CN109609541A - A method of improvement crops character - Google Patents

Abstract

The invention discloses a kind of method for improveing crops character, the method is the importing cytochrome p450 protein encoding gene into crops, realizes that crops plant height reduces, disease resistance improves or lodging resistance improves；The cytochrome p450 protein includes the derived peptides that amino acid sequence shown in the polypeptide or SEQ ID NO:1 of amino acid sequence shown in SEQ ID NO:1 is formed by 1-24 replacing, missing or adding for amino acid residue.The plant height (reducing 3%-30%) of plant can be reduced by the technical program or improve the disease resistance (scab, which reduces, compared with non-transgenic control perhaps reduces 5%-80%) of plant or improve plant lodging resistance (lodging rate reduces 10%-80% compared with non-transgenic control).

Description

A method of improvement crops character

(1) technical field

The present invention relates to a kind of reduction crops plant height, improve disease resistance, or the method for improving lodging resistance.

(2) background technique

Plant height refers to that plant rootstock arrives the distance between top, wherein top refers at the top of stem.Plant height determines plant Form, biomass, lodging resistance and yield etc., be very crucial plant trait.The 1960s to 90 years " green revolution " in generation has benefited from the application of semi-dwarf mutant character.The half of plant height downgrades so that the harvest index of plant significantly improves, The ability that plant resists wind and rain is significantly enhanced, and then realizes huge raising (the Sasaki A, Ashikari of crop yield M,Ueguchitanaka M,et al.Green revolution:A mutant gibberellin-synthesis gene in rice[J].Nature,2002,416(6882):701-702.)。

Wherein, the Semi dwarfism gene SD1 of rice is gibberellin GA20 oxidase gene GA20ox-2.When GA20ox-2 gene After mutation, then gibberellin GA4 and GA1 (the M Ashikari etal.Loss-of- for normally having bioactivity cannot be synthesized function of a rice gibberellin biosynthetic gene,GA20 oxidase(GA20ox-2),led to the rice'green revolution',Breeding Science,2002,52(2)143-150).The half of wheat is short Stalk is as caused by the function gain mutation of Rht (" reduced height ") gene.Rht gene is a gibberellin sense Transcription factor in induction signal access.Cause plant pair gibberellin insensitive after Rht gene mutation, plant become it is short (Peng J, Richards D E,Hartley N M,et al.|[lsquo]|Green revolution|[rsquo]|genes encode mutant gibberellin response modulators[J].Nature,1999,400(6741):256.)。

The application of " green revolution " on rice and wheat achieves unprecedented success, but plant height including plant shape at present The adjustment and optimization of aspect are still to improve an important directions of crop yield.For example, the corn product of China's authorization in 2004 Kind " first jade 335 " is the first big kind in eastern North China Spring Maize Area, is in Huang-Huai-Hai summer corn area from more than ten year so far is released The second largest kind.This corn variety have many advantages such as high yield, dehydration it is fast, but have the shortcomings that one it is bigger be not It is resistant to lodging.If the method by improvement of genes suitably reduces its plant height, its lodging resistance can be improved, improve the product The adaptability and yield stability of kind.

The disease of crops is the major reason that another causes crop yield to lose.Improving disease resistance has weight The value wanted.

Cytochrome P450s (Cytochrome P450s, CYP450s) are ancient more member's supergene families. CYP450s is a kind of single oxygenation catalytic protein enzyme, with reduction-state CO ining conjunction with after, the light absorption value highest at 450nm, therefore be named as carefully Born of the same parents' cytochrome p 450 s.CYP450s is the most huge protease superfamily for participating in biological metabolism, including more than 1 000 families and 2 500 subfamilies, are widely present in living nature.In animal, plant, fungi, protist, bacterium, Archimycetes and virus CYP450s (Murray R I, Fisher M T, Debrunner P G, et al.Structure and is had been found that Chemistry of Cytochrome P-450[M]//Metalloproteins.Palgrave Macmillan UK,1985: 2253-2277；Nelson D,Werckreichhart D.A P450-centric view of plant evolution. [J].Plant Journal,2011,66(1):194-211；Werckreichhart D,Feyereisen R.Cytochromes P450:a success story[J].Genome Biology,2000,1(6):1-9；Chinese patent: CN 101932596 B)。

CYP450s is a kind of monooxygenase, introduces oxygen atom by the specific position in substrate and completes substrate sites The modification of oxygenation atom, member abundant produce the function of multiplicity.On the one hand, homology is very low between CYP450s family member, The CYP450s homology of different biologies can be down to 16%.The CYP450s high for homology, the change meeting of single amino acids Lead to the change of its function, CYP450s generally occurs within sub- functionalization and new function during evolution.On the other hand, CYP450s protein steric structural is similar, and the conservative of such structure is of great significance for its catalysis.CYP450s gene The diversity of sequence and structure determines the diversity of its function.

CYP450s gene depth participates in the basic metabolism of plant, it is not only involved in the metabolic processes of endogenous substance, such as The biosynthesis and degradation, the synthesis of secondary metabolite of fatty acid metabolism, plant hormone, also participate in the degradation of allogenic material Journey, such as herbicide degradation (the plant cytochrome P450 s such as Li Xiangyu, Wang Zhugan, Sun Chunyu and its in plant metabolism Effect, Agriculture of Anhui science, 2016,44 (13): 129-134.).

Bermuda grass crawls to grow on the ground, we have cloned a CYP450 gene 3X from Bermuda grass, before, I Find that 3X gene has the function of being resistant to flazasulfuron and other several herbicides (United States Patent (USP): US09657307B2), this Invention has found that it regulates and controls plant plant height, disease-resistant or resistant to lodging function for the first time.

(3) summary of the invention

It is an object of the present invention to provide the genes that one kind can improve crops economical character, including reduce plant plant height, mention High disease resistance of plant and raising plant lodging resistance, have very important significance to plant improvement and crop yield.

The technical solution adopted by the present invention is that:

The present invention provides a kind of method for improveing crops character, and the method is that cytochromes are imported into crops P450 protein coding gene realizes that crops plant height reduces, disease resistance improves or lodging resistance improves；The Cytochrome P450 Albumen includes amino acid sequence shown in the polypeptide or SEQ ID NO:1 of amino acid sequence shown in SEQ ID NO:1 by 1-24 Replacing, missing or adding for amino acid residue and the derived peptides formed.

Further, it is described reduce crops plant height polypeptid acid sequence be SEQ ID NO:2 shown in, encoding gene Nucleotides sequence is classified as shown in SEQ ID NO:3.

Further, the derived peptides amino acid sequence for reducing crops plant height is shown in SEQ ID NO:6.

Further, the method for the improvement crops character includes:

(1) expression vector of the encoding gene containing cytochrome p450 protein is constructed, and imports Agrobacterium, obtains color containing cell The Agrobacterium of plain P450 protein coding gene；

(2) crop plant cells, tissue or organ are contacted with the Agrobacterium in step (1), so that containing cytochromes P450 protein gene enters crop plant cells, and is integrated on the chromosome of crop plant cells；

(3) crop plant cells, tissue or the organ for being transferred to the gene containing cytochrome p450 protein are filtered out；

(4) by cell, tissue or the neomorph in step (3) at crop；

(5) selection plant height reduces, disease resistance improves or the transgenosis transformant of lodging resistance raising.

Further, the crops include corn and soybean, cotton, wheat or rice.

Cytochrome p450 protein encoding gene of the present invention is the CYP450 gene 3X of Bermuda grass, mediates CYP450 base Because the promoter of 3X expression can be natural promoter, it is also possible to the starting of the element artificial combination using different promoters Son, including constitutive promoter and tissue-specific promoter, tissue-specific promoter are special such as in plant haulm and blade The promoter of opposite sex expression.Natural promoter is generally the nucleotide sequence of 1kb to 2kb before gene start codon, individually Promoter can be especially short or especially long.The CYP450 gene 3X of Bermuda grass is mediated to plant preferably by corn Ubi promoter It is expressed in object, reduces the plant height of plant, perhaps improve the disease resistance of plant or improve the lodging resistance of plant.

The present invention provides it is a kind of using CYP450 gene 3X suitably reduces corn plant height or raising corn disease resistance, Or the method for improving corn lodging resistance, it mainly comprises the steps that

(1) prepare the Agrobacterium of carrying CYP450 gene 3X expression vector；

(2) maize immature embryos are contacted with the Agrobacterium in step (1), so as to enter maize immature embryos thin by the 3X Born of the same parents, and be integrated on the chromosome of maize cell；

(3) maize immature embryos for being transferred to 3X gene are filtered out；

(4) maize immature embryos in step (3) are regenerated into plant.

The present invention provides a kind of methods for suitably reducing Plant Height of Rice using CYP450 gene 3X, mainly include following step It is rapid:

(1) prepare the Agrobacterium of carrying CYP450 gene 3X expression vector；

(2) Rice Callus is contacted with the Agrobacterium in step (1), so that the 3X enters rice callus Histocyte, and be integrated on the chromosome of rice cell；

(3) Rice Callus for being transferred to 3X gene is filtered out；

(4) Rice Callus in step (3) is regenerated into rice plant.

The present invention provides a kind of methods for suitably reducing soybean plant height using CYP450 gene 3X, mainly include following step It is rapid:

(1) prepare the Agrobacterium of carrying CYP450 gene 3X expression vector；

(2) soybean cotyledon is contacted with the Agrobacterium in step (1), so as to enter soybean cotyledon thin by the 3X Born of the same parents, and be integrated on the chromosome of soya cells；

(3) soybean cotyledon for being transferred to 3X gene is filtered out；

(4) soybean cotyledon in step (3) is regenerated into soybean plant strain.

Other aspects of the present invention are apparent to those skilled in the art due to the disclosure 's.

Compared with prior art, beneficial effect of the present invention is mainly reflected in: the invention firstly discloses in Bermuda grass CYP450 gene 3X has the function of reducing plant plant height, improve disease resistance, or improve lodging resistance etc., and for the first time Utilize the plant height of the crops such as 3X gene regulation corn, rice and soybean.The present invention is regulation plant key character-reduction plant Plant height, perhaps improves disease resistance or raising lodging resistance provides reliable gene and method, to plant improvement and crop Volume increase has very important significance.The plant height (reducing 3%-30%) of plant can be reduced by the technical program or is mentioned The disease resistance (scab, which reduces, compared with non-transgenic control perhaps reduces 5%-80%) of high plant or raising plant are resistant to lodging Performance (lodging rate reduces 10%-80% compared with non-transgenic control).

(4) Detailed description of the invention

The structural domain schematic diagram of Fig. 1,3x polypeptide sequence.

Fig. 2,3x expression vector T-DNA structural schematic diagram；It include two expression cassettes in the T-DNA, one of them is Expression cassette during Genetic Transformation in Higher Plants for the marker gene of screening, can be the gene for being resistant to certain chemical substance, Such as glyphosate-tolerant gene or the gene of resistance to glufosinate-ammonium；Another expression cassette is the expression cassette for regulating and controlling the 3X gene of plant plant height.

Fig. 3,3x gene regulation corn plants high effect schematic diagram；CK is non-transgenic corn control；3X is indicated and CK background The transgenic corn plant of the consistent transgenosis for turning 3X gene.

(5) specific embodiment

The present invention is described further combined with specific embodiments below, but protection scope of the present invention is not limited in This:

1 vector construction of embodiment

In order to construct 3X gene binary expression vector, the limited public affairs of work bioengineering (Shanghai) share (are given birth to by our authorized companies Department) the artificial synthesized Ubi promoter of corn, nucleotide sequence is as shown in SEQ ID NO:4, the end of promoter 5 ' and 3 ' end difference It is provided with HindIII and BamHI restriction endonuclease sites；Artificial synthesized 3X gene and its terminator 3X-ter, nucleotide For sequence as shown in SEQ ID NO:5, the end of 3X-ter segment 5 ' and 3 ' ends are respectively arranged with BamHI and KpnI restriction enzyme position Point.

Then it is constructed based on the binary vector conversion carrier pCambia1300-35S-G10 that this laboratory saves and is carried eventually Body (Chinese patent application: 201280061792.2).Marker gene in carrier pCambia1300-35S-G10 is an expression The gene G10 of EPSPS assigns plant glyphosate tolerant.With HindIII and KpnI to pCambia1300-35S-G10 carrier Carry out double digestion；Double digestion is carried out to Ubi promoter with HindIII and BamHI；3X-ter is carried out with BamHI and KpnI double Then digestion carries out three sections of connections to above three segment, obtains the whole carrier pCambia1300-35S- for being used for Plant Transformation G10-pUbi-3X (Fig. 2), this carrier (T-DNA plasmid) is named as 3X by we.

Finally, this T-DNA plasmid is transferred in Agrobacterium LB4404 by the method that electricity turns, by containing 15 μ g/ml The YEP solid medium of the kanamycins of tetracycline and 50 μ g/mL filters out positive colony, and protects bacterium, is used for next plant Object conversion.

2 corn transformation of embodiment

Comparative maturity, such as Frame etc. describe the method using Agrobacterium-mediated Transformation corn to the method for transformation of corn (Frame et al.,(2002)Plant Physiol,129:13-22).Take Agrobacterium (the i.e. carrier containing T-DNA of the 3X containing carrier Agrobacterium) draw plate, choose single colonie inoculation, prepare conversion use Agrobacterium.Take 8-10 days after pollinating Hi-II corncob.It collects All immature embryos (size 1.0-1.5mm).Agrobacterium containing T-DNA carrier and immature embryo are co-cultured 2-3 days (22℃).It shifts on immature embryo to calli induction media (containing the Timentin of 200mg/L in culture medium, for killing Agrobacterium, referring to (Frame et al., (2002) Plant Physiol, 129:13-22)), 28 DEG C dark culture 10-14 days.It will All callus go on the screening and culturing medium with final concentration 2mM glyphosate (Frame et al., (2002) Plant Physiol, 129:13-22), 28 DEG C dark culture 2-3 weeks.

Shift on all tissues to the screening and culturing medium of the fresh glyphosate of 2mM containing final concentration, 28 DEG C dark culture 2-3 weeks. Then, (Frame et al., (2002) Plant are shifted in the embryonal connective tissue to regeneration culture medium survived after all screenings Physiol, 129:13-22), 28 DEG C dark culture 10-14 days, one strain of every ware.Embryonal connective tissue is shifted to train to fresh regeneration Support base on, 26 DEG C illumination cultivation 10-14 days.Shift on all full-grown plants to root media (Frame et al., (2002) Plant Physiol, 129:13-22), 26 DEG C of illumination cultivations are complete until root development, are then transplanted in greenhouse single Strain culture, detects the antiweed ability of transgenic corns.With the 41% gyphosate solution spray of 300 times of Monsanto of dilution It spills, rear blade jaundice in 7 days is withered for feminine gender；It is positive plant as spray clear water control growing way.

The screening of embodiment 3 transgenic corns target plant and objective trait

1, the screening for the corn strain that plant height reduces

By the method in embodiment 2,76 3X carrier conversion strains are had successfully been obtained in we.We are to these transgenosis The plant height of material is analyzed, it is found that the plant height of these strains compared with non-transgenic control plant, has different degrees of It reduces (table 1, Fig. 3).

Table 1, compared with the control transgenic corns plant height reduce situation:

Reduced ratio	< 5%	5%-10%	10%-15%	15%-20%	> 20%
						3X transformant quantity	11	23	20	15	7

Remarks: the plant height of non-transgenic control PH6WC is 215 ± 17cm.

We finally have chosen 3 compared with adjoining tree, and it is respectively 6%, 7%, 8% that plant height, which reduces, and fringe does not have again The transgenic strain of significant difference carries out the research of next step, plans with next production application.

2, the screening for the corn strain that disease resistance improves

In order to filter out the corn strain of disease resistance raising, we respectively turn the 76 3X carriers obtained in embodiment 2 Change strain progress corn southern leaf blight germ and connect bacterium experiment, filters out anti-to the germ compared with Non-transgenic control lines PH6WC The Transgenic corn lines that property significantly improves.

3, the screening for the corn strain that lodging resistance improves

In order to filter out the corn strain of lodging resistance raising, we are respectively to the 76 3X carriers obtained in embodiment 2 It converts strain and carries out wind resistance test, corn is placed in test laboratory resistant to lodging, what is manually manufactured is respectively equivalent to 5, the wind-force of 6,7,8 grades of strong wind tests the wind resistance of each corn strain, filters out than Non-transgenic control lines PH6WC wind resistance The stronger Transgenic corn lines of performance.

4 rice conversion of embodiment

The preparation method of transgenic paddy rice is using the prior art (Lu Xiongbin, Gong ancestral an ancient egg-shaped, holed wind instrument (1998) life science 10:125- 131；Liu Fan etc. (2003) Molecular Plant Breeding 1:108-115).Choose " elegant water -134 " seed decladding of mature and plump, induction Callus is generated as converting material.3X Agrobacterium is taken to draw plate.Single colonie inoculation is chosen, prepares conversion and uses Agrobacterium.It will be wait turn The callus of change is put into the 3X Agrobacterium bacterium solution that OD is 0.6 or so the (preparation of Agrobacterium bacterium solution: by Agrobacterium inoculation to training Base is supported, 28 DEG C of cultures to OD are 0.6 or so；Culture medium composition: 3g/L K₂HPO₄、1g/LNaH₂PO₄、1g/LNH₄Cl、0.3g/L MgSO₄·7H₂O、0.15g/L KCl、0.01g/L CaCl₂、0.0025g/L FeSO₄·7H₂O, 5g/L sucrose, 20mg/L acetyl Syringone, solvent are water, pH=5.8), it allows Agrobacterium to be integrated to callus surface, callus is then transferred to total training Support culture medium (MS+2mg/L 2,4-D+30g/L glucose+30g/L sucrose+3g/L agar (sigma 7921)+20mg/L acetyl Syringone) in, 28 DEG C co-culture 2-3 days.Callus after being converted with aseptic water washing is transferred to screening and culturing medium (MS+2mg/L 2,4-D+30g/L sucrose+3g/L agar (sigma 7921)+20mg/L acetosyringone+2mM glyphosates (Sigma)) on, sieve Choosing two months (intermediate subculture is primary) of culture.After screening, the good callus of growth vigor is transferred to pre- differential medium (MS+ 0.1g/L inositol+5mg/L ABA+1mg/L NAA+5mg/L6-BA+20g/L sorbierite+30g/L sucrose+2.5g/L Gelrite it cultivates 20 days or so, then moves on to the callus broken up in advance on differential medium, the daily 14 small time on) It germinates according to differentiation.After 2-3 weeks, resistance regeneration plant is transferred to root media (1/2MS+0.2mg/L NAA+20g/L sucrose + 2.5g/L gelrite) on strengthening seedling and rooting, regeneration plant is finally washed away into agar transplanting in greenhouse, selects that yield is high, seed is big Or biomass height etc. can be improved the transgenic line of rice yield, cultivate new varieties.It obtains respectively and contains above-mentioned conversion carrier With the transgenic rice plant for the empty carrier for containing only riddled basins EPSPS.

The screening of embodiment 5 transgenic paddy rice target plant and objective trait

By the method in embodiment 4,123 3X carrier conversion strains are had successfully been obtained in we.We turn base to these Because the plant height of material is analyzed, the plant height of these strains is found compared with non-transgenic control plant, is had in various degree Reduction (table 2).

Transgenic paddy rice plant height reduces situation to table 2 compared with the control

Reduced ratio	< 5%	5%-10%	10%-15%	15%-20%	> 20%
						3X transformant quantity	19	29	30	23	22

Remarks: the plant height of non-transgenic control 9311 is 115 ± 10cm.

We finally have chosen 5 compared with adjoining tree, and it is respectively 4%, 5%, 6%, 7%, 8% that plant height, which reduces, and The transgenic strain that grain weight, grain number per spike and tiller are all not significantly different carries out the research of next step, plans with next life Produce application.

6 transformation of soybean of embodiment

The step of acquisition genetically engineered soybean used herein from existing technology (Deng et al., 1998, PlantPhysiology Communications34:381-387；Ma et al.,2008,Scientia Agricultura Sinica41:661-668；Zhou et al.,2001,Journal of Northeast AgriculturalUniversity32:313-319).Healthy, full, mature soybean is chosen, is divided with 80% ethanol disinfection 2 Clock, then with sterile water wash, it is then placed within the drier full of chlorine (reacting generation with the dense HCl of 2ml by 50ml NaClO) Middle 4-6 hour of sterilizing.Soybean after sterilizing is sowed in superclean bench into B5 medium, cultivates 5 under the conditions of 25 DEG C It, with optical densities in 90-150 μm of ol photon/m²S is horizontal.When cotyledon greening and top is broken in the seed coat, and sterile bean sprouts is with regard to president Out.The bean sprouts for eliminating hypocotyl is cut into fifty and fifty percent in length, so that two panels explant all has cotyledon and epicotyl.? It is cut at the node of cotyledon and epicotyl at explant about 7-8, that is, can be used as the destination organization infected.List containing carrier 3X It is stand-by by separated culture to clone Agrobacterium.The OD value that ready explant is immersed in the preparation of 4 method of embodiment is 0.6 or so It is co-cultured 30 minutes or so in 3X agrobacterium suspension.Then, the cell suspending liquid blotting paper that the tissue infected is extra It absorbs cleanly, is then transferred to 25 DEG C dark culture 3-5 days in 1/10B5 co-culture medium.The plant tissue of co-cultivation B5 liquid Culture medium cleaning, to remove extra Agrobacterium, is then placed into B5 solid medium at 25 DEG C and cultivates 5 days, to its germination. Induction occur embryonic tissue be transferred in the B5 screening and culturing medium containing 0.1-0.5mM glyphosate, 25 DEG C illumination cultivation 4 weeks, Period replaces a subculture every two weeks.The embryonic tissue screened is then transferred in solid medium, 25 DEG C of cultures, to it Grow up to seedling.Then, transgenic plant seedling is transferred in 1/2B5 culture medium and carries out rooting induction.Finally, the plantlet grown up to It is planted in the greenhouse after cleaned removal agar.

The screening of embodiment 7 genetically engineered soybean target plant and objective trait

By the method in embodiment 6,92 3X carrier conversion strains are had successfully been obtained in we.We are to these transgenosis The plant height of material is analyzed, it is found that the plant height of these strains compared with non-transgenic control plant, has different degrees of It reduces (table 3).

Genetically engineered soybean plant height reduces situation to table 3 compared with the control:

Reduced ratio	< 5%	5%-10%	10%-15%	15%-20%	> 20%
						3X transformant quantity	13	28	25	14	12

Remarks: grand No. 1 plant height in non-transgenic control day is 60 ± 6cm.

We finally have chosen 4 compared with adjoining tree, and it is respectively 4%, 5%, 6%, 7% that plant height, which reduces, and grain weight, The transgenic strain that knot folder number and every folder grain number are all not significantly different carries out the research of next step, plans with next production Using.

Sequence table

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gtgtgggacg ggccgaccga gttcgtgccg gagcggttcg aggatggcaa ggcagaaggc 1320

cggctgctga tgccgttcgg gatgggacgg cgcaagtgtc ccggcgagac gctcgcgctg 1380

cggacgatcg ggctggtgct cggcacgctg atccagtgtt tcgactggga ccgggttgat 1440

ggtcttgagg tcgacatgac tgaaagtggt gggctcacga tccccagggc tgtcccgttg 1500

gaggccatgt gcaggcctcg tgcgacgatg cgtgaggttt tgcaggagct ctga 1554

<210> 4

<211> 2023

<212> DNA

<213>unknown (Unknown)

<400> 4

aagcttgcat gcctacagtg cagcgtgacc cggtcgtgcc cctctctaga gataatgagc 60

attgcatgtc taagttataa aaaattacca catatttttt ttgtcacact tgtttgaagt 120

gcagtttatc tatctttata catatattta aactttactc tacgaataat ataatctata 180

gtactacaat aatatcagtg ttttagagaa tcatataaat gaacagttag acatggtcta 240

aaggacaatt gagtattttg acaacaggac tctacagttt tatcttttta gtgtgcatgt 300

gttctccttt ttttttgcaa atagcttcac ctatataata cttcatccat tttattagta 360

catccattta gggtttaggg ttaatggttt ttatagacta atttttttag tacatctatt 420

ttattctatt ttagcctcta aattaagaaa actaaaactc tattttagtt tttttattta 480

ataatttaga tataaaatag aataaaataa agtgactaaa aattaaacaa atacccttta 540

agaaattaaa aaaactaagg aaacattttt cttgtttcga gtagataatg ccagcctgtt 600

aaacgccgtc gacgagtcta acggacacca accagcgaac cagcagcgtc gcgtcgggcc 660

aagcgaagca gacggcacgg catctctgtc gctgcctctg gacccctctc gagagttccg 720

ctccaccgtt ggacttgctc cgctgtcggc atccagaaat tgcgtggcgg agcggcagac 780

gtgagccggc acggcaggcg gcctcctcct cctctcacgg cacggcagct acgggggatt 840

cctttcccac cgctccttcg ctttcccttc ctcgcccgcc gtaataaata gacaccccct 900

ccacaccctc tttccccaac ctcgtgttgt tcggagcgca cacacacaca accagatctc 960

ccccaaatcc acccgtcggc acctccgctt caaggtacgc cgctcgtcct cccccccccc 1020

ccctctctac cttctctaga tcggcgttcc ggtccatggt tagggcccgg tagttctact 1080

tctgttcatg tttgtgttag atccgtgttt gtgttagatc cgtgctgcta gcgttcgtac 1140

acggatgcga cctgtacgtc agacacgttc tgattgctaa cttgccagtg tttctctttg 1200

gggaatcctg ggatggctct agccgttccg cagacgggat cgatttcatg attttttttg 1260

tttcgttgca tagggtttgg tttgcccttt tcctttattt caatatatgc cgtgcacttg 1320

tttgtcgggt catcttttca tgcttttttt tgtcttggtt gtgatgatgt ggtctggttg 1380

ggcggtcgtt ctagatcgga gtagaattct gtttcaaact acctggtgga tttattaatt 1440

ttggatctgt atgtgtgtgc catacatatt catagttacg aattgaagat gatggatgga 1500

aatatcgatc taggataggt atacatgttg atgcgggttt tactgatgca tatacagaga 1560

tgctttttgt tcgcttggtt gtgatgatgt ggtgtggttg ggcggtcgtt cattcgttct 1620

agatcggagt agaatactgt ttcaaactac ctggtgtatt tattaatttt ggaactgtat 1680

gtgtgtgtca tacatcttca tagttacgag tttaagatgg atggaaatat cgatctagga 1740

taggtataca tgttgatgtg ggttttactg atgcatatac atgatggcat atgcagcatc 1800

tattcatatg ctctaacctt gagtacctat ctattataat aaacaagtat gttttataat 1860

tattttgatc ttgatatact tggatgatgg catatgcagc agctatatgt ggattttttt 1920

agccctgcct tcatacgcta tttatttgct tggtactgtt tcttttgtcg atgctcaccc 1980

tgttgtttgg tgttacttct gcaggtcgac tctagaggat cca 2023

<210> 5

<211> 1737

<212> DNA

<213>unknown (Unknown)

<400> 5

ggatccaaca atggataagg cctacgtggc cctcctctcc ttcgcctccc tcttcttgct 60

ccactacctc gtttcccgcc gcaatggcac cgggaagggc agcaaggcca agggcgcgct 120

gccgccaagc cctccatccg ttccgttcct gggccacctc caccttgtca agacgccatt 180

ccacgctgcg ctggcacgcc tcgcggactg ccacggcccg gtcttctccc tgcggatggg 240

agcccgcccc gcagttgtgg tgtcctcgcc ggagcacgcc aaggagtgct tcacggagca 300

cgacgtggcc ttcgccaacc ggccgcgctt tccctcgcag cagctcgcct ccttcaacgg 360

tgccgcgctg ggttccgcca gctacggccc gtactggcgc aacctccgcc gcgtcgccac 420

cgtccacctc ctgtccgcgc accgcgtcgc gtgcatgacg gggactatcg cggccgaggt 480

gcgggccatg gtgcgacgga tgaaccgcgc cgcgcaggtg gcatcaggcg gcgcggcgcg 540

catcgagctc aagcggaggc tatttgaggt ctcgctcagc gtgcttatgg agaccatcgc 600

gcggaccaag acgtcacgta cggaggcgga cgacgacacg gacatgtcgc ctgaggcccg 660

ggagttcaag cagatcgtgg atgagctcct gcctcacctc ggcacggcta acttgtggga 720

ctacatgccg gtgttgcggt ggttcgacgt gttcggcgtg aggaagaaga tcgtgtccgc 780

ggtgaggaga agggacgcgt tcctgcggca tcttgtcgac gcagagagga cgaggctgga 840

cgacggcaac gatgcgggcg agaagaagag catcattgct atgctgctca ctctgcagaa 900

gtcagagccg gacgtctact cggataccat gatcatggct ctatgtggga acttgtttgg 960

ggccggcaca gagaccacgt cgacgaccac cgaatgggcc atgtctctcc tcctcaacca 1020

cccggagaag ctcaggaagg cgcaggctga gatcgatgct gtcgtgggca catcccgcct 1080

tcttaccgcc gacgacatgc ctcgtctcac ctacctccgc tgcatcatcg acgagaccat 1140

gcgcctgtac ccggccgcac cacttctgct gccacacgag tcctcgacac actgcaaggt 1200

cggcggctac gacgtgcccg ccggcacgat gctgctcgtc aacgtgtacg ccatccacag 1260

ggaccccgcg gtgtgggacg ggccgaccga gttcgtgccg gagcggttcg aggatggcaa 1320

ggcagaaggc cggctgctga tgccgttcgg gatgggacgg cgcaagtgtc ccggcgagac 1380

gctcgcgctg cggacgatcg ggctggtgct cggcacgctg atccagtgtt tcgactggga 1440

ccgggttgat ggtcttgagg tcgacatgac tgaaagtggt gggctcacga tccccagggc 1500

tgtcccgttg gaggccatgt gcaggcctcg tgcgacgatg cgtgaggttt tgcaggagct 1560

ctgactcgag tttctccata ataatgtgtg agtagttccc agataaggga attagggttc 1620

ctatagggtt tcgctcatgt gttgagcata taagaaaccc ttagtatgta tttgtatttg 1680

taaaatactt ctatcaataa aatttctaat tcctaaaacc aaaatccagt gggtacc 1737

<210> 6

<211> 515

<212> PRT

<213>unknown (Unknown)

<400> 6

Met Asp Lys Ala Tyr Val Ala Ile Leu Ser Phe Ala Phe Leu Phe Val

1 5 10 15

Leu His Tyr Leu Ile Gly Arg Gly Asn Gly Thr Trp Lys Ala Gly Lys

20 25 30

Gly Arg Gln Leu Pro Pro Ser Pro Pro Ala Leu Pro Leu Ile Gly His

35 40 45

Leu His Leu Val Lys Thr Pro Phe His Ala Ala Leu Ala Arg Leu Ala

50 55 60

Ala Cys His Gly Pro Val Phe Ser Met Arg Met Gly Ser Arg Pro Ala

65 70 75 80

Val Val Val Ser Ser Pro Asp Cys Ala Arg Glu Cys Phe Thr Glu His

85 90 95

Asp Val Ala Phe Ala Asn Arg Pro Leu Phe Pro Thr Leu Gln Leu Val

100 105 110

Ser Phe Asn Gly Ala Ala Leu Ser Thr Ala Ser Tyr Gly Pro Tyr Trp

115 120 125

Arg Asp Leu Arg Arg Val Ala Ser Val His Leu Leu Ser Ala His Arg

130 135 140

Val Asn Cys Met Ala Gly Thr Ile Ser Ala Glu Val Arg Ala Met Val

145 150 155 160

Arg Arg Met Ser Arg Ala Ala Ala Ala Ala Pro Ser Gly Ala Ala Arg

165 170 175

Val Glu Leu Lys Arg Arg Leu Phe Glu Leu Ser Leu Ser Val Leu Met

180 185 190

Glu Thr Ile Ala Gln Thr Lys Met Ser Arg Ala Glu Ala Asp Ala Asp

195 200 205

Thr Asp Met Ser Pro Glu Ala Gln Glu Phe Lys Gln Met Val Asp Ala

210 215 220

Leu Val Pro Tyr Leu Gly Thr Ala Asn Leu Trp Asp Tyr Leu Pro Val

225 230 235 240

Leu Trp Trp Phe Asp Val Phe Gly Val Arg Asn Lys Ile Leu Ser Leu

245 250 255

Val Ser Thr Arg Asp Ala Phe Leu Arg Arg Leu Ile Asp Ala Glu Arg

260 265 270

Arg Arg Leu Asp Asp Gly Asn Asp Gly Ala Lys Lys Ser Ile Ile Ala

275 280 285

Val Leu Leu Thr Leu Gln Lys Ser Glu Pro Glu Val Tyr Thr Asp Thr

290 295 300

Thr Ile Met Ala Leu Cys Gly Asn Leu Phe Gly Ala Gly Thr Glu Thr

305 310 315 320

Thr Ser Thr Thr Thr Glu Trp Ala Met Ser Leu Leu Leu Lys His Pro

325 330 335

Glu Ala Leu Lys Lys Ala Gln Ala Glu Ile Asp Ala Ala Val Ser Thr

340 345 350

Ser Arg Leu Leu Thr Ala Asp Asp Met Pro Arg Leu Thr Tyr Leu Arg

355 360 365

Cys Val Ile Asp Glu Ala Met Arg Leu Tyr Pro Ala Val Pro Leu Leu

370 375 380

Leu Pro His Glu Ser Ala Ala Asp Cys Lys Val Gly Gly Tyr Asp Val

385 390 395 400

Pro Ala Gly Thr Met Leu Leu Val Asn Val Tyr Ala Ile His Arg Asp

405 410 415

Pro Ala Val Trp Glu Glu Pro Thr Gln Phe Arg Pro Glu Arg Phe Glu

420 425 430

Asp Gly Asp Val Glu Gly Arg Leu Leu Met Pro Phe Gly Met Gly Arg

435 440 445

Leu Lys Cys Pro Gly Glu Thr Leu Ala Leu Arg Thr Ile Ala Leu Val

450 455 460

Leu Gly Thr Leu Ile Gln Cys Phe Asp Trp Asp Thr Val Asp Gly Val

465 470 475 480

Glu Leu Asp Met Thr Glu Ser Gly Gly Leu Thr Met Gln Arg Ala Val

485 490 495

Pro Leu Glu Ala Met Cys Arg Pro Arg Ala Val Met Arg Glu Val Leu

500 505 510

Glu Lys Leu

515

Claims (6)

1. a kind of method for improveing crops character, it is characterised in that the method is to import Cytochrome P450 into crops Protein coding gene realizes that crops plant height reduces, disease resistance improves or lodging resistance improves；The cytochrome p450 protein Amino acid sequence shown in polypeptide comprising amino acid sequence shown in SEQ ID NO:1 or SEQ ID NO:1 passes through 1-24 amino Sour replacing, missing or adding for residue and the derived peptides formed.

2. the method for improvement crops character as described in claim 1, it is characterised in that the polypeptide for reducing crops plant height Amino acid sequence is shown in SEQ ID NO:2.

3. the method for improvement crops character as claimed in claim 2, it is characterised in that the polypeptide for reducing crops plant height Nucleotides sequence be classified as shown in SEQ ID NO:3.

4. the method for improvement crops character as described in claim 1, it is characterised in that the derivative for reducing crops plant height Polypeptid acid sequence is shown in SEQ ID NO:6.

5. the method for improvement crops character as described in claim 1, it is characterised in that the described method includes:

(1) expression vector of the encoding gene containing cytochrome p450 protein is constructed, and imports Agrobacterium, obtains and contains cytochromes The Agrobacterium of P450 protein coding gene；

(2) crop plant cells, tissue or organ are contacted with the Agrobacterium in step (1), so that containing Cytochrome P450 Protein gene enters crop plant cells, and is integrated on the chromosome of crop plant cells；

(3) crop plant cells, tissue or the organ for being transferred to the gene containing cytochrome p450 protein are filtered out；

(4) by cell, tissue or the neomorph in step (3) at crop；

(5) selection plant height reduces, disease resistance improves or the transgenosis transformant of lodging resistance raising.

6. as described in claim 1 improvement crops character method, it is characterised in that the crops include corn and soybean, Cotton, wheat or rice.