JPH07322882A - Guayule rubber particle protein gene - Google Patents

Abstract

PURPOSE: To obtain the subject gene useful for stimulating the biosynthesis of a rubber or screening a cDNA or a genome DNA from a material from which the rubber can biologically be synthesized.

CONSTITUTION: A guayule rubber particle protein gene clone pRPP30 containing 1692 base pair fragment of the formula starting from a CTCCACATTCA nucleotide and ending at ATTGTTTCACAAAATTTAGAAAAAAAAAAAAAAAAAA nucleotide. The protein gene clone is obtained by screening a cDNA library originated from the bark tissue of the trunk of guayule shrub 11591 line on the basis of amino acid sequence data from a purified rubber particle protein(RPP) and subsequently isolating the cDNA clone of RPP gene.

COPYRIGHT: (C)1995,JPO

Description

Detailed Description of the Invention

[0001]

FIELD OF THE INVENTION This invention relates to biotechnology, and in particular, isolation and clarification of guayule rubber particle protein (RPP), its DNA template, and friendly (frien).
It relates to means, methods and steps for the replication of DNA clones of the guayule RPP gene in both dly and foreign environments. The invention also relates to the use of this gene and protein to promote rubber biosynthesis in both friendly and unfriendly environments.

[0002]

PRIOR ART cDN coding for Guayule RPP
The isolated recombinant vector containing A is transferred to another prokaryotic or eukaryotic host organism, where the RPP cDNA
Is expressed to produce functional RPP for rubber biosynthesis. Rubber biosynthesis depends on recombinant RPP in the host organism
It occurs as a result of the expression of cDNA. The expression is then placed under the control of a promoter DNA sequence selected from the following group. That is, lacZ promoter, trp promoter, trc promoter, T7 promoter, N
CaMV35, another major prokaryotic and eukaryotic operator and promoter region that enhances expression of OS promoters and terminators and other genes in prokaryotic hosts
S promoters, GAL1 and GAL10 promoters and other prokaryotic or eukaryotic promoters which enhance the expressed genes in eukaryotic cells or their viruses.
Gum biosynthesis occurs as a result of increased expression of the guayule RPP gene in these host organisms.

Rubber is found in more than 2,000 species of plants, but is restricted to only a few families (Backha).
us, Israel J. Bot. , 34: 283-2
93 (1985); and Archer et al, C.
chemistry and Physics of Ru
bber-like Substances, Bate
man, L .; et. pp. 41-72, MacLare
n, London (1963))). Rubber is 320
To 35,000 isoprene molecules. They form polyisoprene chains. They are joined in a stepwise cis-1,4-condensation from head to tail. The stepwise addition of isoprene is due to rubber transferase (Ru
T), a rubber polymerase and a prenyl transferase enzyme known as gum cis-1,4 polyprenyl transferase [E. C. 2.5.1.20]. RuT is known as the only enzyme required for gum formation in plants. (Backhau
S. Israel J.S. Bot. 34: 283-29
3, 1985); Berndt, U .; S. Gonver
nm Res. Rep. AD-601-729,
(1963), Archer and Cockbai.
n, Methods in Enzymology, 1
5: 476-480 (1969) Archer and
Audley, Advances in Enzym
29: 221-257, (1967) an
d Mynen, Rubber Res. Inst. M
alaya, 21: a2 (4) 389-406 (196).
9)). However, as the present invention shows, there are other enzymes known as RPPs (rubber particle proteins) that apparently undergo rubber biosynthesis when transferred to other host plants (in our case tobacco). As the present invention shows, RP
Unlike RuT, P is an allene oxide synthase (AOS) rather than a prenyltransferase. This is a member of the family of heme-binding enzymes, cytochrome P450. RPP has similar properties to others of this class of heme-binding enzymes and explains that it is the enzyme responsible for the biosynthesis of gums. The P450 enzyme has never before been used for rubber biosynthesis. PB below
The structure of a binary vector designated IRPP is described. It contains the RPP gene, which produces a functional RPP enzyme when the RPP gene is transferred and expressed in tobacco. RP produced in these light conversion plants
The P enzyme causes rubber biosynthesis, and the rubber thus produced accumulates in the rubber particles in the cells of these plants. The present inventor believes that Guayule RPP and its homologues from other gum-forming species are the enzymes required for rubber biosynthesis in plants.

The enzymatic activity of RPP has never been elucidated. A spectral analysis confirming that RPP is cytochrome P450 will be described. Let us show that RPP is a non-monooxygenase type heme-binding protein and not a phenyltransferase. P in rubber
Knowledge of 450 such roles has never been described. Biochemical analyzes indicate that RPP metabolizes lipid hydroperoxides to lipids and does not undergo prenyltransferase-type reactions. The present invention extends the means and methods for the isolation of full length cDNA for the Guayule RPP gene. The present invention describes the DNA and amino acid sequences of RPP and pBIRR downstream of the RPP cDNA of the strong eukaryotic CaMV35S plant promoter.
The method of insertion into the P two-component vector is shown. The present invention describes how this vector is then used to generate transformed tobacco plants that produce functional RPP. These transformed tobacco plants express the external RPP gene contained in the pBIRPP vector and carry out rubber biosynthesis. Rubber is observed as rubber particles that accumulate in tobacco cells. These particles are similar to those observed in guayule cells. The present invention demonstrates that RPP encoded by a single nuclear gene is responsible for rubber biosynthesis in plants. Furthermore, it is that the mechanism of gum formation is not solely due to the presence of the RuT-type cis-prenyltransferase enzyme as originally thought, but requires the heme-binding enzyme RPP-type cytochrome P450. Suggest.

The first identification of Guayule RPP was 1985.
Made in the year. (Backhausand Chand
Ra, in Alcorn, S .; and Fangme
ier, D (ed) Proceedings of t
he 4th Internal-Guy
ule Conference on Guayule
Research and Development
t, Oct 16-19, (1985); Backha.
us and Bess, In Randall, D .;
D. et al (eds) Current Topic
s in Plant Biochemistry a
nd Physiology, Vol. 5: 186 (1
986)). Subsequent literature describes its purification and characterization and suggests a putative role as prenyltransferase in guayule gum particles. (Ba
ckhaus and Bess, in Benedi
tc, C.I. R. (Ed) Biochemistry a
nd Regulation of cis-Poly
isoprene in Plants, NSFspo
nanoswork public publicati
on, p. 204-220 (1986); Cornis
h and Backhaus, Phytochem.
29: 3809-3813 (1990); and Ba.
ckhaus et al. Phytochem. Three
0: 2493-2497 (1991)). Corn
The research by ish and Siler showed that the RPP uniform protein is He
It was also shown to be present in vea and Ficus.
(Siler and Cornish, Phyto-
cnem. 32: 1097-1102 (1993); C
ornish, et al. J. Nat. Rubb. R
es. (1994) (printing); Cornish, et.
al, Phytochem. (Printing) (1994
0; Siler and Cornish, Phyto
chem. (1994) (printing)). RPP uniform proteins in rubber particles of these other species appear to undergo the same enzymatic reaction as RPP. These are all known publications which specifically mention RPP. References on other proteins isolated from guayule gum particles can be found in Ben.
ddi t et al. Plant Physiol.
92: 816-821 (1990), which describes a prenyl-transferase, but which identified this protein as RPP, despite known techniques by other authors for the presence of RPP in guayule gum particles. None (Benedict, CR (ed) b
e Biochemistry and Regula
region of cis-Polyisoprene
in Plants, NSF sponsored w
orkshop publication, p. 204
-220 (1986)). The initial identification and demonstration of RPP as a cytochrome P450 required for rubber biosynthesis is the present patent application.

Next, the molecular cloning of RPP will be described.
Prior to that, its molecular weight was determined by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) to be 48,500 to 53,000 daltons.
Other specializations of RPP are its amino acid composition, its isoelectric point (pI = 6.2), that it is a glycoprotein, and how it is assembled as a constituent protein in rubber particles. Includes. (Backhaus et a
l. , Bytochem. 30: 2493-2497
(1991). The invention further describes its amino acid sequence, its biochemical function and its occurrence in tobacco (non-rubber producing species).

RPP is the most abundant protein in guayule gum particles (Backhaus et al, P.
hytochem. 30: 2493-2497 (199
1) Therefore, it has been considered to be important for rubber biosynthesis. For this reason, the cDNA of the RPP gene has been targeted for isolation from guayule stem bark with the intent to use genes that induce rubber biosynthesis in a variety of other organisms that normally do not synthesize rubber. . The present invention teaches what happens when: That is, when the RPP gene is present in a binary plasmid under the control of a strong promoter and is transformed into a new host (tobacco) that expresses the RPP gene and produces a functional RPP enzyme. Conversely, the gene may also be guayule or Hev, which already synthesizes rubber with the intention of overexpressing RPP.
It can also be located in other plants such as ea. Overexpressed RPP then enhances rubber production in plants such as these. In addition, RPPs can be transferred and expressed in other eukaryotic or cell cultures, including yeasts, insects or higher animals, or prokaryotes, with the intention of producing rubber. The present invention seeks to protect the invention of an isolated DNA molecule of the RPP gene from guayule, which gene also expresses the rubber biosynthesis in their transformed organisms (ie tobacco) when the external RPP gene is expressed. It shows that can occur.

[0008]

Problems to be Solved by the Invention; Means for Solving the Problems The RPP gene and its DNA template have been isolated and elucidated. A full-length cDNA clone of the existing Guayule RPP gene has been isolated and described. It was named pRPP30.

The present invention relates to RPP from guayule rubber particles.
Indicates that it is not a prenyl transferase. And it is a non-monooxygenase that produces lipid epoxide from lipid hydroperoxide, cytochrome P450, a heme-binding protein. The present invention shows how this enzyme is purified from guayule gum particles and its activity is measured. It also shows how the amino acid sequence of the purified protein was determined and how this sequence information was used for the isolation of RPP cDNA from a guayule stem skin cDNA library. It also shows how a guayule stem skin cDNA library is made and the amino acid sequence and DNA of a full-length cDNA clone of RPP. It also shows similarities and differences between RPP and another non-monooxygenase, a heme-binding protein known as allene oxide synthase (AOS). AOS is an enzyme found in some monocots. These species do not produce rubber and therefore their AOS production is not included in the rubber synthesis of these species. This is probably due to the structural differences between RPP and AOS.

Furthermore, the present invention shows how the RPP cDNA can be formed in a new binary vector by insertion of the strong, essential plant promoter downstream of RPP. This invention also describes how this two-component vector Agrobacte of RPP cDNA into tobacco.
Indicates whether it is used for transfer to riam. The present invention also shows whether the external RPP gene is transcribed, translated and expressed as a fully functional RPP with AOS-like enzymatic activity. The present invention also shows that this activity is only present in tobacco transformed with RPP and not in non-transformed tobacco. The present invention also shows that this functional enzyme causes rubber biosynthesis in these transformed tobacco plants and that the rubber is deposited and stored as rubber particles in the cells of these plants. Finally, the present invention
We show that these rubber particles can be observed by electron and optical microscopy. Due to the ability to cause rubber biosynthesis in tobacco, it is clear that transfer or expression of the RPP gene into other hosts also triggers rubber biosynthesis in these other organisms.

The exact mechanism of the role of RPP in rubber biosynthesis is not yet known. Unlike RuT, RPP
Is not involved in the continuous cis polymerization of isoprene from IPP (see Figure 1). RPP is probably responsible for the epoxide formation of the isoprene group in the polyisoprene chain. Epoxides are ubiquitous in the isoprene chain of natural rubber (Burfield, Nature 2
49: 29-30 (1974); Burfield,
J. Nat. Rubb. Res. 1: 202-208
(1986); Burfield, et al. Pol
ymer 17: 713-716 (1976); Bur.
field and Eng, Polymer 30:
2019-2022 (1989); Burfield
and Gan, Polymer 18: 607-61.
1 (1977); Burfield and Gan,
J. Polymer Sci. , Polymer Ch
em. Edn. 13: 2725-2734 (197)
5): Burfield and Gan, J. et al. Poly
mer Sci, Polymer Chem. Ed
n. 15: 2721-2730 (1977)), it serves as a site for cross-linking in the rubber molecule. Burfi
Eld proposes that the latex enzyme is required for such epoxidation within the rubber molecule. 100,000 or more 10
It has always been believed that a high molecular weight rubber of 0,000 Daltons consists of chain bonds containing one long polyisoprene molecule. This leads to the following theory. That is, the production of gum is due to only a single prenyltransferase enzyme that undergoes this stepwise, chain extension. But one other hypothesis was suggested. That is, the rubber produces high molecular weight isoprenoids that are actually produced as a series of cross-links between small polyisoprene chains (We
stall, Polymr 9: 243-248). This may explain why the bimolecular weight distribution is always found in natural rubber. This is because large and small pools of polyisoprene are separately present in the plant. In principle, the small chains are cross-linked to form large polymers. This is due to a clonally expanded Hevea (subra) with a specific and reproducible bimolecular weight distribution.
maniam, Proceedings of the
International Rubber Con
ference, Kuala Lumpur, vol.
4, p. 3-27,1975) and guayule (Ba
ckhaus and Nakayama, Rubber
Chem. Technol. , 61: 78-85 (1
988) and is partly supported by the genetic evidence that it is obtained for rubber. Now, the inventor believes that the rubber can be assembled by the following mechanism. That is, small chain isoprene cross-links to form a high molecular weight rubber polymer and RPP plays a fundamental role in this process. This means that small chain cispolyisoprene containing 20 to 320 isoprene
We suggest that it exists and is available in plants for this cross-linking reaction. In fact, this has been demonstrated in a number of non-rubber-producing plants that assemble cispolyprenol containing 50 or more isoprene, but do not synthesize rubber (eg, gymnosperms, birch, and a large number of Rosaceae plants). (Sweezewska, et al. Bio
chem. Cell Biol. 70: 448-454
(1992). These species do not seem to produce rubber. Because they do not have an enzyme that cross-links small polyisoprenes to large ones.
We have found that it forms epoxides in small polyisoprenes that act as sites for cross-linked rubber.
I believe it is a PP function. Independent of this hypothetical function, the present invention makes it clear that if the RPP enzyme is introduced into a non-rubber-producing tobacco plant, that plant will synthesize rubber.

The first step in practicing the present invention was to isolate a cDNA clone of the RPP gene. The strategy is to screen a cDNA library for DNA
It was to use the amino acid sequence data from the purified RPP to generate the probe. To do this,
RPP was purified to homogeneity and decomposed with CNBr. R
PP was decomposed into four separate fragments arranged from their N-termini (see Table 1).

[0013]

[Table 1] Table 1 is the amino acid sequence of four different fragments obtained from the cyanogen bromide digest of purified guayule gum particles. Oligonucleotides corresponding to known protein sequences were synthesized (Table 2) and used by the polymerase chain reaction (PCR) for prime plus and minus strand amplification of guayule bark cDNA.

[Table 2] Table 2 shows chemically synthesized oligonucleotide DNA probes and / or primers used in the present invention. 92 bp using primers 5 and 6
When the fragment DNA was obtained and sequenced, RP
There was a perfect match with the known protein sequence of P peptide fragment # 3 (Table 2).

[Table 2] Next, this 92 bp probe is lambda ZA.
P, used to screen the bark cDNA library. Positive clones are at least 2 with radioactive probe
Re-screened twice to demonstrate hybridization. It contains RPP cDNAs of various lengths. 45 cDN
A clone was obtained. Sequence the 4 longest clones,
It was found that their protein coding regions were identical.
All also had DNA sequences encoding the four CNBr fragments shown in Table 1.

The order of these fragments in the protein sequence is from the 5'to the 3'direction of the gene, #
4, # 2, # 1, and # 3 (Tables 3 and 4).

[Table 3]

[Table 4] Table 3 shows pR of guayule gum particle protein gene of the present invention.
3 represents the full length nucleotide sequence of PP30. Table 4 lists restriction endonuclease sites; Primer-P1, P3, P
8, P9, P5, P6 and PCR generation 5/6, 92 bp
Probe; CNB _r peptide fragment # 1, #
2, positions # 3 and # 4; and a map of guayule RPP cDNA in pRPP30 depicting potential N-linked glycosylation sites (*). The RPP cDNA clone described herein was one of the four full length clones isolated. It is named pRPP30 and is shown in Tables 3 and 4.

[Table 3]

[Table 4] This pRPP30 gene has a length of 1692 bp and an open reading frame of 1419 bp for RPP. The 1419 open reading frame encodes a 473 amino acid sequence equivalent to 53,438 daltons and contains 8 methionines and 2 cysteines. This 473
The predicted pI of the amino acid protein is 6.15, which is RP
It agrees with the experimental value of P of 6.2 (Backhaus e
t al. Phytochem. 30: 2493- 24
97 (1991)). There are three active N-linked glycosylation sites in the RPP of the Asn-X-Ser / Thr sequence, where X can be any of the usual 20 amino acids. In addition, pRPP30 contains a stop codon (TGA), a 23 bp 5'-noncoding region and 2
It contains a 50 bp 3'-non-coding region. This pRPP
30cDNA is Bluescript SK- (Str
atage, Inc) consisting of the 1692 bp RPP gene contained in the phagemid. This p
Hagemid is a well-known and commercially available cloning vector. This pR
The PP30 cDNA phagemid is a 4631 bp circular molecule, of which 1692 bp is the RPP gene inserted between the specific EcoR1 and Xho1 cloning sites of the vector. A map of pRPP30 is shown in Table 4.

The raw material for the isolation of this pRPP30 cDNA molecule is the bark tissue of the trunk of the 11591 line of the guayule shrub. Rich in natural rubber, this tissue is readily available from the Guayule forest growing in and around the Phoenix area. The lambda ZAP cDNA cloning system used here is a well known and readily available lambda phage expression vector. Its structure and restriction endonuclease map are described in: Short
t, et al. Nucl. Acids Res, 16:
7583-7600 (1988); Huse and
Hansen, Strategies 1: 1-3; S
orge, Strategies 1: 3-7 (198)
8); Associated Technologies
in Gubler and Hoffman, Gen
e 25: 263 (1983); Young and.
Davis, Proc, Natl. Acad. Sci.
USA 80: 1194-1198 (1983); Wa.
tson and Jackson, DNA Cloni
ng; A Practicl Approach 79
-88 (1985).

In the computer search using the WORD, FASTA and TFASTA algorithms, GENBAN is used.
No significant equivalence of known sequences with pRPP30 in the J / EMBL and SWISSPROT databanks could be found. Sequence of the Hevea rubber elongation factor (Dennis and Light, J. Biol.
Chem, 264: 18618-18626 (198).
9); Attanyaka et al, Plant
Mol. Biol. 16: 1079-1081 (199
1); Goyvaerts et al. , Plant
Physicl. 97: 317-321 (1991)).
A direct comparison with also found no equivalence. RPP
Are one or more known prenyl transferases (Kuntz et al. Plant J. 2:
25-34 (1992)), but share a similarity or structural region, which was negative. BL
Sequence comparison using the AST algorithm (Altsch
ul et al. J. Mol, Biol. 215: 4
03-410 (1990)) finally showed a striking similarity with some parts of cytochrome P450s. R
A small homology with PP was found in two conserved B and C regions of P450s (Kalb and Lope).
r, Proc. Natl. Acad. Sci. USA
85: 7221-7225 (1988)), but RPP lacked homology with the A and B regions. The highly conserved D region is particularly critical for most P450s. This apparent structural anomaly is due to another unique P450 (allene oxide synthase; A) derived from flaxseed.
OS) sequence announced (Song et al, Proc
Natl. Acad. Asic. USA, 90: 851.
9-8512 (1993)).

A comparison of RPP and AOS (Table 5) shows 65% similarity and 85% similarity between the two protein sequences.

[Table 5] Table 5 shows a 65% identity and 80% similarity between the amino acid sequences of RPP and flaxseed AOS. Both are
In particular, it has several structures common to specific heme binding sites in the D region. However, unlike AOS, RPP does not have transit signal peptides. It is believed that AOS localizes in the plasmid, while RPP does not. This difference in N-terminals is due to the RPP
Could be an explanation as to whether or not is inside the rubber particles and not inside the plastid. Allene oxide synthase is a non-monooxygenase-type P450 for the production of nioxide in lipids (Son and Brush, Science,
253: 781-783 (1991); Lau, et.
al. , Biochem. , 32: 1945-1950
(1994); Vick, in Lipid Metab
olism in Plants, T.W. S. Moor
e, Ed. (CRC Press, Boca Rato
n, p. 167-191 (1993): Zimmermer
an, Biochem. Biophys, Res. Co
mm. 23: 398-403 (1966); Zimme.
rman and Feng, Lipids 13:31.
3-316 (1978): Zimmermanand
Vick, Plant. Physilo. 46: 445
-453 (1970)). Its function in these plants is believed to be involved in the production of prostaglandin-like compounds in plants, such as jasmonic acid.

Since RPP may be a member of this group of enzymes, biochemical analysis was performed on RPP purified from rubber particles. The rubber particles, which had been washed three times, were treated with the above-mentioned Cornish a from crude homogenate of guayule stem bark
Produced by centrifugation according to nd Backhaus (1990). RPP is 0.5% CHA
Sonicate the washed particles in PS and add the solution to 0.2
All residual rubber particles were removed and solubilized through a 2 micrometer filter. Protein gels range from crude homogenate (FIGS. 1A, 1) to washed rubber particles (FIGS. 1A, 2) to the final filtered CHAPS extract (FIG. 1A).
A, the degree of purification of each step up to 3) was clarified. Prenyltransferase activity was measured for each purification step by measuring the incorporation of ¹⁴ C-isopentenyl pyrophosphate into the isoprenoid derivative analyzed by TLC (benzene / ethyl acetate; 90/10) and autoradiography. (Dogbo and
Camara, Plant Sci. 49: 103-
109 (1987); Kuntz et al. Pla
nt J. 2: 25-34 (1992)). Dimethylallyl pyrophosphate (DMAPP) was used as an initiator for aliquot activity of 100 microliters of protein from each of the three steps (FIGS. 1B, 1-3). TLC analysis shows a complete lack of prenyltransferase activity in the filtered RPP formulation with all rubber particles removed. However, prenyltransferase activity is present in both the crude homogenate and the gum-containing cleaning formulation (Fig. 1B). These prenyltransferases synthesize several isoprenoid derivatives such as geranyl pyrophosphate, farnesyl pyrophosphate, geranylgeranyl pyrophosphate, phytoene, squalene and other unidentified higher isoprenes. But any isoprenoid is CHA
It cannot be made with PS solubilized and filtered RPP formulation (FIG. 1B).

Spectral analysis of this CHAPS solubilized, filtered RPP formulation showed that the RPP was cytochrome P450. The difference spectrum of this extract clearly shows a characteristic peak at 450 nm (arrow, 1C). To confirm that RPP is an allene oxide synthase, an aliquot of this formulation was used in an assay for the degradation measurement of linoleic acid hydroperoxide (LOOH) with a maximum absorption at 234 nm. This substrate was prepared according to the method of Zimmermann (Zimmerman, Biochem. Bi.
opnys. Res. Comm. 23: 398-403
(1966); Zimmerman and Fen.
g, Lipids 13: 313-316 (197).
8); Zimmerman and Vick, Pla
nt. Physiol. 46: 445-453 (197)
0)). The reaction was initiated by the addition of purified RPP to a LOOH-containing cuvette followed by a series of UVs at 10 second intervals.
The cuvette was scanned for spectra (Song a
nd Brush, Science, 253: 781-.
784 (1991)). As a result, the purified RPP has a high A
It was found to have OS activity (Fig. 1D). This biochemical activity is similar to that of flaxseed AOS and the sequence data are also identical, suggesting similar functions of these two proteins. Furthermore, this data clearly shows that RPP is not a prenyl transferase.

In order to determine the effect of RPP expression in transformed tobacco, a 12.8 kb binary vector was added to
It was constructed with the RPP gene downstream of the 35S promoter and the NOS terminator downstream. pBIRPP (Table 6)
This vector, designated as Tobacco cv. Samsun (s
amaun) Agrobacte used in plants
rum tumefa-ciens (LBA4404
Fixed in the strain).

[Table 6] Table 6 shows pBIR used to transform tobacco.
A map of PP, a 12.8 kb binary plasmid. It contains the RPP cassette in the sense configuration. The RPP cDNA from pRPP30 is pB112
Replacement of the GUS gene from 1 inserts downstream of the 35S promoter and upstream of the NOS terminator. A second construct containing the antisense coordinated RPP was also made. Transformed tobacco plants were selected and matured with kanamycin. The results of the analysis showed the presence and expression of the external RPP gene (Fig. 2) in the transformed tobacco plants. The total tobacco protein SDS gel (FIG. 2A) showed the presence of RPP (arrow) in four different transformants (# 3, # 6, # 14 and # 17) and a non-transformed control (C) and Antisense (A) showed its absence in tobacco. An equivalent gel probed with a mono-specific RPP antibody (Fig. 2
Western hybridization of A) is a sense construction (# 3,
# 6, # 14 and # 17) each single RPP band (arrow), control (C) and antisense (A)
It showed its absence in the tobacco construct. Enzymatic analysis of protein extracts made from control (C) and transformed (# 3) tobacco showed strong AOS activity due to expression and production of functional RPPs from foreign genes.
It was shown only in D) and completely absent in the control tobacco (Fig. 2C). Control plants do not contain RPP genes and do not produce any functional RPP enzyme and therefore cannot metabolize LOOH. However, a UV scan of an extract made from transformed tobacco
High AOS because they express recombinant RPP gene
It was shown to have activity.

Since transformed tobacco plants were established to produce RPP, their tissues were examined microscopically for any signs of rubber formation. Electron microscopy is the most sensitive means for detecting rubber particles.
Electron micrographs of tobacco stems showed that rubber particles were not produced in untransformed control plants (FIG. 3A) but in transformed plants (FIGS. 3B and 4C). Tobacco gum particles (arrows) are very similar to guayule gum particles (Backhaus and Walsh,
Bot. Gaz. 144: 391-400 (198
3)), first appearing in the vacuoles of plants. This is the first report of rubber biosynthesis in non-rubber-producing foods by transformation procedures.

A further demonstration of rubber production in transgenic tobacco is the highly specific staining for rubber (Addicot).
t, Tain Technol. 19: 99-102
(1944)). This stain is only effective when a relatively high concentration of rubber is produced in the tissue. No rubber was found in the cells of the non-transformed plant (FIG. 5A) and in the cells of the transformed plant (FIG. 5B) as well as in the light microscope, as in the electron microscope. . Rubber is
Appear in the cells as dark blue stained spheres (arrows). These rubber-producing cells are not evenly distributed in the plant but are concentrated in the outer layer of the trunk. It is believed that these cells produce the precursors (probably small chain polyisoprenes) required for the synthesis of high molecular weight rubber. However, it is clear that RPP is necessary to induce this type of rubber biosynthesis.

As described herein, cloning of the RPP cDNA and elucidation of its protein sequence and structural region allows for structural-functional analysis of its role in the biosynthesis of rubber in guayule and other organisms. It will provide the basis for the decision. As mentioned herein, RPP is not a prenyltransferase, but a non-monooxygenase type of cytochrome P450. RPP can generate lipid epoxide from lipid hydroperoxide,
This seems to be crucial for the biosynthesis of high molecular weight rubber. Also, expression of RPP in natural or exogenous hosts can be regulated by incorporating the pRPP30 cDNA downstream of the appropriate 5'promoter and transferring the recombinant gene into the genome of another host. Expression of the functional RPP enzyme can then be performed by a sensitive biochemical assay that monitors the rapid degradation of LOOH in the presence of RPP.
The effect of RPP gene expression on rubber biosynthesis is also
Shown by electron and light microscopy of transformed plants. Thus, the present invention is important to agriculture, biology and chemistry and provides the basis for the production of natural rubber in a wide range of organisms, including prokaryotes and eukaryotes, using recombinant DNA methods. Disclosed are DNA sequences encoding RPP and its proteins.

Accordingly, a first object of the present invention is to provide a recombinant DNA molecule and a DNA sequence encoding RPP.

Another object of the present invention is to provide a new and improved method for producing RPP polypeptides from recombinant DNA molecules encoding RPP and DNA sequences.

Yet another object of the present invention is to provide a vector containing a suitable DNA molecule encoding RPP,
And to provide a culture producing recombinant rubber particle protein.

Yet another object of the present invention is the use of recombinant RPP to promote rubber synthesis in other host organisms.

To fully understand the invention described below, some definitions are provided.

Nucleotide is a monomer unit of DNA or RNA consisting of a sugar moiety (pentose), a phosphate and a nitrogen heterocyclic base. The base is attached to the sugar moiety by the glycosidic carbon (the 1'-carbon of the pentose), and such a base-sugar linkage is called a nucleoside. The base characterizes the nucleotide. The four DNA bases are adenine (A), guanine (G), cytosine (C) and thymine (T). The four RNA bases are A, C, G and uracil (U).
Only one strand is shown, as is commonly done for convenience in structural representation of DNA nucleotide sequences, where A on one strand contains T on its complement and G is C
including. Amino acids are represented by the following three-letter or one-letter abbreviations.

A = Ala = alanine; C = Cys = cysteine; D = Asp = aspartic acid; E = Glu = glutamic acid; F = Phe = phenylalanine; G = Gl
y = glycine; H = His = histidine; I = Ile =
Isoleucine; K = Lys = Lysine; L = Leu = Leucine; M = Met = Methionine; N = Asn = Asuraparagine; P = Pro = Proline; G = Gln = Glutamine; R = Arg = Arginine; S = Ser = Serine; T
= Thr = Threonine; V = Val = Valine; W = Tr
p: tryptophan; Y = Tyr = tyrosine.

By DNA sequence is meant an alignment of linear nucleotides linked one after another by phosphodiester bonds between the 3'and 5'carbons of adjacent pentoses.

A codon is an amino acid through mRNA,
By three nucleotide (triplet) DNA sequences encoding a translation initiation signal or a translation termination signal is meant. For example, Nucleotide Triplet TT
A, TTG, CTT, CTC, CTA and OTG encode the amino acids leucine (Leu), and TAG, TA
A and TGA are translation stop signals, and AT
G is a translation initiation signal.

By open reading frame is meant the grouping of codons during translation into mRNA amino acid sequence. Appropriate reading frames must be maintained during translation. For example, GC
The DNA sequence TGGTTTGTAAG must be expressed in three reading frames, each of which gives a different amino acid sequence.

GTC GGT TCT AAG- Al
a-Gly-Cys-Lya; G CTG GTT GT
A AG -Leu-Vla-Val; GO TGG
TTG TAA G -Trp-Leu- (STOP)

Polypeptide and peptide refer to a linear alignment of amino acids that are linked one after the other by a peptide bond between the alpha-amino and carboxyl groups of adjacent amino acids.

The genome is the whole DN of a cell or virus.
Means A. Among other things, it means the constitutive genes encoding the polypeptide of the substance, as well as the operators, promoters, terminators, enhancers, and ribosome binding and interaction sequences.

A gene is a template or messenger RNA that has a sequence of amino acids characteristic of a particular peptide.
(MRNA) means a DNA sequence encoded by.

[0036] cDNA means m as a template for synthesizing the first strand of DNA using reverse transcriptase, a suitable oligonucleotide primer and a mixture of nucleotides.
It means complementary or copy DNA made with RNA.

PCR means the following polymerase chain reaction. That is, a specific DNA sequence of genomic or cDNA, synthetic oligonucleotide sense and antisense primers (they specify the target sequence), nucleotide mixture and temperature thermocycling conditions (which target between the primers). Sequential denaturation of DNA,
It can be preferentially amplified by the enzyme Taq polymerase (which allows annealing and synthesis).

Transcription means m from a gene or DNA sequence.
It means the process of producing RNA.

[0039] Translation means a process of producing a polypeptide from mRNA.

By expression is meant the process performed by a gene or DNA sequence to produce a polypeptide,
It consists of transcription and translation.

By plasmid or phagemid is meant a non-chromosomal double stranded DNA sequence consisting of a complete replicon such that the plasmid is replicated in the host cell. When a plasmid is inside a unicellular organism, the properties of that organism can be altered or transduced as a result of the DNA of the plasmid. For example, a plasmid carrying the gene for ampicillin resistance (AMPR) transforms cells initially sensitive to ampicillin into those resistant to it. Cells transformed with the plasmid are called transformed cells.

Phage or bacteriophage
By bacterial viruses, many of which consist of DNA sequences (envelopes) encapsidated in a protein envelope or membrane.

A cloning vehicle is a plasmid, phagemid, binary vector, phage DNA, cosmid or other DNA sequence capable of replicating in a host cell characterized by one or a few endonuclease recognition sites. . Here, at that site, the DNA sequence can be cleaved in a determinable manner without concomitant loss of essential biological functions of DNA (eg, replication, production of membrane proteins or loss of promoters or binding sites). , And the site contains a marker suitably used to identify transformed cells (eg ampicillin resistant). Cloning vehicles are often referred to as vectors.

Cloning means the process of obtaining a sequence or DNA sequence derived from an organism or many organisms by asexual reproduction.

Recombinant DNA molecule or hybrid DN
By A is meant a molecule composed of segments of DNA from different genomes that are held end-to-end outside the living cell and can be retained inside the living cell.

The expression control sequence means a nucleotide sequence that controls and manages the expression of these genes when operably linked to the genes. It includes but is not limited to: That is, lacZ
Promoters, trp promoters, tac and trc promoters, T7 polymerase promoters, other major prokaryotic and eukaryotic promoters that increase expression of exogenous genes in prokaryotic hosts, NOS promoters and terminators, CaMV promoters and eukaryotic cells. Other prokaryotic and eukaryotic promoters that increase the expression of the genes of, or viruses or combinations thereof.

Guayule gum particle protein (RPP) is
It is a 53,500 dalton polypeptide in guayule rubber particles. It is a cytochrome P450 heme-binding protein, a non-monooxygenase with a function essential for the biosynthesis of rubber in plants involved in the production of epoxides.

The present invention is a D encoding guayule RPP.
It relates to NA sequences and recombinant DNA molecules; methods for producing these polypeptides; recombinant expression systems for those sequences; vectors containing them; cultures producing recombinant proteins; and materials necessary for their production. A DNA sequence encoding a guayule RPP or protein having substantially the same biological activity for rubber biosynthesis as guayule RPP is shown.

The isolated recombinant vector containing the cDNA encoding the guayule gum particle protein (RPP) has been characterized in that the RPP DNA is expressed to produce a functional RPP for the biosynthesis of gums, etc. Transformed into a prokaryotic or eukaryotic host organism. Rubber biosynthesis occurs as a result of recombinant RPP vector DNA expression in these host organisms.

Although many selection and DNA cloning techniques have been used successfully in the isolation and cloning of the DNA sequences of the present invention, a strategy of selection based on purified RPP has been adopted.

Purification of RPP and decomposition of CNBr

The raw material of RPP is Cornish described above.
and Backhaus (1990) and Backh
aus et al. Purified rubber particles isolated from guayule type 11591 stem bark tissue by the method described in the article of (1990). Purified rubber particles are SDS-P for preparation
When RPP was purified by AGE, 48500 to 5
It is recognized in the gel as a protein band having a mixed molecular weight of 3500 daltons. RPP is purified from the gel band by electroelution using a Bio-Rad electroelutator (Model 422) according to its manual.

At least 1000 pmole of purified RPP is lyophilized to dryness and dissolved in 150 microliters of formic acid in an Eppendorf tube. The mixture is incubated for 24 hours at room temperature in the dark. The RPP digested with this CNBr was used as Schagger and von.
Jagow, Anal. Biochem. 166: 36
SD using a 16% acrylamide gel in Tris-Tricine in a three-layer system as described in 8-379 (1987).
Attached to S-PAGE. The protein fragments were blotted onto a PVDF (Millipore) membrane by electrophoresis and Plo
ug et al. Anal. Biochem. 18
Visualize according to the method of 1: 33-39 (1989). A region of the PVDF membrane containing the stained peptide was cut out and used for N-terminal amino acid sequencing (Uni
v. Calif. Davis, Protein Str
ucture Lab. ). The amino acid sequences of four distinct peptide fragments of RPP are shown in Table 1.

[Table 1]

Isolation of guayule stem bark mRNA.

Guayule stem bark (10 g) was cut to a size of 1 cm, and the amount of guanidine (8 M guanidine HCl, 20 mM ES pH 7,
20 mM EDTA, 50 mM mercaptoethanol)
Homogenize in. The homogenate was extracted with 1 volume of phenol: chloroform and then at 15 degrees Celsius at 10,000 rpm (Solvall SS-3.
4 rotors) Centrifuge. Transfer the aqueous phase to a new tube, centrifuge at 10,000 rpm for 10 minutes at 15 degrees Celsius and transfer the supernatant to a new tube. To this is added 0.7 volume of pre-chilled 100% ethanol and 0.2 volume of 1M acetic acid. This sample is kept at -20 degrees Celsius overnight
Keep in time. RNA is 10 at 5000 rpm at 4 degrees Celsius
Centrifuge for minutes and collect. The pellet was washed with 3M sodium acetate (pH: 5.2) at room temperature and washed with 10000r.
Centrifuge for 5 minutes at pm. The pellet is washed with 70% ethanol, dried under reduced pressure and dissolved in sterile water. Poly-A mRNA was found in Sam-brook et a
l. Molecular Cloning, A Lab
oratory Manual, 2nd E. 7.26
Purified by fractional solidification on oligo-dT cellulose according to the method described in 7.29 (1989).

Construction of guayule stem bark cDNA library.

Guayule stem bark is used for the generation of cDNA for the construction of the library in Lambda-ZAP available in kit form from Straightgene Inc. according to the manufacturer's instructions. Lambda ZAP, Short et
al, Nucl. , Acids Res. 16:75
83-7600 (1988).

PCR amplification of guayule RPP cDNA.

The guayule stem bark cDNA library was originally screened with the sequence P5 / 6 (Table 2) of the 92 bp probe generated by PCR amplification of the guayule cDNA.

First strand cDNA synthesis was performed using the Straight Gene cDNA synthesis kit according to the manufacturer's instructions. About 1.5 micrograms of guayule stem bark was incubated for 1 hour at 37 degrees Celsius in the presence of oligo-dT, reverse transcriptase, and nucleotide mixture. Single-stranded cDNA is 0.5M final concentration of ammonium acetate,
Then 2 volumes of ethanol are added to effect precipitation. The mixture is microcentrifuged for 10 minutes at room temperature with 200 microliters of 70% ethanol at 14000 rpm, dried under reduced pressure and redissolved in 50 microliters of sterile water.

Sequence P5 (Table 2) of the degradant, which is a 20-mer oligonucleotid primer corresponding to the sense single strand of CNBr fragment # 3, and the antisense single strand of CNBr fragment # 3 (Table 1). The sequence P6 (Table 2) of the degradation product, which is a 20-mer oligonucleotid primer corresponding to DNA, was changed to 92bpP5 / 6.
Used to generate an array. 20 microliters of PCR reaction mixture [contains: each primer 10
0 pmol; 1 microliter of the above mentioned cDNA mixture; 0.2 mM of each nucleotide; and 20 mM
0.5 microliters of replinase in 50 mM Tris-HCl buffer (pH: 9.0) containing ammonium sulphate and 1.5 mM magnesium chloride]
Amplify for 39 cycles under heat cycling conditions of 55 minutes for 2 minutes, 55 degrees for 2 minutes and 72 minutes for 3 minutes, and finally for 10 minutes at 72 degrees.

[Table 1]

[Table 2] 9 when the obtained mixture was applied to agarose gel
It turns out to be a 2 bp fragment. DNA sequence analysis of this fragment revealed a CNB as shown in Figures 2 and 3.
It can be seen that the amino acid sequences of the r fragments match exactly.

Following this, the P5 / 6 radiolabeled ³² P probe was applied to 50 of each primer P5 and P6.
P5 / with Dmol and purified as template DNA
Regenerate by PCR reaction utilizing 6. This is the same as that described in Sambrook, et al. Results in the highly specific active probes used to screen the lambda ZAP cDNA library.

Following sequencing of the pRPP30 clone, P5 / 6 was found to encode the 3'-terminal region of the RPP gene (Table 3). That is, when the cDNA library was screened with P5 / 6, 900b
Only partial cDNA clones shorter than the length of p are mainly isolated. Conversely, a small number of these partial cDNAs were used to isolate the full length pRPP30 cDNA clone (Table 3).

[Table 3]

Screening of the cDNA library.

Plaques containing the recombinant lambda ZAP cDNA were transferred to a membrane filter (DuPont, colony / plaque screen), 6X SSPC, 5X.
Denhardt's solution, 50% formaldehyde, 0.5% S
An initial screen is performed by hybridization to radiolabeled P5 / 6 with DS, 200 microgram / ml salmon sperm DNA and 10% sodium dextran sulfate for 18 hours at 42 degrees Celsius. 2XSSC
Wash twice for 10 minutes in the well, then with 2X SSC and 0.1
Wash twice in 20% SDS for 20 minutes. Positive plaques from the first screening were hybridized to the radioactive label P5 / 6 in 6XSSPE, 5X Dunhardt's solution, 0.5% SDS and 100 microgram / ml salmon sperm DNA by nitrocellulose (Schleicher & Schleicher & Schleicher & Schleicher & Schleicher & D. Schuell Schleicher and
Rescreen on Shuell) at 42 degrees Celsius for 18 hours and perform the same wash as the initial screen.

Clones showing a positive response to the second screen are then cleaved and circularized according to the lambda ZAP protocol. Phagemid DNA,
Isolate by standard miniprep method and separate on agarose gel. Bluescript containing the largest cDNA insert (Bluescript)
t) These colonies producing phagemid are further characterized by DNA sequencing.

DNA sequencing.

The DNA sequence was determined by the dideoxy chain growth termination method using Sequenase (US Biochemical Corporation) according to the manufacturer's instructions.

Cleavage of RPP with CNBr yielded four separate peptide fragments that could be sequenced from the N-terminus (Table 1).

The amino acid sequence spans 17 to 36 residues in length. Of these, the peptide fragments # 3, # 4 and # 2 yield a deduced DNA stretch of at least 17 bp with low resolution (Table 2).

[Table 2] Large guayule cDNA by PCR amplification by synthesizing oligonucleotides corresponding to these parts
Used to generate fragments. Of these, two PCR products were successfully obtained. They are known C
P of 92 bp sequence that matches NBr fragment # 3
5/6 (Table 2); CNBr fragments # 4 and #
It was a 434 bp sequence (Table 3) between the P1 and P9 primers that included the region of the RPP gene joining 3. Sequence P5 /
6 is a large cD showing c17 containing an 823 bp insert encoding the 3'end of the RPP gene (not shown).
NA clone 2 was used as a probe for successful isolation.

[Table 2]

[Table 3]

A full-length cDNA clone was isolated by screening against both a 92 bp probe and a 434 bp probe. The analysis result of full-length pRPP30 is
1, 44, 56, 97, 129, 252, 327 and 3
8 methionines at position 71, and 274 and 426
An open reading frame containing two cysteines at the position is shown (Table 3).

Table 3 pRPP30 polypeptide also binds 132,
It probably contains three N-linked glycosylation sites at positions 318 and 354. This is consistent with the structure of guayule RPP, which is known as a glycoprotein. pRPP3
The 0 code region is negatively charged with a deduced pKa value of 6.15 which matches the experimentally derived value of 6.2. This pRPP30 clone contains a TAG stop codon and an AT-rich 250b expected in the terminal sequence.
It contains the 3'-noncoding sequence of p. The 5'-noncoding region contains 23 bp and the common plant gene translation initiation site.

GEN using the BLAST algorithm
A computer study of BAK / EMBL and SWISSPROT showed homology with several cytochrome P450s in a small dispersed region with the amino acid sequence of the 473 amino acid pRPP30 open reading frame. It was subsequently found that the RPP protein sequence shows considerable homology with the flaxseed AOS sequence (see Song et al., Supra).

Analysis of RPP as cytochrome P450.

RPP is AOS type cytochrome P4
In order to demonstrate that it has the properties of 50, Song and Brach (199
1); Zimmerman (1966); Zimmer
man and Vick (1970); and Zim
It was analyzed according to the method of merman and Fen (1978).

Biochemical analysis of prenyl transferase activity.

Washed rubber particles were removed from the crude homogenate of guayule stem bark from Cornish and Back-h.
made by centrifugation according to the method of Aus (1990) (supra). Sonicate washed particles in 0.5% CHAPS and remove all remaining rubber particles M
illi-pore, Millex-GV, U. The solution was passed through a 22 micrometer 3LGV to solubilize the RPP. Aliquot 100 of protein extract
Method using dimethylallyl pyrophosphate (DMAPP) as an initiator for measuring activity in microliters (Dogbo and Camara, supra)
(1987) and Kuntz et al (1992).
Measured at each purification step by measuring the transfer of ¹⁴ C isopentenyl pyrophosphate to the isoprenoid derivative according to.

Construction of the pBIRPP binary vector.

One means of using the present invention is the pBI121 two-component plasmid vector of the 35S promoter (J
efferson R.E. A. et al. EMBO
J. 6: 3901-3907 (1986)) R downstream
This is the insertion of the PP code area. This recombinant plasmid is an Agrobacterium used to inoculate one of many plants such as tobacco, sunflower and guayule.
It is transplanted to iumtumetfacies LBA4404 strain. The RPP-encoding region containing the 35S promoter is then transferred into the chromosomal DNA of the plant and the plant is replicated. The resulting transformed plant becomes a large amount of RPP, which then undertakes rubber synthesis in its cells. Rubber synthesis is a substrate (IPP,
DMAPP, EPP, Mg, etc.) are present. The rubber thus produced is collected from plant tissue.

Transformation of tobacco is carried out using a binary vector derived from pBI121 in which the GUS gene replaces the RPP downstream of the strong CaMV35S promoter. This new vector was originally pBI12
1 was digested with SmaI and SstI to give a 1.9 kb GU
It is constructed by removing the S insert and then filling in the SstI site with T4 polymerase. The resulting linear 10 kb fragment is gel purified and cyclized in a blunt end ligation reaction. This gives the cyclic GUS minus pBI121. Then, this was cut with BamHI and Klenow (Kl
Fill with enow) and cut again with XbaI. The linear fragment containing one sticky XbaI terminus and the BamHI blunt end is gel purified. at the same time,
PRPP30 in the SK-vector is digested with XhoI and loaded with Klenow. This linear plasmid was then digested with XbaI to blunt 3'end and RPP.
Gives an RPP fragment with one attached XbaI site at the 5'end of It is linked to a complementary GUS-minus fragment and contains the 12.8 kb pB containing the RPP gene downstream of the sense configuration of the CaMV35S promoter.
Generates an IRPP binary plasmid vector (Table 9).
Tobacco plants are leaf disc methods.
method; Horsch et al. Scie
, 227: 1229-1231 (1985)), selected and propagated.

Microscopic observation of transformed tobacco.

The transformed tobacco plants are the above-mentioned Add.
After staining a piece of tissue according to the method of Icott (1944), it is then examined under a light microscope or as described above in Backhaus.
It was observed with an electron microscope according to the method of And Walsh (1983).

Other uses of the present invention are as follows.

Another means of utilizing the present invention is to use the RPP coding region for the GAL10 promoter in the pYEDP60 plasmid vector (Turan et al. Gene).
125: 490-55 (1933)) insertion downstream. This plasmid is then transferred to yeast and allowed to grow for some time before being galactose-induced to produce high levels of RPP. Eukaryotes were cytochrome P-450 (Urban et al. Bioch).
Yeast compatible with imi 72: 463-472 (1990)) can be selected. Gum biosynthesis occurs because the substrates for IPP and FPP are synthesized by yeast. The gum so produced is then harvested from the cells.

Another means of utilizing the present invention is RP
The P coding region was cloned into the T7 promoter pET3 plasmid vector (Sturdier, et al. M).
methods in Enzymol. 185: 60-
98 (1990)) is to insert downstream. This plasmid was then transformed into E. E. coli (BL21 strain) and grown for several hours to induce the production of T7 polymerase which gives rise to higher levels of RPP. Substrate I into medium
When PP and FPP are supplied, rubber biosynthesis occurs. The rubber thus produced is then harvested from the cells.

The cDNA molecules of the present invention can be operably linked to expression control sequences and used to produce RPP in a variety of eukaryotic and prokaryotic host cells. The cDNA sequences of the present invention also include cDNAs derived from other sources capable of rubber biosynthesis.
Alternatively, it is useful as a probe for screening genomic DNA. Similar to the above cDNA sequences of the present invention, these genomic sequences are also useful for isolating the RPP gene from other organisms.

[Brief description of drawings]

FIG. 1 shows the results of biochemical analysis of RPP purified from guayule rubber particles. A is Coomassie blue (Coomassie B) showing each of the purification steps for obtaining purified RPP.
SDS gel stained with lute). 1 contains crude homogenate; 2 contains rubber particles washed three times;
3 is a millipore that removes all the remaining rubber particles,
Millex GV, 0.22um, containing 0.5% CHPS solubilized RPP filtered through a SLGV filter; and M contains a molecular weight marker. B is an autoradiograph of a TCL plate, which is 1, 2, and 3 of A.
3 represents the product of phenyltransferase activity from each of the purification steps of Aliquots from each purification step
¹⁴ C-IPP, DMAPP, Mg ⁻² and Mn ⁻²
The isoprene of various chain lengths is obtained by culturing with. Where SQ is squalene; P is phytoene;
X is unknown isoprene; GG is geranylgeraniol;
F is farnesol; G is geraniol; and O is the origin. Note that the purified RPP in 3 is 1
Compared with the crude homogenate in 2 and washed rubber particles in 2,
The point is not to synthesize isoprene. C is the difference spectrum of filtered RPP soluble in purified CHAPS from A-3. The reference and authentic cuvettes are reduced with sodium dithionate and blown with authentic cuvette CO, producing a characteristic peak at 450 nm (arrow). D shows AOS-like activity in an aliquot of filtered RPP soluble in purified CHAPS from A-3. The activity is measured by repeated UV scans at 10 second intervals and shows the disappearance of linoleic acid hydroperoxide (LOOH) with an absorption maximum at 234 nm. When purified RPP was added to the cuvette, LOOH was rapidly metabolized to allene oxide, which was 234n
Observed by a decrease in the absorption of m. Complete disappearance of LOOH occurs after 10 minutes.

FIG. 2 shows the results of biochemical analysis of tobacco plants transformed with the pBIRPP binary vector producing functional pPP. A represents a Coomassie-stained SDS gel of total protein extracted from RPP-transformed tobacco expressed in the sense configuration. It shows the RPP (arrows) of four different transformants (# 3, # 16, # 14 and # 17). Untransformed control tobacco (C), and R
Tobacco (A) with antisense construction of PP cDNA is RP
Do not produce P. The position of the standard from RPP in the molecular weight marker (M) and washed guayule gum particles (RPP) is indicated. B shows Western hybridization of the same gel as A. The blot is probed with a monospecific RPP-1 antibody and stained with a second antibody conjugated with alkaline phosphatase. The blot shows a single band of RPP (arrow) for each tobacco transformant (# 3, # 6, # 14 and # 17) in the sense configuration. Control tobacco (C) and antisense construct (A) do not produce RPP. C has an activity of functional RPP similar to AOS,
It is shown to be absent in the protein extract of untransformed control tobacco (C). These plants do not produce RPP,
Therefore, it does not metabolize LOOH. The spectrum shows repeated scans performed at 10 second intervals for a few minutes without loss of LOOH in the standard cuvette. D shows high AOS activity in the protein made from Tobacco Transformant # 3.
These plants express the external guayule RPP gene,
Produces a functional RPP. This is 234 nm scanned at 10 second intervals after the next addition of tobacco protein extract.
Observed due to the rapid disappearance of the LOOH peak. 1
Scans after 0 and 30 seconds are indicated and labeled with arrows, respectively. 10 repeated scans over a few minutes
Shows complete disappearance of LOOH in the standard cuvette after minutes.

FIG. 3. Untransformed (A) and functional Guayule RPP.
3 is an electron micrograph of pBIRPP-transformed (B and C) tobacco (transformant # 3) that produces Escherichia coli. The non-transformed plant (A) does not contain rubber particles, but the transformed plant (B
Cells from C and C) contain rubber particles (arrows) found mainly in vacuoles. The bar corresponds to 2 um.

FIG. 4 is a continuation of FIG.

[Fig. 5] Rubber-specific Calco Oil Blue (Calc)
FIG. 3 is a light micrograph of non-transformed (A) and pBIRPP transformed (B) tobaccos stained with o Oil Blue). Cells of non-transformed plants do not contain rubber, whereas cells of transformed plants do contain rubber as dark blue-stained spheres (arrows) in the cells.

Continuation of the front page (51) Int.Cl. ⁶ Identification number Office reference number FI technical display location C12P 5/02 9548-4B (C12N 15/09 ZNA C12R 1:91) (C12N 5/10 C12R 1:91) (C12N 9/02 C12R 1:91) (C12P 5/02 C12R 1:91) (C12N 15/00 ZNA A C12R 1:91) (C12N 5/00 C C12R 1:91)

Claims (9)

[Claims] 1. As shown in Table 3, CTCACATTCA
Starts with a nucleotide, ATTGTTTCCACAAAA
Guayule gum particle protein gene clone pRPP containing a 1692 base pair fragment ending in TTTAGAAAAAAAAAAAAAAAAAA nucleotides
30. [Table 3] 2. A single nucleotide sequence shown in Table 3 which encodes a guayule gum particle protein having a peptide sequence of 473 amino acids beginning with Met-Asp-Pro-Ser and ending with Arg-Ala-Ser-Ile. Separated cDNA molecule. [Table 3] 3. An isolated cDNA molecule having a nucleotide sequence encoding a guayule gum particle protein having the amino acid sequence shown in Table 3. [Table 3] 4. The isolated cDNA molecule of claim 2 having a DNA promoter operably linked to affect expression of said molecule. 5. The DNA promoter is a lacZ promoter, a trp promoter, a trc promoter,
tac promoter, T7 polymethase promoter,
GAL1 and GAL10 promoters and prokaryotic hosts and N
The group consisting of the OS promoter and terminator, the 35S promoter and other prokaryotic and eukaryotic promoters that increase the expression of foreign genes in eukaryotic cells or other prokaryotic and eukaryotic promoters. A recombinant DNA molecule according to item 4, which is selected from 6. An isolated recombinant vector comprising a DNA fragment encoding a 53,500 dalton gum particle protein of guayule or a naturally occurring variant thereof, wherein the gum particle is a guayule gum particle protein shown in Table 2. At 45 ° C. to the DNA encoding
M NaCl, 0.045 M sodium citrate and 0.1
% Sodium dodecyl sulphate wash, and shall be encoded by DNA that hybridizes under the conditions shown. [Table 2] 7. The isolated recombinant vector of claim 6, wherein said vector is transferable to the host and reproducible in the host. 8. The isolated recombinant vector of claim 7, wherein said host expresses said DNA fragment upon receiving said hybrid. 9. The isolated recombinant vector of claim 8, wherein said host is selected from the group consisting of plants, bacteria and fungi.