CN1167797C - Cinnamoyl-CoA reductase gene associated with wheat stem development - Google Patents

Abstract

The invention provides the cDNA sequence of the cinnamoyl-CoA reductase gene (Ta-CCR1) related to wheat stem development and the amino acid sequence of the protein encoded by it. The cDNA sequence of this gene has 1317 bases, and the protein encoded by it The protein has 349 amino acids; further, the present invention provides an in vitro expression plasmid constructed from the above-mentioned Ta-CCR1 gene, and the gene is translated into a protein with expected functions by Escherichia coli. The gene and its encoded protein have the characteristics of specific expression in wheat stems, and are closely related to the lignin synthesis of wheat and the lodging resistance of stems.

Description

Cinnamoyl-CoA reductase gene associated with wheat stem development

The technical field to which the present invention belongs is the technical field of plant genetic engineering. In particular, the present invention relates to the cinnamoyl-CoA reductase gene and its associated protein product associated with wheat stem development. More specifically, the present invention relates to the isolation of the wheat cinnamoyl-CoA reductase gene, the use of the gene to construct a plasmid that can be expressed in Escherichia coli, and the translation of the gene into a protein and the functional identification of the protein. The gene and protein can be used to control the growth and development of wheat stems.

In vascular plants, including ferns, gymnosperms, and angiosperms, the appearance of lignin has played an important role in the evolution of plants to land, allowing plants to adapt to drier external environments and develop into larger spaces , thus forming a more diverse plant group (Facchini, P.J, 1999, Trends in Plant Sci, 4: 382-384). The synthesis of lignin also plays an important role in the growth and development of plants. Lignin is mainly deposited on the cell walls of plant vessels, rays, thick-walled cells, etc., which can increase the mechanical strength of the cell wall and reduce its permeability. Therefore, the synthesis of lignin enhances the mechanical support of plants. When plants are injured and attacked by diseases and insect pests, plant cells can rapidly lignify to effectively protect their internal tissues from further damage, thus enhancing the resistance of plants to external mechanical damage and attack by diseases and insect pests (Whetten, R.W. et al., 1998, Annu Rev Plant Physiol Plant Mol Biol. 49:585-609).

Lignin is a macromolecular organic phenolic compound, and its content ranks second after cellulose among all macromolecular organic compounds on earth. It usually accounts for 10-20% of the dry weight in plants, and even up to 30% in trees. Lignin synthesis is based on phenylalanine as a precursor, and through a series of enzymatic reactions, several simple phenolic alcohol derivatives are formed, which are called monolignols, and then polymerized by these lignin monomers Form high molecular weight lignin. There are three main types of monomers that make up lignin, namely p-coumaryl alcohol, coniferyl alcohol and sinapyl alcohol. The structural difference between these three classes of compounds is the number of methoxyl groups.

The biosynthesis reaction of lignin involves the following basic processes: (1) phenylalanine undergoes aminolysis to form cinnamic acid (phenylacrylic acid, cinnamic acid), (2) hydroxylation and transmethylation, in the benzene ring (3) has been acylated, that is, the carboxyl side chain has been acylated, (4) reduction reaction, so that the carboxyl side chain passes through the aldehyde group and finally forms an alcohol. Wherein, the first three-step reaction also involves the synthesis of other secondary metabolites, such as phytoalexin, anthocyanidin, etc., and the reduction reaction of the fourth step is unique to lignin (Baucher, M.B., etc., 1998, Crit.Rev.Plant Sci., 17:125-197).

Cinnamoyl-CoA reductase (EC 1.2.1.44, CCR) catalyzes the first reduction reaction in lignin synthesis and is responsible for catalyzing the conversion of cinnamoyl-CoA to cinnamaldehyde. It determines whether the phenylalanine metabolites in plants enter the lignin synthesis pathway, and is a key enzyme that controls the lignin synthesis reaction.

Since the 1970s, researchers have carried out the separation and purification of cinnamoyl-CoA reductase, successively from soybean suspension culture cells (Wegenmayer, H et al., 1976, Eur.JBiochem, 65:529-536. ), spruce (Luderitz, T et al., 1981, Eur.J.Biochem, 119:115-124) and poplar (Sarni, F et al., 1984, Eur.J.Biochem, 139:259-265) obtained Partially purified enzyme protein. However, because these proteins have not been completely purified, there is still a lack of in-depth research on their biochemical characteristics and biological properties. In 1994, French scientists obtained relatively pure cinnamoyl-CoA reductase from Eucalyptus and proved that it belongs to NADP oxidoreductase (Goffner, D et al., 1994, Plant Physiol, 106:625-687).

On this basis, French scientists first cloned the cinnamoyl-CoA reductase gene from Eucalyptus in 1997. Sequence analysis showed that the enzyme encoded by this gene is the same as the dihydroflavonol reductase (dihydroflavonol-4- reducease) has high homology, and has homology with 3-hydroxysteroid dehydrogenase (3-hydroxysteroid dehydrogenase) in mammals and UDP-galactose-4-epimerase (UDP-galactose-4-epimerase) in bacteria , indicating that this type of enzyme is a relatively ancient class of enzymes in the process of biological evolution. In the eucalyptus genome, CCR is a single-copy gene, and the expression of the gene is mainly in the forming xylem of tissues such as roots and stems, indicating that this gene is closely related to the synthesis of plant lignin and the lignification of tissues (Lacombe, E et al., 1997, Plant J, 11:429-441). Thereafter, the CCR gene isolated from maize also proved that in monocotyledonous plants, the expression of CCR gene is closely related to the synthesis and lignification of lignin in stem tissue (Pichon, M et al., 1998, Plant Mol Biol, 38 : 671-676).

Transgenic experiments have been carried out using the model plant tobacco. The results show that if the activity of cinnamoyl-CoA reductase in transgenic tobacco is reduced to less than 50% of that in normal plants by antisense inhibition, the lignin content in transgenic tobacco will be reduced by more than 40%, and the lignin composition will also change significantly , among which, the content of syringol increased, the growth of tobacco stems was seriously affected, the plant height of transgenic tobacco was only two-thirds of that of normal plants, and the vessels in stems showed signs of collapse (Piquemal, J. et al., 1998, Plant J ., 13:71-83). This proves from the negative side that CCR, as a key enzyme gene of lignin synthesis, plays an important role in controlling the growth and development of plant stems, and thus has wide application potential in the field of plant genetic engineering technology.

French scientists have isolated CCR genes from eucalyptus (Eucalyptus gunnii), poplar (Populus trichocara), tobacco (Nicotiana tabacum), corn (Zea mays), fescue grass (Festuca arundinacea) and alfalfa (Medicago sativa), and applied for the Invention patent (WO 9527790). However, the isolation and identification of CCR genes in wheat (Triticum aestivum) is still blank.

Whether in China or in the world, wheat is one of the most important crops. In production practice, the lodging caused by insufficient mechanical strength of wheat stalks can lead to yield loss of more than 20%. In addition, the losses caused by the invasion of diseases and insect pests are also very huge. Utilizing the wheat cinnamoyl-CoA reductase gene and regulating the expression activity of the gene through genetic engineering means, it is possible to obtain new wheat strains with high yield and resistance to lodging and pests and diseases.

The invention provides the wheat cinnamoyl-CoA reductase gene and the protein coded by it, so as to control the synthesis of lignin in wheat and further control the growth and development of wheat stems. Another object of the present invention is to provide an expression plasmid capable of translating the above-mentioned gene into an active protein.

The purpose of the present invention is achieved like this:

1. Extract total RNA from wheat stems, and isolate PolyA RNA, use this as a template, and amplify through RACE (rapid amplification of cDNA ends) to obtain the wheat cinnamoyl-CoA reductase gene (CCR) for isolating wheat molecular probes.

The primers used were:

(1) A1: 5'-GACTCGAGTCGACATCGA(T) ₁₇ -3';

(2) A2: 5'-GACTCGAGTCGACATCG-3';

(3) A3: 5'-GA(A/G)AA(A/G)GG(T/C/A/G)TA(T/C)AC(T/C/A/G)GT-3' ;

(4) A4:

5'-AG(A/G)TG(T/C/A/G)CC(T/C)TT(T/C)TC(T/C)TG(T/C/A/G)AG-3 ';

(5) A5:

5'-GT(T/C/A/G)AC(T/C/A/G)GA(T/C)GA(T/C)CC(T/C/A/G)GA(A/G )

CA-3'.

First, under the guidance of primer A1, the first strand cDNA is synthesized by reverse transcription; then primers A2 and A3 are added for PCR amplification; then primers A4 and A5 are added for the second round of PCR amplification; the PCR product is passed through agar After purified by sugar gel electrophoresis, it was connected to a T-vector and transformed into Escherichia coli; positive clones were screened out, and probes were obtained after enzyme digestion and sequence analysis.

2. Use the PolyA RNA of wheat stem as a template to synthesize cDNA, add XhoI and EcoRI linkers, insert into ZAP vector (Stratagene Company, La Jolla, CA, USA), and construct a cDNA library of wheat stem through in vitro packaging.

3. After the probe was labeled with ³² P-dCTP, it was subjected to plaque hybridization with the cDNA library; after three rounds of screening, a single positive plaque was selected; after excision in vitro, the positive clone was isolated from the phage DNA; the obtained After restriction analysis and sequence analysis of the recombinant plasmid, the full-length cDNA encoding the wheat cinnamoyl-CoA reductase gene (CCR) was obtained and named Ta-CCR1.

4. Analysis of the full-length cDNA sequence of the Ta-CCR1 gene: the full-length cDNA sequence of the Ta-CCR1 gene was analyzed to obtain its sequence, expressed as SEQ ID NO1. The sequence consists of a total of 1317 bases, of which the 5' untranslated region includes 72 bases, the 3' untranslated region includes 195 bases (including PolyA consisting of 24 bases), and the coding region consists of 1050 bases, of which A accounts for 17.81% (187), C accounts for 34.48% (352), G accounts for 33.62% (353), T accounts for 14.10% (148), A+T accounts for 31.90% ( 335), C+G accounted for 68.10% (715).

The gene homology analysis in the international gene sequence database (GenBank\EMBL\DDBJ) shows that the Ta-CCR1 gene is a new gene that has not been reported in wheat, and it is related to eucalyptus (Eucalyptus gunnii), poplar (Populus trichocara), The cinnamoyl-CoA reductase genes isolated from tobacco (Nicotiana tabacum), maize (Zea mays), fescue grass (Festuca arundinacea) and alfalfa (Medicago sativa) share homology.

5. Sequence analysis of the protein encoded by the full-length cDNA sequence of Ta-CCR1 gene: from the above-mentioned full-length cDNA sequence of Ta-CCR1 gene, the protein sequence encoded by it was obtained. The protein sequence comprises 349 amino acids, as shown in SEQ ID NO 2. In the protein sequence, there are 139 hydrophobic amino acids, 74 hydrophilic amino acids, 36 basic amino acids, and 36 acidic amino acids. The protein has a molecular weight of 37.4KD and an isoelectric point of 6.3.

6. Synthesize primers on both sides of the Ta-CCR1 gene coding region, and introduce HindIII and NotI restriction sites respectively, and the primer sequences are respectively:

5'-Primer: 5'-CACAAGCTTATGACCGTCGTCGCCGCCGC-3';

3'-Primer: 5'-ATAAGAATGCGGCCGCTCACGCTGTTGCACCGTCCAG-3'.

The full-length coding region of the Ta-CCR1 gene was amplified by PCR, cut out with HindIII and NotI double enzymes, connected to the corresponding restriction site of the expression vector pET-29b (see Figure 1), and analyzed by enzyme digestion and sequence Prove the integrity and correctness of this expression vector.

7. The expression vector was transformed into E. coli BL21(DE3)pLysS strain. After IPTG induction, the E. coli protein was analyzed by SDS-PAGE gel electrophoresis, which proved that the Ta-CCR1 gene had been translated into the correct protein in E. coli. After the above-mentioned Escherichia coli strain was cultured at 37°C and induced by IPTG, the cell was lysed, the protein of the cell was isolated, and the protein was analyzed for cinnamoyl-CoA reductase enzyme activity, and the substrate used was ferulic acid-CoA (feruloyl CoA), its enzymatic activity is 1.67nmol/sec/mg, which proves that this protein has the catalytic activity of cinnamoyl-CoA reductase.

8. Extract total RNA from wheat root, stem, and leaf tissue, transfer to nylon membrane by capillary method after electrophoresis, hybridize with wheat CCR gene probe, and detect the hybridization result by autoradiography, using rRNA probe as control Normalize the hybridization signal. This method was used to detect the expression of Ta-CCR1 gene in different parts of wheat. The results obtained indicated that the gene was mainly expressed in wheat stems.

9. Northern hybridization of the Ta-CCR1 gene in the stems of different developmental stages (from jointing stage to milk maturity stage) of lodging-prone and lodging-resistant wheat varieties to detect the expression of the gene in different varieties, the results show that the gene The expression level in lodging-resistant varieties was significantly higher than that in lodging-easy varieties, and the difference was more obvious in the late growth period of stems. The results indicated that the Ta-CCR1 gene not only controlled the synthesis of wheat lignin, but also was closely related to the lodging resistance of wheat stems.

The invention provides a cinnamoyl-CoA reductase gene related to wheat stem development and a protein product related thereto. Using this gene, transgenic plants can be developed to control lignin synthesis in the stem, thereby changing the mechanical support ability of the stem and the ability to resist the adverse external environment. For example, as shown in a preferred embodiment of the present invention, the use of transgenic wheat to obtain lodging-resistant high-yielding varieties increases the ability of wheat to resist diseases and resist external adverse environments; Transfer to existing wheat production varieties.

Examples of the present invention are described below. It should be noted that the embodiments of the present invention are only illustrative but not limiting to the present invention.

Example 1. Isolation and sequence analysis of wheat cinnamoyl-CoA reductase gene probe

Wheat was planted in the greenhouse, watered and fertilized normally, until it grew to 2-3 internodes, roots, stems, and leaf tissues were collected, and total RNA was extracted with TRI reagent (Molecular Research Center, Inc, Cincinnati, USA). PolyAT tract mRNA Isolation Kit (Promega, Madison, USA) was used to separate Poly(A) ⁺ RNA, and the content and purity of RNA were analyzed by UV spectrophotometer.

The conditions for reverse transcription to synthesize the first strand of cDNA are: Poly(A) ⁺ RNA 1ug, 5μmol/L primer 5'-GACTCGAGTCGACATCGA(T) ₁₇ -3', 0.5mmol/L dNTP, 50 units of RNasin, incubated at 65°C After cooling on ice for 10 minutes, add 200U SuperScript ^™ II RNaseH ^- Reverse Transcriptase (Gibco), incubate at 42°C for 1 hour, add EDTA to a final concentration of 10mM, keep at 95°C for 10 minutes, and cool on ice.

The conditions for the first round of PCR are: 3 μL cDNA reaction solution, 1 μmol/L primer, 0.4 mmol/LdNTP, 2.5U Taq DNA polymerase (Gibco Company, Grand Island, NY, USA), denatured at 95°C for 5 minutes, and 50°C Refolding for 2 minutes, 72°C extension for 40 minutes, then 40 cycles (95°C for 1 minute, 55°C for 1 minute, 72°C for 3 minutes), and finally 72°C for 10 minutes.

The second round of PCR: Use 1 μL of the first round of PCR reaction solution, denature at 95°C for 5 minutes, and other reactions are the same as the first round. The PCR product was subjected to 1.0% agarose gel electrophoresis using ^GlassMAX® DNA Isolation Kit (Gibco, Grand Island, NY, USA). After purification, it was connected to the pGEM-TEasy vector (Promega, Madison, USA). The ligation condition was 16°C for 12 hours. The recipient bacteria were transformed into E.coli DH5α, and the competent cells were prepared using the CaCl ₂ treatment method (Sambrook, J. et al. (eds), Molecular cloning.A laboratory manual, 2 ^nd ed., Cold Spring Harbor Laboratory Press, Cold Spring Harbor , NY, 1989). The ligation product was added to competent cells, ice-bathed for 30 minutes, heat-shocked at 42°C for 90 seconds, added to LB liquid medium, incubated at 37°C for 45 minutes, and coated on a mixture containing ampicillin (100ug/ml) and X-Gal ( 20ug/ml) on LB plates, and cultured overnight at 37°C. The white clone was inoculated on LB liquid medium (containing ampicillin 100ug/ml), cultured overnight at 37°C, the plasmid was extracted and analyzed by enzyme digestion, followed by sequence analysis with ABI 377 DNA sequence analyzer to determine the wheat CCR probe.

The results of sequence analysis showed that the isolated probe had 693 nucleotides, which were compatible with eucalyptus (Eucalyptus gunnii), poplar (Populus trichocara), tobacco (Nicotiana tabacum), corn (Zea mays), fescue grass (Festuca arundinacea) and The homology of the cinnamoyl-CoA reductase gene isolated from alfalfa (Medicago sativa) is more than 60%, and the encoded protein has the catalytic active center NWYCY for the reduction of cinnamoyl-CoA.

Example 2. Screening and sequence analysis of wheat cinnamoyl-CoA reductase gene

Using wheat stem Poly(A) ⁺ RNA as a template, cDNA was synthesized with ZAP vector (Stratagene, La Jolla. CA, USA), and packaged in vitro to construct a wheat stem cDNA library. Take 50ng of probe DNA, add 50uCi ³² P-dCTP for labeling, incubate at 37°C for 1 hour, add EDTA to a final concentration of 10mM to terminate the reaction. After the probe passed through the Sephadex G-50 column, it was boiled at 100° C. for 10 minutes, and immediately cooled on ice for hybridization.

Take 50000pfu and spread it on the plate, and transfer it to nitrocellulose membrane, denature in 0.5M NaOH plus 1.5M NaCl for 2 minutes, 1.5M NaCl plus 0.5M Tris-HCl (pH8.0) for 5 minutes, 0.2MTris- Rinse with HCl (pH7.5) plus 2×SSC for 30 seconds, sandwich the membrane between two layers of Whatman filter paper, bake in vacuum at 80°C for 2 hours, then add hybridization solution (6×SSC, 5×Denhardt, 0.5% SDS, 100 μg /mL protist DNA, 50% formamide), incubated at 42°C for 2 hours, added the labeled probe, hybridized overnight at 42°C, washed twice at room temperature with 6×SSC plus 0.1% SDS, each time for 30 minutes, and then used Wash twice in 0.1×SSC plus 0.1% SDS at 65°C for 20 minutes each time, and then perform autoradiography at -80°C. After confirming positive clones, remove the plaques and extract them in SM solution overnight at 4°C. Follow the same procedure for two rounds of screening.

For the obtained positive phage plaques, they were converted into plasmid DNA according to the method provided by Stratagene Company, and after analysis by enzyme digestion, sequence analysis was carried out by ABI 377 DNA sequence analyzer.

The measured DNA sequence is shown in SEQ ID NO1, which is named Ta-CCR1. It is a wheat cinnamoyl-CoA reductase gene, which has not been reported so far.

Example 3. Translation of wheat cinnamoyl-CoA reductase gene-encoded protein

Artificially synthesize a pair of primers:

5'-Primer: 5'-CACAAGCTTATGACCGTCGTCGCCGCCGC-3';

3'-Primer: 5'-ATAAGAATGCGGCCGCTCACGCTGTTGCACCGTCCAG-3'.

HindIII and NotI restriction sites were introduced at both ends of the primers, and Ta-CCR1 gene was used as a template for PCR amplification. Amplification conditions: 10ng DNA, 1μmol/L primers, 0.4mmol/L dNTP, 2.5U Taq DNA polymerization Enzyme (Gibco company, Grand Island, NY, USA). Denaturation at 95°C for 5 minutes, followed by 30 cycles (1 minute at 95°C, 1 minute at 55°C, 1.5 minutes at 72°C), and a final extension at 72°C for 10 minutes.

The PCR product was subjected to 1.0% agarose gel electrophoresis using ^GlassMAX® DNA Isolation Kit (Gibco, Grand Island, NY, USA). After purification, it was digested with HindIII and NotI, connected to the vector pET-29b, and transformed into Escherichia coli BL21(DE3)pLysS competent cells. After the transformed cells were ice-bathed for 30 minutes, they were heat-shocked at 42°C for 50 seconds, and then added to LB liquid. The culture medium was incubated at 37° C. for 45 minutes, and spread on LB plates containing kanamycin (50 ug/ml). Culture the resistant colonies in LB liquid medium (containing kanamycin 50ug/ml) at 37°C overnight, then dilute 1/100, culture at 37°C for 2 hours, add IPTG to a final concentration of 1mM, and continue to cultivate After 3 hours, centrifuge at 12000 g for 10 minutes to collect the cells, add 100 ul lysate (50 mM Tris-HCl pH6.8, 100 mM dithiothreitol, 2% SDS, 0.1% bromophenol blue, 10% glycerol) to the cells, 100 Boil at ℃ for 5 minutes, and the bacterial protein was subjected to SDS-PAGE electrophoresis (gel concentration 12%) according to the conventional method. The result showed that there was expression of cinnamoyl-CoA reductase protein in the bacteria, and its structure was shown in SEQ ID NO 2.

Embodiment four, the determination of cinnamoyl-CoA reductase activity

Centrifuge 50ml of IPTG-induced Escherichia coli at 4000g for 10 minutes, add 4ml of cell lysate (20mM Tris-HCl pH7.5, 1mM PMSF, 2mM dithiothreitol, 100ug/ml lysozyme), and incubate at 30°C for 15 minutes , treated with ultrasonic waves 20 times, centrifuged at 4000g for 10 minutes, and the supernatant was taken for enzyme activity determination. The enzyme activity assay reaction solution includes 100mM sodium phosphate buffer (pH6.25), 0.1mM NADPH, 70uM ferulic acid coenzyme A, 50ul protein solution, the total volume is 500ul, and the decrease of A ₃₆₆ is measured at 30°C to calculate cinnamoyl coenzyme A Reductase activity. The protein content was determined according to the method of Bio-Rad (Bradford, MM, 1976, Anal. Biochem., 72: 248-254).

The results show that with ferulic acid coenzyme A (feruloyl CoA) as substrate, its enzyme activity is 1.67nmol/sec/mg, indicating that the protein encoded by the Ta-CCR1 gene has the function of catalyzing the synthesis of cinnamaldehyde, thus it can control wheat lignin synthesis.

Embodiment 5, detection of Ta-CCR1 gene expression product in wheat tissue

Northern blot hybridization was used to detect the expression products of Ta-CCR1 gene in different wheat tissues. The total RNA samples isolated from different wheat tissues were separated on 1.4% agarose/formaldehyde denaturing gel electrophoresis according to conventional methods, and separated by 20×SSC Transfer to nylon membrane by capillary method (transfer time 12 hours), nylon membrane sandwiched between two layers of Whatman filter paper, 80 ℃ vacuum baked for 2 hours, put the membrane in the hybridization bottle, then add hybridization solution (6×SSC, 5×Denhardt, 0.5% SDS, 100 μg/mL protidine DNA, 50% formamide) 10ml, pre-hybridize at 42°C for 2 hours, add the labeled cinnamoyl-CoA reductase gene probe, hybridize overnight at 42°C, after 6× Wash twice with SSC plus 0.1% SDS at room temperature for 30 minutes each time, then wash twice with 0.1×SSC plus 0.1% SDS at 65°C for 10 minutes each time, and perform autoradiography at -80°C for one week. After developing, add 0.1×SSC plus 0.1% SDS to wash the membrane twice at 95°C for 15 minutes each time, then add hybridization solution containing 18S rRNA, and hybridize overnight at 42°C. The membrane was taken out, washed according to the same procedure, and autoradiographed at -80°C for 1 hour.

The hybridization results are shown in Table 1.

Table 1. The expression level of Ta-CCR1 gene in different wheat tissues (take the stem as 100) organize stem leaf root relative expression 100 23.9 10.0

The results showed that the wheat cinnamoyl-CoA reductase gene Ta-CCR1 was mainly expressed in the stem.

The expression of Ta-CCR1 in the stems of two different varieties at different growth stages was compared, and the results are shown in Table 2.

Table 2. The expression level of TaCCR1 gene in different development stages of wheat stem

(taking the jointing period of C6001 as 100) Variety puberty TaCCR1 level C6001 jointing stage heading stage milk ripening stage 10015.544.0 H4565 jointing stage heading stage milk ripening stage 170112188

It can be seen from the results that the expression level of Ta-CCR1 in the lodging-resistant variety (H4564) is significantly higher than that in the lodging-easy variety (C6001), and the difference is more obvious in the late growth period. It indicated that the Ta-CCR1 gene was closely related to the lodging resistance of wheat. Therefore, controlling the expression of Ta-CCR1 gene can control the lodging resistance traits of wheat.

Attachment: Nucleotide sequence and amino acid sequence involved in the present invention:

SEQ ID NO1 (cDNA sequence of wheat cinnamoyl-CoA reductase gene):

GTAGCTCGTACGTGGCATCTCCATCTCACCAACAATCTTGTTTAGACAAGCAGTGTA

AAAGTGACAGCAACAATGACCGTCGTCGCCGCCGCCGCCGCCGCCGCGGCGCAGGAG

CTGCCCGGGCACGGGCAGACCGTGTGCGTCACCGGCGCCGCCGGGTACATCGCGTCG

TGGCTCGTCAAGCTGCTCCTGGAGCGAGGCTACACCGTCAAGGGCACCGTGAGGAAC

CCAGATGATCCGAAGAACGCGCATCTGAAGGCGCTGGACGGCGCCGCCGAGAGGCTG

GTCCTCTGCAAGGCCGACCTCCTCGACTACGACGCCATCTGCGCCGCCGTCGAGGGC

TGCCACGGCGTGTTCACACCGCCTCCCCCGTCACCGACGACCCCGAGCAGATGGTG

GAGCCGGCGGTGAGGGGCACGGAGTACGTGATCAACGCGGCGGCGGACGCCGGCACC

GTGCGCCGGGTGGGTGTTACGTCGTCCATCGGCGCCGTCACCATGGACCCCAACCGT

GGTCCCGACGTGGTCGTCGACGAGTCCTGCTGGAGCGACCTTGAATTCTGCAAGAAA

ACCAAGAACTGGTACTGCTACGGCAAGGCGGTGGCGGAGCAGGCGGCGTGGGAGAAG

GCCGCGGCGCGCGGCGTCGACCTCGTCGTGGTGAACCCGGTGCTGGTGGTCGGGCCG

CTGCTGCAGCCGACGGTGAACGCCAGCGCCGCGCACATCCTCAAGTACCTCGACGGC

TCCGCCAAGTAGTACGCCAACGCGGTGCAGGCGTACGTGAACGTGCGCGACGTCGCC

GCCGCGCACGTCCGGGTCTTCGAGGCGCCCGGGGCCTCCGGCCGGCACCTCTGCGCC

GAGCGCGTCCTGCACCGCGAGGACGTCGTCCACATCCTCGGCAAGCTCTTTCCCGAG

TACCCCGTCCCAACAAGGTGCTCTGACGAGGTGAACCCACGGAAGCAGCCTTACAAG

ATGTCCAACCAGAAGCTGCAGGATCTTGGCCTCCAGTTCACTCCTGTCAACGACTCT

CTGTACGAGACGGTGAAGAGCCTCCAGGAGAAGGGGCACCTCCCGGCGCCGAGGAAA

GATATCCTCCCAGCGGAACTGGACGGTGCAACAGCGTGATGGTGCTGAAGAAACAGC

GCGGTTCACGTTTTTCTGTAACGCGGTGGGACATCGTATGTGGTCTGTTTGTGTATA

CATTCTATCTAATATCGTGTTATTTAAGTGGACTAAGCAAATATGGTAATGTATCGG

CTTCGATGATCGACACTTAAAAGTGAGCTTTCGCAAACTAAAAAAAAAAAAAAAAAAA

AAAAAAA

SEQ ID NO2 (protein sequence encoded by wheat cinnamoyl-CoA reductase gene):

MTVVAAAAAAAAAQELPGHGQTVCVTGAAGYIASWLVKLLLERGYTVKGTVRNPDDPK

NAHLKALDGAAAERVLCKADLLDYDAICAAVEGCHGVFHTASPVTDDPEQMVEPAVR

GTEYVINAAADAGTVRRVGVTSSIGAVTMDPNRGPDVVVDESCWSDLEFCKKTKNWY

CYGKAVAEQAAWEKAAARGVDLVVVNPVLVVGPLLQPTVNASAAHILKYLDGSAKKY

ANAVQAYVNVRDVAAAHVRVFEAPGASGRHLCAERVLHREDVVHILGKLFPEYPVPT

RCSDEVNPRKQPYKMSNQKLQDLGLQFTTPVNDSLYETVKSLQEKGHLPAPRKDILPA

ELDGATA.

Accompanying drawing 1 is the structural diagram of the Escherichia coli expression plasmid containing the cinnamoyl-CoA reductase gene.

Claims (4)

1. the cDNA sequence of a cinnyl CoA reductase gene is characterized in that this cDNA sequence has the nucleotide sequence shown in SEQ ID NO 1.

2. a proteinic aminoacid sequence is characterized in that this proteinic aminoacid sequence is coded by the cDNA sequence of claim 1, and has the aminoacid sequence shown in SEQ ID NO 2.

3. a colibacillary expression plasmid is characterized in that this plasmid is constructed by the cDNA sequence of claim 1.

4. the expression vector of a kind of plant is characterized in that this carrier is constructed by the cDNA sequence of claim 1.

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