CN107299102B

CN107299102B - The positive regulatory factor OsWRKY53 gene of rice BR signal and its coding albumen

Info

Publication number: CN107299102B
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: 2019-11-08
Anticipated expiration: 2037-07-20
Also published as: CN107299102A

Abstract

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

Description

The positive regulatory factor OsWRKY53 gene of rice BR signal and its coding albumen

Technical field

The present invention relates to the positive regulatory factor OsWRKY53 gene of rice BR signal and its coding albumen.

Background technique

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 of great significance to grain security and development of stabilizing the economy is improved.And plant type of rice is influence rice crop yield one A key factor, rice leaf intersection angle are 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, improves crop yield to a certain extent；And Leaf angle is small, blade is in upright state, can To improve planting density to a certain extent, and then it also can be improved crop yield.So the Leaf angle of systematic research 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, genetic improvement is carried out to it by transgenic approach with great significance.

Summary of the invention

The present invention provides a kind of white birch gene and its coding albumen and application.

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

The present invention encodes the amino acid sequence such as sequence table of the albumen of the positive regulatory factor OsWRKY53 gene of rice BR signal Shown in middle SEQ ID NO:2.

BR, i.e. brassinosteroid are a kind of important sterols plant hormones, participate in the regulation to plant Leaf angle, water Rice BR deficient mutants such as d61-2, d11, BZR1-RNAi, OsGSK2-OE etc., Leaf angle significantly becomes smaller；And BR function obtains Property mutant such as M107, bzr1-D, OsGSK2-RNAi etc., Leaf angle significantly increases, and sufficiently shows that BR and plant Leaf angle regulate and control Between there is direct relationships.

The invention discloses the positive regulatory factor OsWRKY53 genes of rice BR signal and its coding albumen, the present invention to utilize PCR method clones rice transcription factor OsWRKY53 gene from rice.The gene coding region OsWRKY53 that the present invention obtains The LOC_Os05g27730.1 announced in full length sequence and Rice Genome Annotation Project is corresponding.The present invention By genetic transformation means, by OsWRKY53 gene overexpression in rice, and it was found that OsWRKY53 gene overexpression turns Trans-genetic hybrid rice Leaf angle significantly increases, seed increases, and shows the phenotype of BR signal enhancing；Skill is knocked out by CRISPR/Cas9 Art obtain OsWRKY53 knock out mutants body, oswrky53 mutant show Leaf angle becomes smaller, seed becomes smaller, plant height become it is short Etc. a series of phenotype of BR signal defects.BR biosynthesis gene detection, external source BR to Leaf angle sensitivity analysis test and OsWRKY53 tests the response that external source BR is handled, and all sufficiently shows that OsWRKY53 being capable of positive regulation BR signal.

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

Detailed description of the invention

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

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

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

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

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

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

Fig. 7 is 3 BR biosynthesis gene D2, OsDWF4, D11 in OsWRKY53 gene overexpression 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 body；

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

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

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

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

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

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 body；

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

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

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

Figure 18 is the picture that the BR of external source tests oswrky53 mutant Leaf angle sensitivity analysis；Wherein WT is wild Type rice, OsWRKY53 are OsWRKY53 knock out mutants body；

Figure 19 is the statistical result that the BR of external source tests the sensitivity analysis of oswrky53 mutant Leaf angle；Wherein a For wild rice, b is OsWRKY53 knock out mutants body；

Figure 20 is the expression characteristic analysis of OsWRKY53 gene pairs BR response on transcriptional level；Wherein b is OsWRKY53 base Cause, c are OsDWF4 gene；

Figure 21 is on protein level, and OsWRKY53 analyzes the expression characteristic that BR is responded.

Specific embodiment

Specific embodiment 1: the nucleotide sequence of the positive regulatory factor OsWRKY53 gene of present embodiment rice BR signal As shown in SEQ ID NO:1 in sequence table.

Present embodiment discloses the positive regulatory factor OsWRKY53 gene of rice BR signal and its coding albumen, this embodiment party Formula clones rice transcription factor OsWRKY53 gene using PCR method from rice.The OsWRKY53 that present embodiment obtains The LOC_Os05g27730.1 phase announced in gene coding region full length sequence and Rice Genome Annotation Project It is corresponding.Present embodiment is by genetic transformation means, by OsWRKY53 gene overexpression in rice, and it was found that OsWRKY53 gene overexpression transgenic paddy rice Leaf angle significantly increases, seed increases, and shows the phenotype of BR signal enhancing；It is logical It crosses CRISPR/Cas9 knockout technology and obtains OsWRKY53 knock out mutants body, oswrky53 mutant shows Leaf angle change Small, seed becomes smaller, plant height becomes the phenotypes of BR signal defects such as short a series of.The detection of BR biosynthesis gene, external source BR press from both sides leaf Angle sensitivity analysis experiment and OsWRKY53 test the response that external source BR is handled, and all sufficiently show that OsWRKY53 can be positive Regulate and control BR signal.

Present embodiment finds that rice transcription factor OsWRKY53 gene being capable of positive regulation BR signal and rice strain for the first time Type.Discovery of the rice transcription factor OsWRKY53 as the positive regulatory factor of BR signal, enriches and perfect water to a certain extent Rice BR signal transduction pathway to transformation plant type of rice, and then improves crop yield and provides Important Theoretic Foundation, has wide answer Use prospect.

Specific embodiment 2: the albumen of the present embodiment coding positive regulatory factor OsWRKY53 gene of rice BR signal Amino acid sequence is as shown in SEQ ID NO:2 in sequence table.

Pass through following experimental verification effect of the invention:

Embodiment 1,

1, the clone of the positive regulatory factor OsWRKY53 gene of rice BR signal and sequencing:

One, using wild rice kind OryzasativaLcv.Nipponbare as experimental material, according to the TRIzol kit of Invitrogen company Operation manual extracts blade total serum IgE；

Two, using the extracted total serum IgE of I processing step of DNase one；

Three, take 1 μ g step 2 treated total serum IgE to be used for the synthesis of cDNA, the synthetic operation of cDNA is according to purchase from BD The BD SMART of Biosciences Clontech company^TMRACE cDNA Amplification Kit kit uses hand Volume carries out, and obtains cDNA；

Four, using the cDNA of above-mentioned acquisition as template, referring to TaKaRa companyHS DNAPolymerase operational manual expands OsWRKY53 gene with forward primer F1 and reverse primer R1.PCR reaction condition It is as follows: 95 DEG C of initial denaturation 5min；95 DEG C of denaturation 30s, 58 DEG C of annealing 30s, 72 DEG C of extension 90s 38 are recycled totally；72 DEG C extend eventually 10min.Finally, PCR product is sequenced in ABI3130 sequenator (ABI company), sequencing result shows that rice BR believes The SEQ ID No:1 of number positive regulatory factor OsWRKY53 gene by 1464 base compositions, in nucleotide sequence such as sequence table It is shown.Encode the protein with the amino acid sequence of SEQ ID NO:2 in sequence table.

Forward primer F1:5'-ATGGCGTCCTCGACGGGG-3'

Reverse primer R1:5'-CTAGCAGAGGAGCGACTCGACG-3'

2, the acquisition of OsWRKY53 gene overexpression transgenic paddy rice

One, vector construction: using OryzasativaLcv.Nipponbare cDNA as template, referring to TaKaRa companyHS DNA Polymerase operational manual expands the volume of OsWRKY53 gene using forward primer F2 and reverse primer R2 as amplimer Code area, and amplified fragments clone is entered into plant over-express vector PC1390U, form a Ubiquitin promoter driving OsWRKY53 gene overexpression carrier.

Forward primer F2:5'-GTTACTTCTGCACTAGGTACCATGGCGTCCTCGACGGGG-3'

Reverse primer R2:5'-TCTTAGAATTCCCGGGGATCCCTAGCAGAGGAGCGACTCGACG-3'

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

Three, after growing bacterium colony, bacterium colony PCR identification 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 cultivates 16h or so, and bacterium solution at this time can use 30% Glycerol is saved by the volume ratio of 1:1, is deposited to -80 DEG C of refrigerators, when infecting callus, is activated i.e. from -80 DEG C of taking-ups It can.

Four, Agrobacterium infects Rice Callus: taking out purpose from -80 DEG C of refrigerators and deposits bacterium, contains in the ratio addition of 1:100 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 can take out to the same color of orange juice (OD=1.0 or so) is visually appeared as from incubator；Take 500ul left Right bacterium solution is into 1.5ml centrifuge tube, 5000rpm, 28 DEG C, is centrifuged 3min, abandons supernatant, it can be seen that tube bottom has the cenobium of white； The liquid for the acetosyringone (purchased from Aldrich) for containing 20ug/ml with 300ul trains culture medium altogether and gently blows and beats tube bottom cenobium, It is set uniformly to suspend in liquid medium；The good callus of growth conditions is selected into 50ml centrifuge tube, about extremely Centrifuge tube scale 5ml or so；The liquid that the acetosyringone that 20ml contains 20ug/ml is added trains culture medium altogether, then will be above-mentioned The 300ul bacterium solution to have suspended is all added in 50ml centrifuge tube；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 extra culture Base, this process take around 1min or so；One layer of culture medium upper berth filter paper is trained altogether in solid, is impregnated with filter paper, it then will be upper The callus infected is stated to shift on so far solid medium；28 DEG C dark culture 2~3 days.

Five, infected the renewal cultivation of Rice Callus: the callus dark culture infected is after 2~3 days, by callus particle It is transferred in 50ml centrifuge tube；With the sterile water wash callus 4 containing 400ug/ml carboxylic Bian penicillin (being purchased from Amresco) ~5 times, continue 1min or so every time, carries out degerming；It uses sterile water wash callus 2~3 times again, is transferred to and is 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 artificial climate incubator (optical culture for 24 hours) renewal cultivation 4~5 days.

Six, 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 (purchased from Roche)；By its It is transferred to culture 30 days or so in 28 DEG C of artificial climate incubators (optical culture for 24 hours).

Seven, the kanamycin-resistant callus tissue on screening and culturing medium the differentiation culture of resistant rice callus: 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 (optical culture for 24 hours) is placed it in, i.e., Transgenic seedling can be differentiated.

Eight, the identification of transgenic seedling: after differentiating transgenic seedling, need to identify it, exclude false positive.First into Row paddy DNA slightly mentions；It is template with the DNA slightly mentioned above, is said according to the EasyTaq DNAPolymerase of Quan Shi King Company 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 rice, it can be seen that be overexpressed and turn Trans-genetic hybrid rice shows the phenotype of BR signal enhancing；Its Leaf angle significantly increases, and the Leaf angle of sword-like leave is more than 100 °, and wild 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 significantly increases, and measures it The wide discovery of grain length grain, compared with wild type both significant elongated (as shown in Fig. 4,5 and 6), OsWRKY53 gene is very likely Positive regulation BR signal.

3, the expression quantity detection of BR biosynthesis gene

One, it cultivates to two weeks sizes, takes for experimental material with OsWRKY53 gene overexpression transgenic paddy rice and its control The blade of same area extracts blade total serum IgE referring to the operation manual of purchase from the TRIzol kit of Invitrogen company；

Two, the total serum IgE extracted using I processing step one of DNase；

Three, take 1 μ g step 2 treated total serum IgE to be used for the synthesis of cDNA, the synthetic operation of cDNA is according to purchase from BD The BD SMART of Biosciences Clontech company^TMRACE cDNA Amplification Kit kit uses hand Volume carries out；

Four, pass through 3 BR biosynthesis gene primers using the cDNA of acquisition as template: D2 gene (forward primer F4 and anti- To primer R4), OsDWF4 gene (forward primer F5 and reverse primer R5) and D11 gene (forward primer F6 and reverse primer 6), Rice internal reference actin (forward primer F7 and reverse primer R7), using SYBR Green PCRmaster mix (TransStart) Quantitative real-time PCR is carried out；Data are from Bio-Rad chromo 4real-time It is obtained on PCR detector；With 2^-△△CTMethod analyzes multiple variation.

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 gene D2, OsDWF4, D11 in OsWRKY53 gene overexpression transgenosis Expression characteristic in rice and its control.The results show that it is raw to be overexpressed 3 BR in transgenic paddy rice compared with compareing wild type The expression quantity of object synthesis gene significantly reduces, and illustrates that its endogenous BR signal is enhancing in being overexpressed transgenic paddy rice, Tentatively illustrate that OsWRKY53 gene being capable of positive regulation BR signal.

4, the acquisition of oswrky53 mutant

One, by the CDS sequence inputting CRISPR Primer Designer software of OsWRKY53 gene, 2 pairs of target position are designed Point primer (F8 and R8；F9 and R9), the building for subsequent 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'

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

Three, this 2 target practice connectors are connected respectively on each gRNA expression cassette.

PCR program are as follows: 37 DEG C of 5min, 20 DEG C of 5min, 5 circulations.

Four, gRNA expression cassette is expanded by the method for nest-type PRC

First round PCR amplification

PCR program: 98 DEG C of 2min；98 DEG C of 10s, 60 DEG C of 10s, 72 DEG C of 20s, totally 25 recycle；72℃5min.Second wheel PCR amplification:

PCR program: 98 DEG C of 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 are as follows:

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'

Five, the segment for obtaining above-mentioned nest-type PRC connects on pYLCRSPR/Cas9-MT carrier framework, that is, completes to strike Except the building of carrier.

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

T₄0.1 μ L of DNA enzymatic (NEB)

10x DNA ligase buffer(NEB) 1.5μL

PCR program: 37 DEG C of 5min, 10 DEG C of 5min, 20 DEG C of 5min, totally 15 recycle.

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

Seven, purpose carrier converts Agrobacterium EHA105；

Eight, OsWRKY53 knock out mutants body is obtained using agrobcterium-mediated transformation.

As shown in figure 8, being the sequencing knot of two kinds of OsWRKY53 knock out mutants bodies oswrky53-1 and oswrky53-2 Fruit, the sequence of WT are as follows: the sequence of 5'-ATCCCGGCT CAGAGGTACGACTGGAAG-3', oswrky53-1 (- CAGAGGT) Are as follows: the sequence of 5'-ATCCCGGCT-------ACGACTGGAAG-3', oswrky53-2 (+A) are as follows: 5'-ATCCCGGCT ACAGAGGTACGACTGGAAG-3' is missing from 7bp and insertion 1bp respectively, causes subsequent frameshift mutation, make the ammonia given expression to Base acid changes, and OsWRKY53 protein function is 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；Firstly, oswrky53 mutant Leaf angle becomes smaller, 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 becomes smaller, and measures its wide discovery of grain length grain, both significantly shorten (such as Figure 12,13 compared with wild type 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 signal.

5, sensitivity analysis of the external source BR to Leaf angle

One, seed disinfection: selecting OsWRKY53 gene overexpression transgenic paddy rice and its control is experimental material, seed stripping Skin impregnates 1min with 70% ethyl alcohol；Then it is impregnated twice with 30% hypochlorous acid, each 15min；Finally use sterile water wash 5 ~7 times；

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

Three, the Leaf angle (include blade, each 1cm of leaf sheath) for choosing second leaf, with various concentration 24-epiBL (Sigma, E1641) the dark culture 48h in 30 DEG C of incubators；

Four, observation is as a result, and carry out the measurement of Leaf angle with ImageJ software.

As shown in figure 16, it is OsWRKY53 gene overexpression transgenic paddy rice and its to impinging upon various concentration 24-epiBL Photo in (Sigma, E1641) after processing 48h, Figure 17 are various concentration 24-epiBL treatment process middle period included angle statistics As a result.The results show that the Leaf angle for being overexpressed transgenic paddy rice reaches 130 ° or so, and right after being handled with 10nM 24-epiBL According to only 60 ° or so；Also, with the raising of 24-epiBL concentration for the treatment of, the phenotype that Leaf angle increases 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 of oswrky53 mutant is pressed from both sides Angle only reaches 50 ° or so, and compares and reach 140 ° or so.Further experiment is provided for OsWRKY53 gene positive regulation BR signal Evidence.

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

One, it selects wild rice kind dragon round-grained rice 11 for experimental material, after presoaking and germinating, the seed sowing of sprouting is being cut It in the 96 hole PCR plates at bottom, is then immersed in 1/2MS fluid nutrient medium, 28 DEG C of illumination box (optical culture 14h；Dark culture 10h) Middle culture 2 weeks；

Two, the consistent rice seedling of growth conditions is selected, handles 0h, 1h, 3h, 6h respectively with 1 μM of 24-epiBL；

Three, the blade of same area is taken, the operation manual from the TRIzol kit of Invitrogen company is bought in reference, Extract blade total serum IgE；

Four, the total serum IgE extracted using I processing step three of DNase；

Five, take 1 μ g step 4 treated total serum IgE to be used for the synthesis of cDNA, the synthetic operation of cDNA is according to purchase from BD The BD SMART of Biosciences Clontech company^TMRACE cDNA Amplification Kit kit uses hand Volume carries out, and obtains cDNA；

Six, using the cDNA of acquisition as template, forward primer F10 and reverse primer R10 are detection primer, detect OsWRKY53 The expression of gene.

Forward primer F10:5'-AGACCTTCAACACCCCTGCTATG-3'

Reverse primer R10:5'-TCACGCCCAGCAAGGTCG-3'

It as shown in figure 20, is to the expression characteristic of BR response on OsWRKY53 gene transcription level, BR can as the result is shown Inhibit the expression on OsWRKY53 transcriptional level, and the BR processing time is longer, inhibition level is more obvious.

7, on protein level, OsWRKY53 analyzes the expression characteristic that BR is responded

One, selecting the homozygous OsWRKY53 gene overexpression transgenic paddy rice with MYC label is experimental material, seed soaking After vernalization, the seed of sprouting is sowed in the 96 hole PCR plates for having cut bottom, is then immersed in 1/2MS fluid nutrient medium, at 28 DEG C Incubator (optical culture 14h；Dark culture 10h) middle culture 2 weeks；

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

Three, the blade for taking same area extracts leaves total protein referring to the SDS lysate specification of green skies company；

Four, 1 × loading buffer is added, boils 5min, high speed centrifugation 10min；

Five, loading carries out SDS-PAGE electrophoresis；

Six, after electrophoresis, transferring film is carried out, it will be on the protein delivery on glue to pvdf membrane (Bio-Rad)；

Seven, after to transferring film, subsequent western hybridization is carried out with MYC antibody (Abmart:M20002L).

It as shown in figure 21, is the expression characteristic that OsWRKY53 responds BR on protein level.The results show that BR can be lured Lead the expression on OsWRKY53 protein level.As the positive regulatory factor of BR signal, the accumulation on protein level is more, BR signal Export stronger, but plant is again there is self negative-feedback balance adjustment mechanism, when BR signal is by force to a certain extent in plant Afterwards, plant is able to suppress the expression on the positive regulatory factor transcriptional level of these BR signals again, to maintain BR signal in plant Balance.

In conclusion the present embodiment is by genetic transformation means, by OsWRKY53 gene overexpression in rice, and It was found that OsWRKY53 gene overexpression transgenic paddy rice Leaf angle significantly increases, seed increases, the table of BR signal enhancing is shown Type；Technology is knocked out by CRISPR/Cas9 and obtains OsWRKY53 knock out mutants body, and oswrky53 mutant shows leaf folder Angle becomes smaller, seed becomes smaller, plant height becomes the phenotypes of BR signal defects such as short a series of.The detection of BR biosynthesis gene, BR pairs of external source Leaf angle sensitivity analysis experiment and OsWRKY53 test the response that external source BR is handled, and all sufficiently show that OsWRKY53 can Positive regulation BR signal.

Sequence table

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

<120>the positive regulatory factor OsWRKY53 gene of rice BR signal and its coding albumen

<160> 28

<210> 1

<211> 1464

<212> DNA

<213>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>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>nucleotide sequence of PCR primer F1.

<400> 3

ATGGCGTCCT CGACGGGG 18

<210> 4

<211> 22

<212> DNA

<213>artificial sequence

<220>

<223>PCR primer R1 nucleotide sequence.

<400> 4

CTAGCAGAGG AGCGACTCGA CG 22

<210> 5

<211> 39

<212> DNA

<213>artificial sequence

<220>

<223>nucleotide sequence of PCR primer F2.

<400> 5

GTTACTTCTG CACTAGGTAC CATGGCGTCC TCGACGGGG 39

<210> 6

<211> 43

<212> DNA

<213>artificial sequence

<220>

<223>nucleotide sequence of PCR primer P2.

<400> 6

TCTTAGAATT CCCGGGGATC CCTAGCAGAG GAGCGACTCG ACG 43

<210> 7

<211> 19

<212> DNA

<213>artificial sequence

<220>

<223>nucleotide sequence of PCR primer F3.

<400> 7

TGCGCCCAAG CTGCATCAT 19

<210> 8

<211> 21

<212> DNA

<213>artificial sequence

<220>

<223>nucleotide sequence of PCR primer R3.

<400> 8

TGAACTCACC GCGACGTCTG T 21

<210> 9

<211> 18

<212> DNA

<213>artificial sequence

<220>

<223>nucleotide sequence of PCR primer F4.

<400> 9

TCGCTGACGG AGCTGATG 18

<210> 10

<211> 20

<212> DNA

<213>artificial sequence

<220>

<223>nucleotide sequence of PCR primer R4.

<400> 10

ACTTGAGGTG GGAGGACTTG 20

<210> 11

<211> 19

<212> DNA

<213>artificial sequence

<220>

<223>nucleotide sequence of PCR primer F5.

<400> 11

CTCCACCTTC TCCGCTCAG 19

<210> 12

<211> 17

<212> DNA

<213>artificial sequence

<220>

<223>nucleotide sequence of PCR primer R5.

<400> 12

GCCGCTCCGT CTCTTCC 17

<210> 13

<211> 21

<212> DNA

<213>artificial sequence

<220>

<223>nucleotide sequence of PCR primer F6.

<400> 13

TGGCGACATT GAGAAGATTG C 21

<210> 14

<211> 21

<212> DNA

<213>artificial sequence

<220>

<223>nucleotide sequence of PCR primer R6.

<400> 14

CAGAAGGCGA TGACATTGAC C 21

<210> 15

<211> 23

<212> DNA

<213>artificial sequence

<220>

<223>nucleotide sequence of PCR primer F7.

<400> 15

AGACCTTCAA CACCCCTGCT ATG 23

<210> 16

<211> 18

<212> DNA

<213>artificial sequence

<220>

<223>nucleotide sequence of PCR primer R7.

<400> 16

TCACGCCCAG CAAGGTCG 18

<210> 17

<211> 24

<212> DNA

<213>artificial sequence

<220>

<223>nucleotide sequence of PCR primer F8.

<400> 17

GGCATTCCAG TCGTACCTCT GAGC 24

<210> 18

<211> 24

<212> DNA

<213>artificial sequence

<220>

<223>nucleotide sequence of PCR primer R8.

<400> 18

AAACGCTCAG AGGTACGACT GGAA 24

<210> 19

<211> 24

<212> DNA

<213>artificial sequence

<220>

<223>nucleotide sequence of PCR primer F9.

<400> 19

GCCGAGCTGG AGGACGGGTA CAAC 24

<210> 20

<211> 24

<212> DNA

<213>artificial sequence

<220>

<223>nucleotide sequence of PCR primer R9.

<400> 20

AAACGTTGTA CCCGTCCTCC AGCT 24

<210> 21

<211> 23

<212> DNA

<213>artificial sequence

<220>

<223>nucleotide sequence of PCR primer F10.

<400> 21

AGACCTTCAA CACCCCTGCT ATG 23

<210> 22

<211> 18

<212> DNA

<213>artificial sequence

<220>

<223>nucleotide sequence of PCR primer R10.

<400> 22

TCACGCCCAG CAAGGTCG 18

<210> 23

<211> 22

<212> DNA

<213>artificial sequence

<220>

<223>nucleotide sequence of PCR primer U-F.

<400> 23

CTCCGTTTTA CCTGTGGAAT CG 22

<210> 24

<211> 20

<212> DNA

<213>artificial sequence

<220>

<223>nucleotide sequence of PCR primer gRNA-R.

<400> 24

CGGAGGAAAA TTCCATCCAC 20

<210> 25

<211> 38

<212> DNA

<213>artificial sequence

<220>

<223>nucleotide sequence of PCR primer B1'.

<400> 25

TTCAGAGGTC TCTCTCGCAC TGGAATCGGC AGCAAAGG 38

<210> 26

<211> 37

<212> DNA

<213>artificial sequence

<220>

<223>nucleotide sequence of PCR primer B2.

<400> 26

AGCGTGGGTC TCGTCAGGGT CCATCCACTC CAAGCTC 37

<210> 27

<211> 38

<212> DNA

<213>artificial sequence

<220>

<223>nucleotide sequence of PCR primer B2'.

<400> 27

TTCAGAGGTC TCTCTGACAC TGGAATCGGC AGCAAAGG 38

<210> 28

<211> 37

<212> DNA

<213>artificial sequence

<220>

<223>nucleotide sequence of PCR primer BL.

<400> 28

AGCGTGGGTC TCGACCGGGT CCATCCACTC CAAGCTC 37

Claims

Application of the 1.OsWRKY53 gene in positive adjusting and controlling rice BR signal, it is characterised in that the nucleotide sequence of the gene such as sequence In list shown in SEQ ID NO:1.
2. application according to claim 1, it is characterised in that the albumen of OsWRKY53 gene coding described in claim 1 Amino acid sequence as shown in SEQ ID NO:2 in sequence table.