CN111662367A - Rice bacterial leaf blight-resistant protein and coding gene and application thereof - Google Patents
Rice bacterial leaf blight-resistant protein and coding gene and application thereof Download PDFInfo
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- A—HUMAN NECESSITIES
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Abstract
The invention discloses a rice bacterial leaf blight resistant protein, and a coding gene and application thereof. The rice bacterial leaf blight resistant protein is Xa7 protein, and the amino acid sequence of the rice bacterial leaf blight resistant protein is shown in SEQ ID NO. 1. The nucleotide sequence of the gene for coding the rice bacterial leaf blight resistant protein is shown as SEQ ID NO. 2. The cloning of the Xa7 functional gene is finally completed by constructing a genome BAC library of a rice variety IRBB7, screening the library, sequencing a candidate insert, predicting an AvrXa7 recognition site by a target insert sequence, and performing a series of transgenic function complementation tests and gene knockout tests. The invention provides the Xa7 functional gene sequence for the first time, which can be used for researching the mechanism of resisting bacterial leaf blight of rice, cultivating rice varieties with disease resistance to bacterial leaf blight of rice or other disease-resistant crops, or breeding rice varieties with disease resistance to bacterial leaf blight of rice.
Description
Technical Field
The invention belongs to the technical field of biology, and particularly relates to a rice bacterial leaf blight resistant protein, and a coding gene and application thereof.
Background
Bacterial leaf blight (Xanthomonas oryzae pv. oryzae) is one of the main diseases of rice in southeast Asia in China and the main rice producing areas in the world, and seriously threatens the safe rice production. The utilization of host resistance is an effective measure for controlling the disease. However, due to pathogenic variation of pathogenic bacteria, the variety resistance is often lost, the persistence of the variety resistance and the mechanism thereof become a key point of the current disease resistance research, the persistent disease resistance molecular mechanism is deeply known, the persistent resistance is obtained for rice varieties, and the important significance is provided for continuously and effectively controlling diseases [ Wushangkai, 1982, rice bacterial blight and prevention thereof, Shanghai science and technology publishers; mew,1987, Current status and future protocols of research on bacterial light of rice, Ann.Rev.Phytopathol, 25:359, 382 ]. Among the identified bacterial blight resistance genes, Xa7 is considered to be a persistent disease resistance gene effective against different pathogenic bacteria and exhibiting excellent and stable resistance in numerous countries throughout the world [ Ona et al, 1998, Epidemic level of bacterial lighting restriction genes Xa-4, Xa-7, and Xa-10.Plant Dis.,82:1337-1340 ]; adhikari et al, 1999, Virus of Xanthomonas oryzae pv. oryzae on cellulose associations single resistance genes and gene combinations plant Dis.,83: 46-50; vera et al, 2000, differentiating duration of a discrete resistance generated on an assessment of the fine loss and elementary local control of the error gene mutation, Proc. Natl.Acad.Sci.,97: 13500-; the study on the resistance of a rice bacterial strain to a southern China bacterial strain by a bacterial strain near isogenic line for resisting bacterial blight, previously listed, 2006, 36:177-180 ℃.
The Xa7 gene was originally identified by the International Rice institute (IRRI) on rice variety DV85 [ Sidhu et al, 1978Genetic analysis of bacterial clearance resistance in the foundation-grassroots of rice, Oryza sativa L.the or apple Genet,53:105-111 ]. Ogawa et al introduced gene Xa7 into the near isogenic line IRBB7 by repeated backcrosses of DV85 with IR24 [ Ogawa et al, 1991, Breedenggof near-isogenic lines of rice with single genes for resistance to microbial light strain J Breed,41:523 pn 529 ]. Research by Hopkins et al showed that Xa7 is a dominant resistance gene directly corresponding to an avirulence gene family [ Hopkins et al, 1992, Identification of a family of genes from Xanthomonas oryzae pv. oryzae. mol Plant Microbe Interact,5: 451. sup. -, Kaji and Ogawa located the gene marker at position 107.5cM on chromosome 6 RGP map, the recombination rate with the G1091 marker was 8% [ Kaji R and ogawa T.identification of the located chromosome of the resistance gene, Xa-7, to bacterial leaf height in rice.Breed.Sci.,1995,45(suppl.1):79.], Porter et al fine-positioned Xa7, however, since the sequencing of rice genomes was now perfect, candidate genes for the target gene region could not be analyzed and predicted [ Porter et al, 2003, Development and mapping of markers linking and the line background gene Xa7.crop Science,43: 1484-. The subject group, by large population analysis, more finely bound the Xa7 gene between the molecular markers GDSSR02 and RM20593, also because of the limitation of representativeness of the sequenced varieties in the rice genome, the gene has a large Gap (Gap) in the located region, and the target gene still cannot be cloned [ Chen et al, High-resolution mapping and gene prediction of Xanthomonas oryzae pv. Oryzae resistance gene Xa7.molecular Breeding,2008,3: 433-.
Disclosure of Invention
The invention aims to overcome the defects in the prior art and provide a rice protein for resisting bacterial leaf blight.
Another object of the present invention is to provide a gene encoding the above rice protein against bacterial blight.
It is still another object of the present invention to provide a promoter region pathogen-inducible regulatory element of the above-mentioned gene.
The invention also aims to provide application of the protein, the gene and the promoter region pathogen induction regulatory element.
The purpose of the invention is realized by the following technical scheme: a rice bacterial leaf blight resistant protein is an Xa7 protein, and the amino acid sequence of the rice bacterial leaf blight resistant protein is as follows:
MAAADHPDRMPVAVAGLRHHYAFPANLRPAARLLTVNSGVFLISTAGAIVLVHTAGNPPAIDNDPAYALVAFVLFLLGIWLMSIALVADQFPRAAGVAVAIARALQDYLIGGN。
the gene for coding the rice bacterial blight resistant protein is named as Xa7 gene, and the nucleotide sequence of the gene is shown as follows:
ATGGCGGCCGCTGATCATCCTGATCGTATGCCCGTTGCAGTTGCAGGCTTGCGCCACCATTACGCCTTCCCTGCAAACCTTCGCCCCGCCGCTCGACTGCTGACCGTCAACTCCGGCGTCTTCCTCATCTCCACCGCCGGGGCCATCGTCCTCGTCCACACCGCCGGTAACCCACCCGCCATCGACAACGATCCAGCCTACGCCTTGGTCGCATTCGTGCTCTTCCTCCTCGGAATCTGGCTCATGTCTATTGCCCTCGTCGCCGACCAGTTCCCGCGCGCCGCTGGGGTCGCCGTGGCCATTGCCAGGGCGCTGCAGGATTACCTCATCGGTGGCAATTAA。
the promoter region pathogen induction regulatory element of the gene for coding the rice bacterial blight-resistant protein has the following nucleotide sequence: TATAACCCCCCCCCCCCCAGATAACCA are provided.
The rice bacterial leaf blight resistant protein can be obtained by chemical synthesis; or cloning the gene for encoding the rice bacterial leaf blight resistant protein into an expression vector to obtain a recombinant expression vector, transforming host cells by the obtained recombinant expression vector, and purifying after expression.
The preparation of the gene for coding the rice bacterial leaf blight resistant protein can be realized by the following modes: obtained by a chemical synthesis mode; or designing a primer, and carrying out PCR amplification by using DV85, IRBB7 or other rice variety genome DNA carrying Xa7 gene as a template; or obtained by enzyme digestion and screening from a plasmid carrying the Xa7 gene.
The application of the gene for coding the protein resisting bacterial blight of rice can be used for researching a mechanism of resisting bacterial blight of rice, can also be used for cultivating rice varieties with disease resistance to bacterial blight of rice or other disease-resistant crops, or can be used as a molecular marker for breeding the rice varieties with disease resistance to bacterial blight of rice.
The steps for cultivating the rice variety with disease resistance to the bacterial blight of the rice or other disease-resistant crops are preferably as follows: introducing the gene for coding the rice bacterial leaf blight resistant protein and the promoter region pathogen induction regulation element into susceptible rice or other crops to obtain rice or disease resistant crops; or connecting the constitutive expression promoter or other pathogenic inducible promoters with the coding sequence of the gene in series, and introducing the promoter into susceptible rice or other crops to obtain rice or disease-resistant crops.
The steps for breeding the rice variety with disease resistance to the bacterial blight of the rice are preferably as follows: the rice variety carrying the gene is used as a donor parent and is subjected to pollen hybridization with a rice variety susceptible to bacterial blight, and a series of obtained offspring are screened by using Xa7 as a molecular marker, so that the bacterial blight resistant rice variety is identified.
The donor parent is preferably DV85 or IRBB 7.
The application of the promoter region pathogen induction regulation element of the gene for coding the rice bacterial leaf blight resistant protein can be used for researching a rice bacterial leaf blight resistant mechanism and can also be used for cultivating rice varieties with disease resistance to rice bacterial leaf blight or other disease resistant crops.
The steps for cultivating the rice variety with disease resistance to the bacterial blight of the rice or other disease-resistant crops are preferably as follows: introducing the gene for coding the rice bacterial leaf blight resistant protein and the promoter region pathogen induction regulation element into susceptible rice or other crops to obtain rice or disease resistant crops; or the promoter region pathogen inducing and regulating element is connected serially with other disease resisting gene coding sequence and introduced into rice or other crop susceptible to disease to obtain rice or other disease resisting crop with disease resistance.
Compared with the prior art, the invention has the following advantages and effects:
on the basis of earlier stage research, the cloning of the Xa7 functional gene is finally completed by constructing a genome BAC library of a rice variety IRBB7, library screening, candidate insert sequencing, prediction of an AvrXa7 recognition site by a target insert sequence, and a series of transgenic function complementation tests and gene knockout tests. The invention provides the sequence of the Xa7 functional gene for the first time.
Drawings
FIG. 1 is a schematic diagram of the position of subcloned fragments and the phenotype of disease resistance of transgenic lines; wherein, the picture A is a schematic diagram of the position and the sequence of an overlapping region of a subcloned fragment used for a transgenic function complementation experiment, and the picture B is a photo picture of resistance phenotype of a subcloned transgenic rice line to the bacterial blight strain PXO 86; FIG. C is a statistical result chart of lesion length of the subcloned transgenic rice line against P.albuginea PXO 86.
FIG. 2 is a diagram showing structural features and resistance expression patterns of Xa7 gene; wherein, the picture A is a schematic diagram of the structural characteristics of the Xa7 gene sequence, and the picture B is a pattern diagram of the resistance expression of the Xa7 gene to P.albuginea PXO 86.
FIG. 3 is a diagram showing the result of the knockout function verification of the pathogenic inducing element in the promoter region of the Xa7 gene; wherein, the picture A is a mutation homozygous line sequence after gene editing is carried out on the Xa7 gene promoter region pathogen inducing element, the picture B is a resistance expression pattern picture of each mutation line to the bacterial blight strain PXO86, and the picture C is a picture of a disease-resistant phenotype picture of each mutation line to the bacterial blight strain PXO 86.
FIG. 4 is a diagram showing the result of verifying the knock-out function of the coding region of Xa7 gene; wherein, the picture A is the mutation homozygous line sequence after the Xa7 gene coding region carries on the gene editing, the picture 4 is the resistance expression pattern to the bacterial blight PXO86 of each mutant line, the picture C is the disease-resistant phenotype to bacterial blight PXO86 of each mutant line.
Detailed Description
The present invention will be described in further detail with reference to examples and drawings, but the present invention is not limited to these examples. Unless otherwise specified, the technical means used in the examples are conventional means well known to those skilled in the art. In the examples section of the present invention, isolated cloning of the Xa7 gene, functional characterization and functional verification thereof are illustrated.
EXAMPLE 1 isolation of the Xa7 Gene
On the basis of earlier stage research, the invention finds that the genome sequence near the Xa7 gene locus is greatly different from the common rice reference genomes Nipponbare, 9311, Minghui 63, Zhenshan 97 and the like. The definition of the genome sequence of the region is the prerequisite basis for realizing the gene cloning. The invention obtains the complete and accurate genome sequence of the region by constructing an Xa7 anti-source variety IRBB7 genome BAC library, screening the library by using the tightly linked molecular markers at two sides of Xa7, fishing positive clone and sequencing the insert of the positive clone.
Rice variety IRBB7 has been disclosed in the literature "Ogawa et al, 1991, Breeding of near-isogenilines of rice with single genes for resistance to bacterial clearance, pathognomonas camphoensis pv. oryzae. Japan J Breeding, 41: 523. 529.").
The plant material for constructing the genome BAC library is an isogenic line IRBB7 containing Xa7 gene, and the carrier is CopyControl of Epicentre companyTMpCC1BACTMThe subcloning and transgenic expression vector was pYLTAC747H/sacB (described herein)The document "Xu et al, 2008, Construction and characterization of the transformation-composition specific chromosome (TAC) libraries of science in China (Series C: Life Sciences), (07):604- & 613').
Construction of BAC library: the procedure was followed according to the experimental procedures reported in "Liu et al, 2002, Development of new transformation-composition specific chromogenes vectors and edge genetic libraries for effect gene cloning. Gene,282(1): 247-. Extracting IRBB7 genome DNA in seedling stage, partially digesting with Hind III restriction endonuclease, separating DNA fragment of 120-140kb length by pulsed field electrophoresis, purifying and mixing with BAC vector pCC1BACTMThe connection is made. 75ng BAC vector was mixed with five groups of genomic DNA cleavage products (30 ng, 60ng, 120ng, 160ng, and 350ng, respectively) according to
Preparing a reaction system by a T4 DNA ligase 50 mu L system, and performing ligation reaction by a temperature-variable ligation procedure in a PCR instrument: 3min at 10 ℃, 3min to 16 ℃, 5min at 16 ℃, 30s to 18 ℃, 30s to 20 ℃, 8s to 4 ℃, 3min at 4 ℃, 5min from 4 ℃ to 22 ℃, 1min at 22 ℃ to 10 ℃, and the cycle is repeated for 20 times, and finally 5min at 65 ℃. Ligation product Using MILLIPORETMThe VSWP membrane (0.025. mu.M) was dialyzed at 4 ℃ against 1/4 × TE for about 2 to 3 hours, and the dialyzed product was transformed into DH10B E.coli competent cells (Invitrogen)TMElectroMAX ofTMDH10BTMCells). Mixing 1 μ L of the dialyzate with 20 μ L of electroporation competent cells, transferring into a pre-cooled 0.1cm electric shock cup, and loading in BioRad
The electric shock on the electric shock instrument is converted (the parameters are that the voltage is 2.0kV, the resistance is 200 omega, and the capacitance is 25 muF). After the electric shock, 1mL of S.O.C. medium was quickly added, and the shaking table at 37 ℃ and 200rpm was used to resume the culture for 1 hour. Taking 10-100 mu L of ten gradient bacterial liquid amounts, respectively spreading the bacterial liquid amounts on LB semisolid culture medium containing 25 mu g/mL kanamycin and 5% sucrose, culturing at 37 ℃ for 12-16 h, and calculating the clone number. Then about 500 clones per plateThe bacterial liquid amount is plated and cultured in the same way, mixed bacterial colonies are scraped from the whole plate and are subpackaged into a 96-hole deep pore plate to construct a BAC clone mixing pool. The total number of clones per well was about 45000 based on approximately 450 original single clones, and the average insert length per clone was 100kb, which resulted in a library coverage of greater than 10 fold, calculated relative to 430Mb of the rice genome.
BAC positive clone screening: and (3) extracting plasmid DNA of each BAC clone mixing pool by an alkali cracking method, and amplifying closely linked molecular markers U05-indel and POZ-indel at two sides of Xa7 by using the plasmids of the mixing pool as a template, wherein the primer pair for amplifying the molecular markers U05-indel is U05-indel Fw and U05-indel Rv, and the primer pair for amplifying the molecular markers POZ-indel is POZ-indel Fw and POZ-indel Rv. And (3) after detecting a positive mixed pool, diluting the positive pool by 40000 times, subpackaging the positive pool into 384-hole plates according to the multiplying power of 5 times per hole, taking a bacterial liquid as a template after culturing overnight, and performing PCR amplification screening by using a corresponding molecular marker primer until positive single clones are screened. After multiple rounds of screening, three positive clones were identified and isolated: P1-10G, P3-12F and P2-9D. The inserts of the three BAC clones were 150kb, 125kb and 107kb, respectively, by restriction and electrophoresis.
U05-indel Fw:5’-CAGACAAGTGTTGTTCATGTTCG-3’；
U05-indel Rv:5’-GAAGTCCGAGCTGGGGACGATGTAC-3’；
POZ-indel Fw:5’-CCAAGAAAGGTCCAACTCGCTTAG-3’；
POZ-indel Rv:5’-GAACAGTCCTCAGAATTCGACCAC-3’。
Sequencing and sequence analysis of positive clone BAC plasmid: plasmids of P1-10G, P3-12F and P2-9D clones were extracted with the BAC/PAC DNA Maxi Kit from Omega to construct a 350bp fragment library, which was subjected to secondary sequencing using the HiSeq PE150 sequencing platform, and the sequencing data volume of each clone was 1 Gb. Sequencing data for Denovo parameterless assembly and eliminating vector pCC1BACTMThe sequences of the three positive plasmids were obtained as insert sequences, respectively. The three insert sequences are assembled by splicing to obtain 307.5kb fragments which are mutually overlapped and staggered end to end. Comparing with international general rice genome sequence, finding that the fragment corresponds to japonica rice varietyThe Japan clear genome is increased by 101.1kb, and the genome of corresponding indica rice variety Minghui 63 is increased by 80 kb. Using the software tool from Softberry (http:// www.softberry.com/berry. phtml&group＝programs&subfroup gfnd) the 307.5kb splicing sequence was predicted, yielding 82 Open Reading Frames (ORFs). To further narrow the target range, the recognition binding site of AvrXa7 (AvrXa7EBE) was predicted in the splice sequence by the TALgetter tool from Galaxy (http:// Galaxy2.informatik. uni-hale. de: 8976). As a result, it was found that the P-Value of four recognition binding sites was less than 1.0E-6Wherein only one AvrXa7EBE is located in the promoter region upstream of the ORF. The ORF is numbered 52, the candidate gene is named CG52, and the sequence is shown in SEQ ID NO. 4.
Construction and functional complementation experiment of BAC subclone library: the inserts of BAC plasmids P1-10G and P3-12F carrying the CG52 gene were incompletely digested with BamH I and Sau3A I, respectively, and ligated to the subcloning vector pYLTAC 747H/sacB. And (3) screening libraries by using CG52 promoter region and CDS region amplification primers respectively, extracting plasmids from positive subclones respectively for end sequencing, and determining the sequence of the insert. Four subcloned BAC plasmids were selected on the basis of sequence information and were mediated by Agrobacterium EHA105 (available from Beijing Huayu Biotechnology Ltd., plasmid transformation Agrobacterium procedure as described in "Hood et al, 1993, New Agrobacterium rhizogenes for gene transfer to plants Res., 2, 208-218"), transformation of indica rice susceptible variety IR24 (rice variety IR24 in "organic et al, 1991.Breeding of rice susceptible genes for gene transfer. Japan J. 41:523-529. published in" Breeding and microorganism strain "547. genetic transformation of indica rice variety J. for gene transfer. expressing. J. 41:523-529. for expression), genetic transformation of indica rice variety reported in" Linhang and culture Collection, eye 2005, culture Collection, 23. for expression of rice variety J. for expression ". Transgenic Rice at the booting stage was inoculated with Rice bacterial strain P.solani PXO86 (the strain is disclosed in "Mew TW et al 1982, Patholomyces of Xanthomonas compensae pv. oryzae in Asia. IRRI Research papers Series, No75") by "leaf cutting" (conducted in "Wood well, et al 1985, Wakan and Philippine Rice bacterial strains pathogenicity comparative study, Phytopathology report, 15-2: 65-72"), and disease resistance phenotype was investigated 21 days after inoculation and evaluated by reference to the method of International Rice Institute INGER (conducted in "International Genetic Resources, 1996, Standard Evaluation for (4th edition) [ M ]. Interilips: Research Rice plant Press 20).
The positional information of the inserts of the four subclones used for functional complementation genetic transformation is shown in FIG. 1A. The full length of the genes CG52 of the cloning coverage rate of L235 and L239, and the two cloned transgenic rice strains both show disease resistance; the sequence of the L236 clone is coincided with the 5 'end of the L235 fragment, the 3' end only comprises a partial promoter region of CG52 gene, and the transgenic rice strain shows susceptibility at the upstream 213bp position of AvrXa7 EBE; and the 3 'end of the L240 clone is coincided with the L235 clone, the 5' end only comprises a CDS region of CG52 and a 13bp UTR sequence, an AvrXa7EBE sequence is deleted, and the transgenic rice strain shows susceptibility. Therefore, CG52 gene is the disease resistance gene, and the promoter region AvrXa7EBE and the complete CDS sequence are the indispensable parts of the gene function. (FIG. 1B and FIG. 1C).
Example 2 sequence Structure and expression characteristics of Xa7 Gene
Sequence structure analysis of Xa7 gene: total RNA was extracted using the near isogenic line IRBB7 carrying the Xa7 gene and passed through InvitrogenTMGeneRacer of (1)TMKit amplified the 5 'and 3' full length of Xa7, respectively. Wherein, the 5 'RACE amplification specific primer is 5'-TGCCACCGATGAGGTAATCCTGC-3', and the 3' RACE amplification specific primer is 5'-CCTCCTCGGAATCTGGCTCATGTC-3'. RACE products by
pEASY ofTMCloning with the Blunt Zero cloning kit. The Transcription Start Site (TSS) of Xa7 was determined to be 104bp upstream of the start codon (ATG) after sequencing; the 3' UTR length of Xa7 was also determined to be 253bp (FIG. 2A).
Expression pattern analysis of Xa7 gene: IRBB7 and IR24 inoculated with Bacillus subtilis (PXO86) by "leaf-cutting method" at booting stageSamples were taken after 0, 1, 3, 5 days, respectively, and total RNA was extracted. With TakaraTMPrimeScript ofTMRT reagentKit with gDNA Eraser reverse transcribes into cDNA
Premix Ex TaqTMII (Tli RNaseHPlus) reagent in Bio-Rad fluorescent quantitative PCR instrument CFX96TMQuantitative analysis of genes was performed as above. By using 2－△△CTThe method calculates the relative expression level of the gene.
The amplification primer pair of the target gene Xa7 is as follows:
Xa7Fw:5’-GATCGTATGCCCGTTGCAGTTGC-3’；
Xa7Rv:5’-GGAGTTGACGGTCAGCAGTCGAG-3’。
the amplification primer pair of the internal reference gene TF2 is as follows:
TF2Fw:5’-GCCTGAAGTGTACTGTACCACCAC-3’；
TF2Rv:5’-CAAAGGGTTCAGAAATGAGGAA GG-3’。
as a result, as shown in FIG. 2B, the expression level of Xa7 gene was very low before inoculation, and the expression level started to be up-regulated 1 day after inoculation, reached a peak 3 days after inoculation, and began to fall back 5 days after inoculation. Thus, the expression pattern of the Xa7 gene is pathogen-inducible.
Example 3 Gene knockout mediated by CRISPR/Cas9 validating the Key functional site of Xa7 Gene
In order to further verify the functions of the Xa7 promoter region AvrXa7EBE and CDS region sequences, the invention also utilizes a CRISPR/Cas9 system to construct gene knockout transgenic strains of the two functional regions. The vector used for gene knockout is a binary expression vector pYLCRISPR/Cas9P provided by Liu dazzling light laboratory of southern China agricultural universityubiH (disclosed in the literature "Ma et al 2015, Aroust CRISPR/Cas9 system for compatible high-efficiency multiplex gene encoding in monol and diot plants. mol. plant.8, 1274-1284.), the intermediate vector pYLsgRNA-OsU6aL (disclosed in the literature" Ma et al 2015, A robust CRISPR/Cas9 system for compatible high-efficiency multiplex gene encoding in monol and diot plants. mol. plant.8, 1274-1284.), pYLsgRNA-OsU3aL (disclosed in the literature "Ma et al 2015, A robust CRISPR/Cas9 system for dependent high-efficiency multiplex gene expression in monocot and diot plants. mol. plant.8, 1274-1284.") and pYLsgRNA-OsU6c (disclosed in the literature "Ma et al 2015, A robust CRISPR/Cas9 system for dependent high-efficiency multiplex gene expression in monocot and diot plants. mol. plant.8, 1274-1284."). The selection and design of the sgRNA targeting sequence of the editing site are assisted by using an online tool CRISPR-P (http:// CRISPR. hzau.edu.cn/CRISPR /).
Firstly, a PAM (polyacrylamide) (proline-cleaving motif) sequence is searched in an Xa7 promoter region AvrXa7EBE and a CDS region through CRISPR-P, an edited target site is selected, and an sgRNA target joint is designed according to the sequence of the target site. The corresponding target adaptor sequences are as follows:
target1 (targeting AvrXa7EBE, Target sequence at positions-126 to-107 in fig. 3A):
OsU6aT1F:5’-gccgTATGTGGTTATCTGGGGGGG-3’；
OsU6aT1R:5’-aaacCCCCCCCAGATAACCACATA-3’；
target2 (targeting AvrXa7EBE, Target sequence located at positions-121 to-102 of fig. 3A):
OsU6aT2F:5’-gccgTTCGTATGTGGTTATCTGG-3’；
OsU6aT2R:5’-aaacCCAGATAACCACATACGAA-3’；
target3 (targeting the CDS region, with the Target sequence at positions +22 to +41 in fig. 4A):
OsU3T3F:5’-ggcaCTGCAACGGGCATACGATC-3’；
OsU3T3R:5’-aaacGATCGTATGCCCGTTGCAG-3’；
target4 (targeting CDS region, Target sequence at positions +94 to +113 in fig. 4A):
OsU6cT4F:5’-tcagCGACTGCTGACCGTCAACTC-3’；
OsU6cT4R:5’-aaacGAGTTGACGGTCAGCAGTCG-3’。
according to the experimental procedures reported in Ma et al 2015, A robust CRISPR/Cas9 system for compatible high-efficiency multiplex gene editing in monocot and dioctotopplants mol.8, 1274-1284, target linkers are respectively connected to corresponding Bsa I enzyme-digested sgRNA intermediate vectors, and a specific sgRNA expression cassette template is obtained through two-round PCR reaction, product annealing and nested PCR amplification. The expression cassette is then assembled into a binary expression vector by ligation. Wherein, Target1 and Target2 construct binary expression vectors respectively and independently, and Target3 and Target4 construct the same binary expression vector together. The three gene editing expression vectors are respectively transferred into IRBB7 varieties through the mediation of EHA105 agrobacterium.
Primers (QCFw: 5'-GAACTGCTCTGCTCAAGTGCCTC-3'; QCrv: 5'-TGCCACCGATGAGGTAATCCTGC-3') for each gene knockout transgenic home line were sequenced after PCR amplification of the target site, and T0Generation, T1And (4) selecting and editing successful homozygous lines.
As shown in FIG. 3A, W6-4 and W7-4 homozygous lines obtained by the editing of transgenes by Target1, and W8-7 and W9-6 homozygous lines obtained by the editing of transgenes by Target2 all produced base deletions in the Xa7 promoter region ArvXa7EBE element, respectively. Analysis of gene expression in these transgenic lines showed (FIG. 3B) that the absence of the ArvXa7EBE element results in a loss of the function of Xa7 expressed by the pathogen, thus conferring susceptibility to the pathogen (FIG. 3C).
The W12-1, W12-6, W13-4 and W15-3 homozygous lines obtained by editing transgenes by Target3 and Target4 generate different types of base deletion, insertion and substitution mutations in the CDS region of an Xa7 gene (FIG. 4A), so that the coded protein after the Xa7 mutation has mutations such as premature termination, frame shift, substitution and the like. Although the CDS region of the gene is mutated so that its transcription is still activated by pathogenic bacteria (FIG. 4B), the resistance of these mutant homozygous lines to pathogenic bacteria is still lost (FIG. 4C).
This example reversely verifies the functional gene of Xa7 on one hand, and clarifies that the disease-resistant function of Xa7 gene is composed of two essential factors: one is the transcriptional activation function of its promoter region, AvrXa7EBE element, under pathogen induction, and the other is the complete Xa7 gene-encoded protein that performs an anti-disease response.
The above embodiments are preferred embodiments of the present invention, but the present invention is not limited to the above embodiments, and any other changes, modifications, substitutions, combinations, and simplifications which do not depart from the spirit and principle of the present invention should be construed as equivalents thereof, and all such changes, modifications, substitutions, combinations, and simplifications are intended to be included in the scope of the present invention.
Sequence listing
<110> institute for plant protection of academy of agricultural sciences of Guangdong province
<120> protein for resisting bacterial blight of rice, and coding gene and application thereof
<130>1
<160>22
<170>SIPOSequenceListing 1.0
<210>1
<211>113
<212>PRT
<213> Rice (Oryza sativa)
<220>
<223> Xa7 protein
<400>1
Met Ala Ala Ala Asp His Pro Asp Arg Met Pro Val Ala Val Ala Gly
1 5 10 15
Leu Arg His His Tyr Ala Phe Pro Ala Asn Leu Arg Pro Ala Ala Arg
20 25 30
Leu Leu Thr Val Asn Ser Gly Val Phe Leu Ile Ser Thr Ala Gly Ala
35 40 45
Ile Val Leu Val His Thr Ala Gly Asn Pro Pro Ala Ile Asp Asn Asp
50 55 60
Pro Ala Tyr Ala Leu Val Ala Phe Val Leu Phe Leu Leu Gly Ile Trp
65 70 75 80
Leu Met Ser Ile Ala Leu Val Ala Asp Gln Phe Pro Arg Ala Ala Gly
85 90 95
Val Ala Val Ala Ile Ala Arg Ala Leu Gln Asp Tyr Leu Ile Gly Gly
100 105 110
Asn
<210>2
<211>342
<212>DNA
<213> Rice (Oryza sativa)
<220>
<223> Xa7 gene
<400>2
atggcggccg ctgatcatcc tgatcgtatg cccgttgcag ttgcaggctt gcgccaccat 60
tacgccttcc ctgcaaacct tcgccccgcc gctcgactgc tgaccgtcaa ctccggcgtc 120
ttcctcatct ccaccgccgg ggccatcgtc ctcgtccaca ccgccggtaa cccacccgcc 180
atcgacaacg atccagccta cgccttggtc gcattcgtgc tcttcctcct cggaatctgg 240
ctcatgtcta ttgccctcgt cgccgaccag ttcccgcgcg ccgctggggt cgccgtggcc 300
attgccaggg cgctgcagga ttacctcatc ggtggcaatt aa 342
<210>3
<211>27
<212>DNA
<213> Rice (Oryza sativa)
<220>
<223> Xa7 gene promoter region pathogen induction regulatory element
<400>3
tataaccccc ccccccccag ataacca 27
<210>4
<211>2337
<212>DNA
<213> Rice (Oryza sativa)
<220>
<223> Xa7 gene genome nucleotide sequence
<220>
<222>(337)..(363)
<223> Xa7 gene promoter region pathogen inducing element nucleotide sequence
<220>
<222>(369)..(472)
<223>5'UTR
<220>
<222>(473)..(814)
<223> exon
<220>
<222>(815)..(1057)
<223>3'UTR
<400>4
aataaaaaaa attctcagtt ctaccggggt tcagttcggc tgcggcctgc agtggctggc 60
aaaaatctcg aactgctctg ctcaagtgcc tcaactggca gagataaatc ctaaaaaaac 120
tgaaaaatag gccggtatcc cagctcttct gctgaaaaaa actgaaaaac tgggtgtaag 180
attgatcgca aaagtacttc tggcacttgt cattttcgcc acttctttgg tctcttcgtc 240
ctatttttga catttctctt cgtccttttt tcttttctct ttcaaacgtg cagtcgccgt 300
cgaggacggt gaaagccctg actgctaaaa ccaatatata accccccccc ccccagataa 360
ccacatacga acgaaggctt tgaagcatcg cacacttgaa gagccccctt cccaaccaca 420
gccagcggtt tccaaactcc acgcctcgct caacctgggg gatccatcat ccatggcggc 480
cgctgatcat cctgatcgta tgcccgttgc agttgcaggc ttgcgccacc attacgcctt 540
ccctgcaaac cttcgccccg ccgctcgact gctgaccgtc aactccggcg tcttcctcat 600
ctccaccgcc ggggccatcg tcctcgtcca caccgccggt aacccacccg ccatcgacaa 660
cgatccagcc tacgccttgg tcgcattcgt gctcttcctc ctcggaatct ggctcatgtc 720
tattgccctc gtcgccgacc agttcccgcg cgccgctggg gtcgccgtgg ccattgccag 780
ggcgctgcag gattacctca tcggtggcaa ttaactagaa gcttcgacca tggctctgca 840
cattcctctg ctccagttgt tcccggcttc ccgtacgtgt gcctgatgat tgtctttctc 900
tgtttatttg gctagtattt taggcttgga agttgaaaaa ctgtaaatct gcttcttttt 960
cccctctgta ctactactag actttctttt ttaagctgac gtcatacaca caccccagat 1020
tcataacgtg tcgtatagta aatgtattcg aggcttgtaa taaaaaggca cccgtaggtg 1080
tatgctctgt tcagtctgat gttctaaata atcaagatac tattagtcgt attttctgct 1140
tcatggcggc agtggtggta atatttcaat tccatacgaa gggtcctcgc tgactcactg 1200
tgtggtgtgc gtgctcattt gctgacatgc taaagtcgga ctatagccag gtgtggccgt 1260
gagggcattc ggtgtactga aaatagtgca aaaacatccg ccgcggtcgc gccaagtttg 1320
tcgagaatac gcacaccgaa ccggcctcac aactgatttc gtcggttttc agttgaaact 1380
tcgctcttta tttttggcag ttcttgttcg tattcgtttt ctttctttct tttttaagaa 1440
ttggaatcga aataaaaaac tccagataca aaaaaaatgt aatcgaatat tgtcgaaacc 1500
ggaaacggag cgagtacgaa ttgacgtgga tacggtaacg aaaatttatc agaatgaaaa 1560
acccctcaaa ctgagttcaa actttatcaa atacatcaca aaacacaatc aattaagatg 1620
ctaacttaaa ttggggtacg ctattattat ggtatatgga gttatggaac tccagaaaat 1680
tccaagaaac tgttaggtaa gttttcgacg gattccacgt tccgactgaa actacaccta 1740
tcgtatccgt tttcgtttct gaaaaaaaaa aaaagaaatc tccgaattgg tttttgtttc 1800
taaaaatagg tttggaatcc aaaaggcttt cctatcgttt tcatcctagt tgtaaacctg 1860
aacataccag taaagcctat tagaacaccc cagcagatgc accatccaat ccaccacctg 1920
agaaatagtg cactggaggg gaaaaccggc cttcagggaa atgggaagga atgagacttt 1980
tagatcttcc tcgcaaaaca gggagttaaa tggacttacg aaggaatcat taattttatt 2040
aaagaaaaaa attaagaagt taaaatctaa attaagaagg caatgtatga aacattgaaa 2100
ataaagcgaa aggaaatggg aaggaatttt cgtttatgac aagtcaactt gtaagagtca 2160
attatctcac caattctgga agcacagtgg gacgtgggtc cccctatagg cctctttgat 2220
gagcccattg gaaagcccat ttatacatgg aataagttca tctgaggtct cttaacttgt 2280
caacgaatcc gattttcatc cctcaaccga aaaaccagac acaacgagtc tctcaac 2337
<210>5
<211>23
<212>DNA
<213> Artificial Sequence (Artificial Sequence)
<220>
<223> primer U05-indel Fw
<400>5
cagacaagtg ttgttcatgt tcg 23
<210>6
<211>25
<212>DNA
<213> Artificial Sequence (Artificial Sequence)
<220>
<223> primer U05-indel Rv
<400>6
gaagtccgag ctggggacga tgtac 25
<210>7
<211>24
<212>DNA
<213> Artificial Sequence (Artificial Sequence)
<220>
<223> primer POZ-indel Fw
<400>7
ccaagaaagg tccaactcgc ttag 24
<210>8
<211>24
<212>DNA
<213> Artificial Sequence (Artificial Sequence)
<220>
<223> primer POZ-indel Rv
<400>8
gaacagtcct cagaattcga ccac 24
<210>9
<211>23
<212>DNA
<213> Artificial Sequence (Artificial Sequence)
<220>
<223> primer Xa7 Fw
<400>9
gatcgtatgc ccgttgcagt tgc 23
<210>10
<211>23
<212>DNA
<213> Artificial Sequence (Artificial Sequence)
<220>
<223> primer Xa7 Rv
<400>10
ggagttgacg gtcagcagtc gag 23
<210>11
<211>24
<212>DNA
<213> Artificial Sequence (Artificial Sequence)
<220>
<223> primer TF2 Fw
<400>11
gcctgaagtg tactgtacca ccac 24
<210>12
<211>24
<212>DNA
<213> Artificial sequence (artificacial sequence)
<220>
<223> primer TF2 Rv
<400>12
caaagggttc agaaatgagg aagg 24
<210>13
<211>24
<212>DNA
<213> Artificial Sequence (Artificial Sequence)
<220>
<223> primer OsU6aT1F
<400>13
gccgtatgtg gttatctggg gggg 24
<210>14
<211>24
<212>DNA
<213> Artificial Sequence (Artificial Sequence)
<220>
<223> primer OsU6aT1R
<400>14
aaaccccccc cagataacca cata 24
<210>15
<211>23
<212>DNA
<213> Artificial Sequence (Artificial Sequence)
<220>
<223> primer OsU6aT2F
<400>15
gccgttcgta tgtggttatc tgg 23
<210>16
<211>23
<212>DNA
<213> Artificial Sequence (Artificial Sequence)
<220>
<223> primer OsU6aT2R
<400>16
aaacccagat aaccacatac gaa 23
<210>17
<211>23
<212>DNA
<213> Artificial Sequence (Artificial Sequence)
<220>
<223> primer OsU3T3F
<400>17
ggcactgcaa cgggcatacg atc 23
<210>18
<211>23
<212>DNA
<213> Artificial Sequence (Artificial Sequence)
<220>
<223> primer OsU3T3R
<400>18
aaacgatcgt atgcccgttg cag 23
<210>19
<211>24
<212>DNA
<213> Artificial Sequence (Artificial Sequence)
<220>
<223> primer OsU6cT4F
<400>19
tcagcgactg ctgaccgtca actc 24
<210>20
<211>24
<212>DNA
<213> Artificial Sequence (Artificial Sequence)
<220>
<223> primer OsU6cT4R
<400>20
aaacgagttg acggtcagca gtcg 24
<210>21
<211>23
<212>DNA
<213> Artificial Sequence (Artificial Sequence)
<220>
<223> primer QC Fw
<400>21
gaactgctct gctcaagtgc ctc 23
<210>22
<211>23
<212>DNA
<213> Artificial Sequence (Artificial Sequence)
<220>
<223> primer QC Rv
<400>22
tgccaccgat gaggtaatcc tgc 23
Claims (9)
1. A rice bacterial leaf blight resistant protein is characterized in that: the amino acid sequence is shown as SEQ ID NO. 1.
2. A gene encoding the rice protein of claim 1, which is resistant to bacterial blight and characterized in that: the nucleotide sequence is shown as SEQID NO. 2.
3. The gene according to claim 2, characterized in that: also comprises a promoter region pathogen induction regulatory element with a nucleotide sequence shown as SEQ ID NO. 3.
4. Use of the gene of claim 2, wherein: the gene is used for researching a mechanism of resisting bacterial blight of rice, and is used for cultivating rice varieties with disease resistance to bacterial blight of rice or other disease-resistant crops, or breeding rice varieties with disease resistance to bacterial blight of rice.
5. Use of a gene according to claim 4, characterized in that: the steps for cultivating the rice variety with disease resistance to the bacterial blight of the rice or other disease-resistant crops are as follows: introducing the gene of claim 2 and the promoter region pathogen-induced regulatory element of claim 3 into susceptible rice or other crops to obtain disease-resistant rice or disease-resistant crops; or connecting a constitutive expression promoter or other pathogen inducible promoters with the gene of claim 2 in series, and introducing the gene into susceptible rice or other crops to obtain rice or disease-resistant crops.
6. Use of a gene according to claim 4, characterized in that: the steps for breeding the rice variety with disease resistance to the bacterial blight of the rice are as follows: using a rice variety carrying the gene of claim 2 as a donor parent, performing pollen hybridization with a rice variety susceptible to bacterial blight, screening a series of obtained offspring by using the gene of claim 2 as a molecular marker, and identifying to obtain the rice variety resistant to bacterial blight.
7. The promoter region pathogen-inducible regulatory element of the gene of claim 2, wherein: the nucleotide sequence is shown as SEQ ID NO. 3.
8. Use of the promoter region pathogen-inducible regulatory element of the gene of claim 7, wherein: the promoter region pathogen induction regulation and control element of the gene is used for researching a mechanism of resisting bacterial blight of rice, or is used for cultivating rice varieties with disease resistance to bacterial blight of rice or other disease-resistant crops.
9. Use of the promoter region pathogen-inducible regulatory element of the gene according to claim 8, wherein: the steps for cultivating the rice variety with disease resistance to the bacterial blight of the rice or other disease-resistant crops are as follows: introducing the gene of claim 2 and the promoter region pathogen-induced regulatory element of claim 7 into susceptible rice or other crops to obtain disease-resistant rice or disease-resistant crops; or the promoter region pathogen induction regulation element of claim 7 is connected in series with other disease-resistant gene coding sequences and is introduced into susceptible rice or other crops to obtain rice or disease-resistant crops.
Priority Applications (3)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201910174451.XA CN111662367B (en) | 2019-03-08 | 2019-03-08 | Rice bacterial leaf blight-resistant protein and coding gene and application thereof |
| PCT/CN2020/082732 WO2020182221A1 (en) | 2019-03-08 | 2020-04-01 | Rice bacterial leaf streak-resistant protein, and encoding gene and application thereof |
| AU2020235775A AU2020235775B2 (en) | 2019-03-08 | 2020-04-01 | Rice bacterial blight resistance protein, coding gene thereof and use therefor |
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| CN201910174451.XA CN111662367B (en) | 2019-03-08 | 2019-03-08 | Rice bacterial leaf blight-resistant protein and coding gene and application thereof |
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| CN111662367B CN111662367B (en) | 2021-06-22 |
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| CN (1) | CN111662367B (en) |
| AU (1) | AU2020235775B2 (en) |
| WO (1) | WO2020182221A1 (en) |
Cited By (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN112266921A (en) * | 2020-11-13 | 2021-01-26 | 上海交通大学 | Rice bacterial leaf blight disease-resistant gene Xa7 and application thereof |
| CN112301035A (en) * | 2020-09-10 | 2021-02-02 | 浙江师范大学 | Highly-resistant rice bacterial leaf blight gene Xa7 and application thereof |
| CN114350687A (en) * | 2022-03-01 | 2022-04-15 | 云南省农业科学院生物技术与种质资源研究所 | Rice bacterial leaf blight resistant gene, protein and application thereof |
| CN114438100A (en) * | 2022-03-01 | 2022-05-06 | 云南省农业科学院生物技术与种质资源研究所 | Method for efficiently separating bacterial leaf blight resistant gene with wild rice blood margin and family members thereof |
| CN117070531A (en) * | 2023-08-31 | 2023-11-17 | 中国科学院华南植物园 | Rice OsWAK123 gene and application of encoding protein thereof |
Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| DE69820180D1 (en) * | 1997-09-08 | 2004-01-15 | Director General Of Nat Inst O | Process for producing a disease-resistant plant containing a thioningen |
| CN1807453A (en) * | 2005-01-19 | 2006-07-26 | 中国科学院遗传与发育生物学研究所 | Bacterial leaf spot resistance related protein and its coding gene and uses |
Family Cites Families (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101451141B (en) * | 2008-12-18 | 2010-11-03 | 上海交通大学 | Avirulence gene avrxa5 capable of stimulating rice to generate disease resistance response |
| CN101974079B (en) * | 2010-08-17 | 2013-06-26 | 中国农业科学院作物科学研究所 | avrXa23 protein activating disease-resistant reaction of rice and coding gene thereof |
| CN103333231B (en) * | 2013-05-24 | 2015-06-24 | 中国农业科学院作物科学研究所 | Plant disease-resistant protein Xa23 and its coding gene and use |
| CN108220327B (en) * | 2016-12-12 | 2020-12-11 | 中国科学院遗传与发育生物学研究所 | Method for cultivating bacterial blight resistant plant |
| CN107475210B (en) * | 2017-09-07 | 2021-06-29 | 四川农业大学 | Rice bacterial leaf blight resistance related gene OsABA2 and application thereof |
-
2019
- 2019-03-08 CN CN201910174451.XA patent/CN111662367B/en active Active
-
2020
- 2020-04-01 WO PCT/CN2020/082732 patent/WO2020182221A1/en active Application Filing
- 2020-04-01 AU AU2020235775A patent/AU2020235775B2/en active Active
Patent Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| DE69820180D1 (en) * | 1997-09-08 | 2004-01-15 | Director General Of Nat Inst O | Process for producing a disease-resistant plant containing a thioningen |
| CN1807453A (en) * | 2005-01-19 | 2006-07-26 | 中国科学院遗传与发育生物学研究所 | Bacterial leaf spot resistance related protein and its coding gene and uses |
Non-Patent Citations (3)
| Title |
|---|
| DWINITA WIKAN UTAMI等: "A Relative Expression of Xa7 Gene Controlling Bacterial Leaf Blight Resistance in Indonesian Local Rice Population (Oryza sativa L.)", 《J.CROP SCI.BIOTECH》 * |
| SIRIPORN SOMBUNJITT等: "Searching for and analysis of bacterial blight resistance genes from Thailand rice germplasm", 《AGRICULTURE AND NATURAL RESOURCES》 * |
| 黄章慧等: "抗白叶枯病Xa7基因的精细定位及基因预测", 《中国植物病理学会2007年学术年会论文集》 * |
Cited By (11)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN112301035A (en) * | 2020-09-10 | 2021-02-02 | 浙江师范大学 | Highly-resistant rice bacterial leaf blight gene Xa7 and application thereof |
| WO2022052380A1 (en) * | 2020-09-10 | 2022-03-17 | 浙江师范大学 | Gene xa7 having high resistance against rice bacterial blight and use thereof |
| CN112301035B (en) * | 2020-09-10 | 2022-12-27 | 浙江师范大学 | Highly-resistant rice bacterial leaf blight gene Xa7 and application thereof |
| CN112266921A (en) * | 2020-11-13 | 2021-01-26 | 上海交通大学 | Rice bacterial leaf blight disease-resistant gene Xa7 and application thereof |
| WO2022100031A1 (en) * | 2020-11-13 | 2022-05-19 | 上海交通大学 | Rice bacterial blight disease resistance gene xa7 and application thereof |
| CN114350687A (en) * | 2022-03-01 | 2022-04-15 | 云南省农业科学院生物技术与种质资源研究所 | Rice bacterial leaf blight resistant gene, protein and application thereof |
| CN114438100A (en) * | 2022-03-01 | 2022-05-06 | 云南省农业科学院生物技术与种质资源研究所 | Method for efficiently separating bacterial leaf blight resistant gene with wild rice blood margin and family members thereof |
| CN114350687B (en) * | 2022-03-01 | 2023-08-22 | 云南省农业科学院生物技术与种质资源研究所 | Rice bacterial leaf blight resistance gene, protein and application thereof |
| CN114438100B (en) * | 2022-03-01 | 2023-11-10 | 云南省农业科学院生物技术与种质资源研究所 | Method for efficiently separating bacterial leaf blight-resistant gene with wild rice blood margin and family members thereof |
| CN117070531A (en) * | 2023-08-31 | 2023-11-17 | 中国科学院华南植物园 | Rice OsWAK123 gene and application of encoding protein thereof |
| CN117070531B (en) * | 2023-08-31 | 2024-01-26 | 中国科学院华南植物园 | Rice OsWAK123 gene and application of encoding protein thereof |
Also Published As
| Publication number | Publication date |
|---|---|
| WO2020182221A1 (en) | 2020-09-17 |
| AU2020235775A1 (en) | 2021-11-11 |
| CN111662367B (en) | 2021-06-22 |
| AU2020235775B2 (en) | 2022-12-22 |
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