WO2012158016A1 - Procédé de régulation de la biosynthèse de monolignols - Google Patents

Procédé de régulation de la biosynthèse de monolignols Download PDF

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WO2012158016A1
WO2012158016A1 PCT/MY2012/000014 MY2012000014W WO2012158016A1 WO 2012158016 A1 WO2012158016 A1 WO 2012158016A1 MY 2012000014 W MY2012000014 W MY 2012000014W WO 2012158016 A1 WO2012158016 A1 WO 2012158016A1
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seq
nucleotide sequence
plant
monolignols
set forth
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Maqsudul Alam
Mohd Nazalan MOHD NAJIMUDIN
Jennifer Ann SAITO
Abhilash OTHALATHARA USHARRAJ
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Universiti Sains Malaysia (USM)
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Universiti Sains Malaysia (USM)
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    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
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    • C12N15/8216—Methods for controlling, regulating or enhancing expression of transgenes in plant cells
    • C12N15/8218—Antisense, co-suppression, viral induced gene silencing [VIGS], post-transcriptional induced gene silencing [PTGS]
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    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
    • C12N15/82—Vectors or expression systems specially adapted for eukaryotic hosts for plant cells, e.g. plant artificial chromosomes (PACs)
    • C12N15/8241—Phenotypically and genetically modified plants via recombinant DNA technology
    • C12N15/8242—Phenotypically and genetically modified plants via recombinant DNA technology with non-agronomic quality (output) traits, e.g. for industrial processing; Value added, non-agronomic traits
    • C12N15/8243—Phenotypically and genetically modified plants via recombinant DNA technology with non-agronomic quality (output) traits, e.g. for industrial processing; Value added, non-agronomic traits involving biosynthetic or metabolic pathways, i.e. metabolic engineering, e.g. nicotine, caffeine
    • C12N15/8255—Phenotypically and genetically modified plants via recombinant DNA technology with non-agronomic quality (output) traits, e.g. for industrial processing; Value added, non-agronomic traits involving biosynthetic or metabolic pathways, i.e. metabolic engineering, e.g. nicotine, caffeine involving lignin biosynthesis
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    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/0004—Oxidoreductases (1.)
    • C12N9/0071—Oxidoreductases (1.) acting on paired donors with incorporation of molecular oxygen (1.14)
    • C12N9/0073—Oxidoreductases (1.) acting on paired donors with incorporation of molecular oxygen (1.14) with NADH or NADPH as one donor, and incorporation of one atom of oxygen 1.14.13
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    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/10—Transferases (2.)
    • C12N9/1003—Transferases (2.) transferring one-carbon groups (2.1)
    • C12N9/1007—Methyltransferases (general) (2.1.1.)
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    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/10—Transferases (2.)
    • C12N9/1025—Acyltransferases (2.3)
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    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/93—Ligases (6)
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    • C12P7/00—Preparation of oxygen-containing organic compounds
    • C12P7/02—Preparation of oxygen-containing organic compounds containing a hydroxy group
    • C12P7/22—Preparation of oxygen-containing organic compounds containing a hydroxy group aromatic

Definitions

  • the present invention relates to the specific genes encoding for a series of enzymes involved in the biosynthesis of lignin 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 biosynthesis of the lignin precursors, monolignols, which allow specific genetic intervention to be conducted for regulating the biosynthesis of lignin in H. brasiliensis.
  • Lignin limits various applications of plant material for fibre, chemical and energy production, therefore, strategies for its down-regulation are of considerable interest.
  • lignin precursors also known as monolignols (p-coumaryl alcohol, coniferyl alcohol and sinapyl alcohol)
  • monolignols p-coumaryl alcohol, coniferyl alcohol and sinapyl alcohol
  • monolignols p-coumaryl alcohol, coniferyl alcohol and sinapyl alcohol
  • phenylalanine is the first precursor molecule in the biosynthesis pathway.
  • the first enzyme involved in the phenylpropanoid pathway is phenylalanine ammonia-lyase (PAL) that causes the deamination of phenylalanine producing p- cinnamic acid.
  • PAL phenylalanine ammonia-lyase
  • C4H cinnamate 4-hydroxylase
  • the hydroxylation steps are catalyzed by two cytochrome P450 enzymes, coumarate 3-hydroxylase (C3H) and ferulate 5-hydroxylase (F5H), which respectively perform the C3 and C5 hydroxylation processes.
  • Another two enzymes caffeoyl-CoA O- methyltransferase (CcoAOMT) and caffeic acid/5 -hydroxyconiferaldehyde O- methyltransferase (COMT) carry out the methylation steps.
  • Hydroxycinnamoyl-CoA shikimate hydroxycinnamoyl transferase (HCT) catalyzes the conversion of p- coumaroyl CoA to form caffeoyl CoA.
  • CCR cinnamoyl coenzyme A reductase
  • CAD cinnamyl alcohol dehydrogense
  • CoA ligase (4CL) catalyzes the formation of CoA thioester of cinnamic acids in the biosynthesis of a wide variety of phenolic derivatives, including benzoic acid, condensed tannins, flavonoids, and the cinnamyl alcohols.
  • Coniferaldehyde 5-hydroxylase (CAld5H) is the enzyme responsible for the last hydroxylation of the syringyl-type lignin precursors, it catalyzes hydroxylation of coniferaldehyde to 5-hydroxyconiferaldehyde.
  • genes encoding the PAL subunits were found to show tissue-specific patterns of expression in several angiosperms.
  • a species-specific approach is hence preferred in order to yield a cost-effective result considering the rubber biosynthesis pathway and genetic makeup of each species of plant are potentially varied among one another.
  • the primary object of the present invention is to provide the polynucleotide sequences encoding for a series of enzymes, including PAL, C4H, 4CL, HCT, C3H, CCoAOMT, F5H, COMT, CCR and CAD which are involved in the biosynthesis of lignin, particularly monolignols, 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 biosynthesis of lignin or monolignols in the plant of H. brasiliensis .
  • Still another object of the present invention is to provide the isolated polypeptides or the enzymes encoded by the polynucleotide sequences provided.
  • Further object of the present invention is to produce a transgenic plant of H. brasiliensis with reduced monolignols production, which, potentially, demonstrates increased yield of 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 a method for reducing the synthesis of monolignols in cytoplasm of the plant of H. brasiliensis, comprising the step of down-regulating expression of a nucleic acid template containing at least one nucleotide sequence set forth in SEQ ID NO. 1, SEQ ID NO. 2, SEQ ID NO. 3, SEQ ID NO. 4, SEQ ID NO. 5, SEQ ID NO. 6, SEQ ID NO. 7, SEQ ID NO. 8, SEQ ID NO. 9, SEQ ID NO. 10, SEQ ID NO. 11 or SEQ ID NO. 12 in at least one cell.
  • the down-regulating step is conducted by introducing a dsRNA having a polynucleotide strand containing nucleotide sequence which is at least 60% complementary to the nucleic acid template in the cell.
  • Another preferred embodiment of the present invention discloses that the down- regulating step is conducted by transforming the cell with a recombinant gene construct encoding open reading frame containing nucleotide sequence which is at least 70% identical to SEQ ID NO. 1, SEQ ID NO. 2, SEQ ID NO. 3, SEQ ID NO. 4, SEQ ID NO. 5, SEQ ID NO. 6, SEQ ID NO. 7, SEQ ID NO. 8, SEQ ID NO. 9, SEQ ID NO. 10, SEQ ID NO. 11 or SEQ ID NO. 12 and expressing the nucleotide sequence of the gene construct under expression control of a lignification-associated promoter.
  • RNAi RNA interference
  • Yet another embodiment of the present invention is an isolated polynucleotide encoding enzymes for catalyzing biosynthesis of monolignols in the plant of H. brasiliensis comprising nucleotide sequence set forth in SEQ ID NO. 1, SEQ ID NO. 2, SEQ ID NO. 3, SEQ ID NO. 4, SEQ ID NO. 5, SEQ ID NO.. 6, SEQ ID NO. 7, SEQ ID NO. 8, SEQ ID NO. 9, SEQ ID NO. 10, SEQ ID NO. 11, SEQ ID NO. 12 or any of complementary sequences thereof.
  • the polynucleotide is isolated from the plant of H. brasiliensis clone RRIM 600.
  • Still another preferred embodiment of the present invention discloses a recombinant gene construct containing the isolated polynucleotide as set forth in the preceding description.
  • FIG. 13 Further embodiment of the present invention is an isolated polypeptide for catalyzing biosynthesis of monolignols in the plant of H. brasiliensis comprising amino acid sequence set forth in SEQ ID NO. 13, SEQ ID NO. 14, SEQ ID NO. 15, SEQ ID NO. 16, SEQ ID NO. 17, SEQ ID NO. 18, SEQ ID NO. 19, SEQ ID NO. 20, SEQ ID NO. 21 , SEQ ID NO. 22, SEQ ID NO. 23 or SEQ ID NO. 24.
  • These polypeptides are a series of enzymes involved in the biosynthesis of monolignols, including PAL, C4H, 4CL, HCT, C3H, CCoAOMT, F5H, COMT, CCR and CAD.
  • the present invention is capable of providing the appropriate molecular biology and genetic information of the genes and enzymes involved in the biosynthesis of monolignols, a comprehensive step toward the biosynthetic pathway as well as a basis for further functional analysis.
  • the functional characterization of the polynucleotides and polypeptides of the present invention allows for potential production of plant biomass with expected lignin content and composition for fibre, pulp and biofuel industry.
  • the present invention also provides in silico methods for identification of all related genes and their encoded proteins or enzymes involved in the biosynthesis pathway of monolignols in H. brasiliensis.
  • Figure 2 shows the nucleotide sequence SEQ ID NO. 2 of the polynucleotide encoding enzyme PAL 2 of the plant H. brasiliensis and the amino acid sequence SEQ ID NO. 14 of the polypeptide encoded thereof as described in one of the preferred embodiments of the present invention.
  • Figure 3 shows the nucleotide sequence SEQ ID NO. 3 of the polynucleotide encoding enzyme C4H of the plant H. brasiliensis and the amino acid sequence SEQ ID NO. 15 of the polypeptide encoded thereof as described in one of the preferred embodiments of the present invention.
  • Figure 4 shows the nucleotide sequence SEQ ID NO. 4 of the polynucleotide encoding enzyme C3H 1 of the plant H. brasiliensis and the amino acid sequence SEQ ID NO. 16 of the polypeptide encoded thereof as described in one of the preferred embodiments of the present invention.
  • Figure 5 shows the nucleotide sequence SEQ ID NO. 5 of the polynucleotide encoding enzyme C3H 2 of the plant H. brasiliensis and the amino acid sequence SEQ ID NO. 17 of the polypeptide encoded thereof as described in one of the preferred embodiments of the present invention.
  • Figure 7 shows the nucleotide sequence SEQ ID NO. 7 of the polynucleotide encoding enzyme HCT of the plant H. brasiliensis and the amino acid sequence SEQ ID NO. 19 of the polypeptide encoded thereof as described in one of the preferred embodiments of the present invention.
  • Figure 8 shows the nucleotide sequence SEQ ID NO. 8 of the polynucleotide encoding enzyme F5H of the plant H. brasiliensis and the amino acid sequence SEQ ID NO. 20 of the polypeptide encoded thereof as described in one of the preferred embodiments of the present invention.
  • Figure 9 shows the nucleotide sequence SEQ ID NO. 9 of the polynucleotide encoding enzyme 4CL of the plant H. brasiliensis and the amino acid sequence SEQ ID NO. 21 of the polypeptide encoded thereof as described in one of the preferred embodiments of the present invention.
  • Figure 10 shows the nucleotide sequence SEQ ID NO. 10 of the polynucleotide encoding enzyme CCoAOMT of the plant H. brasiliensis and the amino acid sequence SEQ ID NO. 22 of the polypeptide encoded thereof as described in one of the preferred embodiments of the present invention.
  • Figure 11 shows the nucleotide sequence SEQ ID NO. 11 of the polynucleotide encoding enzyme CCR 1 of the plant H. brasiliensis and the amino acid sequence SEQ ID NO. 23 of the polypeptide encoded thereof as described in one of the preferred embodiments of the present invention.
  • Figure 13 is the electrophoresed agarose gel image showing the polymerase chain reaction (PCR) amplification result of PAL gene 1 , in which lane L is lkbp DNA ladder marker, lanes 1 to 3 are samples of PAL gene 1.
  • PCR polymerase chain reaction
  • Figure 16 is the electrophoresed agarose gel image showing the PCR amplification result of C3H gene, in which lane L is lkbp DNA ladder marker, and lanes 1 and 2 are samples of C3H gene 1 and 2, respectively.
  • Figure 17 is the electrophoresed agarose gel image showing the PCR amplification result of COMT gene, in which 1 to 3 are samples of COMT gene, lane NTC is negative control, and lane L is Ikbp DNA ladder marker.
  • Figure 21 is the electrophoresed agarose gel image showing the PCR amplification result of CCR gene, in which lane L is Ikbp DNA ladder marker, and lanes 1 and 2 are samples of CCR gene.
  • Figure 22 is the electrophoresed agarose gel image showing the PCR amplification result of CCoAOMT gene, in which lane L is Ikbp DNA ladder marker, and lanes 1 and 2 are samples of CCoAOMT gene.
  • Figure 23 is the electrophoresed agarose gel image showing the PCR amplification result of CAD gene, in which lane L is Ikbp DNA ladder marker, lane 1 is negative control and lane 2 is sample of CAD gene.
  • the present invention relates to the specific genes encoding for a series of enzymes involved in the biosynthesis of lignin in the plant of H. brasiliensis . More particularly, the present invention provides the polynucleotide sequences of the genes encoding for the series of enzymes involved in the biosynthesis of the lignin precursors, monolignols, including PAL, C4H, 4CL, HCT, C3H, CCoAOMT, F5H, COMT, CCR and CAD, which allow specific genetic intervention to be conducted for regulating the biosynthesis of lignin in H. brasiliensis.
  • gene is defined as the genomic sequence of the plant H. brasilliensis particularly polynucleotide sequences encoding polypeptide sequence of the series of enzymes involved in the biosynthesis pathway of monolignols.
  • polynucleotide as used herein, is a nucleic acid chain containing a sequence greater than 100 nucleotides in length.
  • polypeptide is a single linear chain of amino acids bonded together by peptide bonds, and having a sequence greater than 100 amino acids in length.
  • isolated polynucleotide or isolated nucleotide sequence used herein refers to polymer of RNA or DNA acquired from biological sample or produced chemically via any known method in the art. The polymer can be single- or double- stranded.
  • primer is an oligonucleotide capable of binding to a target nucleic acid sequence and priming the nucleic acid synthesis.
  • 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.
  • nucleic acids comprising nucleotide sequences are the conventional one-letter abbreviations.
  • the naturally occurring encoding nucleotides are abbreviated as follows: adenine (A), guanine (G), cytosine (C), thymine (T) and uracil (U).
  • A adenine
  • G guanine
  • C cytosine
  • T thymine
  • U uracil
  • nucleic acid sequences presented herein in the 5 ' ⁇ 3' direction 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”.
  • hybridization or “hybridizing” used herein refers to the pairing action of two at least substantially complementary strands under appropriate conditions of temperature and ionic strength. The conditions of temperature and ionic strength also control stringency of the "hybridization” defining the extent of complementary of the two strands needed for successful pairing.
  • '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.
  • siHccr used herein refers to experiments conducted via computer-based simulations or mathematical models relating to animal biochemistry.
  • the present invention discloses a method for reducing the synthesis of monolignols in cytoplasm of the plant of H. brasiliensis, comprising the step of down-regulating expression of a nucleic acid template containing at least one nucleotide sequence set forth in SEQ ID NO. 1, SEQ ID NO. 2, SEQ ID NO. 3, SEQ ID NO. 4, SEQ ID NO. 5, SEQ ID NO. 6, SEQ ID NO. 7, SEQ ID NO. 8, SEQ ID NO. 9, SEQ ID NO. 10, SEQ ID NO. 11 or SEQ ID NO. 12 in at least one cell.
  • the present invention is highly lignin-specific with less inference towards the biosynthesis pathway. In more particular, the present method merely genetically intervenes the monolignol biosynthesis pathway in H. brasiliensis, which is responsible for lignin production.
  • SEQ ID NO. 1 SEQ ID NO. 2, SEQ ID NO. 3, SEQ ID NO. 4, SEQ ID NO. 5, SEQ ID NO. 6, SEQ ID NO. 7, SEQ ID NO. 8, SEQ ID NO. 9, SEQ ID NO. 10, SEQ ID NO. 11 and SEQ ID NO. 12 are respectively shown in Figures 1 to 12.
  • SEQ ID NO. 1 and SEQ ID NO. 2 respectively encode for the isoforms PAL 1 and PAL 2 which can catalyze the deamination of phenylalanine to form cinnamate.
  • Cinnamate can be converted by C4H which is encoded by SEQ ID NO. 3 into j ⁇ -coumarate; whereas />-coumarate can then be ligased into />-coumaroyl CoA, catalyzed by 4CL which is encoded by SEQ ID NO. 9.
  • the biosynthesis pathway of monolignols in H. brasiliensis also involves a series of hydroxylation and methylation steps.
  • SEQ ID NO. 4 and SEQ ID NO. 5 respectively encode for the isoforms of C3H which perform the subsequent C3 hydroxylation steps.
  • SEQ ID NO. 7 encodes for HCT which also helps in the C3 hydroxylation steps.
  • F5H encoded by SEQ ID NO. 8 is involved.
  • the methylation step for C3 can be catalyzed by CCoAOMT encoded by SEQ ID NO. 10; whereas that of C5 can be catalyzed by COMT encoded by SEQ ID NO. 6.
  • SEQ ID NO. 11 and SEQ ID NO. 12 respectively encode for the reductases of CCR and CAD.
  • the present invention is provided to potentially reduce the lignification process of the rubber plant, H.
  • brasiliensis by applying a method for reducing the biosynthesis of monolignols, including the lignin precursors of ⁇ -coumaryl alcohol, coniferyl alcohol and sinapyl alcohol, which respectively forms the ⁇ -hydroxyphenyl (H), guaiacyl (G) and syringyl (S) lignin subunits at the end of the pathway.
  • H ⁇ -hydroxyphenyl
  • G guaiacyl
  • S syringyl
  • the present method aims to disrupt the biosynthesis process of monolignols by genetically intervening the expression of gene encoding the ten families of enzymes involved and reducing the availability of these enzymes.
  • the down-regulation process of the nucleic acid template expression can be conducted by two types of genetic intervention approaches.
  • the down-regulation process can be performed by introducing a dsRNA having a polynucleotide strand containing nucleotide sequence which is at least 60% complementary to the nucleic acid template in the cell.
  • RNAi system is capable of silencing the expression of the related enzymes set forth in the foregoing description.
  • the complementary polynucleotide strand introduced, coupled with the RNA-induced silencing complexes in the transfected cells is able to initiate degradation of the mRNA of the related enzymes and thus prohibits subsequent creation of the corresponding enzymes in the affected cells.
  • the mRNA of the related enzymes can have a nucleotide sequence set forth in SEQ ID NO. 1 , SEQ ID NO. 2, SEQ ID NO. 3, SEQ ID NO. 4, SEQ ID NO. 5, SEQ ID NO. 6, SEQ ID NO. 7, SEQ ID NO. 8, SEQ ID NO. 9, SEQ ID NO. 10, SEQ ID NO. 11 or SEQ ID NO.
  • the down-regulation process is conducted by transforming the cell with a recombinant gene construct encoding open reading frame containing nucleotide sequence which is at least 70% identical to SEQ ID NO. 1, SEQ ID NO. 2, SEQ ID NO. 3, SEQ ID NO. 4, SEQ ID NO. 5, SEQ ID NO. 6, SEQ ID NO. 7, SEQ ID NO. 8, SEQ ID NO. 9, SEQ ID NO. 10, SEQ ID NO. 11 or SEQ ID NO. 12 and expressing the nucleotide sequence of the gene construct under expression control of a lignification-associated promoter.
  • the open reading frame containing nucleotide which is at least 70% identical to SEQ ID NO. 1, SEQ ID NO. 2, SEQ ID NO. 3, SEQ ID NO. 4, SEQ ID NO. 5, SEQ ID NO. 6, SEQ ID NO. 7, SEQ ID NO. 8, SEQ ID NO. 9, SEQ ID NO. 10, SEQ ID NO. 11 or SEQ ID NO. 12 and expressing the nucleotide sequence of the gene construct under expression
  • the antisense mRNA may not need complete complementary sequence to successfully hybridize onto the sense mRNA. It may only require at least 70% sequential nucleotides complimentary to the sense mRNA.
  • the dsRNA applied in this RNAi system may have a length of 20bp to a few hundreds bp.
  • the expression of the antisense mRNA in the present invention is also preferably for the expression of the antisense mRNA in the present invention to be initiated by the lignification-associated promoter located at upper stream of the open reading frame.
  • the expression of the antisense mRNA corresponding to the expression of the related target enzymes mRNA in the transfected cell can be activated upon receipt of the particular signal for generating lignin in the H. brasiliensis.
  • the lignification-associated promoter serves as a regulatory mechanism to control expression of the antisense RNA so that only in the specific cell types is signaled by the plant to produce lignin.
  • the present method is capable of controlling the generation of antisense RNA specifically in only those lignin-producing cells, but not other transfected non-lignin-producing cell types. More preferably, tissue-specific promoters can be used to drive the expression of the recombinant gene construct in the plant, so that the desired events can be limited to only the lignifying tissues or lignin- producing cells, thus preventing the disruption of other physiological pathways in the transgenic plant.
  • the transformed genes could then be stably inherited over generations in the genetically modified plant of Hevea brasiliensis.
  • a transgenic plant of H. brasiliensis with potentially reduced monolignol, or lignin content is provided.
  • the transgenic plant is characterized by its expression of a nucleic acid template containing at least one nucleotide sequence set forth in SEQ ID NO. 1 , SEQ ID NO. 2, SEQ ID NO. 3, SEQ ID NO. 4, SEQ ID NO. 5, SEQ ID NO. 6, SEQ ID NO. 7, SEQ ID NO. 8, SEQ ID NO. 9, SEQ ID NO. 10, SEQ ID NO. 11 or SEQ ID NO. 12 in at least one cell therein, which is down-regulated.
  • the transgenic plant of the present invention may possess secondary wall with reduced thickness compared to wild type plant of H, brasiliensis. Such feature may also be able to ease the rubber- tapping process as well as latex collection from the transgenic plant, thus effecting in the increase of the yield of rubber.
  • the expression of a nucleic acid template containing at least one nucleotide sequence set forth in SEQ ID NO. 1 , SEQ ID NO. 2, SEQ ID NO. 3, SEQ ID NO. 4, SEQ ID NO. 5, SEQ ID NO. 6, SEQ ID NO. 7, SEQ ID NO. 8, SEQ ID NO. 9, SEQ ID NO. 10, SEQ ID NO. 11 or SEQ ID NO. 12 in at least one cell in the transgenic plant of H. brasiliensis is down-regulated.
  • the down- regulated expression process can be achieved by antisense inhibitors or RNA interference (RNAi) system as set forth in the preceding description.
  • tissue-specific promoters can be used to drive the expression of the recombinant gene construct in the plant so that the desired events can be limited to only the lignifying tissues or lignin-producing cells.
  • an isolated polynucleotide encoding enzymes for catalyzing biosynthesis of monolignols in the plant of Hevea brasiliensis comprises nucleotide sequence set forth in SEQ ID NO. 1 , SEQ ID NO. 2, SEQ ID NO. 3, SEQ ID NO. 4, SEQ ID NO. 5, SEQ ID NO. 6, SEQ ID NO. 7, SEQ ID NO. 8, SEQ ID NO. 9, SEQ ID NO. 10, SEQ ID NO. 11 , SEQ ID NO. 12 or any of complementary sequences thereof.
  • the polynucleotide is isolated from the plant of H. brasiliensis 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.
  • Suitable primers or amplification oligonucleotides can be designed from the genomic database available to identify and obtain the polynucleotide of the genes of interest, which encodes the series of monolignols synthesis-associated enzymes.
  • An example of the oligonucleotides sequences used is tabulated in Example 1.
  • the partial or complete sequence of these isolated polynucleotides can be utilized for synthesizing partial or complete polynucleotides of the enzymes PAL, C4H, 4CL, HCT, C3H, CCoAOMT, F5H, COMT, CCR or CAD.
  • Still another preferred embodiment of the present invention discloses a recombinant gene construct containing the isolated polynucleotide as set forth in the preceding description.
  • the recombinant gene construct further comprises a promoter region operably-linked to control the expression of the nucleic acid template.
  • the recombinant gene construct can be used to facilitate the gene silencing process as set forth in the preceding description.
  • polypeptides of the related enzymes can also be synthesized using biological agents such as bacteria or plant.
  • biological agents such as bacteria or plant.
  • polypeptide for catalyzing biosynthesis of monolignols in the plant of Hevea brasiliensis comprising amino acid sequence set forth in SEQ ID NO. 13, SEQ ID NO. 14, SEQ ID NO. 15, SEQ ID NO. 16, SEQ ID NO. 17, SEQ ID NO. 18, SEQ ID NO. 19, SEQ ID NO. 20, SEQ ID NO. 21, SEQ ID NO. 22, SEQ ID NO. 23 or SEQ ID NO. 24.
  • These polypeptides are a series of enzymes involved in the biosynthesis of monolignols. They can either be synthesized chemically or biologically. A particular enzyme of interest synthesized can be used in the production of the target lignin precursor or intermediate molecules in vitro for various biological studies or industrial usage.
  • the gene was amplified from the cDNA by PCR using the gene specific primers for PAL gene 1 , PAL gene 2, C4H, C3H 1, C3H 2, COMT, HCT, F5H, CCoAOMT, 4CL, CCR and CAD respectively, as tabulated in Table 1.
  • the PCR reaction (50 ⁇ ) contained ⁇ ⁇ , of cDNA, 20 pmoles of each primers, 5 ⁇ of 10X Pfu Buffer, 5 of 2.5 mM dNTP mix and 1.0 unit of PfuTurbo® DNA polymerase (Stratagene). PCR was carried out in VeritiTM Thermal Cycler (Applied Biosystems) using the following conditions.
  • the purified PCR product was ligated into pCR® 4 Blunt TOPO® Vector (Invitrogen) and transformed into One Shot® MachlTM-T1R Chemically Competent E. coli cells (Invitrogen). Plasmids were isolated from putative colonies using QIAprep Spin® Miniprep Kit (Qiagen) following the manufacturer's instructions. The presence of the insert was checked by digesting with EcoRI (NEB) and positive plasmids were subjected to sequencing.
  • nucleotide sequences and the amino acid sequences were analysed by BLASTN and BLASTP programmes respectively.
  • the nucleotide sequences of the polynucleotides as well as the amino acid of the polypeptides isolated are shown in Figures 1 to 12.

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Abstract

La présente invention a pour objet un procédé pour la réduction de la synthèse de monolignols dans le cytoplasme de la plante d'Hevea brasiliensis, comprenant l'étape de régulation à la baisse de l'expression d'une matrice d'acide nucléique contenant au moins une séquence de nucléotides présentée dans SEQ ID NO. 1, SEQ ID NO. 2, SEQ ID NO. 3, SEQ ID NO. 4, SEQ ID NO. 5, SEQ ID NO. 6, SEQ ID NO. 7, SEQ ID NO. 8, SEQ ID NO. 9, SEQ ID NO. 10, SEQ ID NO. 11 ou SEQ ID NO. 12 dans au moins une cellule. La présente invention concerne également des séquences de nucléotides codant les enzymes phénylalanine ammoniac-lyase 1 (PAL1), phénylalanine ammoniac-lyase 2 (PAL2), cinnamate 4-hydroxylase (CH4), coumarate 3-hydroxylase 1 (C3H1), coumarate 3-hydroxylase 2 (C3H2), acide caféique/5-hydroxyconiféraldéhyde O-méthyltransférase (COMT), hydroxycinnamoyl-CoA:shikimate hydroxycinnamoyl transférase (HCT), férulate 5-hydroxylase (F5H), 4-coumarate CoA ligase (4CL), 4-coumarate cafféoyl-CoA O-méthyltransférase (CCoAOMT), 4-coumarate cinnamoyl coenzyme A réductase (CCRl), et 4-coumarate alcool cinnamylique déshydrogénase (CAD), toutes issues d'Hevea brasiliensis.
PCT/MY2012/000014 2011-05-19 2012-01-30 Procédé de régulation de la biosynthèse de monolignols Ceased WO2012158016A1 (fr)

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