CN1167797C

CN1167797C - Cinnamon acyl-CoA reductase gene associated with wheat stalk development

Info

Publication number: CN1167797C
Application number: CNB011100559A
Authority: CN
Inventors: 马庆虎
Original assignee: Institute of Botany of CAS
Current assignee: Institute of Botany of CAS
Priority date: 2001-03-28
Filing date: 2001-03-28
Publication date: 2004-09-22
Anticipated expiration: 2021-03-28
Also published as: CN1376797A

Abstract

The present invention provides a cDNA sequence of a cinnamoyl-CoA reductase gene (Ta-CCR1) associated with the development of wheat stalks and an amino acid sequence of a protein coded by the cDNA sequence. The cDNA sequence of the gene has 1317 basic groups, and the coded protein has 349 amino acids. Furthermore, the present invention provides an in-vitro expression plasmid constructed by the Ta-CCR1 gene, and the gene can be translated into protein with prospective functions by colibacillus. The gene and the coded protein have the characteristic of specific expression in wheat stalks and are closely associated with the synthesis of wheat lignin and the lodging resistance of stalks.

Description

Grow relevant cinnyl CoA reductase gene with wheat stalk

Technical field under the present invention is the plant gene engineering technology field.Specifically, the present invention relates to grow relevant cinnyl CoA reductase gene and relevant protein thereof with wheat stalk.More particularly, the present invention relates to wheat cinnyl CoA reductase gene separation, with this gene constructed can be at the plasmid of expression in escherichia coli, and this gene translation gone out protein and protein carried out Function Identification.This gene and protein can be used for controlling the g and D of wheat stalk.

In the vascular plant that comprises fern, gymnosperm and angiosperm, the appearance of xylogen is evolved to Lu Sheng for plant and is had vital role, it can adapt to than the exsiccant external environment plant, and to bigger spatial development, thereby more diversified phyto-group (Facchini, P.J, 1999 have been formed, Trends inPlant Sci, 4:382-384).The synthetic of xylogen also has vital role for growth and development of plant.The xylogen major sedimentary can increase the physical strength of cell walls on the cell walls of the conduit of plant, ray, sclerenchymatous cell etc., reduce its permeability, therefore, and the synthetic machinery support effect that has strengthened plant of xylogen.Injured and be subjected to disease and pest when invasion and attack plant; vegetable cell lignifying is rapidly avoided further harm to protect its interior tissue effectively; therefore strengthened the resistibility (Whetten of plant for extraneous physical abuse and disease and pest invasion and attack; R.W. etc.; 1998, Annu Rev Plant Physiol Plant Mol Biol.49:585-609).

Xylogen belongs to the polymer organic phenolic compound, and in all macromolecular organic compounds, its content occupies after the Mierocrystalline cellulose on earth, occupies second.In plant materials, account for the 10-20% of dry weight usually, in trees even can reach 30%.Xylogen is synthetic to be to be precursor with the phenylalanine, through a series of enzymatic reactions, forms the alcohol derivate of several simple phenols, is called as lignin monomer (monolignol), forms the polymer wood quality by these lignin monomer polymerizations again.The monomer that constitutes xylogen mainly contains three classes, i.e. tonquinol (p-coumaryl alcohol), lubanol (coniferyl alcohol) and syringyl alcohol (sinapyl alcohol).This three compounds difference structurally is the quantity of methoxyl group.

The biosynthesizing reaction of xylogen relates to following several primary process: (1) phenylalanine forms styracin (phenylpropyl alcohol diluted acid through ammonolysis; cinnamic acid); (2) Hydroxyalkyl baseization and transmethylases; at the different sites Xing of phenyl ring Cheng Hydroxyalkyl base and methoxyl group; (3) acidylate; promptly in carboxylic side-chain acidylate, (4) reduction reaction makes carboxylic side-chain form alcohol at last through aldehyde radical.Wherein, preceding three-step reaction also relates to the synthetic of other secondary metabolite, as plant protecting chemical, cyanidin(e) etc., and that the reduction reaction in the 4th step is an xylogen is peculiar (Baucher, M.B. etc., 1998, Crit.Rev.Plant Sci., 17:125-197).

(cinnamoyl CoA reductuase, EC 1.2.1.44 are the first step reduction reaction of catalysis xylogen in synthetic CCR) to the cinnyl CoA-reductase, are responsible for the conversion of catalysis cinnyl coenzyme A to phenylacrolein.Whether the phenylalanine metabolite of its decision plant materials enters the xylogen route of synthesis, is the key enzyme that has control action kou in the xylogen building-up reactions.

From the seventies in 20th century, just there is the researchist to carry out separation and purifying work, successively from the suspended culture cell (Wegenmayer of soybean to the cinnyl CoA-reductase, H etc., 1976, Eur.JBiochem, 65:529-536.), cloud shirt (Luderitz, T etc., 1981, Eur.J.Biochem, 119:115-124) and willow (Sarni, F etc., 1984, Eur.J.Biochem has obtained partially purified zymoprotein in 139:259-265).But because these protein are by complete purifying, so people still lack deep research to its biochemical characteristics and biological property.1994, French scientist obtained purer cinnyl CoA-reductase from eucalyptus, and prove it belong to NADP class oxydo-reductase (Goffner, D etc., 1994, Plant Physiol, 106:625-687).

On this basis, French scientist has at first been cloned the cinnyl CoA reductase gene from eucalyptus in 1997, sequential analysis shows that the enzyme of this coded by said gene and responsible cyanidin(e) synthetic flavanonol reductase enzyme (dihydroflavonol-4-reducase) have higher homology, simultaneously with Mammals in 3-hydroxysteroid dehydrogenase (3-hydroxysteroid dehydrogenase) and the UDP-semi-lactosi isomerase (UDP-galactose-4-epimerase) in the bacterium have homology, show that this fermentoid is a class of enzymes more ancient in the organic evolution process.In the eucalyptus genome, CCR belongs to single copy gene, and expression of gene is mainly organized the xylem that is forming at root, stem etc., the lignifying closely related (Lacombe, E etc., 1997 that show the synthetic and tissue of this gene and plant lignin, Plant J, 11:429-441).Thereafter, isolating CCR gene also proves from corn, in monocotyledons, in CCR expression of gene and the cane tissue the synthetic and lignifying of xylogen in close relations (Pichon, M etc., 1998, Plant Mol Biol, 38:671-676).

People utilize the model plant tobacco to carry out transgenic experiments.The result shows, if the activity by cinnyl CoA-reductase in the Antisense Suppression render transgenic tobacco is reduced to below 50% of normal plants, then the content of lignin in the transgene tobacco reduces more than 40%, obvious variation also takes place in the xylogen composition, wherein, syringyl alcohol content increases, the growth of tobacco stem is had a strong impact on, the plant height of transgene tobacco only is 2/3rds of a normal plants, and the sign that caving in appears in conduit in the stem (Piquemal, J. etc., 1998, Plant J., 13:71-83).This proves that from the negative CCR as xylogen synthetic key gene, has vital role in the growth and development process of controlling plant stem, thereby can have application potential widely in the plant gene engineering technology field.

French scientist is isolated the CCR gene from eucalyptus (Eucalyptus gunnii), willow (Populus trichocara), tobacco (Nicotiana tabacum), corn (Zea mays), fescue (Festuca arundinacea) and clover (Medicago sativa), and has applied for patent of invention (WO 9527790).But separation and evaluation work to wheat (Triticum aestivum) CCR gene are still blank at present.

No matter in China still worldwide, wheat all is one of most important farm crop.In production practice, because the lodging that wheat cane physical strength deficiency causes can make production loss more than 20%.In addition, because the loss that the disease and pest invasion and attack are caused is also very huge.Utilize wheat cinnyl CoA reductase gene, by genetic engineering means, the expression activity of regulatory gene then may obtain the new strain of wheat of the resistant to lodging and disease and insect resistance of high yield.

The present invention by wheat is provided the cinnyl CoA reductase gene and by its encoded protein matter, reach in the control wheat synthetic, and the further growth of control wheat stalk and the purpose of growing of xylogen.Another object of the present invention provides the expression plasmid that said gene can be translated into active protein.

The object of the present invention is achieved like this:

1. from wheat stalk, extract total RNA, and separate PolyA RNA,,, obtain being used to separate the molecular probe of wheat coding wheat cinnyl CoA reductase gene (CCR) through RACE (the terminal rapid amplifying method of cDNA) amplification as template.

Employed primer is:

(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’。

At first under the guiding of A1 primer, article one chain cDNA is synthesized in reverse transcription; Add A2 and A3 primer again, carry out pcr amplification; And then add A4 and A5 primer, carry out second and take turns pcr amplification; Behind the PCR product process agarose gel electrophoresis purifying, be connected on the T-carrier transformed into escherichia coli; Filter out positive colony, through enzyme cut with sequential analysis after obtain probe.

2. the PolyA RNA with wheat stalk is a template, synthetic cDNA, add XhoI and EcoRI joint after, (CA USA), through external packing, is built into the cDNA library of wheat stalk for Stratagene company, La Jolla to insert ZAP vector.

3. probe is used ³²Behind the P-dCTP mark, carry out plaque hybridization with the cDNA library; Through the three-wheel screening, choose single positive bacterial plaque; Through external excision, positive colony is separated from phage DNA; Behind the recombinant plasmid process restriction analysis and sequential analysis that obtains, obtain the full-length cDNA of coding wheat cinnyl CoA reductase gene (CCR), and called after Ta-CCR1.

4.Ta-CCR1 full length gene cDNA sequential analysis: Ta-CCR1 full length gene cDNA sequence is analyzed, obtained its sequence, represent with SEQ ID NO1.This sequence is altogether by 1317 based compositions, wherein, the untranslated district of 5 ' end comprises 72 bases, and the untranslated district of 3 ' end comprises 195 bases (comprising the PolyA of 24 based compositions), and the coding region is by 1050 based compositions, wherein A accounts for 17.81% (187), C accounts for 34.48% (352), and G accounts for 33.62% (353), and T accounts for 14.10% (148), A+T accounts for 31.90% (335), and C+G accounts for 68.10% (715).

Dna homolog analysis revealed in international geneseq database (GenBank EMBL DDBJ), the Ta-CCR1 gene is the new gene of not reported in the wheat, and it has homology with isolated cinnyl CoA reductase gene from eucalyptus (Eucalyptus gunnii), willow (Populus trichocara), tobacco (Nicotiana tabacum), corn (Zea mays), fescue (Festuca arundinacea) and clover (Medicago sativa).

5.Ta-CCR1 the proteinic sequential analysis of full length gene cDNA sequence encoding:, obtain by its encoded protein matter sequence by above-mentioned Ta-CCR1 full length gene cDNA sequence.This protein sequence comprises 349 amino acid, shown in SEQ ID NO 2.In this protein sequence, hydrophobic amino acid accounts for 139, and hydrophilic amino acid accounts for 74, and basic aminoacids accounts for 36, and acidic amino acid accounts for 36, and this proteinic molecular weight is 37.4KD, and iso-electric point is 6.3.

6. at Ta-CCR1 gene coding region both sides synthetic primer, and introduce HindIII and NotI restriction enzyme site respectively, its primer sequence is respectively:

5 '-primer: 5 '-CACAAGCTTATGACCGTCGTCGCCGCCGC-3 ';

3 '-primer: 5 '-ATAAGAATGCGGCCGCTCACGCTGTTGCACCGTCCAG-3 '.

Go out the full length coding region of Ta-CCR1 gene through pcr amplification, under HindIII and NotI double digestion, be connected to (see figure 1) on the corresponding restriction enzyme site of expression vector pET-29b, cut integrity and the exactness that proves this expression vector with sequential analysis by enzyme.

7. with expression vector transformed into escherichia coli BL21 (DE3) pLysS bacterial strain, after process IPTG induces, e. coli protein through the SDS-PAGE gel electrophoresis analysis, is proved that the Ta-CCR1 gene has translated correct protein in intestinal bacteria.After above-mentioned coli strain induced through 37 ℃ of cultivations and IPTG, carry out cellular lysate, separating thallus protein, and this protein carried out cinnyl CoA-reductase enzyme activity assay, used substrate is forulic acid coenzyme A (feruloyl CoA), obtaining its enzymic activity is 1.67nmol/sec/mg, proves that this protein has the catalytic activity of cinnyl CoA-reductase.

8. extract total RNA respectively from the root of wheat, stem, leaf texture, transfer on the nylon membrane with capillary tube technique through behind the electrophoresis, with the hybridization of wheat CCR gene probe, radioautograph detects results of hybridization, serves as that contrast makes the hybridization signal stdn with the rRNA probe.Detect of the expression of Ta-CCR1 gene with this method at the wheat different sites.The result who obtains shows that this gene mainly expresses in wheat stalk.

9. the Ta-CCR1 gene in the stem (from the jointing stage to the milk stage) of the different development stage of commute lodging and wheat breed resistant to lodging carries out Northern hybridization, to detect the expression of this gene in different varieties, the result shows the expression amount of this gene in kind resistant to lodging apparently higher than easy lodging kind, and is more obvious in the growth later stage of stem difference.This result shows that the Ta-CCR1 gene not only controlling the synthetic of wheat xylogen, and closely related with the characteristic resistant to lodging of wheat stalk.

The invention provides with wheat stalk and grow relevant cinnyl CoA reductase gene and relative protein.Use this gene, can develop transgenic plant, synthetic with the xylogen in the control stem, thus the mechanical holding capacity of change stem and the ability of resisting extraneous poor environment.For example, indicated as a preferred embodiment of the present invention, utilize transgenic wheat to obtain high-yield variety resistant to lodging, increase the disease-resistant of wheat and the ability of resisting extraneous poor environment; Certainly, also can by the sexual hybridization method with this transgenosis in existing Wheat Production kind.

Below narrate embodiments of the invention.Should be noted that embodiments of the invention only to have illustration and effect without limits for the present invention.

The separation and the sequential analysis of embodiment one, wheat cinnyl CoA reductase gene probe

With wheat planting in the greenhouse, normally water and apply fertilizer, to growing into 2-3 internode, gather root, stem, leaf texture, with TRI reagent (Molecular Research Center, Inc, Cincinnati USA) extracts total RNA, with PolyAT tract mRNA Isolation test kit (Promega company, Madison USA) separates Poly (A) ⁺RNA is with content and the purity of ultraviolet spectrophotometer analysis RNA.

The condition of the synthetic cDNA article one chain of reverse transcription is: Poly (A) ⁺RNA 1ug, 5 μ mol/L primer 5 '-GACTCGAGTCGACATCGA (T) ₁₇-3 ', 0.5mmol/L dNTP, 50 RNasin of unit, 65 ℃ are incubated 10 minutes, after cooled on ice, add 200U SuperScript ^TMII RNaseH ^-ReverseTranscriptase (Gibco), 42 ℃ are incubated 1 hour, add EDTA to final concentration 10mM, and in 95 ℃ of insulations 10 minutes, in cooled on ice.

The condition of first round PCR is: 3 μ L cDNA reaction solutions, 1 μ mol/L primer, 0.4mmol/LdNTP, 2.5U Taq archaeal dna polymerase (Gibco company, Grand Island, NY, USA), in 95 ℃ of sex change 5 minutes, 50 ℃ of renaturation 2 minutes, 72 ℃ were extended 40 minutes, carried out 40 circulations then (95 ℃ 1 minute, 55 ℃ 1 minute, 72 ℃ 3 minutes), last 72 ℃ were extended 10 minutes.

Second takes turns PCR: use 1 μ L first round PCR reaction solution, in 95 ℃ of sex change 5 minutes, other reacted the same first round.The PCR product adopts GlassMAX through 1.0% agarose gel electrophoresis ^The DNAIsolation test kit (Gibco company, Grand Island, NY, USA).Be connected behind the purifying on the pGEM-TEasy carrier (Promega company, Madison, USA).Condition of contact be 16 ℃ 12 hours.The transformation receptor bacterium is E.coli DH5 α, and CaCl is adopted in the preparation of competent cell ₂Treatment process (Sambrook, J. etc. (eds), Molecular cloning.A laboratory manual, 2 ^NdEd., Cold Spring HarborLaboratory Press, Cold Spring Harbor, NY, 1989).To connect product and join in the competent cell, ice bath 30 minutes, 42 ℃ of heat shocks 90 seconds add the LB liquid nutrient medium, and 37 ℃ of insulations 45 minutes are coated on the LB flat board that contains penbritin (100ug/ml) and X-Gal (20ug/ml) 37 ℃ of overnight incubation.White clone is inoculated on the LB liquid nutrient medium and (contains penbritin 100ug/ml), and 37 ℃ of overnight incubation are extracted plasmid, carry out restriction analysis after, carry out sequential analysis with ABI 377 dna sequence analysis instrument, determine the CCR probe of wheat.

The result of sequential analysis shows, isolating probe has 693 Nucleotide, with the homology of isolated cinnyl CoA reductase gene in eucalyptus (Eucalyptusgunnii), willow (Populus trichocara), tobacco (Nicotiana tabacum), corn (Zea mays), fescue (Festuca arundinacea) and the clover (Medicago sativa) more than 60%, and its encoded protein matter has the catalytic active center NWYCY of cinnyl coenzyme A reductase.

The screening and the sequential analysis of embodiment two, wheat cinnyl CoA reductase gene

With wheat stalk Poly (A) ⁺RNA is a template, and (USA) synthetic cDNA through external packing, is built into the cDNA library of wheat stalk for Stratagene company, La Jolla.CA with ZAP vector.Get the 50ng dna probe, add 50uCi ³²P-dCTP carries out mark, and 37 ℃ are incubated 1 hour, add EDTA to final concentration 10mM termination reaction.Probe is by behind the Sephadex G-50 post, boils 10 minutes in 100 ℃, immediately in cooled on ice, hybridizes.

Get 50000pfu and pave plate, and it is transferred on the nitrocellulose filter, add 1.5M NaCl sex change 2 minutes in 0.5M NaOH, 1.5M NaCl adds 0.5M Tris-HCl (pH8.0) renaturation 5 minutes, 0.2MTris-HCl (pH7.5) added 2 * SSC rinsing 30 seconds, film is clipped in the two-layer Whatman filter paper, and 80 ℃ of vacuum bakeouts 2 hours add hybridization solution (6 * SSC afterwards, 5 * Denhardt, 0.5%SDS, 100 μ g/mL milt DNA, 50% methane amide), 42 ℃ are incubated 2 hours, add the probe of mark, 42 ℃ of hybridization are spent the night, and add under the 0.1%SDS room temperature through 6 * SSC and wash twice, each 30 minutes, use 0.1 * SSC to add 0.1%SDS again and wash twice in 65 ℃, each 20 minutes, afterwards in-80 ℃ of following radioautograph, after determining positive colony, plaque is taken off, and 4 ℃ of extractions are spent the night in SM solution, carry out the two-wheeled screening by same program again.

For the positive plaque that obtains, the method that provides by Stratagene company is translated into plasmid DNA, through behind the restriction analysis, adopts ABI 377 dna sequence analysis instrument to carry out sequential analysis.

The dna sequence dna that records is shown in SEQ ID NO1, with its called after Ta-CCR1.It is a wheat cinnyl CoA reductase gene, is the gene of not reporting as yet so far.

The translation of embodiment three, wheat cinnyl CoA reductase gene coded protein

The a pair of primer of synthetic:

5 '-primer: 5 '-CACAAGCTTATGACCGTCGTCGCCGCCGC-3 ';

3 '-primer: 5 '-ATAAGAATGCGGCCGCTCACGCTGTTGCACCGTCCAG-3 '.

Introducing HindIII and NotI restriction enzyme site respectively at the primer two ends, is template with the Ta-CCR1 gene, carries out pcr amplification, amplification condition: 10ng DNA, 1 μ mol/L primer, 0.4mmol/L dNTP, 2.5U Taq archaeal dna polymerase (Gibco company, Grand Island, NY, USA).In 95 ℃ of sex change 5 minutes, carry out then 30 circulations (95 ℃ 1 minute, 55 ℃ 1 minute, 72 ℃ 1.5 minutes), last 72 ℃ were extended 10 minutes.

The PCR product adopts GlassMAX through 1.0% agarose gel electrophoresis ^DNA Isolation test kit (Gibco company, Grand Island, NY, USA).Behind the purifying, behind HindIII and NotI double digestion, be connected on the carrier pET-29b, transformed into escherichia coli BL21 (DE3) pLysS competent cell, behind the transformant ice bath 30 minutes, 42 ℃ of heat shocks 50 seconds add the LB liquid nutrient medium, 37 ℃ are incubated 45 minutes, are coated on the LB flat board that contains kantlex (50ug/ml).With resistance bacterium colony 37 ℃ of concussion overnight incubation in LB liquid nutrient medium (containing kantlex 50ug/ml), after carrying out 1/100 dilution again, 37 ℃ of concussions were cultivated 2 hours, add IPTG to final concentration 1mM, continue to cultivate 3 hours, centrifugal 10 minutes of 12000g collects thalline, and thalline adds 100ul lysate (50mM Tris-HCl pH6.8, the 100mM dithiothreitol (DTT), 2%SDS, 0.1% tetrabromophenol sulfonphthalein, 10% glycerine), 100 ℃ were boiled 5 minutes, tropina carries out SDS-PAGE electrophoresis (gel strength 12%) according to a conventional method, and the result is presented at the expression that cinnyl CoA reductase protein matter is arranged in the bacterium, and its structure is shown in SEQ ID NO 2.

Embodiment four, the active mensuration of cinnyl CoA-reductase

Intestinal bacteria 50ml after IPTG induced centrifugal 10 minutes in 4000g, add 4ml cellular lysate liquid (20mMTris-HCl pH7.5,1mM PMSF, the 2mM dithiothreitol (DTT), the 100ug/ml N,O-Diacetylmuramidase), 30 ℃ are incubated 15 minutes, use ultrasonication 20 times, centrifugal 10 minutes of 4000g gets supernatant liquor and carries out enzyme activity determination.The enzyme activity determination reaction solution comprises 100mM sodium phosphate buffer (pH6.25), 0.1mMNADPH, and 70uM forulic acid coenzyme A, the 50ul protein solution, cumulative volume 500ul measures A down at 30 ℃ ₃₆₆Reduction calculate the activity of cinnyl CoA-reductase.Protein content is pressed method (Bradford, M.M, 1976, Anal.Biochem., 72:248-254) mensuration of Bio-Rad company.

The result shows that (feruloyl CoA) is substrate with the forulic acid coenzyme A, and its enzymic activity is 1.67nmol/sec/mg, show that the protein of Ta-CCR1 coded by said gene has the function of the synthetic phenylacrolein of catalysis, thereby the xylogen that can control in the wheat is synthetic.

The detection of Ta-CCR1 gene expression product in embodiment five, the wheat tissue

Adopt the Ta-CCR1 expression of gene product in the Northern blot hybridization detection wheat different tissues, isolating total RNA sample from different wheat tissues, in 1.4% agarose/denaturing formaldehyde gel electrophoresis, separate according to a conventional method, transfer to (12 hours transfer time) on the nylon membrane with 20 * SSC by capillary tube technique, nylon membrane is clipped in the two-layer Whatman filter paper, 80 ℃ of vacuum bakeouts 2 hours, film is placed in the hybridization bottle, add hybridization solution (6 * SSC afterwards, 5 * Denhardt, 0.5%SDS, 100 μ g/mL milt DNA, 50% methane amide) 10ml, 42 ℃ of prehybridizations 2 hours, add the cinnyl CoA reductase gene probe of mark, 42 ℃ of hybridization are spent the night, and add under the 0.1%SDS room temperature through 6 * SSC and wash twice, each 30 minutes, use 0.1 * SSC to add 0.1%SDS again and wash twice in 65 ℃, each 10 minutes, in-80 ℃ of following one weeks of radioautograph.Film after the development adds 0.1 * SSC and adds 0.1%SDS and wash twice in 95 ℃, and each 15 minutes, add the hybridization solution that contains 18S rRNA afterwards, 42 ℃ of hybridization are spent the night.Film is taken out, wash film by identical program, in-80 ℃ of following radioautograph 1 hour.

Results of hybridization is as shown in table 1.

Ta-CCR1 expression of gene amount in the table 1. wheat different tissues (is 100 with stem)

Tissue	Stem	Leaf	Root
Tissue	Stem	Leaf	Root	Relative expression quantity	100	23.9	10.0

The result shows that wheat cinnyl CoA reductase gene Ta-CCR1 mainly expresses in stem.

Relatively the expression of two different varieties Ta-CCR1 in the stem of different growing stage the results are shown in Table 2.

Table 2. wheat stalk different development stage TaCCR1 expression of gene amount

(jointing stage with C6001 is 100)

Kind	Growth period	The TaCCR1 level
Kind	Growth period	The TaCCR1 level	C6001	Milk stage heading stage jointing stage	100 15.5 44.0
H4565	Milk stage heading stage jointing stage	170 112 188	C6001	Milk stage heading stage jointing stage	100 15.5 44.0

Can be found out that by the result in kind resistant to lodging (H4564), the expression amount of Ta-CCR1 is apparently higher than easy lodging kind (C6001), difference is more obvious at later stages.The characteristic resistant to lodging that shows Ta-CCR1 gene and wheat is closely related.Therefore, control Ta-CCR1 expression of gene just can be controlled the proterties resistant to lodging of wheat.

Attached: nucleotide sequence that the present invention relates to and aminoacid sequence:

SEQ ID NO1 (the cDNA sequence of wheat cinnyl CoA reductase gene):

GTAGCTCGTACGTGGCATCTCCATCTCACCAACAATCTTGTTTAGACAAGCAGTGTA

AAAGTGACAGCAACAATGACCGTCGTCGCCGCCGCCGCCGCCGCCGCGGCGCAGGAG

CTGCCCGGGCACGGGCAGACCGTGTGCGTCACCGGCGCCGCCGGGTACATCGCGTCG

TGGCTCGTCAAGCTGCTCCTGGAGCGAGGCTACACCGTCAAGGGCACCGTGAGGAAC

CCAGATGATCCGAAGAACGCGCATCTGAAGGCGCTGGACGGCGCCGCCGAGAGGCTG

GTCCTCTGCAAGGCCGACCTCCTCGACTACGACGCCATCTGCGCCGCCGTCGAGGGC

TGCCACGGCGTGTTCCACACCGCCTCCCCCGTCACCGACGACCCCGAGCAGATGGTG

GAGCCGGCGGTGAGGGGCACGGAGTACGTGATCAACGCGGCGGCGGACGCCGGCACC

GTGCGCCGGGTGGGTGTTACGTCGTCCATCGGCGCCGTCACCATGGACCCCAACCGT

GGTCCCGACGTGGTCGTCGACGAGTCCTGCTGGAGCGACCTTGAATTCTGCAAGAAA

ACCAAGAACTGGTACTGCTACGGCAAGGCGGTGGCGGAGCAGGCGGCGTGGGAGAAG

GCCGCGGCGCGCGGCGTCGACCTCGTCGTGGTGAACCCGGTGCTGGTGGTCGGGCCG

CTGCTGCAGCCGACGGTGAACGCCAGCGCCGCGCACATCCTCAAGTACCTCGACGGC

TCCGCCAAGAAGTACGCCAACGCGGTGCAGGCGTACGTGAACGTGCGCGACGTCGCC

GCCGCGCACGTCCGGGTCTTCGAGGCGCCCGGGGCCTCCGGCCGGCACCTCTGCGCC

GAGCGCGTCCTGCACCGCGAGGACGTCGTCCACATCCTCGGCAAGCTCTTTCCCGAG

TACCCCGTCCCAACAAGGTGCTCTGACGAGGTGAACCCACGGAAGCAGCCTTACAAG

ATGTCCAACCAGAAGCTGCAGGATCTTGGCCTCCAGTTCACTCCTGTCAACGACTCT

CTGTACGAGACGGTGAAGAGCCTCCAGGAGAAGGGGCACCTCCCGGCGCCGAGGAAA

GATATCCTCCCAGCGGAACTGGACGGTGCAACAGCGTGATGGTGCTGAAGAAACAGC

GCGGTTCACGTTTTTCTGTAACGCGGTGGGACATCGTATGTGGTCTGTTTGTGTATA

CATTCTATCTAATATCGTGTTATTTAAGTGGACTAAGCAAATATGGTAATGTATCGG

CTTCGATGATCGACACTTAAAAGTGAGCTTTCGCAAACTAAAAAAAAAAAAAAAAAA

AAAAAA

SEQ ID NO2 (wheat cinnyl CoA reductase gene encoded protein matter sequence):

MTVVAAAAAAAAQELPGHGQTVCVTGAAGYIASWLVKLLLERGYTVKGTVRNPDDPK

NAHLKALDGAAERLVLCKADLLDYDAICAAVEGCHGVFHTASPVTDDPEQMVEPAVR

GTEYVINAAADAGTVRRVGVTSSIGAVTMDPNRGPDVVVDESCWSDLEFCKKTKNWY

CYGKAVAEQAAWEKAAARGVDLVVVNPVLVVGPLLQPTVNASAAHILKYLDGSAKKY

ANAVQAYVNVRDVAAAHVRVFEAPGASGRHLCAERVLHREDVVHILGKLFPEYPVPT

RCSDEVNPRKQPYKMSNQKLQDLGLQFTPVNDSLYETVKSLQEKGHLPAPRKDILPA

ELDGATA.

Accompanying drawing 1 is the structure iron that contains the colibacillus expression plasmid of cinnyl CoA reductase gene.

Claims

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.