Method for creating broad-spectrum fine-streak-disease-resistant crop new germplasm through gene editing and application thereof
阅读说明：本技术 通过基因编辑创制广谱抗细条病作物新种质的方法及其应用 (Method for creating broad-spectrum fine-streak-disease-resistant crop new germplasm through gene editing and application thereof ) 是由 黄大辉 秦钢 张月雄 马增凤 刘驰 岑贞陆 韦敏益 罗同平 李振经 韦维 于 2019-10-29 设计创作，主要内容包括：本发明公开一种通过基因编辑创制广谱抗细条病作物新种质的方法及其应用,对水稻OsMPK6基因全长cDNA进行克隆,利用CRISPR/Cas9技术对OsMPK6进行编辑,构建转基因载体,利用农杆菌介导法进行转基因,获得T0转基因阳性苗,通过测序和野生型的目的片段进行比对确定所得转基因阳性苗目的基因已被编辑,种植T0转基因苗,利用分子标记辅助选择,获得基因型纯合的T1代转基因苗,对T1、T2代转基因苗进行人工接种鉴定,若表现出广谱细条病抗性,则获得了广谱抗细条病的水稻新种质。本发明利用基因编辑技术,对细条病抗性主效基因BLS1(OsMPK6)进行修饰,创制广谱抗细条病新种质,可以大大提高这一种重要资源的利用效率。(The invention discloses a method for creating a broad-spectrum slim disease-resistant crop new germplasm through gene editing and application thereof, wherein full-length cDNA of a rice OsMPK6 gene is cloned, a CRISPR/Cas9 technology is used for editing OsMPK6, a transgenic vector is constructed, transgenosis is performed by an agrobacterium-mediated method to obtain a T0 transgenic positive seedling, the comparison between sequencing and a wild-type target fragment is performed to determine that the target gene of the obtained transgenic positive seedling is edited, a T0 transgenic seedling is planted, molecular marker-assisted selection is used to obtain a T1 transgenic seedling with homozygous genotype, artificial inoculation identification is performed on T1 and T2 transgenic seedlings, and if broad-spectrum slim disease resistance is expressed, the broad-spectrum slim disease-resistant crop new germplasm is obtained. The invention utilizes gene editing technology to modify the main effective gene BLS1(OsMPK6) for the bacterial leaf streak resistance, creates a novel broad-spectrum bacterial leaf streak resistance germ plasm and can greatly improve the utilization efficiency of the important resource.)
1. A method for creating a novel broad-spectrum anti-leptospirosis germplasm through gene editing is characterized in that full-length cDNA of a rice OsMPK6 gene is cloned, the gene OsMPK6 is edited by using a CRISPR/Cas9 technology to construct a transgenic vector, transgenosis is performed by using an agrobacterium-mediated method to obtain a T0 transgenic positive seedling, the comparison between sequencing and a wild-type target fragment is performed to determine that the target gene of the obtained transgenic positive seedling is edited, a T0 transgenic seedling is planted, molecular marker-assisted selection is used to obtain a T1 transgenic seedling with a homozygous genotype, artificial inoculation identification is performed on T1 and T2 transgenic seedlings, and if the resistance of the broad-spectrum leptospirosis is shown, the novel broad-spectrum anti-leptospirosis rice germplasm is obtained.
2. The method for creating broad-spectrum new germplasm for resisting fine streak disease by gene editing according to claim 1The method is characterized in that: in the cloning step of the full-length cDNA of the rice OsMPK6 gene, the cDNA is reversely transcribed, and the primer sequences used are as follows: left end primer sequence cagtggtctcacaacatggacgccggggcgcagcc, left primer sequence: cagtggtctcatacactggtaatcagggttgaacg。
3. The method for creating a novel germplasm of broad spectrum against leptospirosis by gene editing according to claim 1, wherein the method comprises the following steps: the constructed transgenic vector has the following target primers designed according to the constructed vector:
(1) 1 st target primer:
7082-Y+：cagtggtctcaggcactggatgaacctcccgcca；
7082-Y-：cagtggtctcaaaactggcgggaggttcatccag；
(2) target primer 2:
7082-B+：cagtggtctcaggcaagccccccatcctccccat；
7082-B-：cagtggtctcaaaacatggggaggatggggggct。
4. the method for creating a novel germplasm of broad spectrum against leptospirosis by gene editing according to claim 1, wherein the method comprises the following steps: the method comprises the following steps:
(1) locating candidate genes: positioning BLS1 in the physical range of 21-kb between RM19400 and RM510, logging in RiceGenome Annotation Project website (http:// rice. plant biology. msu. edu /), querying the positioning region (Chr.62810860-2831635) to obtain 4 candidate genes including OsMPK6 gene (LOC _ Os06g06090), dihydroneopterin aldolase (LOC _ Os06g06100), transposon (LOC _ Os06g06110) and unknown gene (LOC _ Os06g 06115);
(2) analysis of candidate gene expression: taking an anti-infection parent plant and a progeny plant as materials, respectively inoculating leptospira tenuissima, sampling at different time points, extracting RNA, performing reverse transcription to obtain cDNA, performing expression analysis on candidate genes in a positioning region, and selecting genes related to leptospira tenuissima resistance;
(3) fine localization area sequencing: using an Agilent capture platform to perform sequencing on rice materials DP3, 9311, 708-2 and 810 in a fine positioning region and a 200kb range of a flank, obtaining a large amount of data of a target section for bioinformatics analysis, and using IGV software to perform comparative analysis on a fine positioning sequencing result to find that rich gene structure difference exists between an anti-susceptible parent and an anti-susceptible progeny single plant in an OsMPK6 gene region;
(4) cloning of candidate gene cDNA: carrying out reverse transcription on cDNA by using a designed primer, cloning the full-length CDNA of the candidate gene OsMPK6, and obtaining sequences of disease-resistant plants DP3 and 708-2 and disease-susceptible plants 9311 and 810;
(5) constructing a transgenic vector by using CRISPR/Cas9 gene editing: selecting a gene editing material 708-2 with resistance to the bacterial streak disease between that of common DP3 and 9311, and then constructing a transgenic vector through PCR amplification, enzyme digestion connection and sequencing analysis;
(6) obtaining transgenic seedlings: utilizing agrobacterium-mediated method to make gene transfer to obtain T0 positive seedlings of 23 strains; extracting genome DNA of the T0 positive seedling, carrying out PCR detection on the T0 positive seedling by using a target gene primer, sequencing an amplified PCR product, comparing a sequencing result with a wild type target fragment to determine that a target gene of the obtained transgenic seedling is edited, planting a T0 transgenic seedling, and carrying out detection by using the target gene primer to obtain a T1 generation transgenic seedling with homozygous genotype;
target gene primers: 7082-6200+: cacctgctcccccgtctc, 7082-6633-: cctccactttcccttccc, respectively;
(7) the creation of a novel broad-spectrum multi-resistance leptospirosis germplasm: through artificial inoculation identification, a strain E136-27-1 which shows better resistance than the wild type 708-2 is obtained by screening T1 generation transgenic seedlings; functional complementation experiments show that the OsMPK6 gene (LOC _ Os06g06090) is the target gene BLS 1; the progeny V902 of the E136-27-1 is detected by using a protein immunoblotting (Western Blot, WB) technology, and the translation amount of the target protein OSMPK6 is greatly reduced compared with that of the wild type 708-2 by the V902; v902 and wild type 708-2 are inoculated and identified by using leptospira I (with the strain number of QC-1), II (with the strain number of HG-3) and III (with the strain number of JZ-8), and the result shows that V902 has broad-spectrum leptospira resistance compared with wild type 708-2, thereby proving that the broad-spectrum leptospira-resistant innovative germplasm is obtained.
5. Use of the gene editing according to any one of claims 1 to 4 for creating a new germplasm of broad-spectrum stringy-resistant rice.
[ technical field ] A method for producing a semiconductor device
The invention relates to the technical field of crop breeding by gene editing technology, in particular to a method for creating a novel broad-spectrum twill disease resistant crop germplasm by gene editing and application thereof.
[ background of the invention ]
Bacterial streak disease (bacterial streak disease for short) is caused by pathogenic bacterium Xanthomonas oryzae pv. oryzicola (Xoc), and is an important disease of rice. The disease can cause 32% of the rice yield loss in the outbreak epidemic years. Cultivation measures and pesticide application are effective methods for preventing and treating the leptospirosis. However, the cultivation of resistant varieties using resistant resources is the most cost-effective way to control the disease. The molecular biotechnology such as gene editing and the like is used for improving the breeding efficiency by taking excellent resistance gene resources as objects, and has important significance. At present, few researches on creating a novel broad-spectrum anti-leptospirosis germplasm by using gene editing are available.
The rice seeds are rich in anti-leptospira resources, but most of the anti-leptospira resources mainly come from tropical regions, most of the anti-leptospira resources are ancient local varieties, and the agronomic characters are poor, so that the rice seeds are improved by a conventional breeding method, the period is long, and the resource utilization efficiency is low. In addition, the insufficient development of the resistance source is also one of the reasons for the slow progress of the anti-leptospirosis breeding. Among the resistance sources of leptospirosis, resistance controlled by major genes is an important type of resistance source.
[ summary of the invention ]
In view of the above, the present application provides a method for creating a broad-spectrum new germplasm of a crop resistant to a streak disease by gene editing and an application thereof, which modifies a main effective gene BLS1(OsMPK6) for the resistance of the streak disease to create a broad-spectrum new germplasm resistant to the streak disease, and can greatly improve the utilization efficiency of the important resource.
In order to achieve the purpose, the technical scheme adopted by the invention is as follows:
a method for creating a novel broad-spectrum anti-leptospirosis germplasm through gene editing comprises the steps of cloning a full-length cDNA of a rice OsMPK6 gene, editing the gene OsMPK6 by using a CRISPR/Cas9 technology, constructing a transgenic vector, carrying out transgenosis by using an agrobacterium-mediated method, determining that a target gene of an obtained transgenic positive seedling is edited by sequencing and comparing a wild-type target fragment, planting a T0 transgenic seedling, obtaining a T1 generation transgenic seedling with a homozygous genotype by using molecular marker for auxiliary selection, carrying out artificial inoculation identification on T1 and T2 generation transgenic seedlings, and obtaining the novel broad-spectrum anti-leptospirosis rice germplasm if the broad-spectrum resistance to the leptospirosis is expressed.
Further, in the cloning step of the full-length cDNA of the rice OsMPK6 gene, the cDNA is reverse transcribed, and the primer sequences used are as follows: left end primer sequence cagtggtctcacaacatggacgccggggcgcagcc, left primer sequence: cagtgg tctcatacactggtaatcagggttgaacg。
Further explaining, the constructed transgenic vector and the designed vector construction target primers are respectively as follows:
(1) 1 st target primer:
7082-Y+：cagtggtctca ggca ctggatgaacctcccgcca；
7082-Y-：cagtggtctca aaac tggcgggaggttcatccag；
(2) target primer 2:
7082-B+：cagtggtctca ggca agccccccatcctccccat；
7082-B-：cagtggtctca aaac atggggaggatggggggct。
further, the method for creating the broad-spectrum new germ plasm for resisting the bacterial streak disease through gene editing comprises the following steps:
(1) locating candidate genes: positioning BLS1 in the physical range of 21-kb between RM19400 and RM510, logging in Rice Genome Annotation Project website (http:// Rice. plant biology. msu. edu /), querying the positioning region (Chr.62810860-2831635) to obtain 4 candidate genes including OsMPK6 gene (LOC _ Os06g06090), dihydroneopterin aldolase (LOC _ Os06g06100), transposon (LOC _ Os06g06110) and unknown gene (LOC _ Os06g 06115);
(2) analysis of candidate gene expression: taking an anti-infection parent plant and a progeny plant as materials, respectively inoculating leptospira tenuissima, sampling at different time points, extracting RNA, performing reverse transcription to obtain cDNA, performing expression analysis on candidate genes in a positioning region, and selecting genes related to leptospira tenuissima resistance;
(3) fine localization area sequencing: using an Agilent capture platform to perform sequencing on rice materials DP3, 9311, 708-2 and 810 in a fine positioning region and a 200kb range of a flank, obtaining a large amount of data of a target section for bioinformatics analysis, and using IGV software to perform comparative analysis on a fine positioning sequencing result to find that rich gene structure difference exists between an anti-susceptible parent and an anti-susceptible progeny single plant in an OsMPK6 gene region;
(4) cloning of candidate gene cDNA: carrying out reverse transcription on cDNA by using a designed primer, and cloning the full-length cDNA of the candidate gene OsMPK6 to obtain sequences of disease-resistant plants DP3 and 708-2 and disease-sensitive plants 9311 and 810;
(5) constructing a transgenic vector by using CRISPR/Cas9 gene editing: selecting a gene editing material 708-2 with resistance to the bacterial streak disease between that of common DP3 and 9311, and then constructing a transgenic vector through PCR amplification, enzyme digestion connection and sequencing analysis;
(6) obtaining transgenic seedlings: utilizing agrobacterium-mediated method to make gene transfer to obtain T0 positive seedlings of 23 strains; extracting genome DNA of T0 positive seedlings, carrying out PCR detection on T0 positive seedlings by using target gene primers, sequencing amplified PCR products, comparing sequencing results with wild type target fragments to determine that the target genes of the obtained transgenic seedlings are edited, planting T0 transgenic seedlings, and carrying out detection by using the target gene primers to obtain T1 generation transgenic seedlings with homozygous genotypes;
the target gene primer comprises the following components: 7082-6200+: cacctgctcccccgtctc, 7082-6633-: cctccactttcccttccc, respectively;
(7) the creation of a novel broad-spectrum multi-resistance leptospirosis germplasm: through artificial inoculation identification, from T1 transgenic seedlings, a strain E136-27-1 which shows better resistance than the wild type 708-2 is obtained through screening. The function complementation experiment shows that the OsMPK6 gene (LOC _ Os06g06090) is the
The application of the broad-spectrum anti-leptospirosis rice new germplasm is created through gene editing.
The invention has the following beneficial effects:
1. the invention utilizes gene editing technology to modify the main effective gene BLS1(OSMPK6) for the resistance of the bacterial streak disease, creates a new bacterial germplasm for resisting the bacterial streak disease, and can greatly improve the utilization efficiency of the important resource.
2. The new broad-spectrum bacterial streak disease resistant crop variety created by gene editing provided by the invention can powerfully supplement the existing bacterial streak disease resistant rice breeding material.
[ description of the drawings ]
FIG. 1 is a lesion length distribution diagram of an isolated population of the near isogenic line F2;
FIG. 2 is a diagram showing the preliminary mapping of
reference numerals: calculating map genetic distance, LOD value and PEV value by integrating genotype and phenotype data of F2 population by using MapQTL5 software; the LOD value and the PEV value correspond to the left side and the right side of the Y axis respectively; the molecular marker is on an X axis, and the genetic unit distance is 10 kb; the LOD significance level was set to 3.0; dark and light curves indicate LOD and PEV values, respectively;
FIG. 3 is a fine mapping of the BLS1 gene;
reference numerals: a indicates that BLS1 is located between RM19391 and RM 510; b indicates that BLS1 is in the range of 21-kb between RM510 and RM 19400; the upper row of numbers in the genetic maps a and b represent the genetic distance between the molecular markers, and the lower row of data in the genetic maps a and b represent the genetic recombination individuals between the molecular markers and the
FIG. 4 is a diagram of genes contained in a fine localization region;
FIG. 5 is an OsMPK6 gene expression analysis;
remarking: DP 3-disease resistant wild rice; 9311-susceptible parents; 708-2-disease resistant progeny; 810-affected offspring;
FIG. 6 is a diagram of amino acid mutations;
FIG. 7 is a CRISPR/Cas9 gene editing transgene vector;
FIG. 8 is a schematic diagram of a T0 positive shoot;
FIG. 9 is a diagram showing the alignment of the OsMPK6 gene editing seedlings with 708-2 sequences;
FIG. 10 is a graph showing the results of Western blotting detection of OsMPK6 on V902.
[ detailed description ] embodiments
All of the features disclosed in this specification, or all of the steps in any method or process so disclosed, may be combined in any combination, except combinations of features and/or steps that are mutually exclusive.
Any feature disclosed in this specification (including any accompanying claims, abstract) may be replaced by alternative features serving equivalent or similar purposes, unless expressly stated otherwise. That is, unless expressly stated otherwise, each feature is only an example of a generic series of equivalent or similar features.
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