AU2020235775A1 - Rice bacterial blight resistance protein, coding gene thereof and use therefor - Google Patents

Abstract

Provided is a rice bacterial leaf streak-resistant protein Xa7 protein, the amino acid sequence thereof being as shown in SEQ ID NO.1; the nucleotide sequence of the gene encoding said rice bacterial leaf streak-resistant protein is as shown in SEQ ID NO.2; and same is applicable in researching the mechanisms of rice bacterial leaf streak resistance, and is used for cultivating rice varieties resistant to rice bacterial leaf streak or for cultivating other disease-resistant crops.

Description

Rice bacterial blight resistant protein, coding gene thereof and use therefor

Technical field

The invention belongs to the field of biotechnology, in particular to a rice

bacterial blight resistant protein, a coding gene thereof and use therefor.

Background

Bacterial blight (Xanthomonas oryzae pv. oryzae (Xoo)) is one of the main

diseases of rice in China and Southeast Asia, the main rice-producing region of the

world, which seriously threatens the safety of rice production. Host resistance is an

effective method to control this disease. However, due to pathogenic variants of the

bacteria, durable resistance is often lost, and therefore the durable resistance and its

mechanism have become a key point in current research of disease resistance.

In-depth understanding of the molecular mechanism of durable disease resistance is of

great significance to provide durable resistance for rice variety and control the disease

sustainably and effectively. [Wu Shangzhong, 1982, "Bacterial blight of rice and its

control", Shanghai Science and Technology Press; Mew, 1987, Current status and

future prospects of research on bacterial blight of rice. Ann. Rev. Phytopathol., 25:

359-382.]. Among the identified bacterial blight resistant genes, Xa7 is considered to

be a durable disease resistant gene, which can effectively resist different pathogenic

pathogens, and has shown excellent and stable resistance in many countries around

the world [Ona et al. ,1998, Epidemic development of bacterial blight on rice carrying

resistance genes Xa-4, Xa-7, and Xa-10. Plant Dis., 82: 1337-1340.; Adhikari et al.,

1999, Virulence of Xanthomonas oryzae pv. oryzae on rice lines containing single

resistance genes and gene combinations. Plant Dis., 83: 46-50; Vera et al., 2000,

Predicting durability of a disease resistance gene based on an assessment of the fitness

loss and epidemiological consequences of avirulence gene Mutation. Proc. Natl. Acad.

Sci., 97: 13500-13505.; Zeng Liexian et al., 2006, Resistance of rice bacterial

blight-resistant near-isogenic lines to South China bacteria. Acta Phytopathology, 36:

177-180].

The Xa7 gene was firstly identified by the International Rice Research Institute

(IRRI) from the rice variety DV85 [Sidhu et al., 1978 Genetic analysis of bacterial

blight resistance in seventy-four cultivars of rice, Oryza sativa L. Theor Appl Genet,

53: 105-111.]. Ogawa et al. introduced Xa7 gene into a near-isogenic line IRBB7

through repeated backcrossing of DV85 with IR24 as a recurrent parent [Ogawa et al.,

1991, Breeding of near-isogenic lines of rice with single genes for resistance to

bacterial blight pathogen Xanthomonas campestris pv. oryzae . Jpn J Breed, 41:

523-529.]. Hopkins et al. have shown that Xa7 is a dominant resistant gene directly

corresponding to a family of avirulence [Hopkins et al., 1992, Identification of a

family of avirulence genes from Xanthomonas oryzae pv. oryzae. Mol Plant Microbe

Interact, 5: 451-459.], Kaji and Ogawa mapped the gene to 107.5 cM on the RGP map

of chromosome 6, which exhibited a recombination rate with the G1091 marker of 8%

[Kaji R and Ogawa T. Identification of the located chromosome of the resistance gene,

Xa-7, to bacterial leaf blight in rice. Breed. Sci., 1995, 45(Suppl. 1):79.], Porter et al.

has fine-mapped Xa7, but due to the imperfect rice genome sequencing at that time, it

was impossible to analyze and predict candidate genes in target genome regions

[Porter et al., 2003, Development and mapping of markers linked to the rice bacterial

blight resistance gene Xa7. Crop Science, 43: 1484-1493.]. Through the analysis of

large populations, the present research group has mapped the Xa7 gene more finely

between the molecular markers GDSSR02 and RM20593. However, also due to the

incomplete rice genome sequence, there was a large gap in the region where the gene

was mapped, leading to failure of cloning the target gene [Chen et al., High-resolution

mapping and gene prediction ofXanthomonas oryzae pv. Oryzae resistance gene Xa7.

Molecular Breeding, 2008, 3: 433-441.].

Summary of the invention

A primary object of the present invention is to overcome the shortcomings and

deficiencies of the prior art and provide a rice bacterial blight resistant protein.

Another object of the present invention is to provide a gene encoding the

above-mentioned rice bacterial blight resistant protein.

Another object of the present invention is to provide a pathogen-inducible regulatory element in a promoter region of the above-mentioned gene.

Another object of the present invention is to provide use of the above-mentioned

protein, gene and pathogen-inducible regulatory element in the promoter region.

The objects of the present invention are achieved through the following technical

solutions: a rice bacterial blight resistant protein named Xa7 protein has an amino acid

sequence as follows:

MAAADHPDRMPVAVAGLRHHYAFPANLRPAARLLTVNSGVFLISTAGAI VLVHTAGNPPAIDNDPAYALVAFVLFLLGIWLMSIALVADQFPRAAGVAVAI ARALQDYLIGGN.

The gene encoding the above-mentioned rice bacterial blight resistant protein

named Xa7 gene has a nucleotide sequence as follows:

ATGGCGGCCGCTGATCATCCTGATCGTATGCCCGTTGCAGTTGCAGGC TTGCGCCACCATTACGCCTTCCCTGCAAACCTTCGCCCCGCCGCTCGACTG CTGACCGTCAACTCCGGCGTCTTCCTCATCTCCACCGCCGGGGCCATCGTC CTCGTCCACACCGCCGGTAACCCACCCGCCATCGACAACGATCCAGCCTA CGCCTTGGTCGCATTCGTGCTCTTCCTCCTCGGAATCTGGCTCATGTCTATT GCCCTCGTCGCCGACCAGTTCCCGCGCGCCGCTGGGGTCGCCGTGGCCATT GCCAGGGCGCTGCAGGATTACCTCATCGGTGGCAATTAA. The nucleotide sequence of a pathogen-inducible regulatory element in a

promoter region of the gene encoding the above-mentioned rice bacterial blight

resistance protein is as follows: TATAACCCCCCCCCCCCCAGATAACCA.

The rice bacterial blight resistance protein can be synthesized by a chemical

synthesis method; or by cloning the gene encoding the rice bacterial blight resistant

protein into an expression vector, transforming the host cells by the obtained

recombinant expression vector, and performing purification after expression.

The preparation of the gene encoding the rice bacterial blight resistant protein

can be achieved by a chemical synthesis method; or by designing a primer, using

genome DNA of DV85, IRBB7 or other rice varieties carrying the Xa7 gene as a

template, and performing PCR amplification; or by enzymatic digestion and screening

from a plasmid carrying the Xa7 gene.

The gene encoding the rice bacterial blight resistance protein is used to study the

mechanism of rice bacterial blight resistance, breed rice varieties resistant to rice

bacterial blight or other disease-resistant crops, function as a molecular marker to

select rice varieties that are resistant to rice bacterial blight.

Steps for breeding rice varieties resistant to rice bacterial blight or other

disease-resistant crops are preferably as follows: introducing the above-mentioned

gene encoding the rice bacterial blight resistance protein and the above-mentioned

pathogen-inducible regulatory element in a promoter region into susceptible rice or

other susceptible crops to obtain disease-resistant rice or disease-resistant crops; or

connecting constitutive expression promoters or other pathogen-inducible promoters

in tandem with the coding sequences of the above genes to introduce into susceptible

rice or other susceptible crops, so as to obtain disease-resistant rice or

disease-resistant crops.

Steps for breeding rice varieties resistant to rice bacterial blight are preferably as

follows: using rice varieties carrying the above-mentioned genes as a donor parent to

hybridize with the rice varieties susceptible to bacterial blight by pollen, screening the

progenies obtained using Xa7 as a molecular marker, and identifying the rice varieties

resistant to bacterial blight.

The donor parent is preferably DV85 or IRBB7.

The pathogen-inducible regulatory element in a promoter region of the gene

encoding the above-mentioned rice bacterial blight resistance protein can be used to

study the mechanism of resistance to rice bacterial blight, and can also be used to

breed rice varieties resistant to rice bacterial blight or other disease-resistant crops.

Steps for breeding rice varieties resistant to rice bacterial blight or other

disease-resistant crops are preferably as follows: introducing the above-mentioned

gene encoding rice bacterial blight resistance protein and the above-mentioned

pathogen-inducible regulatory element in a promoter regions into susceptible rice or

other susceptible crops to obtain disease-resistant rice or disease-resistant crops; or

connecting the above-mentioned pathogen-inducible regulatory element in a promoter

regions in tandem with coding sequences of other disease-resistant genes to introduce into susceptible rice or other susceptible crops, so as to obtain disease-resistant rice or disease-resistant crops. Compared with the prior art, the present invention has the following advantages and effects: Based on previous research, the present invention achieves cloning of the Xa7 functional gene through the construction of a genomic BAC library of rice variety IRBB7, BAC library screening, sequencing of candidate BAC clones, AvrXa7 binding site prediction on the target genome region, a series of candidate genes functional complementation assays and gene knockout assays. The present invention provides the sequence ofXa7 functional gene for the first time. Brief description of the drawings Figure 1 is a schematic diagram of the location of the subclones and the disease resistant phenotype of the transgenic lines; wherein figure A is a schematic diagram of the location and sequence of the overlapping regions of the subclones used in the transgenic functional complementation assays, and figure B is a photograph showing the disease phenotype of the subclones transgenic rice lines after inoculation with bacterial blight pathogen PX86; and figure C shows the statistical results of the lesion length of the subclones transgenic rice lines after inoculation with bacterial blight pathogen PX086. Figure 2 is a diagram showing the structural characteristics and expression pattern of Xa7 gene for disease resistance; wherein, figure A is a schematic diagram of the sequence structural characteristics of Xa7 gene, and figure B is a diagram showing the expression pattern of Xa7 gene for resistance to bacterial blight pathogen PX086. Figure 3 is a diagram showing the function verification results after knockout of the pathogen-inducible element of in the promoter region of the Xa7 gene; wherein figure A shows the sequence of the mutant homozygous line after gene editing of the pathogen-inducible element in the promoter region of the Xa7 gene, figure B shows a diagram of the expression pattern of the mutant lines after inoculation with bacterial blight pathogen PX086, and figure C is a photograph showing the disease-resistant phenotype of each mutant line against bacterial blight pathogen PX086.

Figure 4 is a diagram showing the function verification results after knockout of

the Xa7 gene coding region; wherein figure A is the sequence of the mutant

homozygous line after gene editing of the Xa7 gene coding region, figure B shows the

expression pattern of each mutant line after inoculation with bacterial blight

pathogen PX086, and figure C shows the disease-resistant phenotype of each mutant

line against bacterial blight pathogen PX086.

Detailed description

Hereinafter, the present invention will be further described in detail with

reference to the embodiments and the drawings, but the embodiments of the present

invention are not limited thereto. Unless otherwise specified, the technical means used

in the embodiments are conventional means well known to those skilled in the art. In

the embodiment of the present invention, the isolation and cloning, functional

characteristics and functional verification of the Xa7 gene are described.

Example 1 Isolation and cloning ofXa7 gene

Based on the previous research of the present invention, it was found that the

genome sequence near the Xa7 gene was very different from the common rice

reference genomes including Nipponbare, 9311, Minghui 63 and Zhenshan 97.

Clarifying the genomic sequence of this region is a prerequisite basis for cloning the

gene. The present invention constructed a genomic BAC library of a Xa7-containing

disease-resistant variety IRBB7, screened the library with close-linked molecular

markers on both sides of Xa7, identified positive clones, and sequenced the inserts of

these positive clones, thereby obtaining a complete and accurate genomic sequence of

the region.

The rice variety IRBB7 has been disclosed in "Ogawa et al., 1991, Breeding of

near-isogenic lines of rice with single genes for resistance to bacterial blight pathogen

Xanthomonascampestrispv. oryzae. Japan J Breed, 41: 523-529.").

The plant material for constructing the genomic BAC library is the near-isogenic

line IRBB7 containing the Xa7 gene, the vector is Epicentre's CopyControlTM

pCC1BACTM , and the subcloning and transgenic expression vector is pYLTAC747H/sacB (disclosed in "Xu et al., 2008, Construction and characterization of the transformation-competent artificial chromosome (TAC) libraries of

Leymusmulticaulis. Science in China (Series C: Life Sciences), (07): 604-613."

BAC library construction: according to experimental steps reported in "Liu et al.,

2002, Development of new transformation-competent artificial chromosome vectors

and rice genomic libraries for efficient gene cloning. Gene, 282(1):247-255.", IRBB7

genomic DNA was extracted at the seedling stage, which was partially digested with

HindIII restriction endonuclease; 120-140kb DNA fragments were separated by

pulsed field electrophoresis, which were then purified and ligated with the BAC

vector pCC1BACTM . 75 ng BAC vector was mixed with five sets of genomic DNA

digestion products (A: 30 ng, B: 60 ng, C: 120 ng, D: 160 ng, E: 350 ng), and

according to NEB@ T4 DNA ligase 50 L system, a reaction system was prepared;

ligation reaction was carried out under a variable temperature ligation program in a

PCR machine: 1 0 Cfor 3 min, rise to 16C in 3 min, 6Cfor 5 min, rise to 18°C in

30 s, 18°C for 30 s, rise to 20 °C in 30 s, 20 °C for 30 s, decrease to 4 °C in 8 s, 4 °C

for 3 min, rise from 4 °C to 22 °C in 5 min, decrease from 22 °C to 10 °C in 1

min;the procedures were cycled for 20 times, and finally maintain at 65 °C for 5 min.

The ligation product was dialyzed with VSWP membrane (0.025[tM) of

MILLIPORE TMon a 1/4xTE solution at 4°C for about 2 - 3 hours. The dialyzed

product was transformed into E. coli competent cells of DH1OB (ElectroMAXTM

DH1OB TM Cells of nvitrgenTM) by electroporation. 1 L of the dialysis product and

20 L of competent cells to be electrotransformed were mixed and transferred into a

pre-cooled 0.1cm shock cup, and a BioRad GenePulser@ electric shock meter was

used for electroporation (parameters: voltage 2.0 k, resistance 200 Q, capacitance 25

[F) . After that, 1 mL of S.O.C. medium was quickly added, and recovery incubation

was carried out for 1 hour at 37C and 200 rpm shaker. Ten gradients of bacterial

solution between 10 and 100 L were applied to LB semi-solid medium containing 25

[g/mL of kanamycin and 5% of sucrose, and were incubated at 37C for 12 - 16 hours

followed by calculating the number of clones. Then, the bacterial solutions were

plated and cultured in the same mannerat an amount of about 500 clones per plate.

After that, the mixed colonies were entirely scraped off from the plate, and distributed to 96-well deep-well plates to construct a "BAC clone mixing pool". Based on about 450 original single clones per well, the total number of clones was about 45,000, and the average insert fragment per clone was 100kb. Relative to 430Mb of the whole rice genome, the library coverage is more than 10 times. Screening of BAC positive clones: alkaline lysis was used to extract plasmid DNA from each "BAC clone mixing pool"; using plasmids from the mixing pools as templates, the close-linked molecular markers U05-indel and POZ-indel on both sides of Xa7 were amplified, wherein the primer pair for amplifying molecular markers U05-indel was U05-indel Fw and U05-indel Rv, and the primer pair for amplifying molecular markers POZ-indel was POZ-indel Fw and POZ-indel Rv. After a positive mixed pool was detected, it was diluted by 40,000 times and distributed to 384-well plates at an amount of 5x per well. After culturing overnight, the bacterial solutions were used as templates for screening by PCR amplification using the corresponding molecular marker primers, until a positive single clone was obtained. After multiple rounds of screening, three positive clones were identified: P1-10G, P3-12F and P2-9D. After restriction enzyme digestion and electrophoresis detection, the insert fragments of these three BAC clones were 150 kb, 125 kb and 107 kb, respectively. U05-indel Fw: 5'-CAGACAAGTGTTGTTCATGTTCG-3'; U05-indel Rv: 5'-GAAGTCCGAGCTGGGGACGATGTAC-3'; POZ-indelFw: 5'-CCAAGAAAGGTCCAACTCGCTTAG-3'; POZ-indelRv: 5'-GAACAGTCCTCAGAATTCGACCAC-3'. Plasmid sequencing and sequence analysisof the BAC positive clones: the plasmids cloned from P1-10G, P3-12F and P2-9D were extracted by Omega's BAC/PAC DNA Maxi Kit, a 350bp small fragment library was constructed, and HiSeq PE150 sequencing platform was used for second-generation sequencing, the amount of sequencing data for each clone being 1Gb. Denovo assembly without reference genome was conducted based on the sequencing data, and the backbone sequence of the vector pCC1BACTM was removed to obtain the insert sequences of the three positive plasmids respectively. After assembly of the three insert sequences,

307.5 kb fragments was obtained, ends of each of the sequences being overlapped.

Comparing with the internationally accepted rice genome sequence, it was found that

this fragment is 101.1 kb longer than the genome of the japonica rice variety

Nipponbare, and is 80 kb longer than the genome of the indica rice variety Minghui

63. Using Softberry's Fgenesh tool

(http://www.softberry.com/berry.phtml?topic=fgenesh&

group=programs&subgroup=gfind) to predict the 307.5kb assembled sequence, 82

open reading frames (ORF) were obtained. In order to further narrow the target range,

Galaxy's TALgetter tool (http://galaxy2.informatik.uni-halle.de:8976) was used to

predict the recognition and binding site of AvrXa7 (AvrXa7EBE) in the assembled

sequence. As a result, the P-Value of four binding sites was identified to be less than 6 1.0E- , and only one of the AvrXa7EBE was located in the promoter region in the

upstream of an ORF.The number of this ORF was 52, and the candidate gene was

named CG52, whosesequence is shown in SEQ ID NO.4.

BAC subcloning library construction and functional complementation assays: the

insert fragments of BAC plasmids P1-1OG and P3-12F carrying the CG52 gene were

incompletely digested with BamHI and Sau3A I , respectively, and were ligated to a

subcloning vector pYLTAC747H/sacB. Amplification primers of promoter region and

CDS region of CG52 were used to screen the library respectively, and plasmids of the

positive subclones were then extracted for end sequencing to determine the sequence

of the inserted fragments. According to the sequence information, four subcloned

BAC plasmids were selected and transferred to susceptible indica rice variety IR24

(disclosed in "Ogawa et al. 1991. Breeding of near-isogenic lines of rice with single

genes for resistance to bacterial blight pathogen Xanthomonas campestrispv. oryzae..

Japan J Breed. 41:523-529.) mediated by Agrobacterium EHA105 (purchased from

Beijing Huayueyang Biotechnology Co., Ltd., and the transformation of

Agrobacterium by plasmids were in accordance with the literature "Hood et al., 1993,

New Agrobacterium helper plasmids for gene transfer to plants. Transgenic Res, 2,

208-218").The genetic transformation of the indica rice variety was operated in

accordance with "Lin and Zhang, 2005, Optimising the tissue culture conditions for high efficiency transformation of indica rice. Plant Cell Rep, 23: 540-547.".The transgenic rice was inoculated with rice bacterial blight pathogen PX086 (disclosed in

"Mew TW et al. 1982, Pathotypes of Xanthomonas compestris pv. oryzae in Asia.

IRRI Research Paper Series, No 75.") at the booting stage by a "leaf cutting method"

in accordance with "Wu Shangzhong et al., 1985, Comparative Study on

Pathogenicity of Bacterial Blight Pathogens of Rice in South China and the

Philippines, Acta Phytopathology, 15-2: 65-72."). The disease-resistant phenotype

was investigated 21 days after the inoculation according to the INGER method of the

International Rice Research Institute (operated according to "INGER Genetic

Resources Center, 1996, Standard Evaluation System for rice (4th edition) [M].

Philippines: International Rice Research Institute Press, p20- 21.").

The insert positions of the four subclones used for functional complementary

genetic transformation are shown in Figure 1A. The L235 and L239 clones cover the

full length of the CG52 gene. The transgenic rice lines of these two clones are

resistant to disease; the sequence of the L236 clone overlaps with the 5'end of the

L235 fragment, while its 3'end only contains part of the promoter region of the CG52

gene at 213bp upstream of AvrXa7EBE, the transgenic rice line of which is

susceptible; the 3'end of clone L240 overlaps with clone L235, while its 5'end only

contains the CDS region of CG52 and 13bp of UTR sequence without the

AvrXa7EBE sequence, the transgenic rice line of which is susceptible. therefore, the

CG52 gene is the disease-resistant functional gene, and the promoter region

AvrXa7EBE and complete CDS sequence are both indispensable parts for the gene

function. (Figure lB and Figure IC).

Example 2 Sequence structure and expression characteristics ofXa7 gene

Sequence and structure analysis of Xa7 gene: total RNA was extracted from the

near-isogenic line IRBB7 carrying the Xa7 gene, and the full 5'end and 3'end of Xa7

were amplified by GeneRacerTM Kit of Invitrogen TM. Among them, the specific

primer for 5'RACE amplification was 5'-TGCCACCGATGAGGTAATCCTGC-3',

and the specific primer for 3'RACE amplification was

5'-CCTCCTCGGAATCTGGCTCATGTC-3'. The RACE products were cloned by the

TM

pEASY -Blunt Zero Cloning Kit of TRANS. After sequencing, the transcription

start site (TSS) ofXa7 was determined to locate at 104 bp upstream of the start codon

(ATG); at the same time, it was determined that the 3'UTR of Xa7 was 253 bp in

length (Figure 2A).

Analysis of the expression pattern of Xa7 gene: IRBB7 and IR24 were inoculated

with bacterial blight pathogen (PX86) at the booting stage by a "leaf-cutting

method" and samples were collected at 0, 1, 3, and 5 days later to extract total RNA.

PrimeScriptTM RT reagent Kit with gDNA Eraser of TakaraTM was used to conduct

reverse transcription to obtain cDNA, and SYBR@ Premix Ex TaqTM II (TliRNaseH

Plus) reagent was used for gene quantitative analysis on a Bio-Rad fluorescent

quantitative PCR instrument CFX96TM. 2-AACT method was used to calculate the

relative expression of genes.

The amplification primer pairs of the target gene Xa7 are as follows:

Xa7 Fw: 5'-GATCGTATGCCCGTTGCAGTTGC-3';

Xa7 Rv: 5'-GGAGTTGACGGTCAGCAGTCGAG-3'.

The amplification primer pairs of the internal reference gene TF2 are as follows:

TF2 Fw: 5'- GCCTGAAGTGTACTGTACCACCAC-3';

TF2 Rv: 5'-CAAAGGGTTCAGAAATGAGGAA GG-3'.

As shown in Figure 2B, the expression level of Xa7 gene was very low before

inoculation, which began to increase one day after inoculation, reached a peak after 3

days, and began to fall after 5 days. Therefore, the expression of Xa7 gene is induced

and activated by pathogenic bacteria.

Example 3 CRISPR/Cas9-mediated gene knockout for verification of the key

functional sites of the Xa7 gene

In order to further verify the functions of AvrXa7EBE in promoter region and

CDS region of Xa7, the present invention also used a CRISPR/Cas9 system to

construct gene knockout transgenic lines of these two functional regions. The vectors

used for gene knockout includeda binary expression vector pYLCRISPR/Cas9Pubi-H

provided by Liu Yaoguang laboratory of South China Agricultural University

(disclosed in "Ma et al. 2015, A robust CRISPR/Cas9 system for convenient high-efficiency mutiplex genome editing in monocot and diocot plants. Mol. Plant. 8,

1274-1284."), and intermediate vectors pYLsgRNA-OsU6aL (disclosed in "Ma et al.

2015, A robust CRISPR/Cas9 system for convenient high-efficiency mutiplex genome

editing in monocot and diocot plants. Mol. Plant. 8, 1274-1284."),

pYLsgRNA-OsU3aL (disclosed in "Ma et al. 2015, A robust CRISPR/Cas9 system

for convenient high-efficiency mutiplex genome editing in monocot and diocot plants.

Mol. Plant. 8, 1274-1284.") and pYLsgRNA-OsU6c (disclosed in "Ma et al. 2015, A

robust CRISPR/Cas9 system for convenient high-efficiency mutiplex genome editing

in monocot and diocot plants. Mol. Plant. 8, 1274-1284."). Selection and design of the

sgRNA target sequences corresponding to editing sites were achieved using the online

tool CRISPR-P (http://crispr.hzau.edu.cn/CRISPR/).

Firstly, PAM (protospacer adjacent motif) sequences in AvrXa7EBE in Xa7

promoter region and CDS region were searched through CRISPR-P, the editing target

sites were selected, and the sgRNA target adapters according to the target site

sequences were designed. The corresponding target adapter sequences are as follows:

Target (targeting AvrXa7EBE, the target sequence is located at positions

-126~--107 in Figure 3A):

OsU6aT1F: 5'- gccgTATGTGGTTATCTGGGGGGG-3';

OsU6aT1R: 5'- aaacCCCCCCCAGATAACCACATA-3';

Target2 (targeting AvrXa7EBE, the target sequence is located at -121~-102 in

Figure 3A):

OsU6aT2F: 5'- gccgTTCGTATGTGGTTATCTGG-3';

OsU6aT2R: 5'-aaacCCAGATAACCACATACGAA-3';

Target3 (targeting CDS region, the target sequence is located at positions +22~

+41 in Figure 4A):

OsU3T3F: 5'- ggcaCTGCAACGGGCATACGATC-3';

OsU3T3R: 5'-aaacGATCGTATGCCCGTTGCAG-3';

Target4 (targeting CDS region, the target sequence is located at positions +94~

+113 in Figure 4A):

OsU6cT4F: 5'- tcagCGACTGCTGACCGTCAACTC-3';

OsU6cT4R: 5'- aaacGAGTTGACGGTCAGCAGTCG-3'.

According to the experimental procedure in "Ma et al. 2015, A robust

CRISPR/Cas9 system for convenient high-efficiency mutiplex genome editing in

monocot and diocot plants. Mol. Plant. 8, 1274-1284.", the target adapters were

respectively connected to BsaI digested sgRNA intermediate vectors, and specific

sgRNA expression cassette templates were obtained after two rounds of PCR reaction,

product annealing, and nested PCR amplification. The expression cassettes were then

assembled into the binary expression vector through a ligation reaction. Among them,

Target and Target2 were independently constructed in a binary expression vector,

while Target3 and Target4 were constructed together into the same binary expression

vector. These three gene editing expression vectors were respectively mediated by

Agrobacterium strain EHA105 and transferred into the variety IRBB7.

Primers (QC Fw: 5'-GAACTGCTCTGCTCAAGTGCCTC-3'; QC Rv:

5'-TGCCACCGATGAGGTAATCCTGC-3') were used to amplify the target sites of

each gene knockout transgenic family by PCR followed by sequencing, and

successfully edited homozygous lines in To and Ti generations were screened.

As shown in Figure 3A, the W6-4 and W7-4 homozygous lines obtained by

transgenic editing of Target1, and the W8-7 and W9-6 homozygous lines obtained by

transgenic editing of Target2 all had base deletion in the AvrXa7EBE element of

theXa7 promoter region. Gene expression analysis of these transgenic lines (Figure

3B) shows that the absence of the AvrXa7EBE element would lead to the loss of

Xa7's function of pathogen-induced expression, and thus become susceptible to the

pathogen (Figure 3C).

The W12-1, W12-6, W13-4 and W15-3 homozygous lines obtained by

transgenic editing of Target3 and Target4 had different types of mutations including

base deletion, insertion and substitution in the CDS region of the Xa7 gene

respectively (Figure 4A), resulting in early termination, frameshift, replacement and

other mutations in the encoded protein of mutated Xa7. Although the transcription

could still be activated by the pathogens (Figure 4B), these mutant homozygous lines

lost their resistances to pathogens (Figure 4C).

On the one hand, this example reversely verifies the functional gene of Xa7, on

the other hand, it clarifies that the disease resistance function ofXa7 gene is provided

by two indispensable factors: one is the pathogen-inducing transcriptional activity of

the AvrXa7EBE element in the Xa7 promoter region, and the other one is the intact

encoded protein ofXa7 gene for executing disease resistance.

The above-mentioned embodiments are preferred embodiments of the present

invention, but the embodiments of the present invention are not limited by the

above-mentioned embodiments, and any other changes, modifications, substitutions,

combinations, and simplifications made without departing from the spirit and

principle of the present invention should be equivalent replacement methods, and they

are all included in the protection scope of the present invention.

Sequence listing

<110> Institute of Plant Protection, Guangdong Academy of Agricultural Sciences

<120> Rice bacterial blight resistant protein, coding gene thereof and use therefor

<130> 1

<160> 22

<170> SIPOSequenceListing 1.0

<210> 1 <211> 113 <212> PRT <213> 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 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> Oryza sativa

<220> <223> Xa7 protein

<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> Oryza sativa

<220> <223> Pathogen-inducible regulatory element in the promoter region of Xa7 gene

<400> 3 tataaccccc ccccccccag ataacca 27

<210> 4 <211> 2337 <212> DNA <213> Oryza sativa

<220> <223> Nucleotide sequence of Xa7 gene

<220> <222> (337)..(363) <223> Nucleotide sequence of pathogen-inducible element in the promoter region of Xa7 gene

<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

<220> <223> Primer U05-indel Fw

<400> 5 cagacaagtg ttgttcatgt tcg 23

<210> 6

<211> 25 <212> DNA <213> Artificial Sequence

<220> <223> Primer U05-indel Rv

<400> 6 gaagtccgag ctggggacga tgtac

<210> 7 <211> 24 <212> DNA <213> Artificial Sequence)

<220> <223> Primer POZ-indel Fw

<400> 7 ccaagaaagg tccaactcgc ttag 24

<210> 8 <211> 24 <212> DNA <213> Artificial Sequence

<220>

<223> Primer POZ-indel Rv

<400> 8 gaacagtcct cagaattcga ccac 24

<210> 9 <211> 23 <212> DNA <213> Artificial Sequence

<220> <223> Primer Xa7 Fw

<400> 9 gatcgtatgc ccgttgcagt tgc 23

<210> 10 <211> 23 <212> DNA <213> Artificial Sequence

<220> <223> Primer Xa7 Rv

<400> 10 ggagttgacg gtcagcagtc gag

<210> 11 <211> 24 <212> DNA <213> Artificial Sequence

<220> <223> Primer TF2 Fw

<400> 11 gcctgaagtg tactgtacca ccac 24

<210> 12 <211> 24 <212> DNA <213> Artificial Sequence

<220> <223> Primer TF2 Rv

<400> 12 caaagggttc agaaatgagg aagg 24

<210> 13 <211> 24 <212> DNA

<213> Artificial Sequence

<220> <223> Primer OsU6aT1F

<400> 13 gccgtatgtg gttatctggg gggg 24

<210> 14 <211> 24 <212> DNA <213> Artificial Sequence

<220> <223> Primer OsU6aT1R

<400> 14 aaaccccccc cagataacca cata 24

<210> 15 <211> 23 <212> DNA <213> Artificial Sequence

<220> <223> Primer OsU6aT2F

<400> 15 gccgttcgta tgtggttatc tgg 23

<210> 16 <211> 23 <212> DNA <213> Artificial Sequence

<220> <223> Primer OsU6aT2R

<400> 16 aaacccagat aaccacatac gaa 23

<210> 17 <211> 23 <212> DNA <213> Artificial Sequence

<220> <223> Primer OsU3T3F

<400> 17 ggcactgcaa cgggcatacg atc 23

<210> 18 <211> 23 <212> DNA <213> Artificial Sequence

<220> <223> Primer OsU3T3R

<400> 18 aaacgatcgt atgcccgttg cag 23

<210> 19 <211> 24 <212> DNA <213> Artificial Sequence

<220> <223> Primer OsU6cT4F

<400> 19 tcagcgactg ctgaccgtca actc 24

<210> 20 <211> 24 <212> DNA <213> Artificial Sequence

<220> <223> Primer OsU6cT4R

<400> 20 aaacgagttg acggtcagca gtcg 24

<210> 21 <211> 23 <212> DNA <213> Artificial Sequence

<220> <223> Primer QC Fw

<400> 21 gaactgctct gctcaagtgc ctc 23

<210> 22 <211> 23 <212> DNA <213> Artificial Sequence

<220> <223> Primer QC Rv

<400> 22 tgccaccgat gaggtaatcc tgc 23

Claims (9)

1. A rice bacterial blight resistant protein, characterized in having an amino acid sequence as shown in SEQ ID NO.1.

2. A gene encoding the rice bacterial blight resistant protein according to claim 1, characterized in having a nucleotide sequence as shown in SEQ ID NO.2.

3. The gene according to claim 2, characterized in further comprising a pathogen-inducible regulatory element in a promoter region having a nucleotide sequence as shown in SEQ ID NO.3.

4. Use of the gene according to claim 3, characterized in that, the gene is used to study the mechanism of rice bacterial blight resistance, cultivate rice varieties resistant to rice bacterial blight or other disease-resistant crops, or select rice varieties that are resistant to rice bacterial blight.

5. Use of the gene according to claim 4, characterized in that, steps for cultivating rice varieties resistant to rice bacterial blight or other disease-resistant crops comprises: introducing the gene according to claim 2 and the pathogen-inducible regulatory element in the promoter region into susceptible rice or other susceptible crops to obtain disease-resistant rice or disease-resistant crops; or connecting constitutive expression promoters or other pathogen-inducible promoters in tandem with the gene according to claim 2 to introduce into susceptible rice or other crops, so as to obtain disease-resistant rice or disease-resistant crops.

6. Use of the gene according to claim 4, characterized in that, steps for cultivating rice varieties resistant to rice bacterial blight comprises: using rice varieties carrying the gene according to claim 2 as a donor parent to hybridize with the rice varieties susceptible to bacterial blight by pollen, screening progenies obtained using the gene according to claim 2as a molecular marker, and identifying the rice varieties resistant to bacterial blight.

7. A pathogen-inducible regulatory element in the promoter region of the gene according to claim 2,characterized in having a nucleotide sequence as shown in SEQ ID NO.3.

8. Use of the pathogen-inducible regulatory element in the promoter region of the gene according to claim 7, characterized in that, the pathogen-inducible regulatory element in the promoter region of the gene is used to study the mechanism of resistance to rice bacterial blight, or cultivate rice varieties resistant to rice bacterial blight or other disease-resistant crops.

9. Use of the pathogen-inducible regulatory element in the promoter region of

the gene according to claim 7, characterized in that, steps for cultivating rice varieties

resistant to rice bacterial blight or other disease-resistant crops comprises: introducing

the gene according to claim 2 and the pathogen-inducible regulatory element in the

promoter region according to claim 7 into susceptible rice or other susceptible crops

to obtain disease-resistant rice or disease-resistant crops; or connecting the

pathogen-inducible regulatory element in the promoter region according to claim 7 in

tandem with coding sequences of other disease-resistant genes to introduce into

susceptible rice or other susceptible crops, so as to obtain disease-resistant rice or

disease-resistant crops.

Lesion length/cm

1/4 Fig. 1

Fig.2

2/4

Relative expression of Xa7 gene

Control and treatment groups of transgenic lines

Fig. 3

3/4

Relative expression of Xa7 gene

Control and treatment groups of transgenic lines

Fig. 4

4/4