WO2014075401A1

WO2014075401A1 - Method for cloning plant disease resistance genes in high-throughput manner

Info

Publication number: WO2014075401A1
Application number: PCT/CN2013/070487
Authority: WO
Inventors: 田大成; 杨四海; 张小辉; 王娇; 谭生军; 仲岩
Original assignee: 南京大学
Priority date: 2012-11-13
Filing date: 2013-01-15
Publication date: 2014-05-22
Also published as: CN103805595A

Abstract

Disclosed is a method for cloning plant disease resistance genes in a high-throughput manner. In the method, candidate disease resistance genes are obtained by means of bioinformatics; the candidate disease resistance genes are cloned in a high-throughput manner by using a high resistance variety or a relative species as a resistant gene source, and are transferred into a infected variety; and disease resistance capabilities of the genes are evaluated by using a method of pathogen infection, so as to ultimately find a broad-spectrum plant disease high resistance genes having importance production value. Further disclosed is a rice blast resistance gene obtained by using the method.

Description

High-throughput method for cloning plant disease resistance genes

The invention belongs to the field of cloning and utilization of plant functional genes, and particularly relates to a novel method for high-throughput cloning of plant disease resistance genes based on molecular genetic and evolutionary analysis of disease resistance genes. Background technique

Plant disease resistance genes are the result of long-term interactions between plants and pathogens, and play a very important role in the evolution of plants. Since the first plant disease resistance gene Hml was isolated from corn by Johal and Briggs in 1992 (Tanksley et al. 2007; Dong Yuxi, 2001), so far, nearly 100 plant disease resistance genes have been isolated from different plants. Can be cloned and isolated, wherein the main type of disease resistance gene is a disease resistance gene of NBP-LRR structure type (Nucleotide-binding site and leucine-rich-repeat, ie, nucleotide binding site and leucine-rich repeat protein); In addition, a small number of disease resistance genes are mainly eLRR-TM-pkinase, eLRR-TM, STK and the like. These disease resistance genes encode resistance proteins to viruses, bacteria, fungi, oomycetes, and even nematodes and insects, respectively.

Since plant diseases bring huge losses to agriculture and forestry production every year, rational and effective control of plant diseases is one of the key issues that must be solved to ensure the sustainable development of agriculture and forestry. A large number of practices have proved that the development and utilization of existing plant disease-resistant germplasm resources is one of the most effective methods for controlling plant diseases (Tanksley et al. 2007; Dong Yuxi, 2001). Traditional disease-resistant varieties are usually crossed with high-quality and high-yielding varieties and disease-resistant materials, and then selected through continuous backcrossing, and finally obtain new varieties that are resistant to disease and high quality. Although this method is very effective, it is extremely time consuming. After the new variety is cultivated, it may be susceptible to the newly evolved strains or races of pathogens (Tanksley et al. 2007); the use of classical map cloning methods The disease-resistant and susceptible varieties are crossed, and then a large number of isolated offspring are inoculated with pathogens, identification resistance, genetic mapping, etc., and finally cloned based on precise genetic localization. This method has achieved good results and plays a very important role in the cloning of plant disease resistance genes (such as the isolation and utilization of the resistance to bacterial blight gene Xa21, Song et al. 1995), but because of its long cycle The time-consuming and laborious, separation and allelic detection are difficult, and the like, and are not suitable for the large-scale cloning of disease resistance genes. At the same time, the unique genetic pattern of disease resistance genes has also greatly limited the application of this method. Taking rice blast resistance genes as an example, such genes are usually distributed in the genome (Wang et al. 1999; Liu et al. 2002; Lin et al. 2007), homologous chromosomes in different varieties. The position and structure are also asymmetrically distributed, equipotential Unclear, copy number variation is large (Yang et al. 2007; Ding et al. 2007a; Sun et al. 2008; Li et al. 2010). These genetic phenomena greatly increase the difficulty of map cloning and seriously affect the cloning efficiency of disease resistance genes.

In recent years, with the breakthrough of research on the molecular level of plant disease resistance and disease resistance related genes, we have fundamentally recognized the structure, function, origin, variation and preservation of plant disease resistance and disease resistance related genes. Variety. That is, the plant disease resistance gene is mainly a kind of gene containing LRR domain, and the NBS-LRR (nucleic acid binding-leucine-rich) type disease resistance gene is mainly used. Because these genes are highly similar in structure and function, combined with their unique genetic and evolutionary features, it facilitates the rapid cloning and identification of these genes, and provides a new way to efficiently utilize disease-resistant germplasm resources. opportunity.

The main object of the present invention is to make full use of the structural similarity of the plant disease resistance gene and the unique genetic variation and evolutionary characteristics to provide an efficient and rapid cloning method for the plant disease resistance gene. In addition, although there are many kinds of plants, the basic laws are very consistent. Therefore, the present invention is mainly described by taking high-throughput clones of rice blast resistance genes as an example.

Rice (O ^^flrivo) is one of the most important food crops in the world and one of the most important cultivated crops in China. However, it suffers from serious pests and diseases every year, causing huge losses to agricultural production. . Among them, rice blast is one of the most widespread and endangered diseases of rice, which seriously affects rice yield. The annual rice yield loss caused by rice blast is 11-30% (about 157 million US dollars, http:/ /www.fungalgenomics.ncsu.edu), directly threatening the increase in rice farmers and food security in the country. Whether in the world or in China, solving the rice blast problem has always been a priority research topic to ensure sustainable rice production.

One of the biggest difficulties in the control of rice blast is the rapid variation and differentiation of the bacteria itself (Ling Zhong, 2004). The new races of Magnaporthe oryzae are endless, and the existing disease resistance (R gene) quickly loses resistance. It is increasingly difficult to find new disease-resistant materials, and the threat of rice blast on rice production. It is getting bigger and bigger. For susceptible pathogens, rice must have many disease resistance genes. From the 70-80 gene loci that have been located, there are numerous rice blast resistance genes. However, only 15 blast resistance genes have been cloned so far. Although the already located genes can be utilized by molecular marker-assisted selection methods, this breeding process is cumbersome and time consuming. After the new varieties are cultivated, the newly generated mutant strains may be susceptible to cloning and direct transformation. The disease gene may be the most direct and effective way to breed resistant varieties. In this context, using new ideas, research and development can be high-throughput grams The new technology against rice blast genes is particularly important and has incalculable value. Summary of the invention

The object of the present invention is to establish a cloning technology system for many-to-many plant disease resistance genes (multiple clones correspond to the detection of multiple pathogens), and to exemplify a high-throughput clone of rice blast resistance genes as an example. Our research shows that plant resistance genes have the following characteristics in genetics and evolution: (I) high genetic variability within species; (2) mostly in the form of gene families and gene clusters, and showing copies Number change

(Copy number variation); (3) It is subject to positive selection during evolution; (4) The same type of disease resistance gene has certain clustering characteristics in the phylogenetic tree. Based on these genetic evolution laws, this method uses bioinformatics methods, combined with clustering on the phylogenetic tree and the distribution characteristics of gene families, to select genes with the same disease resistance function in the species and between species. As a candidate disease resistance gene; or based on the principle of co-evolution of host disease resistance genes and corresponding pathogens, select a host gene with more mutations, more copy number, and faster evolution as a candidate resistance gene against the disease. . Conversely, a relatively conservative host gene is selected as a candidate resistance gene. Using molecular biology techniques, these candidate genes are cloned in large numbers and quickly, and the effectiveness of their functions is verified by transgenics. The experiment proves that the method is simple, efficient and convenient to identify. It is suitable for a large number of cloning resistance genes, and can supply new disease resistance genes to plants more, faster, better and more.

The solution of the technology of the present invention is: using a principle of many-to-many between disease resistance gene and pathogenic bacteria to establish a high-throughput method for cloning plant disease resistance genes, and clarifying high-throughput cloned rice blast resistance genes as an example . It mainly includes the following steps:

(1) Based on the genetic variation characteristics and natural evolution laws of plant disease resistance genes, based on the phylogenetic tree, select genes with the following characteristics as candidate genes for resistance to certain diseases: (1) Selection of species and species It is known that disease resistance functional gene homologous branches or nearby branch genes are candidate resistance genes; (2) According to the principle of co-evolution of host disease resistance genes and corresponding pathogens, for the pathogens with rapid evolution, more mutations and more copy numbers are selected. The fast-growing host gene acts as a candidate resistance gene against this disease. Conversely, a relatively conservative host gene is selected as a candidate resistance gene. Specifically, the rice blast resistance gene is selected from the phylogenetic tree as a candidate gene with a gene known to be resistant to the rice blast or antifungal gene or a nearby branch, or a gene family with a rapid evolution in the rice genome (eg, Multiple copies and changes in copy number, obvious selection, etc.) As a candidate gene group. These genes were cloned in resistant or related species and verified for their effectiveness by transgenesis.

(2) Selecting suitable plant-resistant varieties as materials for gene cloning, taking rice blast resistance as an example, the selected closely related species are mainly species of gramineous plants and rice closely related: such as wild rice, corn, sorghum, short handle Grasses, oats, rye, wheat, barley, etc., or highly resistant varieties within rice species, such as Tetep, Gumei, Digu, Minghui 63 and wild rice.

(3) The selected candidate genes were cloned by long-length PCR (Long-PCR), and the integrity of the genes was detected by sequencing; these genes were constructed into a bifunctional vector.

(4) Select the susceptible variety as the recipient plant, and transfer the candidate gene to the susceptible variety by a suitable Agrobacterium-mediated transgenic method.

(5) Inoculate infections with natural pathogens, screen and identify disease-resistant individuals, and identify new disease resistance genes for disease-resistant individuals. Taking high-throughput clones of rice blast resistance genes as an example, strains of rice blast that were collected from different sources, at different times, or produced were selected as pathogens, and resistant plants were screened.

(6) The marker gene in the transgenic process of the selected individual is removed by a system such as Cre-lox recombinase, so that the plant with the new disease resistance gene retains only the natural DNA sequence.

(7) Combine the screening of agronomic traits to breed new varieties with promotional value.

Another object of the present invention is to provide DNA sequences and encoded protein sequences of the rice blast resistance gene (RMglO-RMg36) in Example 1, Example 2 and Example 3.

Another object of the present invention is to provide a vector comprising the above resistance gene.

Another object of the present invention is to provide a transgenic plant transformed with the above vector.

Another object of the present invention is to provide a use of the above protein for the preparation of a drug against rice blast fungus. The present invention relates to the cloning and identification of a DNA fragment comprising a resistance gene (RMglO-RMg36), which encodes a protein which produces a specific disease-resistance response to rice diseases caused by Magnaporthe oryzae. Wherein the fragment is represented by SEQ ID NO: 1 - SEQ ID NO: 27 of the Sequence Listing or substantially corresponding to SEQ ID NO: 1 - SEQ ID NO: 27, or the function thereof corresponds to SEQ ID NO: - A subfragment of the sequence shown as SEQ ID NO:27. The proteins encoded by these DNA sequences belong to the NBS-LRR class of proteins. The amino acid sequences thereof are shown in the Sequence Listing SEQ ID NO: 28 - SEQ ID NO: 54, respectively.

The invention also encompasses fragments encoding different domains in the resistance gene (RMglO-RMg36) (eg NBS or LRR) recombines with other nucleotide fragments to form a chimeric gene or protein, giving it a new function. Modification or modification of the resistance gene (RMglO-RMg36) can alter or increase a certain function of the gene. For example, replacing the LRR region with a domain of another resistance gene, or site-directed mutagenesis of the NBS domain, may result in loss of gene resistance or altered resistance.

The present invention also encompasses a chimeric gene formed by efficiently ligating the main structure of the resistance gene (RMglO-RMg36) with a suitable regulatory sequence, and a plant comprising such a gene in the genome. Such genes can be natural or chimeric.

The rice blast resistance gene (RMglO-RMg36) provided by the invention has important application value. The resistance gene (RMglO-RMg36) sequence is introduced into rice or other plant cells by any one of the transformation methods, and a transgenic disease-resistant variety expressing the gene can be obtained, thereby being applied to production. The gene of the present invention is constructed into a plant transformation vector, and the gene or its regulatory sequence may be appropriately modified, and other promoters may be substituted for the original promoter of the gene, thereby broadening the spectrum or enhancing resistance.

The present invention has the following beneficial effects: The transfer of the cloned rice blast resistance gene into susceptible plants facilitates the acquisition of new disease resistant plants. The cloned disease resistance gene can be transferred and utilized among different species, thereby overcoming the difficulties of distant hybridization in traditional disease resistance breeding. In addition, transgenic technology can be used to accumulate multiple disease resistance genes in plants to shorten the breeding cycle. The present invention can further provide or use the disease-resistant transgenic plants obtained by the above DNA fragments and the corresponding seeds, and the plants transformed with the genes of the present invention or the recombinants based on the genes or the seeds obtained from such plants. The gene of the present invention can be transferred to other plants by sexual hybridization.

The invention is characterized in that according to the genetic evolution rule of plant disease resistance genes, bioinformatics methods are used to screen potential disease resistance genes, and molecular biological techniques are used to rapidly and rapidly clone candidate disease resistance genes. The method has the advantages of simple method, high efficiency and convenient identification, and is suitable for a large number of cloning of disease resistance genes, and effectively overcomes the shortcomings of long-term, time-consuming and labor-intensive, separation and isotope detection of traditional maps, and thus can be more, faster, better, and the like. The province continues to supply new resistance genes for rice production. DRAWINGS

Figure 1 is a schematic flow chart of the present invention. Figure 1A: Determination of candidate sequences for resistance to rice blast; Figure 1 B-E: Isolation and cloning of rice blast resistance genes; Figure 1F-G: Genetic transformation of rice blast resistance genes; Figure 1H: Identification of transformants. Fig. 2 is a PCR detection electrophoresis map of the hygromycin resistance gene and the CaMV 35S promoter of the transformant of the rice blast resistance gene in the embodiment; Fig. 1 A is a PCR amplification product of the CaMV 35S promoter, and lanes 1-6 are The PCR amplification product of the transformant, Lane 7 is the PCR amplification product of the vector pCAMBIA1300-AscI plasmid, and Lane 8 is the PCR amplification product of the non-transformer of Receptor New 2; Figure 1B is the hygromycin resistance gene The PCR product, lanes 1-6 are PCR amplification products of the transformants, lane 7 is the PCR amplification product of the vector pCAMBIA1300-Asd plasmid, and lane 8 is the PCR amplification product of the non-transformer of the receptor No. 2.

Figure 3 is a diagram showing the identification of plant resistance in the examples. Fig. 3A: Identification map of resistance of control plants; Fig. 3B: Identification map of resistance of transformed plants of rice blast resistance gene. detailed description

The principle of determining candidate resistance genes: based on the genetic variation characteristics and natural evolution laws of plant disease resistance genes, on the basis of phylogenetic tree, select the same disease resistance function gene or nearby branch genes within and between species. As a candidate disease resistance gene; or according to the co-evolution relationship and the distribution of the gene family, candidate disease resistance genes in the genome are selected.

Specific to the determination of rice blast resistance candidate genes: based on the complete sequencing of the genome of Gramineae, such as rice whole genome sequencing variety 93-11 and Nipponbare, corn, sorghum, Brassica, etc., first identify all NBS in the genome. -LRR type of disease resistance gene, and the construction of phylogenetic tree, based on the phylogenetic tree, locate all disease resistance genes known to be antifungal (rice pathogens as fungi) in phylogenetic tree, or rice A gene family with many variations in the genome, a large number of copies, and a fast evolution is used as a candidate gene group. According to this principle, we have selected nearly 100 candidate rice blast resistance loci, through primer design, in rice related species such as corn, sorghum, Brassica, and rice varieties resistant to rice blast and wild Long-fragment PCR, vector construction, transgenic and resistance identification were carried out in rice cultivars. Finally, 27 rice blast resistance genes were identified by 30 rice blast pathogen systems from all major rice producing areas in mainland China.

The technical solution of the present invention will be further specifically described below through specific embodiments. The methods used in the following examples are all conventional methods unless otherwise specified.

Example 1: Cloning and identification of candidate resistance to rice blast gene loci Rpl/Pi37 and Rp3/Pc 1. Identification of candidate locus resistance lines Rpl/Pi37 and Rp3/Pc: at the Rpl/Pi37 locus: Rpl The gene is a maize rust-resistant (fungus) gene, and Pi37 is a rice blast resistance (fungi) gene. From the phylogenetic tree, the two clusters are clustered in the same clades, and at the same time, in this evolution In the branches, both maize and sorghum have different degrees of gene expansion and newly generated gene clusters, showing a fast evolutionary feature. At the Rp3/Pc locus: Rp3 is a maize rust (fungi) gene, and Pc is a gene that is resistant to root rot (fungi) in sorghum. From the phylogenetic tree, the two cluster in the same clades, with Significant clustering; At the same time, in this clades, both maize and sorghum have different degrees of gene expansion and newly generated gene clusters, showing a fast evolutionary feature. Both of these loci conform to the basic characteristics of the candidate site for disease resistance genes, so they are cloned and identified as candidate sites.

2. Isolation and cloning of candidate blast resistance loci Rpl/Pi37 and Rp3/Pc genes: using public database sequencing species as reference sequences (rice: Nipponbare and 93-11, sorghum: BTx623, maize: B73, Brachypodium : Bd21 ) Design primers. Primer sequences are shown in the Sequence Listing SEQ ID NO: 55 - SEQ ID NO:68.

The DNA of each Gramineae plant is used as a template, including various sorghum (SSQ, P49, Jinzall, saozhouB, X622, T607), corn (B73, Mol7, Qi 319, 414, Huang Zao Si, Qi 205, Shen 317, Btl , 178 ), Brassica napus (Bd21) and rice (Tetep, Gumei 2, Minghui 63, Tadukan) and other DNA, using long-sequence PCR technology (Long-PCR) to amplify candidate gene fragments. The PCR procedure was as follows: pre-denaturation at 95 °C for 5 minutes, denaturation at 95 °C for 30 seconds, renaturation for 45 seconds at 60 °C, extension of 6 minutes at 68 °C for 35 cycles, followed by incubation at 72 °C for 10 minutes, and finally 10 ° C constant temperature. The PCR product is then subjected to gel extraction purification and electrophoresis.

3. Preparation of the bifunctional base vector: The rare enzyme cleavage site Ascl was introduced into the bifunctional vector pCAMBIA1300, the multiple cloning site BamHI and Sail, and the substitution site Xbal was substituted to form the basic vector pCAMBIA1300-AscI.

The promoters of Pi9 gene were amplified by primers with BamHI and Ascl at the 5' end, and the PCR product of the promoter was digested with restriction endonucleases BamHI and Ascl, and the basic vector pCAMBIA1300-Asd was ligated to obtain Pi9-containing promoter. Subcarrier Pi9-pro-vector. The primers with Ascl and Sail were used to amplify the terminator sequence of Pi9 gene, and the restriction endonuclease Ascl and Sail were simultaneously digested with the PCR product of the terminator and the vector Pi9-pro-vector. The base vector Pi9-vector of ΡΪ9 was obtained.

4. Linkage of candidate resistance gene to basic vector: restriction endonuclease Ascl, simultaneous digestion PCR The product was ligated with the base vector plasmid and purified. Under the action of T4 ligase, the candidate gene fragment was ligated into the basic vector pCAMBIA1300-AscI, and the positive monoclonal was picked by colony PCR, and the plasmid was extracted by shaking, and sequenced and detected. From this we have successfully cloned 30 genes.

5. Genetic transformation of candidate resistance genes: The bifunctional vector carrying the candidate gene was introduced into Agrobacterium tumefaciens EHA105, and the candidate gene was transformed into rice sensitization variety No. 2 and TP309 by Agrobacterium-mediated method. A total of 55 independent transformants were obtained in the end.

6. PCR Molecular Detection: PCR was carried out using the transformant DNA as a template and specific primers for the hygromycin resistance gene and its upstream CaMV35S promoter. The primer sequences of the hygromycin resistance gene are shown in the Sequence Listings SEQ ID NO: 109 and SEQ ID NO: 110. The primer sequences of the CaMV35S promoter are shown in the sequence listing SEQ ID 111: 111 and SEQ ID NO: 112.

7. Identification of resistance of transformants: 12 different provenances of rice blast fungus were selected as the pathogens for different pathogens. The T2 transgenic plants and the control varieties were inoculated with rice blast fungus races to screen for transformants with altered disease resistance. A total of 11 resistant strains and 7 new resistant genes (RMglO-RMgl6) were identified.

8. Structural analysis of candidate resistance to rice blast gene loci Rpl/Pi37 and Rp3/Pc protein: The DNA sequence of the obtained disease resistance gene was sequenced by walking, and the seven resistant new genes RMglO-RMgl6 were sequenced. The DNA sequences are shown in SEQ ID ΝΟ: 1-7, respectively, and the proteins encoded are shown in SEQ ID NOS: 28-34, respectively. Example 2: Cloning and identification of candidate resistance to rice blast gene locus AC134922

1. Determination of candidate resistance locus AC134922: AC134922 locus is one of the most resistant copy gene family sites in the rice genome, which is located in 4 gramineous relative species (rice, maize, sorghum and There are different copy numbers in rice and maize, and there are obvious gene expansions and newly generated gene clusters. There are obvious gene copy number changes among different individuals in rice, showing the characteristics of rapid evolution. It conforms to the basic characteristics of candidate sites for disease resistance genes, and thus is cloned and identified as candidate sites.

2. Isolation and cloning of the candidate resistance to rice blast gene locus AC134922: Primers were designed using the public database sequencing species Nipponbare and 93-11 as reference sequences. The primer sequences are shown in SEQ ID NO: 69 - SEQ ID NO: 72 of the Sequence Listing. The candidate gene fragments were amplified by long-sequence PCR (Long-PCR) using the genomic DNA of the resistant rice varieties Tetep, Gumei 2, Minghui 63 and Tadukan as templates. The PCR procedure was as follows: pre-denaturation at 95 °C for 5 minutes, denaturation at 95 °C for 30 seconds, renaturation for 45 seconds at 60 °C, extension of 6 minutes at 68 °C for 35 cycles, followed by incubation at 72 °C for 10 minutes, and finally 10 ° C constant temperature. The PCR product was subsequently subjected to gel extraction purification and electrophoresis.

4. Linkage of the candidate resistance gene to the basic vector: The restriction endonuclease Ascl is used to simultaneously digest the PCR product and the base vector plasmid, and purify. Under the action of T4 ligase, the candidate gene fragment was ligated into the basic vector pCAMBIA1300-AscI, and the positive monoclonal was picked by colony PCR, and the plasmid was extracted by shaking, and sequenced and detected. From this we have successfully cloned 8 genes.

5. Genetic transformation of candidate resistance genes: The bifunctional vector carrying the candidate gene was introduced into Agrobacterium tumefaciens EHA105, and the candidate gene was transformed into rice sensitization variety No. 2 and TP309 by Agrobacterium-mediated method. A total of 14 independent transformants were obtained in the end.

7. Identification of resistance of transformants: 12 different provenances of rice blast fungus were selected as the pathogens for different pathogens. The T2 transgenic plants and the control varieties were inoculated with rice blast fungus races to screen for transformants with altered disease resistance. Finally, a total of 4 resistant strains and 2 new resistant genes (RMgl7 and RMgl8) were identified.

8. Structural analysis of candidate resistance to rice blast gene locus AC134922 resistance gene protein: The DNA sequence of the disease resistance gene was sequenced by walking, and the DNA sequences of the two resistant new genes RMgl7 and RMgl8 were respectively SEQ ID NOS: 8-9, the proteins encoded are shown in SEQ ID NOs: 35-36, respectively. Example 3: Cloning and Identification of Candidate Resistance to Rice Blast Gene Loci in Rice Genome-wide Range 1. Determination of candidate sites for resistance to rice blast: According to the selection principle, the disease resistance genes of the known disease resistance functional gene homologous branches or nearby branch genes within the species and between the species are selected as candidates; or according to the distribution of the gene family, A family of genes showing rapid evolutionary traits (such as more variants and more copy number) were selected as candidate resistance genes. A total of 90 candidate gene loci were selected and systematic disease resistance gene cloning was carried out in rice resistant varieties.

2. Isolation and cloning of candidate resistance to rice blast disease loci: Primers were designed using a public database sequencing species as a reference sequence (rice: Nipponbare Wo B 93-11). The primer sequence is shown in the Sequence Listing SEQ ID NO: 73 - SEQ ID NO: 108.

The candidate gene fragments were amplified by long-length PCR (Long-PCR) using genomic DNA of rice Tetep, Gumei 2, Minghui 63 and Tadukan as templates. The PCR procedure was as follows: pre-denaturation at 95 °C for 5 minutes, denaturation at 95 °C for 30 seconds, renaturation for 45 seconds at 60 °C, extension of 6 minutes at 68 °C for 35 cycles, followed by incubation at 72 °C for 10 minutes, and finally 10 ° C constant temperature. The PCR product is then subjected to gel extraction purification and electrophoresis.

4. Linkage of the candidate resistance gene to the basic vector: The restriction endonuclease Ascl is used to simultaneously digest the PCR product and the base vector plasmid, and purify. Under the action of T4 ligase, the candidate gene fragment was ligated into the basic vector pCAMBIA1300-AscI, and the positive monoclonal was picked by colony PCR, and the plasmid was extracted by shaking, and sequenced and detected. As a result, we successfully cloned 101 genes.

5. Genetic transformation of candidate resistance genes: The bifunctional vector carrying the candidate gene was introduced into Agrobacterium tumefaciens EHA105, and the candidate gene was transformed into rice sensitization variety No. 2 and TP309 by Agrobacterium-mediated method. A total of 180 independent transformants were obtained in the end.

7. Identification of resistance of transformants: Select 12 different origins, and distinguish the physiological races of independent rice blast fungus as the pathogens for detection. Inoculation of T2 transgenic plants and inoculation with rice blast fungus races According to the variety, the transformants with altered disease resistance were screened. A total of 33 resistant strains and 18 resistant new genes (RMgl9-RMg36) were identified.

8. Structural analysis of rice blast resistance gene protein: The DNA sequence of the disease resistance gene was sequenced by walking. The DNA sequences of the 18 resistant new genes RMgl9-RMg36 are shown in SEQ ID NOS: 10-27, respectively, and the proteins encoded are shown in SEQ ID NOS: 37-54, respectively.

Claims

Rights request

A method for high-throughput cloning of a plant disease resistance gene, comprising the steps of:

(1) Selecting candidate resistance genes by means of genetic evolution of disease resistance genes and bioinformatics: using bioinformatics methods to identify all NBS-LRR type disease resistance genes in the genome of the target plant genome or its related species; Construct a phylogenetic tree, divide gene families, and analyze the distribution of gene clusters on chromosomes. According to the distribution of disease resistance genes with known functions on the phylogenetic branches, select genes with known functional resistance genes and adjacent branch genes as candidates. The disease resistance gene; and according to the clustering of the system clade and the gene family distribution, a gene family with multiple copies in the same genome and a protein similarity >70% between copies is selected as a candidate disease resistance gene;

The target plant may be rice, corn, sorghum, wheat, barley, soybean, cotton, tomato, potato, tobacco, or grape, apple, orange, poplar or the like; or the selected disease is fungus. a disease caused by bacteria, viruses, or nematodes that is recognized by plant disease resistance genes and produces a disease-resistant response;

(2) selecting suitable plant resistant varieties as materials for gene cloning, which may be resistant varieties or ecotypes within the same species, or may be related species of this species;

(3) cloning the selected candidate genes by long-segment PCR, and detecting the integrity of the genes by sequencing; constructing these genes into a bifunctional vector;

(4) selecting a high-sensitivity and susceptible genetically modified variety, or a variety having excellent agronomic traits as a receptor, and using Agrobacterium-mediated or other transgenic methods to transfer candidate genes therein;

(5) Inoculation of infection by plant-specific pathogenic strains, screening and identification of transgenic plants, and identification of candidate resistance genes.

2. A method according to claim 1, characterized in that the disease-resistant plant variety or its related species are selected as materials for gene cloning.

3. Method according to claim 2, characterized in that a plant variety of high sensibility is selected as a transgenic receptor material.

The method according to claim 2 or 3, wherein the target plant is rice, the disease is rice blast, and the cloning donor materials are rice Tetep, Gumei 2, Gumei 4, Minghui 63 and Wild rice and related species maize, sorghum, and Brachypodium.

The method according to claim 4, wherein a material susceptible to rice blast is selected as a transgenic receptor material, and the susceptible rice blast material comprises a rice variety Lijiang New Group Black Valley, C039, New No. 2, Taipei 309 and Su Yuxi.

The method according to any one of claims 1 to 5, characterized in that in the step (1), the candidate gene is further screened, and the gene coding region is required to be longer than 2000 bp, the full length of the gene is less than 20000 bp, and the routine is used. The software uses it as a promoter and terminator prediction.

7. A rice blast resistance gene having a DNA sequence as shown in any one of SEQ ID NO: 1 to SEQ ID NO: 27, respectively.

8. A vector comprising the rice blast resistance gene according to claim 7.

9. Use of a vector according to claim 8 for transforming rice to produce a transgenic rice blast resistant rice.

The protein encoded by the rice blast resistance gene according to claim 7, which has an amino acid sequence as shown in any one of SEQ ID NO: 28 to SEQ ID NO: 54, respectively.

The use of the protein encoded by the rice blast resistance gene according to claim 10 for the preparation of a drug resistant to rice blast fungus.