CN103333231B - Plant disease-resistant protein Xa23 and its coding gene and use - Google Patents

Abstract

The invention discloses a rice bacterial blight resistance protein Xa23 and its coding gene, its nucleotide sequence is shown in SEQ ID NO:1, and its amino acid sequence is shown in SEQ ID NO:2. The invention also relates to the application of the gene in cultivating disease-resistant crop varieties, especially cultivating rice varieties resistant to rice bacterial blight.

Description

Plant disease resistance protein Xa23 and its coding gene and application

technical field

The invention belongs to the field of biotechnology, and relates to a rice disease resistance protein and its coding gene, in particular to a rice bacterial blight resistance gene and its application.

Background technique

Rice is the most important food crop in my country. About 60% of the population takes rice as a staple food. The sustainable development of rice production is directly related to my country's food security. Rice bacterial blight (Bacterial blight, BB) is one of the major diseases in rice production in the world. It was first discovered in Fukuoka, Japan in 1884. Since the 1950s, the incidence range has continued to expand. The current occurrence range of bacterial blight All over the rice producing areas in the world (Mew TW. Current status and future prospects of research on bacterial blight of rice. Annu Rev Phytopathol, 1987, 25:359-382; Ou SH. Rice disease, Commonwealth Agricultural Bereaux, England, 1985 380pp.). Rice bacterial blight is especially serious in Asia. In my country, from south to north, it is distributed in all rice regions except Xinjiang.

Rice bacterial blight is a vascular disease caused by the Gram-negative bacterium Xanthomonas oryzae pv . Sometimes even fail. Because pathogenic bacteria invade vascular bundles through wounds or water holes to cause harm, the effect of chemical control is not good, which not only costs money and labor, but also pollutes the environment. Practice has proved that planting disease-resistant varieties is the most economical and effective method to control rice bacterial blight. Therefore, since the 1960s, the discovery and utilization of new genes resistant to bacterial blight has been one of the hotspots of research at home and abroad. So far, 38 rice bacterial blight resistance genes have been reported, and most of these resistance genes have been mapped to chromosomes, and 8 of them have been cloned successively, including: Xa1 (Yoshimura S et al. Expression of Xa1, a bacterial blight-resistance gene in rice, is induced by bacterial inoculation. Proc Natl Acad Sci USA, 1998, 95:1663-1668) , Xa3/Xa26 (same gene) (Sun XL, et al. Xa26, a gene conferring resistance to Xanthomonas oryzae pv oryzae in rice, encodes an LRR receptor kinase-like protein. The Plant Journal, 2004, 37:517-527; Xiang Y, et al. Xa3, conferring resistance for rice bacterial blight and encoding a receptor -like protein, is the same as Xa26. Theoretical and Applied Genetics, 2006,113:1347–1355.) 、xa5 (Iyer AS, McCouch SR. The rice bacterial blight resistance gene xa5 encodes a novel form of disease resistance. Molecular Plant Microbe Interactions, 2004, 17(12): 1348-1354; Jiang GH, et al. Testing the rice bacterial blight resistance gene xa5 by genetic complementation and further analyzing xa5 (Xa5) in comparison with its homolog TFIIAg1. Molecular Genetics, and Genomics 2006, 275 : 354–366.) , xa13 (Chu Z, et al. Promoter mutations of an essential gene for pollen development result in disease resistance in rice. Genes Dev, 2006, 20:1250–1255) , Xa21 (Song WY, et al . A receptor kinase-like protein encoded by the rice disease resistance gene, Xa21. Science, 1995, 270:1804-1806) 、xa25 (Liu QS, et al. A paralog of the MtN3/saliva family recessively confers race-specific resistance to Xanthomonas oryzae in rice. Plant Cell and Environment, 2011, 34:1958-1969) and Xa27 (Gu KY, et al. R gene expression induced by a type-III effector triggers disease resistance in rice. Nature, 2005, 435, 23 June, 1122-1125). Among the above-mentioned disease resistance genes, most of them have narrow resistance spectrum, poor resistance persistence or recessive genes, which are inconvenient to use in production. At present, only a few such as Xa3, Xa4, Xa7 and Xa21 are effectively used. The long-term widespread utilization of a small number of bacterial blight resistance genes always faces the risk of loss of resistance due to the continuous variation of the pathogenicity of pathogenic bacteria. In order to sustainably control the harm of rice bacterial blight, it is necessary to continuously excavate new resistance resources and find new genes with breeding value from them, so as to lay the foundation for breeding excellent new rice varieties with disease resistance.

In the previous work of the present inventors, a Xa23 gene that was highly resistant, fully dominant, and resistant to bacterial blight during the whole growth period was identified and excavated from common wild rice in China. Previous studies have found that the Xa23 gene is a broad-spectrum disease resistance gene (resistance to all representative bacterial blight strains from China, the Philippines, Japan, South Korea, Bangladesh, etc., and no bacterial blight that can overcome the resistance of the Xa23 gene has been found so far. Natural strains of pathogens, Wang CL et al., 2009, Generation and characterization of Tn5-tagged Xanthomonasoryzae pv. oryzae mutants that overcome Xa23 -mediated resistance to bacterial blight of rice. European Journal of Plant Pathology, 123 (3): 343-351 ), and the resistance mediated by the Xa23 gene has the characteristics of full dominance and disease resistance throughout the growth period, so the genetic improvement of rice, especially hybrid rice, including molecular marker-assisted breeding and transgenic engineering breeding has very broad application prospects . The principle of Xa23 gene disease resistance is particular: Xa23 gene is not expressed under normal circumstances, but has a promoter trap. An invention patent, patent name: avrXa23 protein and its encoding gene that stimulates rice to produce disease resistance, patent number: ZL 201010256332.8) specifically activates the expression of Xa23 gene, and its encoded protein Xa23 leads to programmed death of rice cells ( PCD) or allergic necrosis (HR), which limit the reproduction and spread of pathogens in plant tissues, so as to achieve the purpose of disease resistance. Therefore, cloning the Xa23 gene is of great value. In the early stage of the present invention, the Xa23 gene has been transferred to the susceptible cultivar King Kong 30, and the near-isogenic line CBB23 (Zhang Q, et al. Development of Near-Isogenic Line CBB23 with a New Resistance Gene to Bacterial Blight in Rice and Its Application. Chinese J Rice Sci, 2002, 16(3): 206-210). Then Xa23 was located between the RFLP marker 69B and the EST marker CP02662 on the long arm of the rice chromosome 11, so that the Xa23 gene was framed within 1.7 cM (Wang Chunlian et al., using genome libraries to accelerate chromosome walking of Xa23 gene positioning. China Rice Science, 2006, 4(20): 355-360). Afterwards, it was finely positioned, and 4 molecular markers closely linked to the Xa23 gene were found, one of which was Lj74 (a patent application was submitted on the same day as this application, and its invention title is: Molecular marker and application of rice disease resistance gene Xa23 ) co-segregates with Xa23 , this molecular marker provides the basis for cloning Xa23 gene, and provides a way for molecular marker-assisted breeding. The present invention is accomplished on the basis of above-mentioned work just.

Contents of the invention

On the basis of the previous research of the present invention, in order to effectively use the Xa23 gene in rice molecular breeding, the BAC library of the rice material variety CBB23 was further constructed and screened, and then through a series of functional complementation tests, the Xa23 gene was finally cloned, thus completing this invention.

Therefore, the present invention provides a rice disease resistance protein, characterized in that its amino acid sequence is as shown in SEQ ID NO:2.

Correspondingly, the present invention also provides the above rice disease resistance protein coding gene, including its genome sequence. Preferably, its nucleotide sequence is shown in SEQ ID NO:1.

Furthermore, the present invention also provides an expression vector comprising the above-mentioned gene. Preferably it is a plant expression vector, preferably the gene is operably linked to an inducible promoter.

In addition, the present invention further provides the application of the gene in breeding disease-resistant crop varieties. Preferably, the vector is introduced into plants, and resistant plants are obtained by screening. The plant is a monocotyledonous plant or a dicotyledonous plant, especially an agricultural crop. Most preferably, the plant is rice, and the disease resistance refers to rice bacterial blight resistance.

The invention has the following beneficial effects: the invention successfully clones the Xa23 gene, which is a broad-spectrum disease resistance gene. At the same time, it has been proved (see Example 3): Xa23 gene expression in dicotyledonous plants such as tobacco can also cause hypersensitive necrosis (HR) in dicotyledonous plant cells. If other inducible promoters are used to control the expression of Xa23 gene, Just can be used for the disease control of other plants, therefore has very extensive application. Using other inducible promoters to control the expression of the Xa23 gene can be used to control other diseases of rice, such as bacterial streak disease, rice blast and the like. For example, not only the promoter induced by rice bacterial blight can be used, but also the OsQ16p promoter induced by rice blast fungus can be used (Wang Guang et al., Cloning and functional analysis of the rice blast fungus-induced expression promoter OsQ16p, Acta Crops 2012 Issue 6 of 2009), when rice is infected by rice blast, it will induce the expression of Xa23 gene in transgenic plants, trigger allergic necrosis and develop resistance to rice blast. Another example, containing other variants of Xanthomonas ( Xanthomonas oryzae pv. oryzicola causing rice bacterial spot, Xanthomonas campestris pv. Xanthonwnas campestris pv.m alvacearum of cotton angular leaf spot, etc.) artificial inducible promoter of effector protein recognition sequence (Humme AW, Doyle EL and Bogdanove AJ, Addition of transcription activator-like effector binding sites to a pathogen strain -specific rice bacterial blight resistance gene makes it effective against additional strains and against bacterial leaf streak. New Phytologist, 2012,195(4):883-93) driving Xa23 gene can be used to control rice bacterial blight, pepper or Tomato spot, citrus canker, cotton horn spot, etc.

Description of drawings

Figure 1 is the quality map of DNA gel blocks detected by pulse electrophoresis.

Among them: 1, 2: electrophoresis detection of high molecular weight genomic DNA extracted from etiolated seedlings of CBB23, M: 50kb marker.

Figure 2 is a graph showing the size and ligation efficiency of exogenous inserts in the BAC library constructed by pulse electrophoresis detection.

Among them: 1-28: monoclonal plasmid DNA of CBB23 BAC library, respectively digested with Not I, M1: 50kb marker M2: 1kb marker.

Figure 3 Screening of BAC clones by molecular marker Lj74.

Figure 4 Screening of the TAC expression gene library, A: Lj74 marker screening of the TAC expression gene library B: Restriction endonuclease Bam HI digestion of the TAC clone.

Figure 5 The resistance response of transgenic plants to PXO99.

Fig. 6 The resistance response of transgenic plants (T189-11) to PXO99, MDJ8: transformed recipient plants. S: disease resistant R: susceptible.

Figure 7 Southern detection of the copy number of exogenous insert in T189 transgenic plants.

Figure 8 Xa23 gene expression in CBB23 was induced by bacterial blight PXO99, and associated with disease resistance response, A: symptoms of disease-resistant leaves (CBB23) and disease-resistant leaves (JG30), B: quantitative detection of Xa23 gene by q-RTPCR Express

Figure 9 The expression of Xa23 gene in transgenic plants is induced by bacterial blight PXO99, and is associated with disease resistance response, A: disease-resistant transgenic plants (T114-151) and susceptible recipient plants (MDJ8), B: q-RTPCR Detection of Xa23 Gene Expression in Transgenic Plants

Figure 10 The resistance response of RNAi transgenic plants to PXO99.

Fig. 11 Schematic diagram of constructing Xa23 gene plant expression vector.

Fig. 12 Expression and HR detection of Xa23 gene in tobacco. A, sampled leaves 2 days after injection; B, leaves after depigmentation.

Detailed ways

The present invention will be further clarified through the detailed description of specific embodiments below, but it is not intended to limit the present invention, but only for illustration.

Example 1 Isolation Containing Xa23 Candidate Gene Sequence Clones

1 plant material

A near isogenic line CBB23 containing the Xa23 gene.

carrier

The vector used to construct the BAC library of CBB23 is CopyControl ^TM pCC1BAC ^TM of Epicentre Company, the transformable artificial expression vector pYLTAC747 (provided by Dr. Liu Yaoguang of South China Agricultural University), and the RNAi interference expression vector pTCK303 (WANG Z et al. A Practical Vector for Efficient Vector for Efficient Knockdown of Gene Expression in Rice ( Oryza sativa L.). Plant Molecular Biology Reporter 22: 409-417).

3 BAC library construction

Cultivate the seeds of the near-isogenic line CBB23 carrying the Xa23 gene in a dark room at 28-30°C to obtain etiolated seedlings, grind them into powder with liquid nitrogen, and gently stir in the pre-cooled extraction buffer at 50 mesh and 200 mesh successively. , 400 mesh nylon mesh suspension filter. Finally, the nuclei were embedded in agarose gel to obtain high-molecular-weight genomic DNA of CBB23 (Figure 1). To select effective DNA, use Hind Ⅲ enzyme to partially digest the high molecular weight genomic DNA embedded in the agarose gel block, select a 100-150Kb DNA fragment, and elute the DNA by electroelution.

The 100-150Kb DNA fragment was ligated with the vector pCC1BAC ^TM under the action of T4 ligase, and the ligated product was transformed into competent cells DH10B. On LB solid medium, cultivate at 37°C for 20-30 hours, grow a single clone, randomly pick a single clone, extract plasmid DNA, use Not Digest the plasmid DNA, and detect the transformation efficiency and insert size by pulse electrophoresis. The results show that the quality of the library is reliable, the size of the inserts is distributed between 70-150Kb, and the average insert is 108Kb (Figure 2). A total of 36,096 clones were selected to form the library. Based on the 430Mb rice genome, the library covers 9.06 times the rice genome and can be used for library screening.

Screen positive BAC clones

Use a toothpick to pick the white single colony into the 384-well plate medium, then dip the toothpick into the 1.5ml centrifuge tube containing LB medium, that is, each 1.5ml centrifuge tube contains 384 different single colonies, and establish BAC clones pool. After recovery, the mixed plasmid DNA was extracted by alkaline lysis. The co-segregation marker Lj74 (such as SEQ ID NO: 4) with the Xa23 gene has been screened in the early stage (a patent application was submitted on the same day as this application, and its invention title is: molecular marker and application of rice disease resistance gene Xa23 ), so each The mixed plasmid DNA of two BAC clone pools was used as a template, amplified with Lj74 marker, and the DNA of CBB23 was set as a control, and the clone pool whose amplified product was the same size as the CBB23 amplified product was selected. After agarose gel electrophoresis detection, there was a The amplified band pattern of the clone pool was consistent with CBB23 as BAC6. Then use a toothpick to re-take the 384 monoclonal bacterial liquids stored in the 384-well plate (BAC6), and amplify them with Lj74 markers respectively, and set the DNA of CBB23 as a control, and select the monoclonal whose amplification is the same size as that of CBB23 , After agarose gel electrophoresis detection, there was a clone with the same amplified band pattern as CBB23, named B6O8 (Figure 3).

Shotgun sequencing and bioinformatic analysis of clones

The B6O8 clone was sequenced by shotgun method (Shotgun) and a 72114bp continuous rice genomic DNA sequence was obtained. According to gene prediction, it contains 12 open reading frames (ORFs), which encode different proteins respectively. According to the homology comparison of ORF encoded products and the position of molecular markers such as Lj74, 6 ORFs were finally identified as candidate genes.

Expression vector construction and screening

Because the B6O8 clone cannot be directly used for plant genetic transformation, and the rice genome DNA fragment inserted by exogenous sources is relatively long. Therefore, the artificial chromosome expression vector pYLTAC747, which can be used for plant transformation, was used to construct a plant expression vector containing the Xa23 gene. The carrying capacity of pYLTAC747 is more than 50Kb. In view of the fact that small fragments are easy to transform and can include more than 2 candidate genes, BAC6O8 was constructed into a TAC expression library of about 15-20Kb. Directly use the bacterial liquid of each single clone as a template, and use Lj74 marker to amplify, screen a total of 282 clones, and obtain 43 positive clones, some clones were identified as shown in Figure 4A, the plasmid DNA was extracted by alkaline lysis, and restriction endonuclease Enzyme digestion with Bam HI, partial clone digestion is shown in Figure 4B. According to the position size of the actual restriction fragment and the predicted restriction fragment of the gene (according to the obtained 72114bp restriction site), and considering the spanning range of the candidate gene, finally select 10 candidate gene TAC clones for transformation, Includes T189 clone. The two ends of these clones were sequenced to obtain the exact position and length of each clone.

Example 2 Obtaining Xa23 gene through rice genetic transformation and expression analysis

1 plant material

Mudanjiang 8 (MDJ8), a susceptible rice variety, and Zhongwu 1, a disease-resistant rice variety (containing the Xa23 gene), were accepted.

Rice bacterial blight

The rice bacterial blight wide pathogen PXO99 used for inoculation identification was quoted from the International Rice Research Institute (IRRI), stored in vacuum at -70°C, rejuvenated on Wakimoto Tetsu's medium before use, and placed at 28°C Cultivate for 48 hours, prepare the inoculum solution with sterile water, and adjust the concentration to OD=1.0.

Agrobacterium-mediated genetic transformation

The above-mentioned 10 TAC clone plasmid DNAs containing Xa23 candidate genes such as T189 were transformed into Agrobacterium EH105 competent cells, and these 10 clones were transformed into Mudanjiang No. . Transgenic plants are obtained through a series of processes of resistance selection, differentiation, rooting and the like. The obtained 1350 transgenic test-tube seedlings (including 258 plants of T189 clone) were all transplanted into rice planting ponds.

Field Inoculation Identification and Molecular Detection of Transgenic Plants

The wide-pathogenic bacteria PXO99 was inoculated on the transgenic plants by artificial leaf-cut inoculation method, and the donor susceptible variety Mudanjiang No. 8 was used as a control. About 2 weeks after inoculation, the investigation was carried out when the condition of the susceptible variety Mudanjiang No. 8 stabilized. The results showed that 23 of the 258 transgenic seedlings transformed with TAC cloning vector T189 were resistant to the disease (Fig. 5, 6). Other 2 candidate gene clones (T30, T180) also obtained 11 and 8 transgenic disease-resistant plants respectively. Molecular detection was used to verify the authenticity of the transgenic disease-resistant plants. The partial sequence of the hygromycin gene was selected as a probe, and Southern hybridization was performed on some resistant and susceptible plants. There are double copies (Figure 7), which further confirms that the disease-resistant plants are indeed caused by the insertion of foreign genes. The results showed that the DNA fragment contained in the T189 clone carried the disease resistance gene Xa23 .

Cloned subcloned library construction

The foreign insert fragment of T189 clone is 18Kb in full length and contains 2 ORFs. So is one gene at work, or both? Therefore, the T189 clone was subcloned to construct a TAC subcloning library with a fragment length of about 7-9Kb. From them, 58 clones were selected for the sequencing of the two ends of the insert fragment, and according to the sequencing results, 4 candidate clones such as W17 were selected for genetic transformation.

Functional verification of Xa23 gene

Four TAC subclones including W17 were transformed into the susceptible variety Mudanjiang No. 8 by the Agrobacterium-mediated method (the method is the same as that in Example 2.3), and 98 transgenic seedlings were obtained by transformation of W17, and 13 strains were identified by inoculation with PXO99. Disease resistance (Fig. 9A). The results showed that the gene contained in the W17 clone was the target gene Xa23. The W17 clone only contains a complete ORF, and the DNA sequence of the gene is 342bp in length (SEQ ID NO: 1), encoding a disease-resistant protein (SEQ ID NO: 2) containing 113 amino acids.

7Expression analysis proves that Xa23 gene transcription is induced by bacterial blight and is associated with disease resistance

 When CBB23 and JG30 were inoculated with bacterial blight PXO99, CBB23 was highly resistant, while JG30 was severely susceptible (Fig. 8A). Take the leaves of inoculation 1d, 2d, and 3d, extract RNA respectively, use the reverse transcription kit of Tiangen Biochemical Technology (Beijing) Co., Ltd. to reverse-transcribe into cDNA, and use the SYBR SELECT MASTER MIX kit of Invitrogen Company for fluorescence real-time quantification PCR (qRTPCR) analysis.

Design fluorescent quantitative PCR primers according to the Xa23 gene sequence:

Xa23BDF3:5'- GTAGAACAGCATGACCGAGAGAC,

Xa23BDF3:5'-GTAGCCGGTATACACATGATCCTC.

Internal reference primers were designed according to the conserved sequence of ubiquitin in the rice genome:

UbiqF:5'- GCTCCGTGGCGGTATCAT

UbiqR: 5'-CGGCAGTTGACAGCCCTAG.

The synthesized primers were diluted to 5 μM. The real-time fluorescence quantitative PCR reaction system is 20 μL: 2×SYBR Mix 10 μL, primer F and R primer 1.6 μL, cDNA template 1 μL, Nuclease-Free Water 7.4 μL. Three replicates were set up for each sample. Reaction program, pre-denaturation at 95°C for 10min; denaturation at 95°C for 15s, annealing at 60°C for 1min, reading the fluorescence value, 40 cycles. The rice Ubiquitin gene was used as the internal standard reference, the relative expression level = 2 ^{- ΔCt} , ΔCt = (Ct _{target gene} - Ct _Actin ), Ct was the fluorescence threshold, and the instrument was 7500 from ABi Company. The results showed that the transcriptional expression of Xa23 gene was induced by PXO99, and the expression level was the highest at 3d, while the expression of Xa23 gene could not be detected by JG30 (Fig. 8B).

The obtained positive transgenic plant T114-151 (Fig. 9A) and the susceptible receptor Mudanjiang No. 8 were analyzed by qRTPCR (the method is the same as above). The expression of Xa23 gene in the transgenic plant T114 was very high (Fig. 9B), but the expression of Xa23 gene was not detectable in the recipient Mudanjiang 8 (Fig. 9B). It was further demonstrated that Xa23 gene was induced by bacterial blight and was associated with disease resistance.

8 RNAi interference experiment further verified the DNA sequence and function of Xa23 gene

The DNA sequence and disease resistance function of Xa23 gene were further verified by gene knockout (RNAi interference) method. Take the Xa23 gene and its 5' non-coding sequence DNA fragment (SEQ ID NO: 3) with a total of 379 bp to construct the RNAi interference expression vector pTCK23, and transform it into a disease-resistant disease-resistant gene containing the Xa23 gene through the Agrobacterium-mediated method (the method is the same as in Example 2). In the genome of the japonica rice variety Zhongwu 1, 105 transgenic seedlings were obtained, and 69 plants were susceptible after inoculation identification (Fig. 10), indicating that after the Xa23 gene expression of the disease-resistant japonica rice variety Zhongwu Turning into a susceptible phenotype further verified the DNA sequence and function of the Xa23 gene.

Example 3 Transient expression proves that Xa23 protein causes allergic reaction in dicotyledonous plant cells

1 plant material

Nicotiana benthamiana was planted in an artificial climate chamber under the following conditions: light 16h/d, light intensity 30~40μmols ^-1 m ^-2 , temperature 25~20℃, humidity 50~60%; Sow in nutrient soil, transplant seedlings after two cotyledons grow, and move to new nutrient soil (add equal volume of vermiculite to facilitate soil ventilation), move 3 to 4 plants in each pot, and pay attention to prevent soil insects during the period , watering and nutrient solution at intervals.

carrier

Fast PCR Clone Kit (Beijing Bioche-Service Sci and Tech Company), plant expression vector pCAMBIA1305.

Construction of Plant Expression Vector of Xa23 Gene

A plant expression vector in which the 35S promoter drives the coding frame of the Xa23 gene was constructed by means of recombination (Fast PCR Clone). As shown in Figure 11, the primers for amplifying the Xa23 gene have Nco I and Pml I restriction sites, and the PCR product is recombined into the Pml I restriction site of the pCAMBIA1305 vector, and the above recombinant plasmid is digested with Nco I to remove the gus gene The fragment was constructed into a Xa23 gene expression vector and named 35S:: Xa23 ATG . Similarly, we constructed two expression vectors 35S:: Xa23 ACG and 35S:: Xa23 AGG with single base mutations in the start codon of Xa23 gene.

The upstream primers for amplifying Xa23 ATG, Xa23 ACG and Xa23 AGG are: CACCATCACCATCACCCATGGCAAATGTTGCATCATCATCTCAAGGAG, CACCATCACCATCCACCCATGGCAAAGCTTGCATCATCTCAAGGAG, CACCATCACCATCCACCCATGGCAAAGGTTGCATCATCTCAAGGAG,

Same downstream primer: GTCACCAATTCACACTCTCAAAACTTTGAGTTATAACATATA.

4 Transient expression of Xa23 gene and allergic reaction in Benming tobacco

The above-mentioned 35S:: Xa23 ATG, 35S:: Xa23 ACG, 35S:: Xa23 AGG and the empty vector pCAMBIA1305 plant expression vector without Xa23 gene were transferred into Agrobacterium EHA105 competent cells, and MMA buffer (10mM MgCl ₂ , 10mM MES pH 5.5, 100μM acetosyringone) resuspended, adjusted OD ₆₀₀ to 0.5~1.0, and then used to inject tobacco leaves after standing at 30°C for 3h.

Tobacco injection and decolorization: Puncture the leaf epidermis with a needle before injection, inject the bacterial liquid into the leaves from the punctured part of the back of the leaves with a 1mL syringe with the needle removed, inject 3 plants, and inject two leaves for each plant, 36 hours after injection , 35S:: Xa23 ATG produces a hypersensitivity response (HR). Take the leaves 2 days after the injection and put them into a petri dish, add absolute ethanol and place them in a 37°C incubator for 3-5 hours for decolorization, then add 70% ethanol and place them in a 37°C incubator to continue decolorization until all the chlorophyll of the leaves are removed, store, take pictures.

5 Protein Xa23 encoded by Xa23 gene causes allergic reaction in plant cells

The results of the above transient expression system of Tobacco Benming showed that the 35S:: Xa23 ATG vector could express the Xa23 gene and cause allergic reactions in the injection area of the leaves of Tobacco Benming (Figure 12) . However, the Xa23 gene start codon single-base mutant expression vectors 35S:: Xa23 ACG and 35S:: Xa23 AGG, like the empty vector pCAMBIA1305 (control), could not cause allergic reactions in tobacco leaves (Figure 12), indicating that The Xa23 gene encodes the protein Xa23 to perform the function of causing allergic reactions in plant cells.

<110> Institute of Crop Science, Chinese Academy of Agricultural Sciences

<120>Plant disease resistance protein Xa23 and its coding gene and application

<160> 4

<210> 1

<211> 342

<212>DNA

<400> 1

ATGTTGCATC ATCTCAAGGA GCTGGCAGCC GTAGCCGGTA TACACATGAT 50

CCTCATCTAC CTCTGCCGCT TTCTCCTCCG CCGCAGCCGC AACGTATTAT 100

TCACCGTTTC CAACAGCCTC CGTTTTCGCC TCAAGGTATT AACTGTATTG 150

TTGTACATAT GTCTCTCGGT CATGCTGTTC TACCTGTTTG GCTCCATCAT 200

GCCGCTGCCG CCGTGGGGCC TCGTGGTCGG TTGGGTCATG GCCCTCATCG 250

CCGTCGAGCT CGCCTACGCC TTCATCTTTC CATATAGCTT TCGCTACATC 300

GCTGACAACG ACGACGACAA GATGGTTATT CTCCCTGTTT AA 342

 

<210> 2

<211> 113

<212> amino acid

<400> 2

Met Leu His His Leu Lys Glu Leu Ala Ala Val Ala Gly Ile His Met Ile Leu Ile Tyr 20

Leu Cys Arg Phe Leu Leu Arg Arg Ser Arg Asn Val Leu Phe Thr Val Ser Asn Ser Leu 40

Arg Phe Arg Leu Lys Val Leu Thr Val Leu Leu Tyr Ile Cys Leu Ser Val Met Leu Phe 60

Tyr Leu Phe Gly Ser Ile MET Pro Leu Pro Pro Trp Gly Leu Val Val Gly Trp Val Met 80

Ala Leu Ile Ala Val Glu Leu Ala Tyr Ala Phe Ile Phe Pro Tyr Ser Phe Arg Tyr Ile 100

Ala Asp Asn Asp Asp Asp Lys Met Val Ile Leu Pro Val 113

       

<210> 3

<211> 379

<212> DNA

<400> 3

CGAAACATCT TCCTCCCGCA TCACTAACAT CAGCTTCTAT AAAAGCCCCTT 50

CCTTGTTGCA TCATCTCAAG GAGCTGCAAG CACTTCCTCT CTGGCAGCAC 100

TTCCTCATCT CAAGGAGTTG CAAATGTTGC ATCATCTCAA GGAGCTGGCA 150

GCCGTAGCCG GTATACACAT GATCCTCATC TACCTCTGCC GCTTTCTCCT 200

CCGCCGCAGC CGCAACGTAT TATTCACCGTTTCCAACAGC CTCCGTTTTC 250

GCCTCAAGGT ATTAACTGTA TTGTTGTACA TATGTCTCTC GGTCATGCTG 300

TTCTACCTGT TTGGCTCCAT CATGCCGCTG CCGCCGTGGG GCCTCGTGGT 350

CGGTTGGGTC ATGGCCCTCA TCGCCGTCG 379

             

the

<210> 4

<211> 983

<212> DNA

<400> 4

the

AAGCCATTTG ATGAGCAACC CTGTAATTTG GTTGAACAAG GTAAATTCAC 50

AAGATAATGA CAGCATGTAT ATTTGCGGAT CAGTTAGTGG AAACTTAAAA 100

AATAGAATAG AATAGACCAA AAAGCTGAGC AACCCCTGTAA TTTGGTTGAA 150

CAAGGTAAAT TCACAAGATA ATGACAGCAT GTATATTTTGC GGATCAGTTA 200

GTGGAAACTT AAAAAATAGA ATAGAATAGA CCAAAAAGCA TATTGATCTT 250

TTAAATGCTC TAATACTGAC ATAATTATTG CAATACGAGA AGTTAAATAT 300

AGCTGGTTAT GAGGTCTCCA AACAACAGAA GAAATAGGAG GTTTAATTTA 350

CCTTGCTCCA TTCTTCAATG ACACAAGTGA ATATTACATG AGAACAACCT 400

TTTCAAGTTCA TACTGGTGG TGCCAACTTT TCTGTTTGAA AGGCCCTGTA 450

AAGAAAAACA AGCTTAGACA AGCTATCAAT AACGCTGGAG CAATGGTAGA 500

CTTCAATTCA GATTTTAATC TCTTAGAGGA GAAGGCAGTG ACTCAGTTAT 550

TAGGACAACA TGAAACAACT AGTATTGAGA GAGCATGATA CAAGAACTAA 600

TATATGTGAT CTTAACAAAA ATACAGCTTG TTATCAAGAC AAAGGTCTTT 650

GTGACTACAG GGTAGCTCAA GCAGAGGTCC TGGTTGTGAT CCTCATCCAT 700

GCAGAGGTGA AAATGAAAAG CAAAGAAACC CTAGTTTTAA AAGCAAAATA 750

GAAAGGACAA TTTCTTTCTG CTGCTCTTGT AGATCAAAAT TATAGCTTCA 800

TCAAGATTTC ATTGACGTCC TCAACCGTTG ACACATACTC ATCGATCAAG 850

TTCTCAACAT GTACACCATT GCAAGCATTC TCTCTTATCT ATAATAATGG 900

AGTTGGAAAA GTTTACTGCA ATCCATTAGC AAACACCAAA TACAGGATTA 950

GAATATACCT AAAGGTTATG CTGAAATGGA TCC 983

the

Claims (9)

1. a paddy disease-resistant albumen, is characterized in that, its aminoacid sequence is as shown in SEQ ID NO:2.

2. the encoding gene of paddy disease-resistant albumen as claimed in claim 1.

3. gene as claimed in claim 2, it is characterized in that, its nucleotide sequence is as shown in SEQ ID NO:1.

4. comprise the expression vector of the gene described in Claims 2 or 3.

5. expression vector as claimed in claim 4, it is characterized in that, described expression vector is plant expression vector.

6. the expression vector as described in claim 4 or 5, is characterized in that, described gene and inducible promoter are operatively connected.

7. the gene described in Claims 2 or 3 is cultivating the application in disease-resistant crop varieties.

8. apply as claimed in claim 7, it is characterized in that: by vector introduction plant as claimed in claim 6, screening obtains resistant plant, and described plant is unifacial leaf or dicotyledons.

9. apply as claimed in claim 8, it is characterized in that: described plant is paddy rice, and described resistance refers to the water resistant bacterial blight of rice.