CN107299102A

CN107299102A - The positive regulatory factor OsWRKY53 genes of paddy rice BR signals and its encoding proteins

Info

Publication number: CN107299102A
Application number: CN201710596891.5A
Authority: CN
Inventors: 卜庆云; 田晓杰; 李秀峰; 王臻昱
Original assignee: Northeast Institute of Geography and Agroecology of CAS
Current assignee: Northeast Institute of Geography and Agroecology of CAS
Priority date: 2017-07-20
Filing date: 2017-07-20
Publication date: 2017-10-27
Anticipated expiration: 2037-07-20
Also published as: CN107299102B

Abstract

The positive regulatory factor OsWRKY53 genes of paddy rice BR signals and its encoding proteins, it is related to a kind of positive regulatory factor OsWRKY53 genes of paddy rice BR signals and its encoding proteins.The purpose is to provide the positive regulatory factor OsWRKY53 genes of paddy rice BR signals and its encoding proteins.SEQ ID NO in the nucleotide sequence of the positive regulatory factor OsWRKY53 genes of paddy rice BR signals such as sequence table：Shown in 1.SEQ ID NO in the amino acid sequence of the albumen of the coding positive regulatory factor OsWRKY53 genes of paddy rice BR signals such as sequence table：Shown in 2.OsWRKY53 genes can positive regulation BR signals and plant type of rice, enrich and perfect paddy rice BR signal transduction pathways, to transformation plant type of rice, and then improve crop yield Important Theoretic Foundation is provided.The present invention is applied to Rice molecular breeding field.

Description

The positive regulatory factor OsWRKY53 genes of paddy rice BR signals and its encoding proteins

Technical field

The present invention relates to the positive regulatory factor OsWRKY53 genes of paddy rice BR signals and its encoding proteins.

Background technology

Paddy rice is a kind of important cereal crops, and the population of more than half improves rice crop using it as staple food in the world Yield is significant to improving grain security and development of stabilizing the economy.And plant type of rice is to influence the one of rice crop yield Individual key factor, rice leaf intersection angle is an important component of plant type of rice again.Leaf angle increases, and is more favorable for blade and catches Light is obtained, and then improves photosynthetic rate, crop yield is improved to a certain extent；And Leaf angle is small, blade is in erectility, can To improve planting density to a certain extent, and then it can also improve crop yield.So, the Leaf angle of systematic research paddy rice There is important theory and application value to moulding plant type of rice and improving crop yield.Forest character improvement is realized in a short time Demand, carrying out genetic improvement to it by transgenic approach has great significance.

The content of the invention

The present invention provides a kind of white birch gene and its encoding proteins and application.

SEQ IDNO in the nucleotide sequence of the positive regulatory factor OsWRKY53 genes of paddy rice BR signals of the present invention such as sequence table： Shown in 1.

The amino acid sequence such as sequence table of the albumen of the present invention coding positive regulatory factor OsWRKY53 genes of paddy rice BR signals Middle SEQ ID NO：Shown in 2.

BR, i.e. brassinosteroid, are a kind of important sterols plant hormones, participate in the regulation and control to plant Leaf angle, water Rice BR deficient mutants such as d61-2, d11, BZR1-RNAi, OsGSK2-OE etc., Leaf angle significantly diminishes；And BR gain-of-functions Property mutant such as M107, bzr1-D, OsGSK2-RNAi etc., Leaf angle is significantly increased, and fully shows that BR regulates and controls with plant Leaf angle Between there is direct relation.

The invention discloses the positive regulatory factor OsWRKY53 genes of paddy rice BR signals and its encoding proteins, the present invention is utilized PCR method clones rice transcription factor OsWRKY53 genes from paddy rice.The OsWRKY53 gene coding regions that the present invention is obtained Full length sequence is corresponding with the LOC_Os05g27730.1 announced in Rice Genome Annotation Project.The present invention By genetic transformation means, by OsWRKY53 genes in paddy rice overexpression, and it was found that OsWRKY53 gene overexpressions turn Trans-genetic hybrid rice Leaf angle is significantly increased, seed increase, shows the phenotype of BR signal enhancings；Skill is knocked out by CRISPR/Cas9 Art obtains OsWRKY53 knock out mutants bodies, and oswrky53 mutant shows that Leaf angle diminishes, seed diminishes, plant height becomes short Etc. a series of phenotype of BR signal defects.BR biosynthesis genes detection, external source BR to Leaf angle sensitivity analysis test and The response experiment that OsWRKY53 is handled external source BR, all fully shows that OsWRKY53 being capable of positive regulation BR signals.

Present invention firstly discovers that rice transcription factor OsWRKY53 genes being capable of positive regulation BR signals and plant type of rice.Water Rice transcription factor OsWRKY53 enriches and perfect paddy rice BR letters to a certain extent as the discovery of the positive regulatory factor of BR signals Number Signal Transduction Pathways, to transformation plant type of rice, and then improve crop yield and provide Important Theoretic Foundation, before wide application Scape.

Brief description of the drawings

Fig. 1 is OsWRKY53 gene overexpression transgenic paddy rice general morphology figures；Wherein WT is wild rice, OsWRKY53-OE is OsWRKY53 gene overexpression transgenic paddy rices；

Fig. 2 is OsWRKY53 gene overexpression transgenic paddy rice Leaf angle aspect graphs；Wherein WT is wild rice, OsWRKY53-OE is OsWRKY53 gene overexpression transgenic paddy rices；

Fig. 3 is OsWRKY53 gene overexpression transgenic paddy rice Leaf angle size statistical results；Wherein WT is wild type water Rice, OsWRKY53-OE is OsWRKY53 gene overexpression transgenic paddy rices；

Fig. 4 is OsWRKY53 gene overexpression transgenic paddy rice seed aspect graphs；Wherein WT is wild rice, OsWRKY53-OE-1, OsWRKY53-OE-2, OsWRKY53-OE-3 are OsWRKY53 gene overexpression transgenic paddy rices；

Fig. 5 is OsWRKY53 gene overexpression transgenic paddy rice seed grain length statistical results charts；Wherein WT is wild type water Rice, 1,2 and 3 be OsWRKY53 gene overexpression transgenic paddy rices；

Fig. 6 is the wide statistical results chart of OsWRKY53 gene overexpressions transgenic paddy rice seed grain；Wherein WT is wild type water Rice, 1,2 and 3 be OsWRKY53 gene overexpression transgenic paddy rices；

Fig. 7 is 3 BR biosynthesis genes D2, OsDWF4, D11 in OsWRKY53 gene overexpressions transgenic paddy rice and open country The expression quantity testing result of raw type；Wherein a is wild rice, and b, c and d are OsWRKY53 gene overexpression transgenosis water Rice；

Fig. 8 is that oswrky53 mutant knocks out type；Wherein WT is wild rice, OsWRKY53-1, OsWRKY53-2 It is OsWRKY53 knock out mutants bodies；

Fig. 9 is oswrky53 mutant general morphology figures；Wherein WT is wild rice, OsWRKY53-1, OsWRKY53- 2 be OsWRKY53 knock out mutants bodies；

Figure 10 is oswrky53 mutant Leaf angle aspect graphs；Wherein WT is wild rice, and OsWRKY53-1 is OsWRKY53 knock out mutants bodies；

Figure 11 is oswrky53 mutant Leaf angle size statistical results；Wherein a is wild rice, and b is OsWRKY53 Knock out mutants body；

Figure 12 is oswrky53 mutant seeds aspect graphs；Wherein WT be wild rice, OsWRKY53-1, OsWRKY53-2 is OsWRKY53 knock out mutants bodies；

Figure 13 is oswrky53 mutant seeds grain length statistical results；Wherein WT is wild rice, and 1 and 2 are OsWRKY53 knock out mutants bodies；

Figure 14 is the wide statistical result of oswrky53 mutant seeds grain；Wherein WT is wild rice, and 1 and 2 are OsWRKY53 knock out mutants bodies；

Figure 15 is the high statistical result of oswrky53 mutant strains；Wherein WT is wild rice, and 1 and 2 be OsWRKY53 bases Because of knockout mutations body；

Figure 16 is figures of the external source BR to the sensitivity analysis experiment of OsWRKY53 gene overexpression transgenic paddy rice Leaf angles Piece；Wherein WT is wild rice, and OsWRKY53-OE is OsWRKY53-OE gene overexpression transgenic paddy rices；

Figure 17 is systems of the external source BR to the sensitivity analysis experiment of OsWRKY53 gene overexpression transgenic paddy rice Leaf angles Count result；Wherein a is wild rice, and b is OsWRKY53-OE gene overexpression transgenic paddy rices；

The picture that Figure 18 tests for the BR of external source to the sensitivity analysis of oswrky53 mutant Leaf angles；Wherein WT is wild Type paddy rice, OsWRKY53 is OsWRKY53 knock out mutants bodies；

Figure 19 is the statistical result that sensitivity analyses of the BR of external source to oswrky53 mutant Leaf angles is tested；Wherein a For wild rice, b is OsWRKY53 knock out mutants bodies；

Figure 20 is the expression characteristic analysis of OsWRKY53 gene pairs BR responses on transcriptional level；Wherein b is OsWRKY53 bases Cause, c is OsDWF4 genes；

Figure 21 is the expression characteristic analysis that OsWRKY53 is responded to BR on protein level.

Embodiment

Embodiment one：The nucleotide sequence of the positive regulatory factor OsWRKY53 genes of present embodiment paddy rice BR signals Such as SEQ ID NO in sequence table：Shown in 1.

Present embodiment discloses the positive regulatory factor OsWRKY53 genes of paddy rice BR signals and its encoding proteins, this embodiment party Formula clones rice transcription factor OsWRKY53 genes using PCR method from paddy rice.The OsWRKY53 that present embodiment is obtained Gene coding region full length sequence and the LOC_Os05g27730.1 phases announced in Rice Genome Annotation Project Correspondence.Present embodiment by genetic transformation means, by OsWRKY53 genes in paddy rice overexpression, and it was found that OsWRKY53 gene overexpression transgenic paddy rice Leaf angles are significantly increased, seed increase, show the phenotype of BR signal enhancings；It is logical Cross CRISPR/Cas9 knockouts technology and obtain OsWRKY53 knock out mutants bodies, oswrky53 mutant shows Leaf angle change Small, seed diminishes, plant height becomes the phenotypes of BR signal defects such as short a series of.The detection of BR biosynthesis genes, external source BR are pressed from both sides to leaf The response experiment that angle sensitivity analysis experiment and OsWRKY53 are handled external source BR, all fully shows that OsWRKY53 can be positive Regulate and control BR signals.

Present embodiment finds that rice transcription factor OsWRKY53 genes being capable of positive regulation BR signals and paddy rice strain first Type.Rice transcription factor OsWRKY53 enriches and perfect water to a certain extent as the discovery of the positive regulatory factor of BR signals Rice BR signal transduction pathways, to transformation plant type of rice, and then improve crop yield offer Important Theoretic Foundation, should with wide Use prospect.

Embodiment two：The albumen of the present embodiment coding positive regulatory factor OsWRKY53 genes of paddy rice BR signals SEQ ID NO in amino acid sequence such as sequence table：Shown in 2.

Pass through the effect of the following experimental verification present invention：

Embodiment 1,

1st, the clone of the positive regulatory factor OsWRKY53 genes of paddy rice BR signals and sequencing：

First, using wild rice kind Nipponbare as experiment material, according to the TRIzol kits of Invitrogen companies Operation manual, extracts blade total serum IgE；

2nd, the total serum IgE extracted using the process steps one of DNase I；

3rd, take 1 μ g step 2 handle after total serum IgE be used for cDNA synthesis, cDNA synthetic operation is according to purchase from BD The BD SMART of Biosciences Clontech companies^TMThe use hand of RACE cDNA Amplification Kit kits Volume is carried out, and obtains cDNA；

4th, using the cDNA of above-mentioned acquisition as template, with reference to TaKaRa companiesHS DNAPolymerase operational manuals, OsWRKY53 genes are expanded with forward primer F1 and reverse primer R1.PCR reaction conditions It is as follows：95 DEG C of pre-degeneration 5min；95 DEG C of denaturation 30s, 58 DEG C of annealing 30s, 72 DEG C of extension 90s, totally 38 circulation；72 DEG C of extensions eventually 10min.Finally, PCR primer is sequenced on ABI3130 sequenators (ABI companies), sequencing result shows, paddy rice BR letters Number positive regulatory factor OsWRKY53 genes are by the SEQ ID No in 1464 base compositions, its nucleotide sequence such as sequence table：1 It is shown.Coding has SEQ ID NO in sequence table：The protein of 2 amino acid sequence.

Forward primer F1：5'-ATGGCGTCCTCGACGGGG-3'

Reverse primer R1：5'-CTAGCAGAGGAGCGACTCGACG-3'

2nd, the acquisition of OsWRKY53 gene overexpressions transgenic paddy rice

First, vector construction：Using Nipponbare cDNA as template, with reference to TaKaRa companiesHS DNA Polymerase operational manuals, using forward primer F2 and reverse primer R2 as amplimer, expand the volume of OsWRKY53 genes Code area, and amplified fragments clone is entered into plant over-express vector PC1390U, form what a Ubiquitin promoter drove OsWRKY53 gene overexpression carriers.

Forward primer F2：5'-GTTACTTCTGCACTAGGTACCATGGCGTCCTCGACGGGG-3'

Reverse primer R2：5'-TCTTAGAATTCCCGGGGATCCCTAGCAGAGGAGCGACTCGACG-3'

2nd, purpose carrier conversion Agrobacterium EHA105：EHA105 competence is taken out from -80 DEG C of refrigerators, is placed in and melts on ice Change；The μ g of 500ng~1 purpose plasmid is added in 100ul EHA105 competence, 30min is placed on ice；It is immediately placed in liquid nitrogen Middle 5min；Taken out from liquid nitrogen, be immediately placed in water-bath 5min in 37 DEG C of pre- pots of water；2min on ice；Add 800ul liquid LB cultures Base, is placed in complete warm oscillator (being purchased from MKN companies), 28 DEG C, 120rpm is incubated 4~5h；Centrifugation, abandons most of supernatant, will be surplus Remaining bacterium solution is applied to containing kanamycins (50ug/ml) (being purchased from Amresco) and rifampin (50ug/ml) (being purchased from Amresco) LB solid mediums on, 28 DEG C cultivate 3 days or so.

3rd, after bacterium colony is grown, bacterium colony PCR identifications is carried out, positive colony is identified；Picking positive colony is to added with corresponding In the LB liquid medium of antibiotic and rifampin, 28 DEG C, 180rpm cultures 16h or so, bacterium solution now can use 30% Glycerine presses 1:1 volume ratio is preserved, and is deposited to -80 DEG C of refrigerators, when infecting callus, is activated i.e. from -80 DEG C of taking-ups Can.

4th, Agrobacterium infects Rice Callus：Purpose is taken out from -80 DEG C of refrigerators and deposits bacterium, by 1:100 ratio, which is added, to be contained In the LB liquid medium for having kanamycins (50ug/ml) and rifampin (50ug/ml), 180rpm, 28 DEG C of overnight incubations；By bacterium Liquid culture to naked eyes appear as the same color of orange juice (OD=1.0 or so), can be taken out from incubator；Take 500ul left Right bacterium solution is into 1.5ml centrifuge tubes, 5000rpm, 28 DEG C, centrifuges 3min, abandons supernatant, it can be seen that ttom of pipe has the cenobium of white； The liquid for containing 20ug/ml acetosyringone (be purchased from Aldrich) with 300ul trains culture medium and gently blows and beats ttom of pipe cenobium altogether, Make it uniformly in suspension liquid medium within；The good callus of growth conditions is selected into 50ml centrifuge tubes, about extremely Centrifuge tube scale 5ml or so；The liquid for adding the acetosyringone that 20ml contains 20ug/ml trains culture medium altogether, then will be above-mentioned The 300ul bacterium solutions suspended are all added in 50ml centrifuge tubes；Continue softly to mix 2~3min, to be infected.By liquid Training culture medium is outwelled altogether, and then the callus infected is transferred in the culture dish for being covered with filter paper, adsorbs unnecessary culture Base, this process takes around 1min or so；The metafiltration paper of culture medium upper berth one is trained altogether in solid, is impregnated with filter paper, then will be upper The callus infected is stated to shift on so far solid medium；28 DEG C of light cultures 2~3 days.

5th, the renewal cultivation of Rice Callus is infected：The callus light culture infected is after 2~3 days, by callus particle It is transferred in 50ml centrifuge tubes；With the sterile water wash callus 4 containing 400ug/ml carboxylic Bians penicillin (being purchased from Amresco) ~5 times, continue 1min or so every time, carry out degerming；Sterile water wash callus is used again 2~3 times, be transferred to and be covered with filter paper On culture dish, excessive moisture is blotted；Above-mentioned callus is transferred to the recovery media containing 400ug/ml carboxylic Bian penicillin On, 28 DEG C of artificial climate incubator (24h optical cultures) renewal cultivations 4~5 days.

6th, the screening and culturing of Rice Callus is infected：After renewal cultivation 4~5 days, by the callus on recovery media Tissue is transferred on the screening and culturing medium containing 400ug/ml carboxylic Bian penicillin and 50ug/ml hygromycin (being purchased from Roche)；By its It is transferred to culture 30 days or so in 28 DEG C of artificial climate incubators (24h optical cultures).

7th, the differentiation culture of resistant rice callus：Kanamycin-resistant callus tissue on screening and culturing medium is transferred to differentiation culture On base, every bottle moves to cluster callus；Culture 30 days or so in 28 DEG C of artificial climate incubators (24h optical cultures) is placed on, i.e., Transgenic seedling can be differentiated.

8th, the identification of transgenic seedling：After transgenic seedling is differentiated, it need to be identified, exclude false positive.Enter first The thick of water-filling rice DNA is carried；The DNA slightly carried more than is template, is said according to the EasyTaq DNAPolymerase of Quan Shi King Companies Bright book, is expanded with hygromycin primer (F3 and R3).

Forward primer F3：5'-TGCGCCCAAGCTGCATCAT-3'

Reverse primer R3：5'-TGAACTCACCGCGACGTCTGT-3'

As shown in figure 1, being the general morphology figure of OsWRKY53 gene overexpression transgenic paddy rices, it can be seen that be overexpressed and turn Trans-genetic hybrid rice shows the phenotype of BR signal enhancings；Its Leaf angle is significantly increased, and the Leaf angle of its sword-like leave is and wild more than 100 ° The Leaf angle of type is only 30 ° or so (as shown in Figure 2,3)；And the seed for being overexpressed transgenic paddy rice is significantly increased, and measures it Grain length grain is wide to be found, compared with wild type both significantly elongated (as shown in Fig. 4,5 and 6), OsWRKY53 genes are very likely Positive regulation BR signals.

3rd, the expression quantity detection of BR biosynthesis genes

First, cultivate to two weeks sizes, take for experiment material with OsWRKY53 gene overexpressions transgenic paddy rice and its control The blade of same area, blade total serum IgE is extracted with reference to purchase from the operation manual of the TRIzol kits of Invitrogen companies；

2nd, the total serum IgE extracted using the process steps one of DNase I；

3rd, take 1 μ g step 2 handle after total serum IgE be used for cDNA synthesis, cDNA synthetic operation is according to purchase from BD The BD SMART of Biosciences Clontech companies^TMThe use hand of RACE cDNA Amplification Kit kits Volume is carried out；

4th, 3 BR biosynthesis gene primers are passed through by template of the cDNA of acquisition：D2 genes (forward primer F4 with it is anti- To primer R4), OsDWF4 genes (forward primer F5 and reverse primer R5) and D11 genes (forward primer F6 and reverse primer 6), Paddy rice internal reference actin (forward primer F7 and reverse primer R7), using SYBR Green PCRmaster mix (TransStart) Quantitative real-time PCR are carried out；Data are from Bio-Rad chromo 4real-time Obtained on PCR detector；With 2^-△△CTMethod analysis multiple change.

Forward primer F4：5'-TCGCTGACGGAGCTGATG-3'

Reverse primer R4：5'-ACTTGAGGTGGGAGGACTTG-3'

Forward primer F5：5'-CTCCACCTTCTCCGCTCAG-3'

Reverse primer R5：5'-GCCGCTCCGTCTCTTCC-3'

Forward primer F6：5'-TGGCGACATTGAGAAGATTGC-3'

Reverse primer R6：5'-CAGAAGGCGATGACATTGACC-3'

Forward primer F7：5'-AGACCTTCAACACCCCTGCTATG-3'

Reverse primer R7：5'-TCACGCCCAGCAAGGTCG-3'

As shown in fig. 7, being 3 BR biosynthesis genes D2, OsDWF4, D11 in OsWRKY53 gene overexpression transgenosis Expression characteristic in paddy rice and its control.As a result show, compared with compareing wild type, be overexpressed 3 BR lifes in transgenic paddy rice The expression quantity of thing synthetic gene is significantly reduced, illustrate be overexpressed transgenic paddy rice in its endogenous BR signal be it is enhanced, Preliminary explanation OsWRKY53 genes being capable of positive regulation BR signals.

4th, the acquisition of oswrky53 mutant

First, by the CDS sequence inputting CRISPR Primer Designer softwares of OsWRKY53 genes, 2 pairs of target position are designed Point primer (F8 and R8；F9 and R9), the structure for follow-up knockout carrier.

Forward primer F8：5'-GGCATTCCAGTCGTACCTCTGAGC-3'

Reverse primer R8：5'-AAACGCTCAGAGGTACGACTGGAA-3'

Forward primer F9：5'-GCCGAGCTGGAGGACGGGTACAAC-3'

Reverse primer R9：5'-AAACGTTGTACCCGTCCTCCAGCT-3'

2nd, 1 μ l are respectively taken to be added in 98 μ l 0.5 × TE solution 100 μM of upstream and downstream primers, 90 DEG C of heat shock 30s are formed Target practice joint, moves to room temperature cooling and completes annealing.

3rd, this 2 target practice joints are connected respectively on each gRNA expression cassettes.

PCR programs are：37 DEG C of 5min, 20 DEG C of 5min, 5 circulations.

4th, gRNA expression cassettes are expanded by the method for nest-type PRC

First round PCR is expanded

PCR programs：98℃2min；98 DEG C of 10s, 60 DEG C of 10s, 72 DEG C of 20s, totally 25 circulations；72℃5min.Second wheel PCR is expanded：

PCR programs：98℃2min；98 DEG C of 10s, 60 DEG C of 10s, 72 DEG C of 30s, 25 circulations；72℃5min.

Universal primer sequence involved by this step operation is：

U-F:5'-CTCCGTTTTACCTGTGGAATCG-3'

gRNA-R:5'-CGGAGGAAAATTCCATCCAC-3'

B1':5'-TTCAGAGGTCTCTCTCGCACTGGAATCGGCAGCAAAGG-3'

B2:5'-AGCGTGGGTCTCGTCAGGGTCCATCCACTCCAAGCTC-3'

B2':5'-TTCAGAGGTCTCTCTGACACTGGAATCGGCAGCAAAGG-3'

BL:5'-AGCGTGGGTCTCGACCGGGTCCATCCACTCCAAGCTC-3'

5th, the fragment for obtaining above-mentioned nest-type PRC, is connected on pYLCRSPR/Cas9-MT carrier frameworks, that is, completes to strike Except the structure of carrier.

Program：37 DEG C, 15min；Then, ligase is added on the basis of above-mentioned system, linked system is：

T₄The μ L of DNA enzymatic (NEB) 0.1

10x DNA ligase buffer(NEB) 1.5μL

PCR programs：37 DEG C of 5min, 10 DEG C of 5min, 20 DEG C of 5min, totally 15 circulations.

6th, the conversion of connection product, the identification of positive colony, sequencing, the extraction of plasmid；

7th, purpose carrier conversion Agrobacterium EHA105；

8th, OsWRKY53 knock out mutants bodies are obtained using agrobcterium-mediated transformation.

As shown in figure 8, being two kinds of OsWRKY53 knock out mutants bodies oswrky53-1 and oswrky53-2 sequencing knot Really, WT sequence is：5'-ATCCCGGCT CAGAGGTACGACTGGAAG-3', oswrky53-1 (- CAGAGGT) sequence For：5'-ATCCCGGCT-------ACGACTGGAAG-3', oswrky53-2 (+A) sequence is：5'-ATCCCGGCT ACAGAGGTACGACTGGAAG-3', is missing from 7bp and insertion 1bp, causes follow-up frameshift mutation, make the ammonia given expression to respectively Base acid changes, and OsWRKY53 protein functions are lost.As shown in figure 9, being the general morphology figure of oswrky53 mutant, from figure In it can be seen that mutant shows the phenotypes of BR signal deletions a series of；First, oswrky53 mutant Leaf angle diminishes, its The Leaf angle of sword-like leave is only 18 ° or so, and the Leaf angle compareed is 38 ° or so (as shown in Figure 10,11)；Secondly, oswrky53 The seed of mutant significantly diminishes, and measures the wide discovery of its grain length grain, both significantly shorten (such as Figure 12,13 compared with wild type With shown in 14)；Again, oswrky53 mutant plant height becomes short (as shown in figure 15).The performance of oswrky53 mutant is said again Bright, OsWRKY53 being capable of positive regulation BR signals.

5th, sensitivity analyses of the external source BR to Leaf angle

First, seed disinfection：It is experiment material, seed stripping from OsWRKY53 gene overexpressions transgenic paddy rice and its control Skin, 1min is soaked with 70% ethanol；Then soaked twice with 30% hypochlorous acid, each 15min；Finally use sterile water wash 5 ~7 times；

2nd, disinfection seed is transferred on 1/2MS solid mediums (1% sucrose, 4%phytagel, pH5.8), 30 DEG C Light culture 8 days or so；

3rd, choose the Leaf angle (include blade, each 1cm of leaf sheath) of second leaf, with various concentrations 24-epiBL (Sigma, E1641) the light culture 48h in 30 DEG C of incubators；

4th, result is observed, and carries out with ImageJ softwares the measurement of Leaf angle.

As shown in figure 16, it is OsWRKY53 gene overexpressions transgenic paddy rice and its to impinging upon various concentrations 24-epiBL Photo in (Sigma, E1641) after processing 48h, Figure 17 is Leaf angle angle statistics in various concentrations 24-epiBL processing procedures As a result.As a result show, after being handled with 10nM 24-epiBL, the Leaf angle for being overexpressed transgenic paddy rice reaches 130 ° or so, and right According to only 60 ° or so；Also, with the raising of 24-epiBL concentration for the treatment of, the phenotype of Leaf angle increase is more obvious, with 1 μ Μ After 24-epiBL processing, the Leaf angle for being overexpressed transgenic paddy rice is even up to 230 ° or so, illustrates that OsWRKY53 is overexpressed and turns Trans-genetic hybrid rice Leaf angle handles hypersensitization to the BR of external source.Opposite, 24- of the oswrky53 mutant Leaf angle to external source EpiBL handles insensitive (as shown in Figure 18,19), i.e., after being handled using 1 μ Μ 24-epiBL, the leaf folder of oswrky53 mutant Angle only reaches 50 ° or so, and compares and reach 140 ° or so.Further experiment is provided for OsWRKY53 gene positive regulation BR signals Evidence.

6th, on transcriptional level, the expression characteristic analysis of OsWRKY53 gene pairs BR responses：

First, after being experiment material, presoaking and germinating from the imperial round-grained rice 11 of wild rice kind, the seed sowing of sprouting is being cut In the 96 hole PCR plates at bottom, then it is immersed in 1/2MS fluid nutrient mediums, 28 DEG C of illumination box (optical culture 14h；Light culture 10h) Middle culture 2 weeks；

2nd, the consistent rice seedling of growth conditions is selected, 0h, 1h, 3h, 6h are handled respectively with 1 μM of 24-epiBL；

3rd, the blade of same area is taken, with reference to the operation manual for buying the TRIzol kits from Invitrogen companies, Extract blade total serum IgE；

4th, the total serum IgE extracted using the process steps three of DNase I；

5th, take 1 μ g step 4 handle after total serum IgE be used for cDNA synthesis, cDNA synthetic operation is according to purchase from BD The BD SMART of Biosciences Clontech companies^TMThe use hand of RACE cDNA Amplification Kit kits Volume is carried out, and obtains cDNA；

6th, using the cDNA of acquisition as template, forward primer F10 and reverse primer R10 is detection primer, detects OsWRKY53 The expression of gene.

Forward primer F10：5'-AGACCTTCAACACCCCTGCTATG-3'

Reverse primer R10：5'-TCACGCCCAGCAAGGTCG-3'

As shown in figure 20, be on OsWRKY53 gene transcription levels to BR respond expression characteristic, as a result show that BR can Suppress the expression on OsWRKY53 transcriptional levels, and BR processing times are longer, and inhibition level is more obvious.

7th, on protein level, the expression characteristic analysis that OsWRKY53 is responded to BR

First, the OsWRKY53 gene overexpressions transgenic paddy rice with MYC labels from homozygosis is experiment material, seed soaking After vernalization, the seed of sprouting is sowed in the 96 hole PCR plates at bottom have been cut, is then immersed in 1/2MS fluid nutrient mediums, at 28 DEG C Incubator (optical culture 14h；Light culture 10h) in culture 2 weeks；

2nd, the consistent rice seedling of growth conditions is selected, 30min is handled respectively with 1 μ Μ 24-epiBL and DMSO；

3rd, the blade of same area is taken, leaves total protein is extracted with reference to the SDS lysates specification of green skies company；

4th, 1 × loading buffer are added, 5min, high speed centrifugation 10min is boiled；

5th, loading, carries out SDS-PAGE electrophoresis；

6th, after electrophoresis terminates, transferring film is carried out, by the protein delivery on glue to pvdf membrane (Bio-Rad)；

7th, after transferring film terminates, with MYC antibody (Abmart：M20002L follow-up western hybridization) is carried out.

As shown in figure 21, it is the expression characteristic that OsWRKY53 is responded to BR on protein level.As a result show, BR can be lured Lead the expression on OsWRKY53 protein levels.As the positive regulatory factor of BR signals, the accumulation on its protein level is more, BR signals Output is stronger, but plant has self negative-feedback balance adjustment mechanism again, when BR signals are strong to a certain extent in plant Afterwards, plant can suppress the expression on the positive regulatory factor transcriptional level of these BR signals again, to maintain BR signals in plant Balance.

In summary, the present embodiment is by genetic transformation means, by OsWRKY53 genes in paddy rice overexpression, and It was found that OsWRKY53 gene overexpression transgenic paddy rice Leaf angles are significantly increased, seed increase, the table of BR signal enhancings is shown Type；Technology is knocked out by CRISPR/Cas9 and obtains OsWRKY53 knock out mutants bodies, oswrky53 mutant shows leaf folder Angle diminishes, seed diminishes, plant height becomes the phenotypes of BR signal defects such as short a series of.The detection of BR biosynthesis genes, BR pairs of external source The response experiment that Leaf angle sensitivity analysis is tested and OsWRKY53 is handled external source BR, all fully shows that OsWRKY53 can Positive regulation BR signals.

Sequence table

<110>Northeast Institute of Geography and Agricultural Ecology, Chinese Academy of Sc

<120>The positive regulatory factor OsWRKY53 genes of paddy rice BR signals and its encoding proteins

<160> 28

<210> 1

<211> 1464

<212> DNA

<213>Paddy rice

<400> 1

atggcgtcct cgacgggggg gttggaccac gggttcacgt tcacgccgcc gccgttcatc 60

acgtcgttca ccgagctgct gtcggggggc ggtggggacc tgctcggcgc cggcggtgag 120

gagcgctcgc cgagggggtt ctccagaggc ggagcgaggg tgggcggcgg ggtgcccaag 180

ttcaagtccg cgcagccgcc gagcctgccg ctctcgccgc cgccggtgtc gccgtcgtcc 240

tacttcgcca tcccgccggg gctcagcccc accgagctgc tcgactcccc cgtcctcctc 300

agctcctccc atatcttggc gtccccgacc accggtgcaa tcccggctca gaggtacgac 360

tggaaggcca gcgccgatct catcgcttct cagcaagatg acagccgcgg cgacttctcc 420

ttccacacca actccgacgc catggccgcg caaccggcct ctttcccttc cttcaaggag 480

caagagcagc aagtggtcga gtcgagcaag aacggcgccg ccgccgcgtc gagcaacaag 540

agcggcggcg gcgggaacaa caagctggag gacgggtaca actggaggaa gtacgggcag 600

aagcaggtga aggggagcga gaacccgagg agctactaca agtgcaccta caacggctgc 660

tccatgaaga agaaggtgga gcgctcgctc gccgacggcc gcatcaccca gatcgtctac 720

aagggcgcac acaaccaccc caagccgctc tccacccgcc gcaacgcctc ctcctgcgcc 780

accgccgccg cctgcgccga cgacctcgcg gcgcccggcg cgggcgcgga ccagtactcc 840

gccgcgacgc ccgagaactc ctccgtcacg ttcggcgacg acgaggccga caacgcatcg 900

caccgcagcg agggcgacga gcccgaagcc aagcgctgga aggaggatgc tgacaacgag 960

ggcagctccg gcggcatggg cggcggcgcc ggcggcaagc cggtgcgcga gccgaggctt 1020

gtggtgcaga cgctgagcga catcgacatc ctcgacgacg gcttccggtg gaggaagtac 1080

ggccagaagg tcgtcaaggg caaccccaac ccaaggagct actacaagtg cacgacggtg 1140

ggctgcccgg tgcggaagca cgtggagcgg gcgtcgcacg acacgcgcgc cgtgatcacc 1200

acctacgagg gcaagcacaa ccacgacgtc ccggtcggcc gcggcggcgg cggcggacgc 1260

gccccggcgc cggcgccgcc gacgtcgggg gcgatccggc cgtcggccgt cgccgccgcc 1320

cagcaggggc cctacaccct cgagatgctc cccaaccccg ccggcctcta cggcggctac 1380

ggcgccggcg ccggcggcgc cgcgttcccg cgcaccaagg acgagcggcg ggacgacctg 1440

ttcgtcgagt cgctcctctg ctag 1464

<210> 2

<211> 487

<212> PRT

<213>Paddy rice

<400> 2

Met Ala Ser Ser Thr Gly Gly Leu Asp His Gly Phe Thr Phe Thr

1 5 10 15

Pro Pro Pro Phe Ile Thr Ser Phe Thr Glu Leu Leu Ser Gly Gly

20 25 30

Gly Gly Asp Leu Leu Gly Ala Gly Gly Glu Glu Arg Ser Pro Arg

35 40 45

Gly Phe Ser Arg Gly Gly Ala Arg Val Gly Gly Gly Val Pro Lys

50 55 60

Phe Lys Ser Ala Gln Pro Pro Ser Leu Pro Leu Ser Pro Pro Pro

65 70 75

Val Ser Pro Ser Ser Tyr Phe Ala Ile Pro Pro Gly Leu Ser Pro

80 85 90

Thr Glu Leu Leu Asp Ser Pro Val Leu Leu Ser Ser Ser His Ile

95 100 105

Leu Ala Ser Pro Thr Thr Gly Ala Ile Pro Ala Gln Arg Tyr Asp

110 115 120

Trp Lys Ala Ser Ala Asp Leu Ile Ala Ser Gln Gln Asp Asp Ser

125 130 135

Arg Gly Asp Phe Ser Phe His Thr Asn Ser Asp Ala Met Ala Ala

140 145 150

Gln Pro Ala Ser Phe Pro Ser Phe Lys Glu Gln Glu Gln Gln Val

155 160 165

Val Glu Ser Ser Lys Asn Gly Ala Ala Ala Ala Ser Ser Asn Lys

170 175 180

Ser Gly Gly Gly Gly Asn Asn Lys Leu Glu Asp Gly Tyr Asn Trp

185 190 195

Arg Lys Tyr Gly Gln Lys Gln Val Lys Gly Ser Glu Asn Pro Arg

200 205 210

Ser Tyr Tyr Lys Cys Thr Tyr Asn Gly Cys Ser Met Lys Lys Lys

215 220 225

Val Glu Arg Ser Leu Ala Asp Gly Arg Ile Thr Gln Ile Val Tyr

230 235 240

Lys Gly Ala His Asn His Pro Lys Pro Leu Ser Thr Arg Arg Asn

245 250 255

Ala Ser Ser Cys Ala Thr Ala Ala Ala Cys Ala Asp Asp Leu Ala

260 265 270

Ala Pro Gly Ala Gly Ala Asp Gln Tyr Ser Ala Ala Thr Pro Glu

275 280 285

Asn Ser Ser Val Thr Phe Gly Asp Asp Glu Ala Asp Asn Ala Ser

290 295 300

His Arg Ser Glu Gly Asp Glu Pro Glu Ala Lys Arg Trp Lys Glu

305 310 315

Asp Ala Asp Asn Glu Gly Ser Ser Gly Gly Met Gly Gly Gly Ala

320 325 330

Gly Gly Lys Pro Val Arg Glu Pro Arg Leu Val Val Gln Thr Leu

335 340 345

Ser Asp Ile Asp Ile Leu Asp Asp Gly Phe Arg Trp Arg Lys Tyr

350 355 360

Gly Gln Lys Val Val Lys Gly Asn Pro Asn Pro Arg Ser Tyr Tyr

365 370 375

Lys Cys Thr Thr Val Gly Cys Pro Val Arg Lys His Val Glu Arg

380 385 390

Ala Ser His Asp Thr Arg Ala Val Ile Thr Thr Tyr Glu Gly Lys

395 400 405

His Asn His Asp Val Pro Val Gly Arg Gly Gly Gly Gly Gly Arg

410 415 420

Ala Pro Ala Pro Ala Pro Pro Thr Ser Gly Ala Ile Arg Pro Ser

425 430 435

Ala Val Ala Ala Ala Gln Gln Gly Pro Tyr Thr Leu Glu Met Leu

440 445 450

Pro Asn Pro Ala Gly Leu Tyr Gly Gly Tyr Gly Ala Gly Ala Gly

455 460 465

Gly Ala Ala Phe Pro Arg Thr Lys Asp Glu Arg Arg Asp Asp Leu

470 475 780

Phe Val Glu Ser Leu Leu Cys

485 487

<210> 3

<211> 18

<212> DNA

<213>Artificial sequence

<220>

<223>PCR primer F1 nucleotide sequence.

<400> 3

ATGGCGTCCT CGACGGGG 18

<210> 4

<211> 22

<212> DNA

<213>Artificial sequence

<220>

<223>PCR primer R1 nucleotide sequences.

<400> 4

CTAGCAGAGG AGCGACTCGA CG 22

<210> 5

<211> 39

<212> DNA

<213>Artificial sequence

<220>

<223>PCR primer F2 nucleotide sequence.

<400> 5

GTTACTTCTG CACTAGGTAC CATGGCGTCC TCGACGGGG 39

<210> 6

<211> 43

<212> DNA

<213>Artificial sequence

<220>

<223>PCR primer P2 nucleotide sequence.

<400> 6

TCTTAGAATT CCCGGGGATC CCTAGCAGAG GAGCGACTCG ACG 43

<210> 7

<211> 19

<212> DNA

<213>Artificial sequence

<220>

<223>PCR primer F3 nucleotide sequence.

<400> 7

TGCGCCCAAG CTGCATCAT 19

<210> 8

<211> 21

<212> DNA

<213>Artificial sequence

<220>

<223>PCR primer R3 nucleotide sequence.

<400> 8

TGAACTCACC GCGACGTCTG T 21

<210> 9

<211> 18

<212> DNA

<213>Artificial sequence

<220>

<223>PCR primer F4 nucleotide sequence.

<400> 9

TCGCTGACGG AGCTGATG 18

<210> 10

<211> 20

<212> DNA

<213>Artificial sequence

<220>

<223>PCR primer R4 nucleotide sequence.

<400> 10

ACTTGAGGTG GGAGGACTTG 20

<210> 11

<211> 19

<212> DNA

<213>Artificial sequence

<220>

<223>PCR primer F5 nucleotide sequence.

<400> 11

CTCCACCTTC TCCGCTCAG 19

<210> 12

<211> 17

<212> DNA

<213>Artificial sequence

<220>

<223>PCR primer R5 nucleotide sequence.

<400> 12

GCCGCTCCGT CTCTTCC 17

<210> 13

<211> 21

<212> DNA

<213>Artificial sequence

<220>

<223>PCR primer F6 nucleotide sequence.

<400> 13

TGGCGACATT GAGAAGATTG C 21

<210> 14

<211> 21

<212> DNA

<213>Artificial sequence

<220>

<223>PCR primer R6 nucleotide sequence.

<400> 14

CAGAAGGCGA TGACATTGAC C 21

<210> 15

<211> 23

<212> DNA

<213>Artificial sequence

<220>

<223>PCR primer F7 nucleotide sequence.

<400> 15

AGACCTTCAA CACCCCTGCT ATG 23

<210> 16

<211> 18

<212> DNA

<213>Artificial sequence

<220>

<223>PCR primer R7 nucleotide sequence.

<400> 16

TCACGCCCAG CAAGGTCG 18

<210> 17

<211> 24

<212> DNA

<213>Artificial sequence

<220>

<223>PCR primer F8 nucleotide sequence.

<400> 17

GGCATTCCAG TCGTACCTCT GAGC 24

<210> 18

<211> 24

<212> DNA

<213>Artificial sequence

<220>

<223>PCR primer R8 nucleotide sequence.

<400> 18

AAACGCTCAG AGGTACGACT GGAA 24

<210> 19

<211> 24

<212> DNA

<213>Artificial sequence

<220>

<223>PCR primer F9 nucleotide sequence.

<400> 19

GCCGAGCTGG AGGACGGGTA CAAC 24

<210> 20

<211> 24

<212> DNA

<213>Artificial sequence

<220>

<223>PCR primer R9 nucleotide sequence.

<400> 20

AAACGTTGTA CCCGTCCTCC AGCT 24

<210> 21

<211> 23

<212> DNA

<213>Artificial sequence

<220>

<223>PCR primer F10 nucleotide sequence.

<400> 21

AGACCTTCAA CACCCCTGCT ATG 23

<210> 22

<211> 18

<212> DNA

<213>Artificial sequence

<220>

<223>PCR primer R10 nucleotide sequence.

<400> 22

TCACGCCCAG CAAGGTCG 18

<210> 23

<211> 22

<212> DNA

<213>Artificial sequence

<220>

<223>PCR primer U-F nucleotide sequence.

<400> 23

CTCCGTTTTA CCTGTGGAAT CG 22

<210> 24

<211> 20

<212> DNA

<213>Artificial sequence

<220>

<223>PCR primer gRNA-R nucleotide sequence.

<400> 24

CGGAGGAAAA TTCCATCCAC 20

<210> 25

<211> 38

<212> DNA

<213>Artificial sequence

<220>

<223>PCR primer B1' nucleotide sequence.

<400> 25

TTCAGAGGTC TCTCTCGCAC TGGAATCGGC AGCAAAGG 38

<210> 26

<211> 37

<212> DNA

<213>Artificial sequence

<220>

<223>PCR primer B2 nucleotide sequence.

<400> 26

AGCGTGGGTC TCGTCAGGGT CCATCCACTC CAAGCTC 37

<210> 27

<211> 38

<212> DNA

<213>Artificial sequence

<220>

<223>PCR primer B2' nucleotide sequence.

<400> 27

TTCAGAGGTC TCTCTGACAC TGGAATCGGC AGCAAAGG 38

<210> 28

<211> 37

<212> DNA

<213>Artificial sequence

<220>

<223>PCR primer BL nucleotide sequence.

<400> 28

AGCGTGGGTC TCGACCGGGT CCATCCACTC CAAGCTC 37

Claims

1. the positive regulatory factor OsWRKY53 genes of paddy rice BR signals, it is characterised in that in the nucleotide sequence of the gene such as sequence table SEQ ID NO：Shown in 1.

2. encode the albumen of the positive regulatory factor OsWRKY53 genes of paddy rice BR signals described in claim 1, it is characterised in that should SEQ ID NO in the amino acid sequence of albumen such as sequence table：Shown in 2.