to collect the rubber
particles bound to the synthesized tPT family protein. Moreover, if
the concentration of rubber particles present in the cell-free
protein synthesis solution exceeds 50 g/L, the rubber particles may
coagulate, so that the synthesized tPT family protein may fail to
bind well to the rubber particles. The concentration of rubber
particles is more preferably 10 to 40 g/L, still more preferably 15
to 35 g/L, particularly preferably 15 to 30 g/L.
[0136] In the protein synthesis in the presence of both rubber
particles and the cell-free protein synthesis solution, additional
rubber particles may be appropriately added as the reaction
progresses. The cell-free protein synthesis solution and rubber
particles are preferably present together during the period when
the cell-free protein synthesis system is active, such as 3 to 48
hours, preferably 3 to 30 hours, more preferably 3 to 24 hours
after the addition of rubber particles to the cell-free protein
synthesis solution.
[0137] The rubber particles do not have to be subjected to any
treatment, e.g., pretreatment, before use in the binding step in
the first invention, preferably before being combined with the
cell-free protein synthesis solution. However, proteins may be
removed from the rubber particles with a surfactant beforehand to
increase the proportion of the tPT family protein desired to be
bound by the method of the first invention, among the proteins
present on the rubber particles. Thus, in another suitable
embodiment of the first invention, the rubber particles used in the
first invention are washed with a surfactant before use in the
binding step in the first invention, preferably before being
combined with the cell-free protein synthesis solution.
[0138] The surfactant is not particularly limited, and examples
include nonionic surfactants and amphoteric surfactants. Nonionic
or amphoteric surfactants, among others, are suitable because they
have only a little denaturing effect on the proteins on the
membrane, and amphoteric surfactants are especially suitable. Thus,
in another suitable embodiment of the first invention, the
surfactant is an amphoteric surfactant.
[0139] These surfactants may be used alone or in combinations of
two or more.
[0140] Examples of the nonionic surfactants include polyoxyalkylene
ether nonionic surfactants, polyoxyalkylene ester nonionic
surfactants, polyhydric alcohol fatty acid ester nonionic
surfactants, sugar fatty acid ester nonionic surfactants, alkyl
polyglycoside nonionic surfactants, and polyoxyalkylene
polyglucoside nonionic surfactants; and polyoxyalkylene alkylamines
and alkyl alkanolamides.
[0141] Polyoxyalkylene ether or polyhydric alcohol fatty acid ester
nonionic surfactants are preferred among these.
[0142] Examples of the polyoxyalkylene ether nonionic surfactants
include polyoxyalkylene alkyl ethers, polyoxyalkylene alkylphenyl
ethers, polyoxyalkylene polyol alkyl ethers, and polyoxyalkylene
mono-, di- or tristyryl phenyl ethers. Among these, polyoxyalkylene
alkylphenyl ethers are suitable. The "polyol" is preferably a
C2-C12 polyhydric alcohol, such as ethylene glycol, propylene
glycol, glycerin, sorbitol, glucose, sucrose, pentaerythritol, or
sorbitan.
[0143] Examples of the polyoxyalkylene ester nonionic surfactants
include polyoxyalkylene fatty acid esters and polyoxyalkylene alkyl
rosin acid esters.
[0144] Examples of the polyhydric alcohol fatty acid ester nonionic
surfactants include fatty acid esters of C2-C12 polyhydric alcohols
and fatty acid esters of polyoxyalkylene polyhydric alcohols. More
specific examples include sorbitol fatty acid esters, sorbitan
fatty acid esters, glycerin fatty acid esters, polyglycerin fatty
acid esters, and pentaerythritol fatty acid esters, as well as
polyalkylene oxide adducts of the foregoing such as polyoxyalkylene
sorbitan fatty acid esters and polyoxyalkylene glycerin fatty acid
esters. Among these, sorbitan fatty acid esters are suitable.
[0145] Examples of the sugar fatty acid ester nonionic surfactants
include fatty acid esters of sucrose, glucose, maltose, fructose,
and polysaccharides, as well as polyalkylene oxide adducts of the
foregoing.
[0146] Examples of the alkyl polyglycoside nonionic surfactants
include those having, for example, glucose, maltose, fructose, or
sucrose as the glycoside, such as alkyl glucosides, alkyl
polyglucosides, polyoxyalkylene alkyl glucosides, and
polyoxyalkylene alkyl polyglucosides, as well as fatty acid esters
of the foregoing. Polyalkylene oxide adducts of any of the
foregoing may also be used.
[0147] Examples of the alkyl groups in these nonionic surfactants
include C4-C30 linear or branched, saturated or unsaturated alkyl
groups. The polyoxyalkylene groups may have C2-C4 alkylene groups,
and may have about 1 to 50 moles of added ethylene oxide, for
example. Examples of the fatty acids include C4-C30 linear or
branched, saturated or unsaturated fatty acids.
[0148] Of the nonionic surfactants, polyoxyethyleneethylene (10)
octylphenyl ether (Triton X-100) or sorbitan monolaurate (Span 20)
is particularly preferred for their ability to moderately remove
membrane-associated proteins while keeping the membrane of rubber
particles stable and, further, having only a little denaturing
effect on the proteins.
[0149] Examples of the amphoteric surfactants include zwitterionic
surfactants such as quaternary ammonium salt group/sulfonate group
(--SO.sub.3H) surfactants, water-soluble quaternary ammonium salt
group/phosphate group surfactants, water-insoluble quaternary
ammonium salt group/phosphate group surfactants, and quaternary
ammonium salt group/carboxyl group surfactants. The acid group in
each of these zwitterionic surfactants may be a salt.
[0150] In particular, such a zwitterionic surfactant preferably has
both positive and negative charges in a molecule. The acid
dissociation constant (pKa) of the acid group is preferably 5 or
less, more preferably 4 or less, still more preferably 3 or
less.
[0151] Specific examples of the amphoteric surfactants include
ammonium sulfobetaines such as
3-[(3-cholamidopropyl)dimethylamino]-2-hydroxy-1-propanesulfonate
(CHAPSO), 3-[(3-cholamidopropyl)-dimethylamino]-propanesulfonate
(CHAPS), N,N-bis(3-D-gluconamidopropyl)-cholamide,
n-octadecyl-N,N'-dimethyl-3-amino-1-propanesulfonate,
n-decyl-N,N'-dimethyl-3-amino-1-propanesulfonate,
n-dodecyl-N,N'-dimethyl-3-amino-1-propanesulfonate,
n-tetradecyl-N,N'-dimethyl-3-amino-1-propanesulfonate
(Zwittergent.TM.-3-14),
n-hexadecyl-N,N'-dimethyl-3-amino-1-propanesulfonate, and
n-octadecyl-N,N'-dimethyl-3-amino-1-propanesulfonate;
phosphocholines such as n-octylphosphocholine,
n-nonylphosphocholine, n-decylphosphocholine,
n-dodecylphosphocholine, n-tetradecylphosphocholine, and
n-hexadecylphosphocholine; and phosphatidylcholines such as
dilauroyl phosphatidylcholine, dimyristoyl phosphatidylcholine,
dipalmitoyl phosphatidylcholine, distearoyl phosphatidylcholine,
dioleoyl phosphatidylcholine, and dilinolenoyl phosphatidylcholine.
Of these, 3-[(3-cholamidopropyl)dimethylamino]-propanesulfonate
(CHAPS) is particularly preferred for its ability to moderately
remove proteins while keeping the membrane of rubber particles
stable.
[0152] The concentration of the surfactant for the treatment is
preferably within three times the critical micelle concentration
(CMC) of the surfactant used. The membrane stability of the rubber
particles may be reduced if they are treated with the surfactant at
a concentration exceeding three times the critical micelle
concentration. The concentration is more preferably within 2.5
times, still more preferably within 2.0 times the CMC. The lower
limit of the concentration is preferably at least 0.05 times, more
preferably at least 0.1 times, still more preferably at least 0.3
times the CMC.
[0153] Examples of protein synthesis protein synthesis reaction
systems or apparatuses for protein synthesis that can be used in
the cell-free protein synthesis include a batch method (Pratt, J.
M. et al., Transcription and Translation, Hames, 179-209, B. D.
& Higgins, S. J., eds, IRL Press, Oxford (1984)), a continuous
cell-free protein synthesis system in which amino acids, energy
sources, and other components are supplied continuously to the
reaction system (Spirin, A. S. et al., Science, 242, 1162-1164
(1988)), a dialysis method (Kigawa et al., 21st Annual Meeting of
the Molecular Biology Society of Japan, WID 6), and an overlay
method (instruction manual of PROTEIOS.TM. wheat germ cell-free
protein synthesis core kit, Toyobo Co., Ltd.). Another method may
be to supply template RNA, amino acids, energy sources, and other
components, if necessary, to the protein synthesis reaction system,
and discharge the synthesis product or decomposition product as
required.
[0154] Among these, the dialysis method is preferred. The reason
for this is as follows. The overlay method has the advantage of
easy operation, but unfortunately rubber particles disperse in the
reaction solution and thus are difficult to efficiently bind to the
synthesized tPT family protein. In contrast, in the dialysis
method, since the amino acids used as raw materials of the tPT
family protein to be synthesized can pass through the dialysis
membrane while rubber particles cannot pass therethrough, it is
possible to prevent dispersal of rubber particles and thus to
efficiently bind the synthesized tPT family protein to rubber
particles.
[0155] The dialysis method refers to a method in which protein
synthesis is carried out using the reaction solution for the
cell-free protein synthesis as an internal dialysis solution, and
an apparatus in which the internal dialysis solution is separated
from an external dialysis solution by a dialysis membrane capable
of mass transfer. Specifically, for example, a translation template
is added to the synthesis reaction solution excluding the
translation template, optionally after pre-incubation for an
appropriate amount of time, and then the solution is put in an
appropriate dialysis container as the internal reaction solution.
Examples of the dialysis container include containers with a
dialysis membrane attached to the bottom (e.g., Dialysis Cup 12,000
available from Daiichi Kagaku) and dialysis tubes (e.g., 12,000
available from Sanko Junyaku Co., Ltd.). The dialysis membrane used
may have a molecular weight cutoff of 10,000 daltons or more,
preferably about 12,000 daltons.
[0156] The external dialysis solution used may be a buffer
containing amino acids. Dialysis efficiency can be increased by
replacing the external dialysis solution with a fresh one when the
reaction speed declines. The reaction temperature and time are
selected appropriately according to the protein synthesis system
used. For example, in the case of a system using a wheat-derived
germ extract, the reaction may be carried out usually at 10 to
40.degree. C., preferably 18 to 30.degree. C., more preferably 20
to 26.degree. C., for 10 minutes to 48 hours, preferably for 10
minutes to 30 hours, more preferably for 10 minutes to 24
hours.
[0157] Since the mRNA coding for a tPT family protein contained in
the cell-free protein synthesis solution is easily broken down, the
mRNA may be additionally added as appropriate during the protein
synthesis reaction to make the protein synthesis more efficient.
Thus, in another suitable embodiment of the first invention, the
mRNA coding for a tPT family protein is additionally added during
the protein synthesis reaction.
[0158] The addition time, the number of additions, the addition
amount, and other conditions of the mRNA are not particularly
limited, and may be selected appropriately.
[0159] In the production method of the first invention, the step of
collecting the rubber particles may optionally be performed after
the step of binding a protein expressed from a gene coding for a
trans-prenyltransferase (tPT) family protein to rubber particles in
vitro.
[0160] The rubber particle collection step may be carried out by
any method that can collect the rubber particles. It may be carried
out by conventional methods for collecting rubber particles.
Specific examples include methods using centrifugation. When the
rubber particles are collected by the centrifugation methods, the
centrifugal force, centrifugation time, and centrifugation
temperature may be selected appropriately so as to be able to
collect the rubber particles. For example, the centrifugal force
during the centrifugation is preferably 15000.times.g or more, more
preferably 20000.times.g or more, still more preferably
25000.times.g or more. Moreover, since increasing the centrifugal
force too much is not expected to produce a correspondingly high
separation effect, the upper limit of the centrifugal force is
preferably 50000.times.g or less, more preferably 45000.times.g or
less. The centrifugation time is preferably at least 20 minutes,
more preferably at least 30 minutes, still more preferably at least
40 minutes. Moreover, since increasing the centrifugation time too
much is not expected to produce a correspondingly high separation
effect, the upper limit of the centrifugation time is preferably
120 minutes or less, more preferably 90 minutes or less.
[0161] From the standpoint of maintaining the activity of the tPT
family protein bound to the rubber particles, the centrifugation
temperature is preferably 0 to 10.degree. C., more preferably 2 to
8.degree. C., particularly preferably 4.degree. C.
[0162] For example, when the cell-free protein synthesis is
performed, the rubber particles and the cell-free protein synthesis
solution are separated into the upper and lower layers,
respectively, by the centrifugation. The cell-free protein
synthesis solution as the lower layer may then be removed to
collect the rubber particles bound to the tPT family protein. The
collected rubber particles may be re-suspended in an appropriate
buffer with a neutral pH for storage.
[0163] The rubber particles collected by the rubber particle
collection step can be used in the same way as usual natural rubber
without the need for further special treatment.
[0164] Moreover, the trans-polyisoprenoid produced by the method
for producing a trans-polyisoprenoid of the first invention can be
recovered by subjecting the rubber particles to the solidification
step described below.
[0165] The method for solidification in the solidification step is
not particularly limited, and examples include a method of adding
the rubber particles to a solvent that does not dissolve the
trans-polyisoprenoid (trans rubber), such as ethanol, methanol, or
acetone; and a method of adding an acid to the rubber particles.
Rubber can be recovered as solids from the rubber particles by the
solidification step. The obtained rubber may be dried if necessary
before use.
[0166] Thus, according to the first invention, by binding a protein
expressed from a gene coding for a trans-prenyltransferase (tPT)
family protein to rubber particles in vitro, trans rubber can be
synthesized in the rubber particles, and therefore it is possible
to efficiently produce trans rubber (one example of
trans-polyisoprenoid) in a reaction vessel (e.g., a test tube or
industrial plant).
[0167] Thus, another aspect of the first invention relates to a
method for synthesizing a trans-polyisoprenoid, which includes
binding a protein expressed from a gene coding for a
trans-prenyltransferase (tPT) family protein to rubber particles in
vitro, for example in a reaction vessel (e.g., a test tube or
industrial plant).
[0168] The step of binding a protein expressed from a gene coding
for a trans-prenyltransferase (tPT) family protein to rubber
particles in vitro is as described above.
[0169] Herein, the term "trans-polyisoprenoid" is a collective term
for polymers containing trans-linked isoprene units
(C.sub.5H.sub.8) (in particular, the content of trans bonds is
preferably at least 90%, more preferably at least 95%, still more
preferably at least 97% of the total bonds). Examples of the
trans-polyisoprenoid include trans-sesterterpenes (C.sub.25),
trans-triterpenes (C.sub.30), trans-tetraterpenes (C.sub.40), trans
rubber such as trans-1,4-polyisoprene, and other polymers. Herein,
the term "isoprenoid" refers to a compound containing an isoprene
unit (C.sub.5H.sub.8), and conceptually includes
polyisoprenoids.
(Method for Producing Rubber Product)
[0170] The method for producing a rubber product of the first
invention includes: kneading a trans-polyisoprenoid produced by the
method for producing a trans-polyisoprenoid of the first invention
with an additive to obtain a kneaded mixture; forming a raw rubber
product from the kneaded mixture; and vulcanizing the raw rubber
product.
[0171] The rubber product is not particularly limited as long as it
is a rubber product that can be produced from rubber, preferably
natural rubber, and examples include pneumatic tires, rubber
rollers, rubber fenders, gloves, and medical rubber tubes.
[0172] In the case where the rubber product is a pneumatic tire; in
other words, in the case where the method for producing a rubber
product of the first invention is the method for producing a
pneumatic tire of the first invention, the raw rubber product
forming step corresponds to the step of building a green tire from
the kneaded mixture, and the vulcanization step corresponds to the
step of vulcanizing the green tire. Thus, the method for producing
a pneumatic tire of the first invention includes: kneading a
trans-polyisoprenoid produced by the method for producing a
trans-polyisoprenoid with an additive to obtain a kneaded mixture;
building a green tire from the kneaded mixture; and vulcanizing the
green tire.
<Kneading Step>
[0173] In the kneading step, the trans-polyisoprenoid produced by
the method for producing a trans-polyisoprenoid is kneaded with an
additive to obtain a kneaded mixture.
[0174] The additive is not particularly limited, and additives used
in production of rubber products may be used. For example, in the
case where the rubber product is a pneumatic tire, examples of the
additive include rubber components other than the
trans-polyisoprenoid, reinforcing fillers such as carbon black,
silica, calcium carbonate, alumina, clay, and talc, silane coupling
agents, zinc oxide, stearic acid, processing aids, various
antioxidants, softeners such as oils, waxes, vulcanizing agents
such as sulfur, and vulcanization accelerators.
[0175] In the kneading step, a rubber kneading machine such as an
open roll mill, a Banbury mixer, or an internal mixer may be used
to perform kneading.
<Raw Rubber Product Forming Step (Green Tire Building Step in
the Case of Tire))>
[0176] In the raw rubber product forming step, a raw rubber product
(green tire in the case of tire) is formed from the kneaded mixture
obtained in the kneading step.
[0177] The method for forming a raw rubber product is not
particularly limited. Methods used to form raw rubber products may
be used appropriately. For example, in the case where the rubber
product is a pneumatic tire, the kneaded mixture obtained in the
kneading step may be extruded according to the shape of a tire
component and then formed in a usual manner on a tire building
machine and assembled with other tire components to build a green
tire (unvulcanized tire).
<Vulcanization Step>
[0178] In the vulcanization step, the raw rubber product obtained
in the raw rubber product forming step is vulcanized to obtain a
rubber product.
[0179] The method for vulcanizing the raw rubber product is not
particularly limited. Methods used to vulcanize raw rubber products
may be used appropriately. For example, in the case where the
rubber product is a pneumatic tire, the green tire (unvulcanized
tire) obtained in the raw rubber product forming step may be
vulcanized by heating and pressing in a vulcanizer to obtain a
pneumatic tire.
Second Invention
(Vector)
[0180] The vector of the second invention contains a nucleotide
sequence in which a gene coding for a trans-prenyltransferase (tPT)
family protein is functionally linked to a promoter having a
promoter activity that drives laticifer-specific gene expression.
By introducing such a vector into a plant for transformation, the
gene coding for a protein involved in trans-polyisoprenoid
biosynthesis in the vector can be expressed specifically in
laticifers, thereby enhancing trans-isoprenoid or
trans-polyisoprenoid production in the plant. This is probably
because, if the expression of an exogenous gene introduced for the
purpose of enhancing latex productivity is promoted in sites other
than laticifers, a certain load is imposed on the metabolism or
latex production of the plant, thereby causing adverse effects.
[0181] Herein, "promoter having a promoter activity that drives
laticifer-specific gene expression" means that the promoter has
activity to control gene expression to cause a desired gene to be
expressed specifically in laticifers when the desired gene is
functionally linked to the promoter and introduced into a plant.
The term "laticifer-specific gene expression" means that the gene
is expressed substantially exclusively in laticifers with no or
little expression of the gene in sites other than laticifers in
plants. Also, "a gene is functionally linked to a promoter" means
that the gene sequence is linked downstream of the promoter so that
the gene is controlled by the promoter.
[0182] The vector of the second invention can be prepared by
inserting the nucleotide sequence of a promoter having a promoter
activity that drives laticifer-specific gene expression and the
nucleotide sequence of a gene coding for a trans-prenyltransferase
(tPT) family protein into a vector commonly known as a plant
transformation vector by conventional techniques. Examples of
vectors that can be used to prepare the vector of the second
invention include pBI vectors, binary vectors such as pGA482, pGAH,
and pBIG, intermediate plasmids such as pLGV23Neo, pNCAT, and
pMON200, and pH35GS containing GATEWAY cassette.
[0183] As long as the vector of the second invention contains the
nucleotide sequence of a promoter having a promoter activity that
drives laticifer-specific gene expression and the nucleotide
sequence of a gene coding for a trans-prenyltransferase (tPT)
family protein, it may contain additional nucleotide sequences.
Usually, the vector contains vector-derived sequences in addition
to these nucleotide sequences and further contains a restriction
enzyme recognition sequence, a spacer sequence, a marker gene
sequence, a reporter gene sequence, or other sequences.
[0184] Examples of the marker gene include drug-resistant genes
such as kanamycin-resistant gene, hygromycin-resistant gene, and
bleomycin-resistant gene. The reporter gene is intended to be
introduced to determine the expression site in a plant, and
examples include luciferase gene, .beta.-glucuronidase (GUS) gene,
green fluorescent protein (GFP), and red fluorescent protein
(RFP).
[0185] The origin of the gene coding for a trans-prenyltransferase
(tPT) family protein is not particularly limited. The gene may be
derived from a microorganism, an animal, or a plant, preferably a
plant, more preferably any of the plants mentioned above, still
more preferably a rubber-producing plant, further preferably at
least one selected from the group consisting of plants of the
genera Hevea, Sonchus, Taraxacum, and Parthenium. In particular, it
is especially preferably derived from at least one species of plant
selected from the group consisting of Hevea brasiliensis, Sonchus
oleraceus, Parthenium argentatum, and Taraxacum kok-saghyz, most
preferably from Hevea brasiliensis.
[0186] The gene coding for a trans-prenyltransferase (tPT) family
protein and the tPT family protein in the second invention are as
described above concerning the first invention.
[0187] As long as the vector of the second invention contains the
nucleotide sequence of a promoter having a promoter activity that
drives laticifer-specific gene expression and the nucleotide
sequence of a gene coding for a trans-prenyltransferase (tPT)
family protein, it may further contain the nucleotide sequences of
genes coding for additional proteins.
[0188] Examples of the genes coding for additional proteins include
those described above concerning the first invention.
[0189] The promoter having a promoter activity that drives
laticifer-specific gene expression is preferably at least one
selected from the group consisting of a promoter of a gene coding
for rubber elongation factor (REF), a promoter of a gene coding for
small rubber particle protein (SRPP), a promoter of a gene coding
for Hevein 2.1 (HEV2.1), and a promoter of a gene coding for MYC1
transcription factor (MYC1).
[0190] Herein, the term "rubber elongation factor (REF)" refers to
a rubber particle-associated protein that is bound to rubber
particles in the latex of rubber-producing plants such as Hevea
brasiliensis, and contributes to stabilization of the rubber
particles.
[0191] The term "small rubber particle protein (SRPP)" refers to a
rubber particle-associated protein that is bound to rubber
particles in the latex of rubber-producing plants such as Hevea
brasiliensis.
[0192] The term "Hevein 2.1 (HEV2.1)" refers to a protein that is
highly expressed in the laticifer cells of rubber-producing plants
such as Hevea brasiliensis. This protein is involved in coagulation
of rubber particles and has antifungal activity.
[0193] The term "MYC1 transcription factor (MYC1)" refers to a
transcription factor that is highly expressed in the latex of
rubber-producing plants such as Hevea brasiliensis and participates
in jasmonic acid signaling. The term "transcription factor" means a
protein having activity to increase or decrease, preferably
increase, gene transcription. In other words, MYC1 herein is a
protein having activity (transcription factor activity) to increase
or decrease, preferably increase, the transcription of a gene
coding for at least one protein among the proteins involved in
jasmonic acid signaling.
(Promoter of Gene Coding for Rubber Elongation Factor (REF))
[0194] The origin of the promoter of a gene coding for REF is not
particularly limited, but the promoter is preferably derived from
any of the plants mentioned above, more preferably a
rubber-producing plant, still more preferably at least one selected
from the group consisting of plants of the genera Hevea, Sonchus,
Taraxacum, and Parthenium. In particular, it is further preferably
derived from at least one species of plant selected from the group
consisting of Hevea brasiliensis, Sonchus oleraceus, Parthenium
argentatum, and Taraxacum kok-saghyz, particularly preferably from
Hevea brasiliensis.
[0195] The promoter of a gene coding for REF is preferably any one
of the following DNAs [A1] to [A3]:
[A1] a DNA having the nucleotide sequence represented by SEQ ID
NO:10; [A2] a DNA which hybridizes under stringent conditions to a
DNA having a nucleotide sequence complementary to the nucleotide
sequence represented by SEQ ID NO:10, and which has a promoter
activity that drives laticifer-specific gene expression; and [A3] a
DNA which has a nucleotide sequence with at least 60% sequence
identity to the nucleotide sequence represented by SEQ ID NO:10,
and which has a promoter activity that drives laticifer-specific
gene expression.
[0196] As used here, the term "hybridize" is as described above.
Also, the stringent conditions are as described above.
[0197] Like the DNAs capable of hybridization under stringent
conditions described above, it is known that promoters with
nucleotide sequences having certain sequence identities to the
original nucleotide sequence can also have promoter activity. In
order to maintain the promoter activity, the sequence identity to
the nucleotide sequence represented by SEQ ID NO:10 is at least
60%, preferably at least 80%, more preferably at least 90%, still
more preferably at least 95%, further preferably at least 98%,
particularly preferably at least 99%.
(Promoter of Gene Coding for SRPP)
[0198] The origin of the promoter of a gene coding for SRPP is not
particularly limited, but the promoter is preferably derived from
any of the plants mentioned above, more preferably a
rubber-producing plant, still more preferably at least one selected
from the group consisting of plants of the genera Hevea, Sonchus,
Taraxacum, and Parthenium. In particular, it is further preferably
derived from at least one species of plant selected from the group
consisting of Hevea brasiliensis, Sonchus oleraceus, Parthenium
argentatum, and Taraxacum kok-saghyz, particularly preferably from
Hevea brasiliensis.
[0199] The promoter of a gene coding for SRPP is preferably any one
of the following DNAs [B1] to [B3]:
[B1] a DNA having the nucleotide sequence represented by SEQ ID
NO:11; [B2] a DNA which hybridizes under stringent conditions to a
DNA having a nucleotide sequence complementary to the nucleotide
sequence represented by SEQ ID NO:11, and which has a promoter
activity that drives laticifer-specific gene expression; and [B3] a
DNA which has a nucleotide sequence with at least 60% sequence
identity to the nucleotide sequence represented by SEQ ID NO:11,
and which has a promoter activity that drives laticifer-specific
gene expression.
[0200] As used here, the term "hybridize" is as described above.
Also, the stringent conditions are as described above.
[0201] Like the DNAs capable of hybridization under stringent
conditions described above, it is known that promoters with
nucleotide sequences having certain sequence identities to the
original nucleotide sequence can also have promoter activity. In
order to maintain the promoter activity, the sequence identity to
the nucleotide sequence represented by SEQ ID NO: 11 is at least
60%, preferably at least 80%, more preferably at least 90%, still
more preferably at least 95%, further preferably at least 98%,
particularly preferably at least 99%.
(Promoter of Gene Coding for HEV2.1)
[0202] The origin of the promoter of a gene coding for HEV2.1 is
not particularly limited, but the promoter is preferably derived
from any of the plants mentioned above, more preferably a
rubber-producing plant, still more preferably at least one selected
from the group consisting of plants of the genera Hevea, Sonchus,
Taraxacum, and Parthenium. In particular, it is further preferably
derived from at least one species of plant selected from the group
consisting of Hevea brasiliensis, Sonchus oleraceus, Parthenium
argentatum, and Taraxacum kok-saghyz, particularly preferably from
Hevea brasiliensis.
[0203] The promoter of a gene coding for HEV2.1 is preferably any
one of the following DNAs [C1] to [C3]:
[C1] a DNA having the nucleotide sequence represented by SEQ ID
NO:12; [C2] a DNA which hybridizes under stringent conditions to a
DNA having a nucleotide sequence complementary to the nucleotide
sequence represented by SEQ ID NO:12, and which has a promoter
activity that drives laticifer-specific gene expression; and [C3] a
DNA which has a nucleotide sequence with at least 60% sequence
identity to the nucleotide sequence represented by SEQ ID NO:12,
and which has a promoter activity that drives laticifer-specific
gene expression.
[0204] As used here, the term "hybridize" is as described above.
Also, the stringent conditions are as described above.
[0205] Like the DNAs capable of hybridization under stringent
conditions described above, it is known that promoters with
nucleotide sequences having certain sequence identities to the
original nucleotide sequence can also have promoter activity. In
order to maintain the promoter activity, the sequence identity to
the nucleotide sequence represented by SEQ ID NO:12 is at least
60%, preferably at least 80%, more preferably at least 90%, still
more preferably at least 95%, further preferably at least 98%,
particularly preferably at least 99%.
(Promoter of Gene Coding for MYC1)
[0206] The origin of the promoter of a gene coding for MYC1 is not
particularly limited, but the promoter is preferably derived from
any of the plants mentioned above, more preferably a
rubber-producing plant, still more preferably at least one selected
from the group consisting of plants of the genera Hevea, Sonchus,
Taraxacum, and Parthenium. In particular, it is further preferably
derived from at least one species of plant selected from the group
consisting of Hevea brasiliensis, Sonchus oleraceus, Parthenium
argentatum, and Taraxacum kok-saghyz, particularly preferably from
Hevea brasiliensis.
[0207] The promoter of a gene coding for MYC1 is preferably any one
of the following DNAs [D1] to [D3]:
[D1] a DNA having the nucleotide sequence represented by SEQ ID
NO:13; [D2] a DNA which hybridizes under stringent conditions to a
DNA having a nucleotide sequence complementary to the nucleotide
sequence represented by SEQ ID NO:13, and which has a promoter
activity that drives laticifer-specific gene expression; and [D3] a
DNA which has a nucleotide sequence with at least 60% sequence
identity to the nucleotide sequence represented by SEQ ID NO:13,
and which has a promoter activity that drives laticifer-specific
gene expression.
[0208] As used here, the term "hybridize" is as described above.
Also, the stringent conditions are as described above.
[0209] Like the DNAs capable of hybridization under stringent
conditions described above, it is known that promoters with
nucleotide sequences having certain sequence identities to the
original nucleotide sequence can also have promoter activity. In
order to maintain the promoter activity, the sequence identity to
the nucleotide sequence represented by SEQ ID NO: 13 is at least
60%, preferably at least 80%, more preferably at least 90%, still
more preferably at least 95%, further preferably at least 98%,
particularly preferably at least 99%.
[0210] Whether the DNA which hybridizes to the above-mentioned DNA
under stringent conditions or the DNA having at least 60% sequence
identity to the above-mentioned DNA is a DNA having a promoter
activity that drives laticifer-specific gene expression or not may
be determined by conventional techniques, such as reporter assays
using .beta.-galactosidase, luciferase, green fluorescent protein
(GFP), and other protein genes as reporter genes.
[0211] Conventional techniques may be employed to identify the
nucleotide sequence of the promoter. For example, a genomic DNA is
extracted from a growing plant by the cetyl trimethyl ammonium
bromide (CTAB) method, then specific and random primers are
designed based on the known nucleotide sequence of the promoter,
and a gene including the promoter is amplified by TAIL (thermal
asymmetric interlaced)-PCR using the extracted genomic DNA as a
template to identify the nucleotide sequence.
[0212] The vector of the second invention (which contains a
nucleotide sequence in which a gene coding for a
trans-prenyltransferase (tPT) family protein is functionally linked
to a promoter having a promoter activity that drives
laticifer-specific gene expression) can be introduced into a plant
to produce a transgenic plant transformed to express a certain
protein involved in trans-polyisoprenoid biosynthesis specifically
in laticifers. In the transgenic plant in which the certain protein
involved in trans-polyisoprenoid biosynthesis is expressed
specifically in the laticifers, a certain function, e.g., enzyme
activity, of the protein newly expressed in the plant transfected
with the vector of the second invention is enhanced in the
laticifers to enhance a part of the trans-polyisoprenoid
biosynthesis pathway. Therefore, trans-isoprenoid or
trans-polyisoprenoid production can be enhanced in the plant.
[0213] The method for preparing the transgenic plant is explained
briefly below, though such a transgenic plant can be prepared by
conventional methods.
[0214] The plant into which the vector of the second invention is
to be introduced to produce the transgenic plant is not
particularly limited, but is preferably a rubber-producing plant,
among others, because improved trans-polyisoprenoid productivity
and increased trans-polyisoprenoid yield can be expected
particularly when a tPT family protein is expressed in plants
capable of biosynthesizing polyisoprenoids. In particular, it is
further preferably derived from at least one species of
rubber-producing plant selected from the group consisting of Hevea
brasiliensis, Sonchus oleraceus, Parthenium argentatum, and
Taraxacum kok-saghyz, particularly preferably from Hevea
brasiliensis.
[0215] The vector of the second invention may be introduced into a
plant (including plant cells, such as callus, cultured cells,
spheroplasts, or protoplasts) by any method that can introduce DNA
into plant cells. Examples include methods using Agrobacterium (JP
S59-140885 A, JP S60-70080 A, WO94/00977), electroporation (JP
S60-251887 A), and methods using particle guns (gene guns) (JP
2606856 B, JP 2517813 B). Among these, it is preferred to use a
method using Agrobacterium (Agrobacterium method) to introduce the
vector of the second invention into a plant to produce a transgenic
plant (transgenic plant cells).
[0216] In addition, the vector of the second invention may also be
introduced into, for example, an organism (e.g., a microorganism,
yeast, animal cell, or insect cell) or a part thereof, an organ, a
tissue, a cultured cell, a spheroplast, or a protoplast by any of
the above-described DNA introduction methods to produce a
trans-isoprenoid or trans-polyisoprenoid.
[0217] The transgenic plant (transgenic plant cells) can be
produced by the above or other methods. The transgenic plant
conceptually includes not only transgenic plant cells produced by
the above methods, but also all of their progeny or clones and even
progeny plants obtained by passaging the foregoing. Once obtaining
transgenic plant cells into which the vector of the second
invention has been introduced, progeny or clones can be produced
from the transgenic plant cells by sexual or asexual reproduction,
tissue culture, cell culture, cell fusion, or other techniques.
Moreover, the transgenic plant cells, or their progeny or clones
may be used to obtain reproductive materials (e.g., seeds, fruits,
cuttings, stem tubers, root tubers, shoots, adventitious buds,
adventitious embryos, callus, protoplasts), which can then be used
to produce the transgenic plant on a large scale.
[0218] Techniques to regenerate plants (transgenic plants) from
transgenic plant cells are already known; for example, Doi et al.
disclose techniques for eucalyptus (JP 2000-316403 A), Fujimura et
al. disclose techniques for rice (Fujimura et al., (1995), Plant
Tissue Culture Lett., vol. 2: p. 74-), Shillito et al. disclose
techniques for corn (Shillito et al., (1989), Bio/Technology, vol.
7: p. 581-), Visser et al. disclose techniques for potato (Visser
et al., (1989), Theor. Appl. Genet., vol. 78: p. 589-), and Akama
et al. disclose techniques for Arabidopsis thaliana (Akama et al.,
(1992), Plant Cell Rep., vol. 12: p. 7-). A person skilled in the
art can regenerate plants from the transgenic plant cells with
reference to these documents.
[0219] Whether a target protein gene is expressed in regenerated
plants or not may be determined by well-known methods. For example,
Western blot analysis may be used to assess the expression of a
target protein.
[0220] Seeds can be obtained from the transgenic plant, for
example, as follows: the transgenic plant is rooted in an
appropriate medium, transplanted to water-containing soil in a pot,
and grown under proper cultivation conditions to finally produce
seeds, which are then collected. Furthermore, plants can be grown
from seeds, for example, as follows: seeds obtained from the
transgenic plant as described above are sown in water-containing
soil and grown under proper cultivation conditions into plants.
[0221] According to the second invention, by introducing the vector
of the second invention into a plant, the gene coding for a protein
involved in trans-polyisoprenoid biosynthesis (particularly
preferably the gene coding for a tPT family protein) in the vector
can be expressed specifically in laticifers, thereby enhancing
trans-isoprenoid or trans-polyisoprenoid production in the plant.
Specifically, a trans-isoprenoid or trans-polyisoprenoid may be
produced by culturing, for example, transgenic plant cells produced
as described above, callus obtained from the transgenic plant
cells, or cells redifferentiated from the callus in an appropriate
medium, or by growing, for example, transgenic plants regenerated
from the transgenic plant cells, or plants grown from seeds
obtained from these transgenic plants under proper cultivation
conditions.
[0222] Thus, another aspect of the second invention relates to a
method for enhancing trans-isoprenoid production in a plant by
introducing the vector of the second invention into the plant.
Furthermore, another aspect of the second invention relates to a
method for enhancing trans-polyisoprenoid production in a plant by
introducing the vector of the second invention into the plant.
(Method for Producing Rubber Product)
[0223] The method for producing a rubber product of the second
invention includes: kneading a trans-polyisoprenoid produced by a
transgenic plant with an additive to obtain a kneaded mixture, the
transgenic plant being produced by introducing the vector of the
second invention into a plant; forming a raw rubber product from
the kneaded mixture; and vulcanizing the raw rubber product.
[0224] The rubber product is as described above concerning the
first invention.
[0225] In the case where the rubber product is a pneumatic tire; in
other words, in the case where the method for producing a rubber
product of the second invention is the method for producing a
pneumatic tire of the second invention, the raw rubber product
forming step corresponds to the step of building a green tire from
the kneaded mixture, and the vulcanization step corresponds to the
step of vulcanizing the green tire. Thus, the method for producing
a pneumatic tire of the second invention includes: kneading a
trans-polyisoprenoid produced by a transgenic plant with an
additive to obtain a kneaded mixture, the transgenic plant being
produced by introducing the vector of the second invention into a
plant; building a green tire from the kneaded mixture; and
vulcanizing the green tire.
<Kneading Step>
[0226] In the kneading step, a trans-polyisoprenoid produced by a
transgenic plant produced by introducing the vector of the second
invention into a plant is kneaded with an additive to obtain a
kneaded mixture.
[0227] The trans-polyisoprenoid produced by a transgenic plant
produced by introducing the vector of the second invention into a
plant can be obtained by harvesting latex from the transgenic
plant, and subjecting the latex to the solidification step
described below.
[0228] The method for harvesting latex from the transgenic plant is
not particularly limited, and ordinary harvesting methods may be
used. For example, latex may be harvested by collecting the
emulsion oozing out from the cuts in the trunk of the plant
(tapping), or the emulsion oozing out from the cut roots or other
parts of the transgenic plant, or by crushing the cut tissue
followed by extraction with an organic solvent.
<Solidification Step>
[0229] The harvested latex is subjected to a solidification step.
The method for solidification is not particularly limited, and
examples include a method of adding the latex to a solvent that
does not dissolve the trans-polyisoprenoid (trans rubber), such as
ethanol, methanol, or acetone; and a method of adding an acid to
the latex. Rubber can be recovered as solids from the latex by the
solidification step. The obtained rubber may be dried if necessary
before use.
[0230] The additive is not particularly limited, and additives used
in production of rubber products may be used. For example, in the
case where the rubber product is a pneumatic tire, examples of the
additive include rubber components other than the rubber obtained
from the latex, reinforcing fillers such as carbon black, silica,
calcium carbonate, alumina, clay, and talc, silane coupling agents,
zinc oxide, stearic acid, processing aids, various antioxidants,
softeners such as oils, waxes, vulcanizing agents such as sulfur,
and vulcanization accelerators.
[0231] In the kneading step, a rubber kneading machine such as an
open roll mill, a Banbury mixer, or an internal mixer may be used
to perform kneading.
<Raw Rubber Product Forming Step (Green Tire Building Step in
the Case of Tire)>
[0232] The raw rubber product forming step is as described above
concerning the first invention.
<Vulcanization Step>
[0233] The vulcanization step is as described above concerning the
first invention.
EXAMPLES
[0234] The present invention is specifically explained with
reference to examples, but the present invention is not limited to
these examples.
Example 1
[0235] [Extraction of Total RNA from Hevea Latex]
[0236] Total RNA was extracted from the latex of Hevea brasiliensis
by the hot phenol method. To 6 mL of the latex were added 6 mL of
100 mM sodium acetate buffer and 1 mL of a 10% SDS solution, and
then 12 mL of water-saturated phenol pre-heated at 65.degree. C.
The mixture was incubated for five minutes at 65.degree. C.,
agitated in a vortex mixer, and centrifuged at 7000 rpm for 10
minutes at room temperature. After the centrifugation, the
supernatant was transferred to a new tube, 12 mL of a
phenol:chloroform (1:1) solution was added, and they were agitated
by shaking for two minutes. After the agitation, the resulting
mixture was centrifuged again at 7000 rpm for 10 minutes at room
temperature. Then, the supernatant was transferred to a new tube,
12 mL of a chloroform:isoamyl alcohol (24:1) solution was added,
and they were agitated by shaking for two minutes. After the
agitation, the resulting mixture was centrifuged again at 7000 rpm
for 10 minutes at room temperature. Then, the supernatant was
transferred to a new tube, 1.2 mL of a 3M sodium acetate solution
and 13 mL of isopropanol were added, and they were agitated in a
vortex mixer. The resulting mixture was incubated for 30 minutes at
-20.degree. C. to precipitate total RNA. The incubated mixture was
centrifuged at 15000 rpm for 10 minutes at 4.degree. C., and the
supernatant was removed to collect a precipitate of total RNA. The
collected total RNA was washed twice with 70% ethanol, and then
dissolved in RNase-free water.
[Synthesis of cDNA from Total RNA]
[0237] cDNA was synthesized from the collected total RNA. The cDNA
synthesis was carried out using a PrimeScript II 1st strand cDNA
synthesis kit (Takara) in accordance with the manual.
[Acquisition of tPT Gene from cDNA]
[0238] The prepared 1st strand cDNA was used as a template to
obtain a tPT gene. PCR was performed using a KOD-plus-Neo (Toyobo
Co., Ltd.) in accordance with the manual. The PCR reaction involved
35 cycles with each cycle consisting of 10 seconds at 98.degree.
C., 30 seconds at 58.degree. C., and 1 minute at 68.degree. C.
[0239] The tPT gene was obtained using the following primers.
TABLE-US-00001 Primer 1:
5'-ctgtattttcagggcggatatgtttcttcgaccaaggcc-3' Primer 2:
5'-caaaactagtgcggccgcgctaatcaatccgttcgagattg-3'
[0240] A tPT gene (HbSDS) was prepared as described above. The
sequence of the gene was isolated to identify the full-length
nucleotide sequence. The nucleotide sequence of HbSDS is given by
SEQ ID NO: 1. The amino acid sequence of HbSDS estimated from the
nucleotide sequence is also given by SEQ ID NO:2.
[Vector Construction]
[0241] The obtained DNA fragment was subjected to dA addition and
then inserted into a pGEM-T Easy vector using a pGEM-T Easy Vector
System (Promega) to prepare pGEM-HbSDS.
[Transformation of Escherichia coli]
[0242] Escherichia coli DH5.alpha. was transformed with the
prepared vector, the transformant was cultured on LB agar medium
containing ampicillin and X-gal, and Escherichia coli cells
carrying the introduced target gene were selected by blue/white
screening.
[Plasmid Extraction]
[0243] The Escherichia coli cells transformed with the plasmid
containing the target gene were cultured overnight at 37.degree. C.
on LB liquid medium. After the culture, the cells were collected,
and the plasmid was collected using a FastGene Plasmid mini kit
(Nippon Genetics Co., Ltd.).
[0244] It was confirmed by sequence analysis that there were no
mutations in the nucleotide sequence of the collected gene inserted
in the plasmid.
[Preparation of Vector for Cell-Free Protein Synthesis]
[0245] The pGEM-HbSDS acquired in the above [Vector construction]
was treated with the restriction enzyme BstZ I, and then a HbSDS
fragment was collected. The fragment was mixed with a
pEU-E01-His-TEV-MCS-N2 cell-free expression vector that had been
treated with the restriction enzymes EcoR I and Kpn I, and they
were connected by in vitro homologous recombination to prepare
pEU-His-N2-HbSDS.
[Transformation of Escherichia coli]
[0246] Escherichia coli DH5.alpha. was transformed with the
prepared vector, the transformant was cultured on LB agar medium
containing ampicillin and X-gal, and Escherichia coli cells
carrying the introduced target gene were screened by colony
PCR.
[Plasmid Extraction]
[0247] The Escherichia coli cells transformed with the plasmid
containing the target gene were cultured overnight at 37.degree. C.
on LB liquid medium. After the culture, the cells were collected,
and the plasmid was collected using a FastGene Plasmid mini kit
(Nippon Genetics Co., Ltd.).
[Preparation of Rubber Particles]
[0248] Rubber particles were prepared from Hevea latex by five
stages of centrifugation. To 900 mL of Hevea latex was added 100 mL
of 1 M Tris buffer (pH 7.5) containing 20 mM dithiothreitol (DTT)
to prepare a latex solution. The latex solution was centrifuged in
stages at the following different speeds: 1000.times.g,
2000.times.g, 8000.times.g, 20000.times.g, and 50000.times.g. Each
stage of centrifugation was carried out for 45 minutes at 4.degree.
C. To the rubber particle layer left after the centrifugation at
50000.times.g was added
3-[(3-cholamidopropyl)dimethylamino]-propanesulfonate (CHAPS) at a
final concentration of 0.1 to 2.0.times.CMC (0.1 to 2.0 times the
critical micelle concentration CMC) to wash the rubber particles.
After the washing, the rubber particles were collected by
ultracentrifugation (40000.times.g, 4.degree. C., 45 minutes) and
re-suspended in an equal amount of 100 M Tris buffer (pH 7.5)
containing 2 mM dithiothreitol (DTT).
[Cell-Free Protein Synthesis Reaction (Step 1: mRNA Transcription
Reaction)]
[0249] Cell-free protein synthesis was performed using a WEPRO7240H
expression kit (CellFree Sciences Co., Ltd.). An mRNA transcription
reaction was performed using the vector acquired in the above
[Preparation of vector for cell-free protein synthesis] as a
template in accordance with the protocol of the WEPRO7240H
expression kit.
[Purification of mRNA]
[0250] After the transcription reaction, the resulting mRNA was
purified by ethanol precipitation.
[Cell-Free Protein Synthesis Reaction (Step 2: Protein Synthesis by
Dialysis)]
[0251] The following amounts of materials were added to a dialysis
cup (MWCO 12000, Bio-Teck). A total amount of 60 .mu.L of a
reaction solution was prepared according to the protocol of the
WEPRO7240H expression kit. To the reaction solution was added 1 to
2 mg of the rubber particles. Separately, 650 .mu.L of SUB-AMIX was
added to a No. 2 PP container (Maruemu container).
[0252] The dialysis cup was set in the No. 2 PP container, and a
protein synthesis reaction was initiated at 26.degree. C. The
addition of the mRNA and the replacement of the external dialysis
solution (SUB-AMIX) were performed twice after the initiation of
the reaction. The reaction was carried out for 24 hours. FIG. 3
shows an outline diagram illustrating the dialysis process.
[Collection of Reacted Rubber Particles]
[0253] The solution in the dialysis cup was transferred to a new
1.5 .mu.L tube, and the reacted rubber particles were collected by
ultracentrifugation (40000.times.g, 4.degree. C., 45 minutes) and
re-suspended in an equal amount of 100 M Tris buffer (pH 7.5)
containing 2 mM dithiothreitol (DTT).
[Measurement of Rubber Synthesis Activity of Reacted Rubber
Particles]
[0254] The rubber synthesis activity of the collected reacted
rubber particles was measured as follows.
[0255] First, 50 mM Tris-HCl (pH 7.5), 2 mM DTT, 5 mM MgCl.sub.2,
15 .mu.M farnesyl diphosphate (FPP), 100 .mu.M 1-14C isopentenyl
diphosphate ([1-14C]IPP, specific activity 5 Ci/mol), and 10 .mu.L
of the rubber particle solution were mixed to prepare a reaction
solution (100 .mu.L in total), which was then reacted for 16 hours
at 30.degree. C.
[0256] After the reaction, 200 .mu.L of saturated NaCl was added to
the solution, and isopentenol and the like were extracted from the
mixture with 1 mL of diethyl ether. Next, polyprenyl diphosphates
were extracted from the aqueous phase with 1 mL of BuOH saturated
with saline, and then a very long chain polyisoprenoid
(trans-1,4-polyisoprene) was further extracted from the aqueous
phase with 1 mL of toluene/hexane (1:1), followed by determination
of radioactivity. The radioactivity of each phase was determined by
.sup.14C counting using a liquid scintillation counter. A higher
radioactivity (dpm) indicates higher production of very long chain
polyisoprenoid (trans-1,4-polyisoprene) and higher rubber synthesis
activity.
[0257] Table 1 shows the results. The "Rubber synthesis activity
difference (dpm)" in Table 1 represents the difference from the
radioactivity of Comparative Example 1 in which the rubber
particles were bound to nothing as described later.
[Measurement of Molecular Weight Distribution of Synthesized Very
Long Chain Polyisoprenoid]
[0258] The molecular weight distribution of the very long chain
polyisoprenoid (trans-1,4-polyisoprene) synthesized as described
above was measured under the following conditions by radio-HPLC.
FIG. 4 shows the results.
HPLC system: a product of GILSON Column: TSK guard column MP(XL)
available from Tosoh Corporation, TSK gel Multipore HXL-M (two
columns) Column temperature: 40.degree. C. Solvent: THF available
from Merck Flow rate: 1 mL/min UV detection: 215 nm RI detection:
Ramona Star (Raytest GmbH)
Comparative Example 1
[Preparation of Rubber Particles]
[0259] The same procedure as in Example 1 was followed.
[Cell-Free Protein Synthesis Reaction (Step 1: mRNA Transcription
Reaction)]
[0260] Cell-free protein synthesis was performed using a WEPRO7240H
expression kit (CellFree Sciences Co., Ltd.). An mRNA transcription
reaction was performed using the cell-free expression vector
pEU-E01-His-TEV-MCS-N2 as a template in accordance with the
protocol of the WEPRO7240H expression kit.
[Purification of mRNA]
[0261] After the transcription reaction, the resulting mRNA was
purified by ethanol precipitation.
[Cell-Free Protein Synthesis Reaction (Step 2: Protein Synthesis by
Dialysis)]
[0262] The same procedure as in Example 1 was followed but using
the prepared mRNA.
[Collection of Reacted Rubber Particles]
[0263] The reacted rubber particles were collected as in Example 1
and re-suspended in an equal amount of 100 M Tris buffer (pH 7.5)
containing 2 mM dithiothreitol (DTT).
[Measurement of Rubber Synthesis Activity of Reacted Rubber
Particles]
[0264] The rubber synthesis activity of the collected reacted
rubber particles was measured as in Example 1.
[0265] Table 1 shows the results.
Comparative Example 2
[0266] [Acquisition of CPT Gene from cDNA]
[0267] The 1st strand cDNA prepared in [Synthesis of cDNA from
total RNA] in Example 1 was used as a template to obtain a CPT
gene. PCR was performed using a KOD-plus-Neo (Toyobo Co., Ltd.) in
accordance with the manual. The PCR reaction involved 35 cycles
with each cycle consisting of 10 seconds at 98.degree. C., 30
seconds at 58.degree. C., and 1 minute at 68.degree. C.
[0268] The CPT gene was obtained using the following primers.
TABLE-US-00002 Primer 3: 5'-tttggatccgatggaattatacaacggtgagagg-3'
Primer 4: 5'-tttgcggccgcttattttaagtattccttatgtttctcc-3'
[0269] A CPT gene (HRT1) was prepared as described above. The gene
was sequenced to identify the full-length nucleotide sequence and
amino acid sequence. The nucleotide sequence of HRT1 is given by
SEQ ID NO:18. The amino acid sequence of HRT1 is given by SEQ ID
NO:19.
[Vector Construction]
[0270] The obtained DNA fragment was subjected to dA addition and
then inserted into a pGEM-T Easy vector using a pGEM-T Easy Vector
System (Promega) to prepare pGEM-HRT1.
[Transformation of Escherichia coli]
[0271] The same procedure as in Example 1 was followed but using
the prepared vector.
[Plasmid Extraction]
[0272] The same procedure as in Example 1 was followed.
[Preparation of Vector for Cell-Free Protein Synthesis]
[0273] The pGEM-HRT1 acquired in the above [Vector construction]
was treated with the restriction enzymes Bam HI and Not I, and
inserted into a pEU-E01-His-TEV-MCS-N2 cell-free expression vector
that had been treated similarly with the restriction enzymes Bam HI
and Not I to prepare pEU-His-N2-HRT1.
[Transformation of Escherichia coli]
[0274] The same procedure as in Example 1 was followed but using
the prepared vector.
[Plasmid Extraction]
[0275] The same procedure as in Example 1 was followed.
[Preparation of Rubber Particles]
[0276] The same procedure as in Example 1 was followed.
[Cell-Free Protein Synthesis Reaction (Step 1: mRNA Transcription
Reaction)]
[0277] Cell-free protein synthesis was performed using a WEPRO7240H
expression kit (CellFree Sciences Co., Ltd.). An mRNA transcription
reaction was performed using the vector pEU-His-N2-HRT1 acquired in
the above [Preparation of vector for cell-free protein synthesis]
as a template in accordance with the protocol of the WEPRO7240H
expression kit.
[Purification of mRNA]
[0278] After the transcription reaction, the resulting mRNA was
purified by ethanol precipitation.
[Cell-Free Protein Synthesis Reaction (Step 2: Protein Synthesis by
Dialysis)]
[0279] The same procedure as in Example 1 was followed but using
the prepared mRNA.
[Collection of Reacted Rubber Particles]
[0280] The reacted rubber particles were collected as in Example 1
and re-suspended in an equal amount of 100 M Tris buffer (pH 7.5)
containing 2 mM dithiothreitol (DTT).
[Measurement of Rubber Synthesis Activity of Reacted Rubber
Particles]
[0281] The rubber synthesis activity of the collected reacted
rubber particles was measured as in Example 1.
[0282] Table 1 shows the results.
[Measurement of Molecular Weight Distribution of Synthesized Very
Long Chain Polyisoprenoid]
[0283] The molecular weight distribution of the very long chain
polyisoprenoid synthesized in the above [Measurement of rubber
synthesis activity of reacted rubber particles] was measured as in
Example 1. FIG. 4 shows the results.
TABLE-US-00003 TABLE 1 Rubber synthesis activity Bound protein
difference (dpm) Comparative None -- Example 1 Comparative HRT1
83700 Example 2 Example 1 HbSDS 93000
[0284] Table 1 demonstrate that a very long chain polyisoprenoid
was synthesized when rubber particles were bound to a tPT family
protein (Example 1), similarly to when rubber particles were bound
to HRT1, a cis-prenyltransferase family protein (Comparative
Example 2). This is in contrast to when rubber particles were bound
to nothing (Comparative Example 1).
[0285] As shown in FIG. 4, the very long chain polyisoprenoid
synthesized in Example 1 is a long chain rubber that showed the
highest peak at a GPC elution time corresponding to a weight
average molecular weight of about 1,000,000. It is also considered
that the very long chain polyisoprenoid synthesized in Example 1
had a molecular weight distribution pattern comparable to that of
the very long chain polyisoprenoid synthesized in Comparative
Example 2. It should be noted that in FIG. 4, peak heights cannot
be used to compare activities because the results were not
standardized among the samples.
<In Silico Estimation of Conserved Regions of tPT Family
Proteins>
[0286] Multiple sequence alignment of the tPT family proteins
derived from various organisms shown in FIG. 5 was performed to
search highly conserved sequence parts (conserved regions). FIG. 5
shows the alignment results around the conserved regions.
[0287] The multiple sequence alignment was carried out using
software called Genetyx Ver. 11.
[0288] In FIG. 5, Erg20p (Yeast TPT (FPPS)) corresponds to a
sequence of positions 91 to 150 or positions 227 to 285 of FPPS
from yeast represented by SEQ ID NO:3;
[0289] EuFPPS (Eucommia ulmoides TPT) corresponds to a sequence of
positions 90 to 149 or positions 225 to 283 of EuFPPS from Eucommia
ulmoides represented by SEQ ID NO:4;
[0290] HbFPPS (HeveaTPT) corresponds to a sequence of positions 84
to 143 or positions 219 to 277 of HbFPPS from Hevea brasiliensis
represented by SEQ ID NO:5;
[0291] Coq1p (Yeast TPT) corresponds to a sequence of positions 82
to 135 or positions 249 to 307 of TPT from yeast represented by SEQ
ID NO:6;
[0292] AtSDS1 (ArabiTPT) corresponds to a sequence of positions 162
to 215 or positions 285 to 343 of AtSDS1 from Arabidopsis thaliana
represented by SEQ ID NO:7;
[0293] HbSDS (HeveaTPT) corresponds to a sequence of positions 174
to 227 or positions 297 to 355 of HbSDS from Hevea brasiliensis
represented by SEQ ID NO:2;
[0294] HsTPT (HumanTPT) corresponds to a sequence of positions 132
to 185 or positions 255 to 313 of HsTPT from human represented by
SEQ ID NO:8; and
[0295] MmTPT (MouseTPT) corresponds to a sequence of positions 92
to 145 or positions 215 to 273 of MmTPT from mouse represented by
SEQ ID NO:9.
[0296] According to literature, such as Andrew H.-J. Wang et al;
Eur. J. Biochem. 269, pp. 3339-3354 (2002), boxA (corresponding to
positions 183 to 187 of HbSDS from Hevea brasiliensis represented
by SEQ ID NO:2) and box B (corresponding to positions 310 to 314 of
HbSDS from Hevea brasiliensis represented by SEQ ID NO:2) in FIG. 5
are parts of highly conserved regions of tPT family proteins
derived from various organisms. In particular, it is considered
that an amino acid sequence at positions corresponding to positions
183 to 187 of HbSDS from Hevea brasiliensis represented by SEQ ID
NO:2, and an amino acid sequence at positions corresponding to
positions 310 to 314 of HbSDS from Hevea brasiliensis represented
by SEQ ID NO:2 are conserved as specific motifs (amino acid
sequences (A1) or (A2) and (B)), and proteins having these motifs
at the respective positions have the functions of tPT family
proteins.
[0297] The following is understood from FIG. 5.
[0298] The conserved region in box A corresponding to positions 183
to 187 of HbSDS from Hevea brasiliensis represented by SEQ ID NO:2
corresponds to:
[0299] positions 100 to 104 of FPPS from yeast represented by SEQ
ID NO:3;
[0300] positions 99 to 103 of EuFPPS from Eucommia ulmoides
represented by SEQ ID NO:4;
[0301] positions 93 to 97 of HbFPPS from Hevea brasiliensis
represented by SEQ ID NO:5;
[0302] positions 91 to 95 of TPT from yeast represented by SEQ ID
NO:6;
[0303] positions 171 to 175 of AtSDS1 from Arabidopsis thaliana
represented by SEQ ID NO:7;
[0304] positions 141 to 145 of HsTPT from human represented by SEQ
ID NO:8; or
[0305] positions 101 to 105 of MmTPT from mouse represented by SEQ
ID NO:9.
[0306] The conserved region in box B corresponding to positions 310
to 314 of HbSDS from Hevea brasiliensis represented by SEQ ID NO:2
corresponds to:
[0307] positions 240 to 244 of FPPS from yeast represented by SEQ
ID NO:3;
[0308] positions 238 to 242 of EuFPPS from Eucommia ulmoides
represented by SEQ ID NO:4;
[0309] positions 232 to 236 of HbFPPS from Hevea brasiliensis
represented by SEQ ID NO:5;
[0310] positions 262 to 266 of TPT from yeast represented by SEQ ID
NO:6;
[0311] positions 298 to 302 of AtSDS1 from Arabidopsis thaliana
represented by SEQ ID NO:7;
[0312] positions 268 to 272 of HsTPT from human represented by SEQ
ID NO:8; or
[0313] positions 228 to 232 of MmTPT from mouse represented by SEQ
ID NO:9.
SEQUENCE LISTING FREE TEXT
[0314] SEQ ID NO:1: Nucleotide sequence of gene coding for HbSDS
from Hevea brasiliensis SEQ ID NO:2: Amino acid sequence of HbSDS
from Hevea brasiliensis SEQ ID NO:3: Amino acid sequence of FPPS
from yeast SEQ ID NO:4: Amino acid sequence of EuFPPS from Eucommia
ulmoides SEQ ID NO:5: Amino acid sequence of HbFPPS from Hevea
brasiliensis SEQ ID NO:6: Amino acid sequence of TPT from yeast SEQ
ID NO:7: Amino acid sequence of AtSDS1 from Arabidopsis thaliana
SEQ ID NO:8: Amino acid sequence of HsTPT from human SEQ ID NO:9:
Amino acid sequence of MmTPT from mouse SEQ ID NO:10: Nucleotide
sequence of promoter of gene coding for rubber elongation factor
from Hevea brasiliensis SEQ ID NO:11: Nucleotide sequence of
promoter of gene coding for small rubber particle protein from
Hevea brasiliensis SEQ ID NO:12: Nucleotide sequence of promoter of
gene coding for Hevien 2.1 from Hevea brasiliensis SEQ ID NO:13:
Nucleotide sequence of promoter of gene coding for MYC1
transcription factor from Hevea brasiliensis
SEQ ID NO:14: Primer 1
SEQ ID NO:15: Primer 2
SEQ ID NO:16: Primer 3
SEQ ID NO:17: Primer 4
[0315] SEQ ID NO:18: Nucleotide sequence of gene coding for HRT1
from Hevea brasiliensis SEQ ID NO:19: Amino acid sequence of HRT1
from Hevea brasiliensis
Sequence CWU 1
1
1911257DNAHevea brasiliensis 1atgatgtcaa tgacatgcta cagtcttgat
tttggaagga ctgtgtttga tttggcggct 60tgtgggtgct cctccaatgc ttcaatagat
aggtgttcag tgaggaatta tgcaaggtcg 120gtttatagga cttgtaatag
agactatgct gctagaagat cgccctattg ccggcgagat 180agtgcttggt
gtcgagtttc ttcgaccaag gcccctgaga ctttacttaa cggggttagt
240caagatcctg ctgtaaattt gaaggagtca agaggcccaa tttcattgat
aaatgtgttt 300gaagcggttg ctggtgatct ccagactctc aaccaaaacc
tccggtcgat tgttggtgca 360gaaaacccag ttttaatgtc tgcagctgat
cagatatttg gtgctggtgg gaaaaggatg 420cgaccagctt tggtattcct
agtgtcaaga gccacagcag aaatagtagg gttaaaagaa 480ctcactacga
aacatcgacg tttagcagag atcattgaga tgatccatac tgcaagctta
540attcatgatg atgtactaga tgaaagtaac atgcgaagag gaaaacaaac
ggttcatcaa 600ctgtatggca cgagggtggc agtactggct ggggatttca
tgtttgctca gtcctcatgg 660tacctagcaa atcttgaaaa cattgaagtc
attaagctta tcagccaggt tattaaagat 720tttgcaagtg gtgaaataaa
gcaagcatct agtttgtttg actgcgatgt tgaactcgag 780gagtacttga
tcaagagcta ttacaaaact gcctctttaa ttgctgcaag taccaaagga
840gctgctattt ttagtggggt ggacagcagt gttgctgaac aaatgtatga
atatggtaag 900aatcttggtc tgtccttcca agttgttgac gacgtactgg
attttacgca gtcagcagag 960cagctgggga agccagctgg cagtgacttg
gcaaaaggga accttaccgc ccctgtaata 1020tttgctctgg agaaagaacc
aaaactgaga gaaatcattg agtctgaatt ctgtgagact 1080ggttctctgg
atgaagctgt tgagttggtt aagcagtgtg ggggtattga aagagcacaa
1140gaattagcga aggagaaagc tgatcttgca atacagaatc ttaattgtct
tcctcggggt 1200gtatttcaat cacatctcaa agaaatggtg ttgtacaatc
tcgaacggat tgattag 12572418PRTHevea brasiliensis 2Met Met Ser Met
Thr Cys Tyr Ser Leu Asp Phe Gly Arg Thr Val Phe1 5 10 15Asp Leu Ala
Ala Cys Gly Cys Ser Ser Asn Ala Ser Ile Asp Arg Cys 20 25 30Ser Val
Arg Asn Tyr Ala Arg Ser Val Tyr Arg Thr Cys Asn Arg Asp 35 40 45Tyr
Ala Ala Arg Arg Ser Pro Tyr Cys Arg Arg Asp Ser Ala Trp Cys 50 55
60Arg Val Ser Ser Thr Lys Ala Pro Glu Thr Leu Leu Asn Gly Val Ser65
70 75 80Gln Asp Pro Ala Val Asn Leu Lys Glu Ser Arg Gly Pro Ile Ser
Leu 85 90 95Ile Asn Val Phe Glu Ala Val Ala Gly Asp Leu Gln Thr Leu
Asn Gln 100 105 110Asn Leu Arg Ser Ile Val Gly Ala Glu Asn Pro Val
Leu Met Ser Ala 115 120 125Ala Asp Gln Ile Phe Gly Ala Gly Gly Lys
Arg Met Arg Pro Ala Leu 130 135 140Val Phe Leu Val Ser Arg Ala Thr
Ala Glu Ile Val Gly Leu Lys Glu145 150 155 160Leu Thr Thr Lys His
Arg Arg Leu Ala Glu Ile Ile Glu Met Ile His 165 170 175Thr Ala Ser
Leu Ile His Asp Asp Val Leu Asp Glu Ser Asn Met Arg 180 185 190Arg
Gly Lys Gln Thr Val His Gln Leu Tyr Gly Thr Arg Val Ala Val 195 200
205Leu Ala Gly Asp Phe Met Phe Ala Gln Ser Ser Trp Tyr Leu Ala Asn
210 215 220Leu Glu Asn Ile Glu Val Ile Lys Leu Ile Ser Gln Val Ile
Lys Asp225 230 235 240Phe Ala Ser Gly Glu Ile Lys Gln Ala Ser Ser
Leu Phe Asp Cys Asp 245 250 255Val Glu Leu Glu Glu Tyr Leu Ile Lys
Ser Tyr Tyr Lys Thr Ala Ser 260 265 270Leu Ile Ala Ala Ser Thr Lys
Gly Ala Ala Ile Phe Ser Gly Val Asp 275 280 285Ser Ser Val Ala Glu
Gln Met Tyr Glu Tyr Gly Lys Asn Leu Gly Leu 290 295 300Ser Phe Gln
Val Val Asp Asp Val Leu Asp Phe Thr Gln Ser Ala Glu305 310 315
320Gln Leu Gly Lys Pro Ala Gly Ser Asp Leu Ala Lys Gly Asn Leu Thr
325 330 335Ala Pro Val Ile Phe Ala Leu Glu Lys Glu Pro Lys Leu Arg
Glu Ile 340 345 350Ile Glu Ser Glu Phe Cys Glu Thr Gly Ser Leu Asp
Glu Ala Val Glu 355 360 365Leu Val Lys Gln Cys Gly Gly Ile Glu Arg
Ala Gln Glu Leu Ala Lys 370 375 380Glu Lys Ala Asp Leu Ala Ile Gln
Asn Leu Asn Cys Leu Pro Arg Gly385 390 395 400Val Phe Gln Ser His
Leu Lys Glu Met Val Leu Tyr Asn Leu Glu Arg 405 410 415Ile
Asp3352PRTSaccharomyces cerevisiae 3Met Ala Ser Glu Lys Glu Ile Arg
Arg Glu Arg Phe Leu Asn Val Phe1 5 10 15Pro Lys Leu Val Glu Glu Leu
Asn Ala Ser Leu Leu Ala Tyr Gly Met 20 25 30Pro Lys Glu Ala Arg Asp
Trp Tyr Ala His Ser Leu Asn Tyr Asn Thr 35 40 45Pro Gly Gly Lys Leu
Asn Arg Gly Leu Ser Val Val Asp Thr Tyr Ala 50 55 60Ile Leu Ser Asn
Lys Thr Val Glu Gln Leu Gly Gln Glu Glu Tyr Glu65 70 75 80Lys Val
Ala Ile Leu Gly Trp Cys Ile Glu Leu Leu Gln Ala Tyr Phe 85 90 95Leu
Val Ala Asp Asp Met Met Asp Lys Ser Ile Thr Arg Arg Gly Gln 100 105
110Leu Cys Trp Tyr Lys Val Pro Glu Val Gly Glu Ile Ala Ile Asn Asp
115 120 125Ala Phe Met Leu Glu Ala Ala Ile Tyr Lys Leu Leu Lys Ser
His Phe 130 135 140Arg Asn Glu Lys Tyr Tyr Ile Asp Ile Thr Glu Leu
Phe His Glu Val145 150 155 160Thr Phe Gln Thr Glu Leu Gly Gln Leu
Met Asp Leu Ile Thr Ala Pro 165 170 175Glu Asp Lys Val Asp Leu Ser
Lys Phe Ser Leu Lys Lys His Ser Phe 180 185 190Ile Val Thr Phe Lys
Thr Ala Tyr Tyr Ser Phe Tyr Leu Pro Val Ala 195 200 205Leu Ala Met
Tyr Val Ala Gly Ile Thr Asp Glu Lys Asp Leu Lys Gln 210 215 220Ala
Arg Asp Val Leu Ile Pro Leu Gly Glu Tyr Phe Gln Ile Gln Asp225 230
235 240Asp Tyr Leu Asp Cys Phe Gly Thr Pro Glu Gln Ile Gly Lys Ile
Gly 245 250 255Thr Asp Ile Gln Asp Asn Lys Cys Ser Trp Val Ile Asn
Lys Ala Leu 260 265 270Glu Leu Ala Ser Ala Glu Gln Arg Lys Thr Leu
Asp Glu Asn Tyr Gly 275 280 285Lys Lys Asp Ser Val Ala Glu Ala Lys
Cys Lys Lys Ile Phe Asn Asp 290 295 300Leu Lys Ile Glu Gln Leu Tyr
His Glu Tyr Glu Glu Ser Ile Ala Lys305 310 315 320Asp Leu Lys Ala
Lys Ile Ser Gln Val Asp Glu Ser Arg Gly Phe Lys 325 330 335Ala Asp
Val Leu Thr Ala Phe Leu Asn Lys Val Tyr Lys Arg Ser Lys 340 345
3504348PRTEucommia ulmoides 4Met Ala Glu Leu Lys Lys Glu Phe Leu
Asn Val Tyr Ser Val Leu Lys1 5 10 15Lys Glu Leu Leu His Asp Pro Ala
Phe Ser Leu Thr Glu Asp Ser Arg 20 25 30Asn Trp Val Glu Arg Met Leu
Asp Tyr Asn Val Pro Gly Gly Lys Leu 35 40 45Asn Arg Gly Leu Ser Val
Val Asp Ser Tyr Lys Leu Leu Lys Glu Leu 50 55 60Ser Ser Ser Lys Lys
Gly Ala Gln Leu Thr Glu Ser Glu Ile Phe His65 70 75 80Ser Ser Val
Leu Gly Trp Cys Ile Glu Trp Leu Gln Ala Cys Ala Leu 85 90 95Val Leu
Asp Asp Ile Met Asp Ser Ser His Thr Arg Arg Gly Gln Met 100 105
110Cys Trp Tyr Lys Leu Pro Lys Val Gly Met Ile Ala Ile Asn Asp Gly
115 120 125Leu Ile Leu Arg Asn His Val Pro Arg Ile Leu Lys Lys His
Phe Arg 130 135 140Ser Lys Pro Tyr Tyr Leu Glu Leu Leu Asp Leu Phe
His Glu Val Glu145 150 155 160Cys Gln Thr Val Gly Gly Gln Met Ile
Asp Leu Ile Thr Thr Leu Val 165 170 175Gly Glu Ile Asp Leu Ser Glu
Tyr Ser Leu Pro Thr His Arg Gln Ile 180 185 190Thr Val Ser Lys Thr
Ser Tyr Tyr Ser Phe Tyr Leu Pro Val Ala Cys 195 200 205Ala Leu Leu
Met Thr Gly Glu Lys Leu Glu Ser His Ser Gly Met Lys 210 215 220Asp
Ile Leu Ile Glu Met Gly Ser Tyr Phe Gln Val Gln Asp Asp Tyr225 230
235 240Leu Asp Cys Phe Gly Asp Pro Glu Val Ile Gly Lys Ile Gly Ser
Asp 245 250 255Ile Glu Asp Phe Lys Cys Thr Trp Leu Val Val Lys Ala
Leu Glu Leu 260 265 270Cys Asn Glu Glu Gln Lys Lys Ile Leu Tyr Asp
Asn Tyr Gly Lys Lys 275 280 285Asp Pro Glu Ser Val Ala Arg Val Lys
Asp Leu Tyr Lys Thr Leu Lys 290 295 300Leu Gln Asp Val Phe Glu Glu
Tyr Glu Lys Lys Thr His Glu Lys Leu305 310 315 320Asn Lys Ser Ile
Asp Ala Tyr Pro Ser Lys Ala Val Gln Ala Val Leu 325 330 335Gln Ser
Phe Leu Ala Lys Ile His Arg Arg Leu Lys 340 3455342PRTHevea
brasiliensis 5Met Ala Asp Leu Lys Ser Thr Phe Leu Lys Val Tyr Ser
Val Leu Lys1 5 10 15Gln Glu Leu Leu Glu Asp Pro Ala Phe Glu Trp Thr
Pro Asp Ser Arg 20 25 30Gln Trp Val Glu Arg Met Leu Asp Tyr Asn Val
Pro Gly Gly Lys Leu 35 40 45Asn Arg Gly Leu Ser Val Ile Asp Ser Tyr
Lys Leu Leu Lys Glu Gly 50 55 60Gln Glu Leu Thr Glu Glu Glu Ile Phe
Leu Ala Ser Ala Leu Gly Trp65 70 75 80Cys Ile Glu Trp Leu Gln Ala
Tyr Phe Leu Val Leu Asp Asp Ile Met 85 90 95Asp Ser Ser His Thr Arg
Arg Gly Gln Pro Cys Trp Phe Arg Val Pro 100 105 110Lys Val Gly Leu
Ile Ala Ala Asn Asp Gly Ile Leu Leu Arg Asn His 115 120 125Ile Pro
Arg Ile Leu Lys Lys His Phe Arg Gly Lys Ala Tyr Tyr Val 130 135
140Asp Leu Leu Asp Leu Phe Asn Glu Val Glu Phe Gln Thr Ala Ser
Gly145 150 155 160Gln Met Ile Asp Leu Ile Thr Thr Leu Glu Gly Glu
Lys Asp Leu Ser 165 170 175Lys Tyr Thr Leu Ser Leu His Arg Arg Ile
Val Gln Tyr Lys Thr Ala 180 185 190Tyr Tyr Ser Phe Tyr Leu Pro Val
Ala Cys Ala Leu Leu Ile Ala Gly 195 200 205Glu Asn Leu Asp Asn His
Ile Val Val Lys Asp Ile Leu Val Gln Met 210 215 220Gly Ile Tyr Phe
Gln Val Gln Asp Asp Tyr Leu Asp Cys Phe Gly Asp225 230 235 240Pro
Glu Thr Ile Gly Lys Ile Gly Thr Asp Ile Glu Asp Phe Lys Cys 245 250
255Ser Trp Leu Val Val Lys Ala Leu Glu Leu Cys Asn Glu Glu Gln Lys
260 265 270Lys Val Leu Tyr Glu His Tyr Gly Lys Ala Asp Pro Ala Ser
Val Ala 275 280 285Lys Val Lys Val Leu Tyr Asn Glu Leu Lys Leu Gln
Gly Val Phe Thr 290 295 300Glu Tyr Glu Asn Glu Ser Tyr Lys Lys Leu
Val Thr Ser Ile Glu Ala305 310 315 320His Pro Ser Lys Pro Val Gln
Ala Val Leu Lys Ser Phe Leu Ala Lys 325 330 335Ile Tyr Lys Arg Gln
Lys 3406371PRTSaccharomyces cerevisiae 6Met Thr Glu Arg Asn His Leu
Lys Ile Asp Lys Ser Asp Val Pro Glu1 5 10 15Asp Pro Ile Tyr Ser Lys
Pro Ser Gln Asn Gln Leu Phe Gln Arg Pro 20 25 30Ala Ser Ser Ile Ser
Pro Leu His Ile Leu His Gly Ile Lys Pro Leu 35 40 45Asn Pro Leu Thr
Lys Gly Pro Glu Pro Leu Pro Glu Glu Thr Phe Asp 50 55 60Lys Gln Arg
Gly Ile Leu Pro Lys Gln Arg Arg Leu Ala Glu Ile Val65 70 75 80Glu
Met Ile His Thr Ala Ser Leu Leu His Asp Asp Val Ile Asp His 85 90
95Ser Asp Thr Arg Arg Gly Arg Pro Ser Gly Asn Thr Ala Phe Thr Asn
100 105 110Lys Met Ala Val Leu Ala Gly Asp Phe Leu Leu Gly Arg Ala
Thr Val 115 120 125Ser Ile Ser Arg Leu His Asn Pro Glu Val Val Glu
Leu Met Ser Asn 130 135 140Ser Ile Ala Asn Leu Val Glu Gly Glu Phe
Met Gln Leu Lys Asn Thr145 150 155 160Ser Ile Asp Ala Asp Ile Asp
Thr Ile Glu Asn Gly His Lys Leu Leu 165 170 175Pro Val Pro Ser Lys
Lys Leu Glu Val Lys Glu His Asp Phe Arg Val 180 185 190Pro Ser Arg
Gln Gln Gly Leu Gln Leu Ser His Asp Gln Ile Ile Glu 195 200 205Thr
Ala Phe Glu Tyr Tyr Ile His Lys Thr Tyr Leu Lys Thr Ala Ala 210 215
220Leu Ile Ser Lys Ser Cys Arg Cys Ala Ala Ile Leu Ser Gly Ala
Ser225 230 235 240Pro Ala Val Ile Asp Glu Cys Tyr Asp Phe Gly Arg
Asn Leu Gly Ile 245 250 255Cys Phe Gln Leu Val Asp Asp Met Leu Asp
Phe Thr Val Ser Gly Lys 260 265 270Asp Leu Gly Lys Pro Ser Gly Ala
Asp Leu Lys Leu Gly Ile Ala Thr 275 280 285Ala Pro Val Leu Phe Ala
Trp Lys Glu Asp Pro Ser Leu Gly Pro Leu 290 295 300Ile Ser Arg Asn
Phe Ser Glu Arg Gly Asp Val Glu Lys Thr Ile Asp305 310 315 320Ser
Val Arg Leu His Asn Gly Ile Ala Lys Thr Lys Ile Leu Ala Glu 325 330
335Glu Tyr Arg Asp Lys Ala Leu Gln Asn Leu Arg Asp Ser Leu Pro Glu
340 345 350Ser Asp Ala Arg Ser Ala Leu Glu Phe Leu Thr Asn Ser Ile
Leu Thr 355 360 365Arg Arg Lys 3707406PRTArabidopsis thaliana 7Met
Met Thr Ser Cys Arg Asn Ile Asp Leu Gly Thr Met Met Met Ala1 5 10
15Cys Gly Cys Gly Arg Arg Gln Phe Pro Ser Leu Ala Lys Thr Val Cys
20 25 30Lys Phe Thr Ser Ser Asn Arg Ser Tyr Gly Gly Leu Val Gly Ser
Cys 35 40 45Lys Ala Val Pro Thr Lys Ser Lys Glu Ile Ser Leu Leu Asn
Gly Ile 50 55 60Gly Gln Ser Gln Thr Val Ser Phe Asp Leu Lys Gln Glu
Ser Lys Gln65 70 75 80Pro Ile Ser Leu Val Thr Leu Phe Glu Leu Val
Ala Val Asp Leu Gln 85 90 95Thr Leu Asn Asp Asn Leu Leu Ser Ile Val
Gly Ala Glu Asn Pro Val 100 105 110Leu Ile Ser Ala Ala Glu Gln Ile
Phe Gly Ala Gly Gly Lys Arg Met 115 120 125Arg Pro Gly Leu Val Phe
Leu Val Ser His Ala Thr Ala Glu Leu Ala 130 135 140Gly Leu Lys Glu
Leu Thr Thr Glu His Arg Arg Leu Ala Glu Ile Ile145 150 155 160Glu
Met Ile His Thr Ala Ser Leu Ile His Asp Asp Val Leu Asp Glu 165 170
175Ser Asp Met Arg Arg Gly Lys Glu Thr Val His Glu Leu Phe Gly Thr
180 185 190Arg Val Ala Val Leu Ala Gly Asp Phe Met Phe Ala Gln Ala
Ser Trp 195 200 205Tyr Leu Ala Asn Leu Glu Asn Leu Glu Val Ile Lys
Leu Ile Ser Gln 210 215 220Val Ile Lys Asp Phe Ala Ser Gly Glu Ile
Lys Gln Ala Ser Ser Leu225 230 235 240Phe Asp Cys Asp Thr Lys Leu
Asp Glu Tyr Leu Leu Lys Ser Phe Tyr 245 250 255Lys Thr Ala Ser Leu
Val Ala Ala Ser Thr Lys Gly Ala Ala Ile Phe 260 265 270Ser Arg Val
Glu Pro Asp Val Thr Glu Gln Met Tyr Glu Phe Gly Lys 275 280 285Asn
Leu Gly Leu Ser Phe Gln Ile Val Asp Asp Ile Leu Asp Phe Thr 290 295
300Gln Ser Thr Glu Gln Leu Gly Lys Pro Ala Gly Ser Asp Leu Ala
Lys305 310 315 320Gly Asn Leu Thr Ala Pro Val Ile Phe Ala Leu Glu
Arg Glu Pro Arg 325 330 335Leu Arg Glu Ile Ile Glu Ser Glu Phe Cys
Glu Ala Gly Ser Leu Glu 340 345 350Glu Ala Ile Glu Ala Val Thr Lys
Gly Gly Gly Ile Lys Arg Ala Gln 355 360 365Gl