WO2013036100A1

WO2013036100A1 - A method for regulating plastidial isoprenoid biosynthesis in hevea brasiliensis

Info

Publication number: WO2013036100A1
Application number: PCT/MY2012/000213
Authority: WO
Inventors: Alam MAQSUDUL; Nazalan Mohd Najimudin MOHD; Ann Saito JENNIFER; Biswavas Misra BISWAPRIYA
Original assignee: Universiti Sains Malaysia
Priority date: 2011-09-05
Filing date: 2012-07-23
Publication date: 2013-03-14

Abstract

The present invention relates to an isolated polypeptide for catalyzing plastidial biosynthesis of isopentenyl diphosphate, dimethylallyl diphosphate or the combination thereof in the plant of Hevea brasiliensis, comprising amino acid sequence set forth in SEQ ID NO. 2, SEQ ID NO. 4, SEQ ID NO. 6, SEQ ID NO. 8, SEQ ID NO. 10 or SEQ ID NO. 12; and a method for catalyzing the biosynthesis of isopentenyl diphosphate, dimethylallyl diphosphate or the combination thereof for enhancing the biosynthesis of natural rubber from its precursors. The present invention also relates to an isolated polynucleotide encoding the polypeptide, a recombinant gene construct comprising the polynucleotide, a transformant and a transgenic plant comprising the recombinant gene construct.

Description

A METHOD FOR REGULATING PLASTIDIAL ISOPRENOID

BIOSYNTHESIS IN HEVEA BRASILIENSIS

FIELD OF INVENTION

The present invention relates to the specific genes encoding for a series of enzymes involved in the biosynthesis of isoprenoids in the plant of Hevea brasiliensis. More particularly, the present invention provides the polynucleotide sequences of the genes encoding for the series of enzymes involved in the plastidial biosynthetic pathway of isoprenoids, which allows specific genetic intervention to be conducted in the plastidial pool of isoprenoids in H. brasiliensis.

BACKGROUND OF THE INVENTION

Rubber tree has been an important industrial crop for natural rubber production throughout the decades. It is reported that the world supply of natural rubber is barely keeping up with the estimated global demand for 12 million tons of natural rubber in 2020. In order to keep up with such increase of the global demand, further improvement of natural rubber production has become a necessary. One of the strategies taken by the industry to promote the natural rubber production is to rapidly expand the rubber plantations throughout the Montane main lands in South East Asia regions. Another strategy developed is a molecular biology approach, whereby the rubber biosynthesis process of the rubber tree is explored and improved.

Natural rubber is cytoplasm of laticifer cell. It is synthesized by at least 2,000 species of plants belonging to 300 genera. Non-Hevea plant species with abilities to produce natural rubber include the Russian Dandelion and Euphorbia lactiflua. However, H. brasiliensis is deemed the only economically- viable source of natural rubber due to its good yield of rubber and the excellent physical properties of the rubber products. Latex is a biodegradable polymer yielding the natural rubber. It is widely used worldwide, because of its excellent properties in terms of high elasticity and mechanical strength including high resistance to impact and tear as well as low heat build-up during deformation. High yields of latex can be obtained from the H. brasiliensis clone RRIM 600, which is a clone evolved by the Rubber Research Institute of Malaysia (RRIM) from parent clones Tjir 1 & PB 86. This clone has an above average initial latex yield and a very high level of subsequent yield. In general, rubber biosynthesis consists of 6 major steps, which include the sucrose import and degradation, glycolysis, acetyl-CoA biosynthesis, prenyl diphosphate synthesis via mevalonate (MVA) pathway in cytosol, geranylgeranyl pyrophosphate synthesis in mitochondria, and rubber polymerization on rubber particle membrane. In recent years, biosynthesis of natural rubber is known to be biochemically related to the biosynthesis of isoprenoids, as the significant metabolites in the biosynthetic pathway of rubber, which are the isopentenyl diphosphate (IPP) and dimethylallyldiphosphate (DMAPP), can be synthesized by these two independent isoprenoid biosynthetic pathways, namely the MVA pathway which takes place in the cytosol, and the methylerythritol phosphate (MEP) pathway which is localized in the plastids.

Isoprenoids, also known as terpenoids, are derived from IPP units, which are the acyclic C₅ precursor, known as 2, 3-methyl butane. The head -to-tail addition of isoprenoid units forms the basis of biosynthesis of terpenoids. The first biosynthetic pathway identified, namely the MVA pathway, is operative in bacteria, fungi, mammals and plants. Whilst, the MEP pathway is conserved in both eubacteria and higher plants. Principal MVA-derived isoprenoid end products in plants are sterols, brassinosteroids (steroid hormones), dolichol (involved in protein glycosylation), and the prenyl groups used for protein prenylation and cytokinin biosynthesis; while the plastidial MEP pathway produces IPP and DMAPP for _. the biosynthesis of photosynthesis-related isoprenoids (carotenoids and the side chains of chlorophylls, plastoquinones, and phylloquinones) as well as hormones (gibberellins and abscisic acid). In spite the compartmentalization of these two pathways, MVA-derived precursors can be used for the synthesis of isoprenoids in the plastid, and MEP- derived precursors can also be exported to the cytosol in at least some plants, tissues 5 or developmental stages.

As natural rubber is a polymer of IPP units condensed sequentially in cis- configuration by the cis-prenyl transferases group of enzymes, and DMAPP is also one of the initiator molecules which is required to prime the subsequent extensive

0 prenyl chain elongation, the regulation of the biosynthesis of rubber can therefore be achieved by intervening the synthesis of the series of enzymes involved in the biosynthesis of isoprenoids of H. brasiliensis. These enzymes include 2C-methyl-D- erythritol 4-phosphate synthase/1 -deoxy-d-xylulose 5-phosphate reductoisomerase (DXR), 2-C-methyl-D-erythritol 4-phosphate cytidylyltransferase (CMS), 4-

5 diphosphocytidyl-2C-methyl-D-erj^thritol kinase (CMK), 2C-methyl-D-erytrithol 2,4- diphosphate synthase (MCS), 2C-methyl-D-erytrithol 2,4-cyclodiphosphate reductase (HDS) and 1 -hydroxy-2-methyl-butenyl 4-diphosphate reductase (HDR).

I the MEP pathway, which is also known variously as non-mevalonate pathwaj', '.0 deoxyxylulose phosphate pathway or Rohmer pathway, the C-5 basic isoprenoid unit is derived from pyruvate and D-glyceraldehyde phosphate (GAP), which are condensed to give 1 -deoxy-D-xylulose 5-phosphate (DXP) by the enzyme DXS. The rearrangement and reduction of DXP to form 2-C-methylerythritol 4-phosphate (MEP), the first committed intermediate in this pathway, is catalyzed by the enzyme !5 DXR. MEP is then appended to CTP to form 4-(cytidine 5'-diphospho)-2-C-methyl-D- erythritol, followed by phosphorylation of the C2 hydroxyl group and elimination of CMP, to form a 2.4-cyclic diphosphate. Subsequent^, 1 -deoxy-d-xylulose 5- phosphate reductoisomerase. also known as 2C-methyl-D-erythritol 4-phosphate synthase, catalyses the first reaction completely specific to the MEP pathway, which is 10 the conversion of DXP into MEP. MCS, the third enzyme in the MEP pathway, catalyzes an Mg-dependent cyclisation step of 4-diphosphocytidyl- 2C-methyl-D-erythritol 2-phosphate into 2C-methyl- Derythritol 2, 4-cyclodiphosphate with release of CMP. This is followed by the HDS, which catalyses the formation of HMBPP from 2-C-methyl-D-erythritol 2, 4- cyclodiphosphate (MEcPP) by an as-yet-unknown mechanism. At a later stage, the HDR catalyzes the last step of the 7-stepped MEP biosynthetic pathways for isoprenoids. It directly converts l-hydroxy-2-methyl-butenyl 4- diphosphate (HMBPP) into a mixture of IPP and DMAPP with the ratio of 5 : 1.

Genes encoding the enzymes involved have been found or cloned from a number of organisms, including plants, and these genes encode polypeptides (enzymes) with certain conserved regions of homology. However, none of the prior art discloses the polynucleotide sequences or polypeptides encoded thereof for these series of isoprenoid biosynthetic genes or enzymes which are specifically found in or derived from the plastidial isoprenoid pool, especially in the plant of H. brasiliensis. There is also no successful technique provided in the prior art to elucidate these related enzymes. In view of the fact that the series of enzymes involved in the MEP pathway could play an important role in the biosynthetic pathway of rubber, it is desirable for the industry to provide a genetic approach relating the biosynthesis of rubber in plant by exploring and utilizing the molecular biology and genetic information of this series of enzymes. Besides, a species-specific approach is also preferable in order to yield a cost-effective result as the rubber biosynthetic pathway and genetic makeup of each species of plant are potentially varied among one another.

SUMMARY OF INVENTION The primary object of the present invention is to provide the polynucleotide sequences encoding for a series of enzymes, including DXR, CMS, CMK, MCS, HDS and HDR which are involved in the plastidial biosynthesis of isoprenoids via MEP pathway, in the plant of H. brasiliensis.

Another object of the present invention is to provide the molecular biology and genetic information of the genes and enzymes set forth in the primary object to be exploited for the regulation of the plastidial biosynthesis of isoprenoids in the plant of H. brasiliensis.

Still another object of the present invention is to provide the isolated polypeptides encoded by the polynucleotide sequences provided, which is the series of enzymes involved in the plastidial biosynthetic pathway of isoprenoids to form IPP and DMAPP.

Yet another object of the present invention is to manipulate or regulate the biosynthesis of cis-polyisoprene (natural rubber) in H. brasiliensis, in vitro or in vivo, by genetically intervening the MEP biosynthetic pathway of isoprenoids, particularly the IPP and DMAPP.

Further object of the present invention is to produce a transgenic plant of H. brasiliensis with increased isoprenoid production, which potentially promotes increased yield of natural rubber. Another further object of the present invention is to provide a potential commercially feasible way to increase the yield of rubber in order to keep up with the increasing global demand on rubber-based products.

At least one of the preceding objects is met, in whole or in part, by the present invention, in which one of the embodiments of the present invention describes an isolated polypeptide for catalyzing plastidial biosynthesis of IPP, DMAPP or the combination thereof in the plant of H. brasiliensis, comprising amino acid sequence set forth in SEQ ID NO. 2, SEQ ID NO. 4, SEQ ID NO. 6, SEQ ID NO. 8, SEQ ID NO. 10 or SEQ ID NO. 12.

5

In accordance with one of the preferred embodiments of the present invention, the plant of H. brasiliensis is clone RPJM 600. The isolated polypeptide is also preferably derived from this clone.

[0 Another embodiment of the present invention is a method for catalyzing the biosynthesis of IPP, DMAPP or the combination thereof for enhancing the biosynthesis of natural rubber from its precursors, comprising the step of introducing an isolated polypeptide having amino acid sequence set forth in the preceding embodiment into a plant cell in vivo or in intro.

15

Still another embodiment of the present invention is an isolated polynucleotide encoding an enzyme for catalyzing plastidial biosynthesis of isopentenyl diphosphate, dimethylallyl diphosphate or the combination thereof in the plant of Hevea brasiliensis, comprising nucleotide sequence set forth in SEQ ID NO. 1, SEQ ID NO. 10 3, SEQ ID NO. 5, SEQ ID NO. 7, SEQ ID NO. 9, SEQ ID NO. 11 or any complementary sequence thereof. Preferably, the plant of H. brasiliensis is clone RRIM 600.

Yet another embodiment of the present invention discloses a recombinant gene 15 construct comprising a polynucleotide template having nucleotide sequence set forth in SEQ ID NO. 1, SEQ ID NO. 3, SEQ ID NO. 5, SEQ ID NO. 7, SEQ ID NO. 9 or SEQ ID NO. 11, wherein the polynucleotide template is expressible in a host cell to produce an enzyme which catalyzes plastidial biosynthesis of isopentenyl diphosphate, dimethylallyl diphosphate or the combination thereof. Preferably, the >0 recombinant gene construct further comprises a promoter region operably-1 inked to enhance expression of the polynucleotide template.

Still another embodiment of the present invention is a transformant comprising a recombinant gene construct as set forth in the preceding embodiments to produce an enzyme which catalyzes plastidial biosynthesis of isopentenyl diphosphate, dimethylallyl diphosphate or the combination thereof.

Further embodiment of the present invention is a transgenic plant of H. brasiliensis with enhanced plastidial biosynthesis of isopentenyl diphosphate, dimethylallyl diphosphate or the combination thereof, comprising a recombinant gene construct having an expressible nucleotide sequence set forth in SEQ ID NO. 1, SEQ ID NO. 3, SEQ ID NO. 5, SEQ ID NO. 7, SEQ ID NO. 9 or SEQ ID NO. 11.

In the present invention, rubber tree isoprenoid biosynthetic pathway genes operative in plastids can be identified from the gene models of the H. brasiliensis genome assembly and transcriptome data, preferably using the program BLASTN with an E- value cut-off at le-20. The present invention provides a complete picture of plastidial isoprenoid biosynthetic pathway in H. brasiliensis, respective sequences of candidate genes and application thereof. This invention provides nucleic acid sequences and characterization of the genes responsible for the biosynthesis of plastidial isoprenoids. In particular, structural and functional annotation is provided for the genes. The enzymes encoded by these genes include the DXR, CMS, CMK, MCS, HDS and HDR involved in the MEP pathway. One skilled in the art will readily appreciate that the present invention is well adapted to carry out the objects and obtain the ends and advantages mentioned, as well as those inherent therein. The embodiments described herein are not intended as limitations on the scope of the invention. BRIEF DESCRIPTION OF THE DRAWINGS

For the purpose of facilitating an understanding of the invention, there is illustrated in the accompanying drawing the preferred embodiments from an inspection of which when considered in connection with the following description, the invention, its construction and operation and many of its advantages would be readily understood and appreciated.

Figure 1 is the nucleotide sequence SEQ ID NO. 1 of the polynucleotide

encoding the enzyme DXR of the plant H. brasiliensis as described in one of the preferred embodiments of the present invention.

Figure 2 is the amino acid sequence SEQ ID NO. 2 of the polypeptide encoded by the SEQ ID NO. 1 of Figure 1.

Figure 3 is the nucleotide sequence SEQ ID NO. 3 of the polynucleotide

encoding the enzyme CMA of the plant H. brasiliensis as described in one of the preferred embodiments of the present invention. Figure 4 is the amino acid sequence SEQ ID NO. 4 of the polypeptide encoded by the SEQ ID NO. 3 of Figure 3.

Figure 5 is the nucleotide sequence SEQ ID NO. 5 of the polynucleotide

encoding the enzyme CMK of the plant H. brasiliensis as described in one of the preferred embodiments of the present invention.

Figure 6 is the amino acid sequence SEQ ID NO. 6 of the pol^ypeptide encoded by the SEQ ID NO. 5 of Figure 5. Figure 7 is the nucleotide sequence SEQ ID NO. 7 of the polynucleotide encoding the enzyme MCS of the plant H. brasiliensis as described in one of the preferred embodiments of the present invention. is the amino acid sequence SEQ ID NO. 8 of the polypeptide encoded by the SEQ ID NO. 7 of Figure 7. is the nucleotide sequence SEQ ID NO. 9 of the polynucleotide encoding the enzyme HDS of the plant H. brasiliensis as described in one of the preferred embodiments of the present invention. is the amino acid sequence SEQ ID NO. 10 of the polypeptide encoded by the SEQ ID NO. 9 of Figure 9. is the nucleotide sequence SEQ ID NO. 11 of the polynucleotide encoding the enzyme HDR of the plant H. brasiliensis as described in one of the preferred embodiments of the present invention. is the amino acid sequence SEQ ID NO. 12 of the polypeptide encoded by the SEQ ID NO . 11 of Figure 11. is the electrophoresed agarose gel image showing the polymerase chain reaction (PCR) amplification result of DXR, in which lane 1 is 1 kbp D A ladder marker and lane 2 is amplicon of DXR (1416 bp). shows the restriction pattern of DXR, in which lane 1 is 1 kbp DNA ladder marker, lane 2 is undigested pJET 1.2 cloning vector harbouring DXR cDNA and lane 3 is Bglll-digested 1.2 cloning vector harbouring DXR cDNA (-1.4 kbp insert). is the electrophoresed agarose gel image showing the PCR amplification result of CMS, in which lane 1 is kbp DNA ladder marker and lane 2 is amplicon of CMS (936 bp).

Figure 16 shows the restriction pattern of HbCMS, in which lane 1 is 1 kbp DNA ladder marker, lane 2 is undigested pJET 1.2 cloning vector harbouring

CMS cDNA and lane 3 is BgHI-digested 1.2 cloning vector harbouring CMS cDNA (~.l kbp insert).

Figure 17 is the electrophoresed agarose gel image showing the PCR

amplification result of CM , in which lane 1 is 1 kbp DNA ladder marker and lane 2 is amplicon of CMK (1167 bp).

Figure 18 shows the restriction pattern of HbCMK, in which lane 1 is 1 kbp DNA ladder marker, lane 2 is Bglll-digested 1.2 cloning vector harbouring CMK cDNA (~ 1200 bp insert) and lane 2 is undigested pJET 1.2 cloning vector harbouring CMK cDNA.

Figure 19 is the electrophoresed agarose gel image showing the PCR

amplification result of MCS, in which lane 1 is 1 kbp DNA ladder marker and lane 2 is amplicon of MCS (726 bp).

Figure 20 shows the restriction pattern of HbMCS, in which lane 1 is 1 kbp DNA ladder marker, lane 2 is undigested pJET 1.2 cloning vector harbouring MCS cDNA and lane 3 is Bglll-digested 1.2 cloning vector harbouring MCS cDNA (-700 bp insert).

Figure 21 is the electrophoresed agarose gel image showing the PCR

amplification result of HDS, in which lane 1 is 1 kbp DNA ladder marker and lane 2 is amplicon of HDS (2223 bp). shows the restriction pattern of HbHDS, in which lane 1 is 1 kbp DNA ladder marker, lane 2 is undigested pJET 1.2 cloning vector harbouring HDS cDNA and lane 3 is Bglll-digested 1.2 cloning vector harbouring HDS cDNA (-2.2 kbp insert). is the electrophoresed agarose gel image showing the PCR amplification result of HDR, in which lane 1 is 1 kbp DNA ladder marker and lane 2 is amplicon of HDR (1389 bp). shows the restriction pattern of HbHDR, in which lane 1 is 1 kbp DNA ladder marker, lane 2 is undigested pJET 1 .2 cloning vector harbouring HDR cDNA and lane 3 is Bglll-digested 1.2 cloning vector harbouring HDR cDNA (-1.3 kbp insert). shoes the MEP or plastidial isoprenoid biosynthetic pathway showing the metabolites and enzymes involved, including DXR, CMS, CMK, MCS, HDS and HDR, in the plastids of H. bmsiliensis to produce IPP and DMAPP. shows an operative MEP pathway in H. brasiliensis plastids in laticifer showing the role of DXR, CMS, CMK, MCS, HDS and HDR constructed using the program of Pathway Studio.

DETAILED DESCRIPTION OF THE INVENTION

The present invention relates to the specific genes encoding for a series of enzymes involved in the biosynthesis of isoprenoids in the plant of Hevea brasiliensis. More particularly, the present invention provides the polynucleotide sequences of the genes encoding for the series of enzymes involved in the plastidial bios)'nthetic pathway of isoprenoids, which allows specific genetic intervention to be conducted in the plastidial pool of isoprenoids in H. brasiliensis.

Hereinafter, the invention shall be described according to the preferred embodiments of the present invention and by referring to the accompanj'ing description and drawings. However, it is to be understood that limiting the description to the preferred embodiments of the invention and to the drawings is merely to facilitate discussion of the present invention and it is envisioned that those skilled in the art may devise various modifications without departing from the scope of the appended claim.

The following terms used throughout the specification have the indicated meanings unless expressly indicated to have a different meaning.

The term "gene", as used herein, is defined as the genomic sequence of the plant H. brasiliensis, particularly polynucleotide sequence encoding polypeptide of the series of enzymes involved in the plastidial biosynthetic pathway of isoprenoids.

The term "polynucleotide", as used herein, is a nucleic acid chain containing a sequence greater than 100 nucleotides in length.

The term "polypeptide", as used herein, is a single linear chain of amino acids bonded together by peptide bonds, and having a sequence greater than 100 amino acids in length. The term "isolated polynucleotide", as used herein, refers to polymer of RNA or DN A acquired from biological sample or produced chemically via any known method in the art. The polymer can be single- or double-stranded.

The term "primer", as used herein, is ah oligonucleotide capable of binding to a target nucleic acid sequence and priming the nucleic acid S3orthesis. An amplification oligonucleotide as defined herein will preferably be 10 to 50, most preferably 15 to 25 nucleotides in length. While the amplification oligonucleotides of the present invention may be chemically synthesized and such oligonucleotides are not naturally- occurring nucleic acids.

The abbreviation used throughout the specification to refer to nucleic acids comprising nucleotide sequences are the conventional one-letter abbreviations. Thus, when included in a nucleic acid, the naturally occurring encoding nucleotides are abbreviated as follows: adenine (A), guanine (G), cytosine (C), thymine (T) and uracil (U). Also, unless otherwise specified, the nucleic acid sequences presented herein in the 5'→3' direction.

As used herein, the term "complementary" and derivatives thereof are used in reference to pairing of nucleic acids by the well-known rules that A pairs with T or U and C pairs with G. Complement can be "partial" or "complete". In partial complement, only some of the nucleic acid bases are matched according to the base pairing rules; while in complete or total complement, all the bases are matched according to the paring rule. The degree of complement between the nucleic acid strands may have significant effects on the efficiency and strength of hybridization between nucleic acid strands as well known in the art. This may be of particular in detection method that depends upon binding between nucleic acids.

The term 'host cell" or "transformed cell" used herein refers to cell received a foreign gene material or a recombinant gene construct and capable of producing a products according to the genetic information presented in the foreign gene material.

The term "operably-linked", as used herein, refers to association of nucleic acid sequence on a single nucleic acid fragment so that the function of one is affected by the other. For example, a promoter can be operably-linked with a coding sequence when it affects the expression of that coding sequence, i.e. that the coding sequence is under the transcriptional control of the promoter.

The term "in vivo^", as used herein, refers to a biological reaction or experimentation conducted in a whole, living organism within a natural setting.

As opposed to "in vivo", the term "in vitro^", as used herein, refers to a biological reaction occurs in an artificial environment outside a living organism, which is usually conducted in a laboratory using components of an organism that have been isolated from their usual biological context in order to permit a more detailed or more convenient analysis to be performed.

The present invention discloses an isolated polypeptide for catalyzing plastidial biosynthesis of IPP, DMAPP or the combination thereof in the plant of H. brasiliensis, comprising amino acid sequence set forth in SEQ ID NO. 2, SEQ ID NO. 4, SEQ ID NO. 6, SEQ ID NO. 8, SEQ ID NO. 10 or SEQ ID NO. 12. These SEQ ID NO. 2, SEQ ID NO. 4, SEQ ID NO. 6, SEQ ID NO. 8, SEQ ID NO. 10 and SEQ ID NO. 12 are the amino acid sequences for the series of enzymes involved in the MEP biosynthetic pathway. In another embodiment of the present invention, an isolated polynucleotide is disclosed, in which the isolated polynucleotide encoding an enzyme for catalyzing plastidial biosynthesis of isopentenyl diphosphate, dimethylallyl diphosphate or the combination thereof in the plant of H. brasiliensis, comprising nucleotide sequence set forth in SEQ ID NO. 1, SEQ ID NO..3, SEQ ID NO. 5, SEQ ID NO. 7, SEQ ID NO. 9, SEQ ID NO. 11 or any complementary sequence thereof.

A simple MEP or plastidial isoprenoid biosynthetic pathway is shown in Figure 25, in which the series of metabolites and enzymes involved are illustrated. This series of enzymes include the DXR, CMS, CMK, MCS, HDS and HDR, which can be found in the plastids of H. brasiliensis for the biosynthesis of IPP and DMAPP in the plastids. As shown in Figure 1 is the polynucleotide sequence SEQ ID NO. 1 which can encode the enzyme of DXR having the polypeptide sequence SEQ ID NO. 2, which is shown in Figure 2. As shown in Figure 25, DXR catalyzes the reduction of DXP to form MEP, which is considered the first intermediate metabolite in this pathway. Therefore, the regulation on the level of DXR in the plant or the plant cell can potentially influence the biosynthesis of the rest of the intermediates as well as the end products of this pathway which is the IPP, DMAPP or the combination thereof.

Figure 3 and Figure 4, respectively, shows the pol^ynucleotide sequence SEQ ID NO. 3 and the polypeptide sequence SEQ ID NO. 4 for the enzyme CMS which catalyzes the conversion of MEP to form 4-diphosphocytidyl-2C-methyl-D-eiytl ritol. Figure 5 shows the polynucleotide sequence SEQ ID NO. 5 of the gene CMK which encodes the enzyme CMK having polypeptide sequence as shown in Figure 6. Catalyzed by CMK, the 4-diphosphocytidyl-2C-methyl-D-erythritol is converted into 4- diphosphoc}^tidyl-2C-methyl-D-erythritol 2-phosphate, which then forms MEcPP with release of CMP. The polynucleotide and amino acid sequences of MCS are respectively shown in Figure 7 and 8 as SEQ ID NO. 7 and SEQ ID NO. 8. Figure 9 and 10 respectively shows the polynucleotide sequence SEQ ID NO. 9 and amino acid sequences SEQ ID NO. 10 of FIDS, which catalyzes the formation of HMBPP from MEcPP. The last step of the MEP pathway is the formation of IPP and DMAPP from HMBPP catalyzed by HDR, wherein the polynucleotide and amino acid sequences of HDR can be found in Figure 11 and 12 respectively as SEQ ID NO. 11 and SEQ ID NO. 12. The presence of IPP and DMAPP can prime or initiate the biosynthetic pathway of natural rubber as these metabolites can act as one of the allylic initiator molecules for priming the biosynthesis process.

In accordance with one of the preferred embodiments of the present invention, the plant of H. brasiliensis used in the present invention is clone RRIM 600. This rubber tree clone is preferably used for the production of natural rubber as it gives higher yield, more adaptable to the environment and known to be less susceptible to climatic variations. Preferably, the isolated polypeptide is also preferably derived from this clone.

Another embodiment of the present invention is a method for catalyzing the biosynthesis of IPP, DMAPP or the combination thereof for enhancing the biosynthesis of natural rubber from its precursors, comprising the step of introducing an isolated polypeptide having amino acid sequence set forth in the preceding embodiment into a plant cell in vivo or in intro. Before the genetic intervention procedure can be conducted, the gene cluster encoding the series of enzymes involved in the plastidial or MEP isoprenoid biosynthetic pathway has to be identified and characterized. As set forth in the preceding embodiment of the present invention, the gene or polynucleotide of the related enzymes can be isolated from H. brasiliensis . Amplification primers are preferably designed targeting the conserved region of each gene. An example of the primer designing strategy is further detailed in Example 1.

Accordingly, total RNA can be isolated from the plant, and the amplification, cloning and sequencing process of the series of enzymes, including the DX , CMS, CMK, MCS, HDS and HDR, can be conducted using a method as detailed in Example 2. RT- PCR is preferably conducted using the cDNA obtained from the total RNA, and the amplification products (amplicons) of the respectively gene encoding these enzymes are shown in Figures 13, 15, 17, 19, 21 and 23. Subsequently, the purified products can be ligated in a commercially obtained cloning vector, and then be transformed into a host cell, such as the commercially available chemically competent E. coli cell. Example 3 shows an example of the plasmid isolation from the transformed cell and the restriction digestion procedure for the confirmation of clones. The restriction digestion patterns of the genes encoding these enzymes are shown in Figures 14, 16, 18, 20, 22 and 24. In Example 4, the analysis of the sequences is disclosed, in which the nucleotide and the amino acid sequences can be analyzed and aligned by the available programs. An operative MEP pathway can be re-constructed by a method as shown in Example 5 using the genetic information obtained from the nucleotide and the amino acid sequences. Comparison of the gene and polypeptide sequences as shown in Example 6 shows that the genes encoding the series of enzymes involved in the biosynthesis of IPP, DMAPP and the combination thereof are unique and specific.

In yet another embodiment of the present invention, a recombinant gene construct comprising a polynucleotide template having nucleotide sequence set forth in SEQ ID NO. 1, SEQ ID NO. 3, SEQ ID NO. 5, SEQ ID NO. 7, SEQ ID NO. 9 or SEQ ID NO. 11 is disclosed. The polynucleotide template is expressible in a host cell to produce an enzyme which catalyzes plastidial biosynthesis of the IPP, DMAPP or the combination thereof, thus potentially enhances the production of the natural rubber in the plant. Preferably, the recombinant gene construct further comprises a promoter region operably-linked to enhance expression of the polynucleotide template.

Still another embodiment, of the present invention is a transformant comprising a recombinant gene construct as set forth in the preceding, embodiments to produce an enzyme which catalyzes plastidial biosynthesis of IPP, DMAPP or the combination thereof. In another embodiment of the present invention, a chimeric gene can be provided, which can be in combinations or multiples. An example of the construction and transformation of the chimeric DXR-HDR fusion product is further detailed in Example 7. Example 8 shows the construction of a fluorescent-labelled fusion vector, as well as its transient and stable expression in a host cell.

Further embodiment of the present invention is a transgenic plant of H. brasiliensis with enhanced plastidial biosynthesis of IPP, DMAPP or the combination thereof, comprising a recombinant gene construct having an expressible nucleotide sequence set forth in SEQ ID NO. 1 , SEQ ID NO. 3, SEQ ID NO. 5, SEQ ID NO. 7, SEQ ID NO. 9 or SEQ ID NO. 11. Example 9 shows a functional assay and biochemical characterization of the transgenic plant, in which the proteins (enzymes) raised, can be quantified. Accordingly, the transgenic plant shall contain a higher expression level of the series of enzymes involved in the plastidial or MEP isoprenoid (IPR DMAPP or the combination thereof) biosynthetic pathway.

The present disclosure includes as contained in the appended claims, as well as that of the foregoing description. Although this invention has been described in its preferred form with a degree of particularity, it is understood that the present disclosure of the preferred form has been made only by way of example and that numerous changes in the details of construction and the combination and arrangements of parts may be resorted to without departing from the scope of the invention.

EXAMPLE

Examples are provided below to illustrate different aspects and embodiments of the present invention. These examples are not intended in any way to limit the disclosed invention, which is limited only by the claims.

Example 1 Designing and synthesis of primers

The primers used in the study were either designed from the manually curated "transcriptome" and the "gene models" predicted from the genomic sequences of Hevea brasiliensis RRIM 600, by choosing the sequences manually with complete ORFs or using databases where similar genes have been successfully isolated from other plants. Comparative and bioinformatic analyses of the nucleotide sequences obtained from transcriptome were carried out online using NCBI BLAST, BLASTP, RPS-BLAST, BLASTX and PSI-BLAST to identify homologues of the related genes and for the proper identification of gene. Nucleotide sequence alignments were performed using Clustal W version 1.82 whenever multiple sequences were found from the "gene pool^"'. The alignment was then edited. Gene-specific primers (both forward and reverse) were selected manually or through Primer 3 Plus Web tool through consensus from highly conserved sequences and the primers were custom synthesized. All oligonucleotides used in this study were synthesized and HPLC- 5 purified by the supplier and procured from IDT. Stock solutions of 100 pmol were prepared in autoclaved ddH O and stored at -20 °C. in aliquots for use. Table 1 depicts the primers or amplification oligonucleotides used in this work and the rationale for their synthesis.

0 Table 1

Gene Name Primers T Amplicon

Sizes (bp)

I DXR 5'- ATGGCGCTCAATTTGCTTT-3' (F) 53.7 1416

5'- TCATGCAAGAACAGGGCTTA-3' (R) 54.2

I CMS 5'- ATGGGTCATCATCTTCTTCACTT-3' (F) 53.9 936

5'- TACTTTGAAGATTCTTCAGAATTCAA-3 ' (R) 51.8

5 CMK 5'- ATGGCTTCCGCTC ATTTC-3 ' (F) 53 . 1167

5'- TCACTC AATAGACTGGGAACGA-3 ' (R) 55.2

1 MCS 5'- ATG AACTCAATGGCTATGGCC-3 ' (F) 55.3 726

5'- TACTTCTTC ATGAGAAGAACC ACTG-3 ' (R) 54:7

5 HDS 5'- ATGGCGACTGGAGCTGTG-3' (F) 57.8 2223

5'- TTACTCTTCTGCAGGAGGATCG-3 ' (R) 56

5 HDR 5'- ATGGCTGTCTCTCTGCAACTC-3 ' (F) .57 , 1389

5'- TTATGCTACTTGCAAAGCTTCG-3' (R) 53.7

Example 2 Amplification, cloning and sequencing of DXR, CMS, CMK, MCS, HDS & HDR from H. brasiliensis RRIM 600

Total RNA was isolated from young leaves of matured, fully grown H. brasiliensis 5 RRIM 600 using QIAGEN-RNeasy Mini Kit following the manufacturer's instructions. The quality as well as quantity, was checked by agarose gel electrophoresis and Thermo Scientific Nano Drop 2000™ (Thermo Scientific- Agilent's). The cDNA first strand was synthesised using Superscript® VILO™ cDNA Synthesis Kit (Invitrogen) according to the manufacturer's instructions. The genes were amplified from the cDNA by PCR using primers and conditions designated in Table. The PCR reaction (50 μί) contained 1 μΐ, of cDNA, 20 pmoles of each primer, 5 μΐ, of 1 OX Pfu Buffer, 5 μί of 2.5 mM dNTP mix and 2.5 units of P uTurbo® DNA polymerase (Stratagene). PCR was carried out in Veriti™ Thermal Cycler (Applied Biosystems) using the following conditions. Initial denaturation for 5 min at 94°C followed by 35 cycles of denaturation at 94°C for 30 sec, annealing at 50°C for 30 sec and extension at 72°C for 1 min/ kbp, with a final extension at 72 °C for 7 min. The PCR product was analysed by 1 % agarose gel and the amplicon was eluted from the gel using GENE CLEAN® TURBO gel band elution kit (MP Biomedicals) following the manufacturer's instructions. Figures 13. 15, 17, 19, 21 and 23 respectively shows the amplification products (amplicons) of the genes of interest, DXR, CMS, CMK, MCS, HDS and HDR, obtained by the RT-PCR from cDNA. The purified PCR product was ligated into pJET 1.2 Blunt Vector (Fermentas) and was transformed into One Shot® Machl™-T1R Chemically Competent E. coli cells (Invitrogen). Plasmids were isolated from putative colonies using QIAprep Spin® Miniprep Kit (Qiagen) according to the manufacturer's instructions. The presence of the insert was checked by digesting the vector harbouring inserts with Bglll (Fermentas) and positive plasmids were subjected to sequencing, in triplicates.

Example 3 Plasmid isolation and restriction digestion for confirmation of clones

Recombinant colonies were cultured and small-scale preparation (mini- preps) of plasmid DNA was performed by the Qiagen Spin Miniprep kit (Qiagen) as per the protocol supplied by the manufacturer. To select positive recombinant colonies, after ligation and transformation, individual colonies were picked up with a sterile tip into 10 iL of sterile water and heated at 95 °C for 20 min for lysis of cells and subsequent amplification of insert DNA by PCR. Presence of insert within the plasmid was reconfirmed with insert flanking restriction enzymes. The presence of the insert was checked by digesting the vector, pJET 1.2 (Fermentas) harbouring inserts with Bglll (Fermentas) and positive plasmids were subjected to sequencing, in triplicates. The digested and undigested recombinant plasmids were separated on a 0.8- 1.5 % agarose gel to identify the correct inserts. Illustrated in Figures 14, 16, 18, 20, 22 and 24 are the respective digested and undigested cloning vectors harbouring DXR, CMS, CMK, MCS, HDS and HDR. Subsequently, the clones were sequenced using vector specific standard Ml 3/ pUC universal primers or pJET 1.2 Forward Sequencing Primer [23- mer, 5'-CGACTCACTATAGGGAGAGCGGC-3'] and p.TET 1.2 Reverse Sequencing Primer [24-mer, 5 ^"' - AAG AAC ATCGATTTTCC ATGGC AG-3 ' ] , supplied by the manufacturers (Fermentas) for sequencing. Automated dye terminator cycle sequencing was done using the ABI BigDye™ (fluorescence-labelled dideoxynucleotide termination method) Terminator Cycle Sequencing Ready reaction kit (Applied Biosystems, USA) in an ABI 3730x1 DNA sequencer (Applied Biosystems, USA) following manufacturer's instructions. Sequences were visualized and analysed using Chromas LiteTM version 2.01 software or BioEdit Tool Version 7.0.8. Glycerol stocks [i.e., 0.5 ml of sterilized {65 % glycerol (v/ v), 0.1 M MgS0₄, 25 mM Tris- HC1, pH 8} and 0.75 ml of culture volume; pipette mixed] of positive clone harbouring cells from mid log phase were stored in aliquots in sterile cryovials and Eppendorf micro centrifuge tubes in -80 °C for future use. Example 4 Analysis of the sequence

The nucleotide sequence and the amino acid sequences were analysed by BLASTN and BLASTP programs respectively. The sequences reported from other plants were aligned with Clustal W. Figures 1, 3, 5, 7, 9 and 11 respectively shows the nucleotide sequence of DXR, CMS, CMK, MCS, HDS & HDR; whereas their amino acid sequences are shown in Figures 2, 4, 6, 8, 10 and 12, respectively.

Example 5 Pathway re-construction showcasing plastidial isoprenoid biosynthetic routes in rubber tree

Automatic metabolic pathway reconstruction was done by identifying orthologs for predicted rubber proteins in Arabidopsis genome and sequence orthologs. Enzymatic reactions encoded within rubber genome towards production of plastidial isoprenoid pools were constructed out of 566 enzymatic reactions available in Resnet-Plant 3.0 database for Pathway Studio and from metabolic pathway databases (MPW). Illustrated in Figure 25 is the operative MEP pathway in H. brasiliensis plastids in laticifer showing the role of DXR, CMS, CMK, MCS, HDS and HDR constructed using the program of Pathway Studio.

Example 6 Comparison with other plant sequences

The HbDXR has a theoretical mol. Wt. 51.014 kDa and pi of 6.09 with glycosylation, myristylation and amidation modification sites. SMART analysis reveals the presence of 2 Pfam domains, i.e., 'DXP_reductoism' [77-205 aa, E value- 3.4 e-73] and 'DXP_ reductoism_C [74-460 aa, E value- 3.4 e-60], with pBLAST matches with PDB: 1 R0L: D [74-460 aa, E value-0] and SCOP: dlk5ha3 [199-377 a, E-value- 3e-70], which shows sequence identities with DXR from H. brasiliensis (DQ473432, DQ437514, AB294701 , 99% for all, AY502937, 94%), Ricinus communis (XM_002511353, 89%), Croton stellatopilosus (EF451544, 88%), Populus trichocarpa (XM_002321563, 87%), Gossypium barbadense (EF051345, 85 %) at the nucleotide levels. At protein levels, HbDXR shows identities to DXR from H. brasiliensis (ABD92702, 99%; BAF98290, 99%, AAS94121, 94%), R. communis (EEF52001, 92%), C. stellatopilosus (AB038177, 91%), Solanum lycopersicum (AF331705, 88%), Rauvolfia verticillata (AAY87151, 88%), Catharanthus roseus (AAF65154, 87%), Nicotiana tabacum (ABH08964, 87%), Glycine max (AEB91528, 86%) and others. The HbCMS has a theoretical mol. Wt. 34.28 kDa and pi of 7.12 with glycosylation, myristylation and amidation modification sites. SMART analysis reveals the presence of 2 overlapping Pfam domains, i.e., 'NTP_transferase^; [83-304 aa, E value- 5.4 e-47] and 'IspD' [84-305 aa, E value-4.4 e-02], with pBLAST matches with PDB: 1W77:A [86-306 aa, E value-0]and SCOP: d li52a [199-377 a, E-value- 9e-30], ], which shows sequence identities with CMS from H. brasiliensis (AB294702, 97 %; AB294703, 95%), P. trichocarpa (EY693021 , 86%), R. communis (XM_002519320, 91 %), Stevia rebaudiana (DQ269452, 82%), Nicotiana langsdorffii x Nicotiana sanderae (EF636807, 82 %), at the nucleotide levels. The conserved domain database (CDD) recognizes, the substrate binding sites, a dimer interface, and classifies as the Glyco_transf_GTA_type superfamily as a CDP-ME synthase. At protein levels, the HbCMS shares identities with CMS's from H. brasiliensis (BAF98291, 97%; BAF98292, 93%), P. trichocarpa (ACD70398, 85%), & rebaudiana (ABB8837, 70%), N. langsdorffii x N. sanderae (ABV02019, 72%), R. communis (XP_002519366, 71 %), C. roseus (ACT16377, 78%) and others.

The HbCMK has a theoretical mol. wt. 43.15 kDa and pi of 7.05 with glycosylation and nvyristylation modification sites. SMART analysis reveals the presence of 2 consecutive Pfam domains, i.e., 'GHMP kinases N' [162-220 aa, E value- 2.8 e-12] and 'GHMP kinases C [275-351 aa, E value-6.6 e-05], with pBLAST matches with PDB: 1UEK: A [81 -313 aa, E value-0] and SCOP: dlkkhal [78-230 aa a, E-value- 4e-20], which shows sequence identities with CMK from H. brasiliensis (EJ217703, 99%: AB294704, 99%;), R. communis (XM_002523170, 90%), P. trichocarpa (EU693022, 86%), C. roseus (DQ848671 , 80%) at nucleotide levels. The CDD recognizes the protein to have the conserved multi-domain known as IspE. At protein levels, the HbCMK shares identities with CMKs from H. brasiliensis (BAF98293, 99%), R. communis (XP_002523216, 88%), _. C. roseus (ABI35992, 74%), P. trichocarpa (ACD70399, 85%), S. lycopersicum (P93841, 78%) and others.

The HbMCS has a theoretical mol. wt. 25.715 kDa and pi of 7.12 with glycosylation and myristylation modification sites, with no significantly defined Pfam domains identifiable. It shows sequence identities with MCS from H. brasiliensis (AB294706, AY502938, 99%; FJ196164, 98%; AB294705, 92%), P. trichocarpa (EU693023, 85%), R. communis (XM_002509813, 91%), 5". rebaudiana (DQ631427, 81%), C. roseus (AF250236, 81%) at the nucleotide levels. The CDD recognizes the protein to belong to the conserved family MECDP synthase, with CDP- and zinc binding sites along with a homotrimer interaction site. At protein levels, the HbMCS shares identities with MCSs from H. brasiliensis (BAF98295, 99%; AAS94122, 98%, ACI22427, 98%; BAF98294, 92%), P. trichocarpa (ACD70400, 89%), Arabidopsis fyrata (EFH62632, 80%), Rauvolfia verticillata (ABV89583, 72%), Citrus jambhiri (BAF73931 , 72%) and others.

The HbHDS has a theoretical mol. Wt. 82.041 kDa and pi of 6.12 with glycosylation, myristylation, two R-G-D sites and amidation modification sites. SMART analysis reveals the presence of a Pfam domain, i.e., 'GcpE' [87-731 aa, E value-2.9 e-280], with pBLAST matches with SCOP: dleuaa [115-186 aa, E value- 1.6e-l] and SCOP: dl cl dal [196-238a, E-value- 1.4e-01] which shows sequence identities with MCS from H. brasiliensis (AB294707, 99%), R. communis (XM_002530240, 91%), P. trichocarpa (EU693024, 89%). Artemisia annua (FJ479720, 82%), S. rebaudiana (DQ768749, 82%) at the nucleotide levels. The CDD recognizes the protein to belong to the conserved super family of proteins, IspG_gcpE. At the protein levels, the HbHDS finds identities to HDSs from H. brasiliensis (BAF98296, 99%), R. communis - (EEF32093, 95%), Arabidopsis thaliana (NP_200868, 87%), S. rebaudiana (ABG75916, 87%), P. trichocarpa (ACD70401, 92%), Ginkgo biloba (ABB78087), Zea mays (AAT70081 , 83%) and others.

The HbHDR has a theoretical mol. Wt. 51.89 kDa and pi of 5.4 with glycosylation, myristylation and amidation modification sites. SMART analysis reveals the presence of a Pfam domain, i.e., 'LYTB' [109-453 aa, E value- 1.4e- 120], with pBLAST matches with SCOP: dlejba [42-68 aa, E value-4.4e-l ] and SCOP: dl hbna2 [361-412aa, E-value- 7.6e-01]. It shows sequence identities with MCS from H: brasiliensis (AB294708, EU881977, 99%), R. communis (XM_002519056, 90%), P. trichocarpa (EU693025, 87%; XM_002305377, 86%), Camptotheca acuminata , (DQ864495, 845), R. verticillata (EU034699, 81%) at the nucleotide levels. The CDD recognizes the protein to belong to the conserved super family of proteins LYTB superfamily. At the protein levels, the HbHDR finds identities to HDRs from H. brasiliensis (BAF98297, 96%; ACG55683, 96%), R. communis (EEF43313, 85%), Camptotheca acuminate (ABI64152, 85%), P. trichocarpa (ACD70402, 87%), R. verticillata (ABV89582, 78%), C. roseus (ABI30631 , 78%) and others. Example 7 Construction and transformation of the chimeric DXR-HDR fusion product

The candidate genes, HbDXR and HbHDR, were amplified and cloned from H. brasiliensis RRIM 600 young leaves, separately using gene specific primers comprising a pair of primers in which a BamHI site was introduced at the 5' end, and a Sacl site at the 3' end. The procedures used to construct the chimeric gene was in such a manner that only the catalytic and most-conserved residues were allowed to be amplified and cloned successively. This chimeric cDNA was then transformed into Agrobacterium tumefaciens LBA4404. H. brasiliensis embryogenic caili were subsequently co-cultured with A. tumefaciens containing the chimeric gene in the dark for 2 days on differentiation medium. Shoots were subsequently regenerated for resistance screening from the proximal portions of the cotyledon hypocotyls on differentiation media supplemented with kanamycin and organogenesis was induced in vitro. Example 8 Construction of GFP fusion vectors, transient and stable expression in Arabidopsis thaliana

The chimeric HbDXR-HDR coding sequences were amplified from the chimeric vector obtained above using standard protocols with the LA Taq PCR system (Takara, Japan), and using specific primers flanked by Gateway recombination cassettes (Invitrogen, California, USA), using specific primers and the PCR products were cloned into pDONR221 according to the manufacturer's instructions. Cloning into the final GFP vectors (pCambia-GFP) was by LR reaction (Invitrogen, California, USA). Floral-dip was followed for GFP fusion vectors to introduce the chimera into Arabidopsis thaliana and leaves of the kanamycin-selected F2 progenies were screened for localization of the chimera. Example 9 Functional assays and biochemical characterization of transgenic plants

Both, transgenic A. thaliana and H. brasiliensis RRIM 600 cell lines were probed for the enzymatic assays for DXR and HDR activities. Localization of DXR and HDR proteins was followed by epiflorescence microcopy of the respective tissues, while anti-DXR and anti-HDR antibodies raised in rabbit were used for ELISA studies for the quantification of the proteins.

Claims

1. An isolated polypeptide for catalyzing plastidial biosynthesis of isopentenyl diphosphate, dimethylallyl diphosphate or the combination thereof in the plant of

5 Hevea brasiliensis, comprising amino acid sequence set forth in SEQ ID NO. 2, SEQ ID NO. 4, SEQ ID NO. 6, SEQ ID NO. 8, SEQ ID NO. 10 or SEQ ID NO. 12.

2. An isolated polypeptide according to claim 1, wherein the plant of Hevea brasiliensis is clone RRIM 600.

0

3. A method for catalyzing the biosynthesis of isopentenyl diphosphate, dimethylallyl diphosphate or the combination thereof for enhancing the biosynthesis of natural rubber from its precursors, comprising the step of introducing an isolated polypeptide having amino acid sequence set forth in SEQ ID NO. 2, SEQ ID NO. 4, SEQ ID NO.

.5 6, SEQ ID NO. 8, SEQ ID NO. 10 or SEQ ID NO. 12 into a plant cell in vivo or in intro. · . .

4. An isolated polynucleotide encoding an enzyme for catalyzing plastidial biosynthesis of isopentenyl diphosphate, dimethylallyl diphosphate or the combination

10 thereof in the plant of Hevea brasiliensis, comprising nucleotide sequence set forth in SEQ ID NO. 1, SEQ ID NO. 3, SEQ ID NO. 5, SEQ ID NO. 7, SEQ ID NO. 9, SEQ ID NO. 11 or any complementary sequence thereof.

5. An isolated polynucleotide according to claim 4, wherein the plant of Hevea 15 brasiliensis is clone RRIM 600.

6. A recombinant gene construct comprising a polynucleotide template having nucleotide sequence set forth in SEQ ID NO. 1 , SEQ ID NO. 3, SEQ ID NO. 5, SEQ ID NO. 7, SEQ ID NO. 9 or SEQ ID NO. 11 , wherein the polynucleotide template is

>0 expressible in a host cell to produce an enzyme which catalyzes plastidial biosynthesis of isopentenyl diphosphate, dimethylallyl diphosphate or the combination thereof.

7. A recombinant gene construct according to claim 6 further comprising a promoter region operably-linked to enhance expression of the polynucleotide template.

8. A transformant comprising a recombinant gene construct capable of expressing a polynucleotide template having nucleotide sequence set forth in SEQ ID NO. 1, SEQ ID NO. 3, SEQ ID NO. 5, SEQ ID NO. 7, SEQ ID NO. 9 or SEQ ID NO. 11 to produce an enzyme which catalyzes plastidial biosynthesis of isopentenyl diphosphate, dimethylallyl diphosphate or the combination thereof.

9. A transgenic plant of Hevea brasiliensis with enhanced plastidial biosynthesis of isopentenyl diphosphate, dimethylallyl diphosphate or the combination thereof, comprising a recombinant gene construct having an expressible nucleotide sequence set forth in SEQ ID NO. 1, SEQ ID NO. 3, SEQ ID NO. 5, SEQ ID NO. 7, SEQ ID NO. 9 or SEQ ID NO. 11.