利用CRISPR/Cas9技术创制番茄青枯病抗性基因Slmlo1/6突变体

史建磊<sup>1; 2</sup>; 熊自立<sup>1</sup>; 苏世闻<sup>1</sup>; 付存念<sup>1</sup>; 宰文珊<sup>1*</sup>

引用本文:	史建磊, 熊自立, 苏世闻, 付存念, 宰文珊.利用CRISPR/Cas9技术创制番茄青枯病抗性基因Slmlo1/6突变体[J].广西植物,2024,44(12):2163-2171.[点击复制]
	SHI Jianlei, XIONG Zili, SU Shiwen, FU Cunnian, ZAI Wenshan.Bacterial wilt resistance gene Slmlo1/6 mutants in tomato created by CRISPR/Cas9 technology[J].Guihaia,2024,44(12):2163-2171.[点击复制]

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*利用CRISPR/Cas9技术创制番茄青枯病抗性基因Slmlo1/6突变体*
史建磊^{1, 2}, 熊自立¹, 苏世闻¹, 付存念¹, 宰文珊^1*
1. 温州科技职业学院浙南作物育种重点实验室, 浙江温州 325006;2. 福建农林大学农学院, 福州 350002

摘要:

青枯病是番茄(Solanum lycopersicum)生产中的一种毁灭性土传病害,致病菌生理小种复杂、易变异,而MLO基因隐性突变mlo具有广谱抗性,前期研究表明Slmlo1/6可能参与番茄青枯病抗性反应。为进一步研究番茄Slmlo1/6青枯病抗性基因功能,该文利用CRISPR/Cas9技术创制Slmlo1/6基因突变材料,并进行表型鉴定。结果表明:(1)设计SlMLO1/6靶点序列gRNA,并与U6启动子组装,再将含高效靶点的U6-gRNA1/6片段通过Bsa I酶切连入CRISPR载体pBGK,构建形成双基因融合敲除载体pBGK-SlMLO1/6。重组质粒经转化大肠杆菌(Escherichia coli)感受态DH5α和平板培养,挑选阳性单克隆。验证正确后,再经根癌农杆菌(Agobacterium tumefaciens)GV3101介导的遗传转化和潮霉素抗性筛选,最终获得9株番茄编辑苗。(2)靶点区测序显示,植株M2和M8分别缺失177 bp和7 bp的SlMLO1片段,M7缺失12 bp的SlMLO6片段,M9发生SlMLO6单碱基T插入,总计4株单基因纯合突变体,其他均为杂合型突变。(3)RT-qPCR分析表明,与野生型相比,突变株SlMLO1/6基因表达水平显著下降,尤其是M2、M7和M8。(4)表型鉴定表明,SlMLO1/6可能是番茄青枯病易感基因。综上,该文成功构建了MLO基因编辑载体并实现了番茄转化,纯合突变体获得了青枯病抗性。氨基酸丢失和移码突变可能是Slmlo1/6抗性功能转变的主要原因。该研究结果为番茄抗青枯病基因功能研究和抗病育种应用提供了理论参考和遗传工程材料。

关键词: 番茄, Slmlo1/6, 基因编辑, 遗传转化, 突变体

DOI：10.11931/guihaia.gxzw202312009

分类号:Q943

文章编号:1000-3142(2024)12-2163-09

基金项目:浙江省农业新品种选育重大科技专项子课题(2021C02065); 浙江省基础公益研究计划(LTGN23C020002); 温州市农业新品种选育协作组项目(ZX2024002-2)。

Bacterial wilt resistance gene Slmlo1/6 mutants in tomato created by CRISPR/Cas9 technology

SHI Jianlei^{1, 2}, XIONG Zili¹, SU Shiwen¹, FU Cunnian¹, ZAI Wenshan^1*

1. Southern Zhejiang Key Laboratory of Crop Breeding, Wenzhou Vocational College of Science and Technology, Wenzhou 325006, Zhejiang, China;2. College of Agriculture, Fujian Agriculture and Forestry University, Fuzhou 350002, China 1. Southern Zhejiang Key Laboratory of Crop Breeding, Wenzhou Vocational College of Science and Technology, Wenzhou 325006, Zhejiang, China; 2. College of Agriculture, Fujian Agriculture and Forestry University, Fuzhou 350002, China

Abstract:

Bacterial wilt is a devastating soil-borne disease in tomato(Solanum lycopersicum)production. The pathogenic species are complex and tend to have a variation, while mlo caused by the recessive mutation of MLO genes has a broad-spectrum resistance. The previous study suggested that Slmlo1/6 may be involved in the resistance response to bacterial wilt in tomato. In order to further study the gene function of Slmlo1/6 in tomato bacterial wilt resistance, the genetic mutant plants were created by CRISPR/Cas9 technology and their phenotypes were identified followed. The results were as follows:(1)gRNA sequences of SlMLO1/6 were designed and assembled with the U6 promoters, then U6-gRNA1/6 fragments containing highly effective targets were ligated to CRISPR vector of pBGK via restriction enzyme Bsa I digestion, to construct the two-gene fusion knockout vector of pBGK-SlMLO1/6. The recombinant plasmid of pBGK-SlMLO1/6 was transformed into Escherichia coli DH5α competent cells and positive monoclonal clones were selected via plate cultivation. Using Agrobacterium tumefaciens GV3101 strains-mediated genetic transformation and resistance screening to hygromycin, a total of nine edited tomato plants were obtained with sequencing validation.(2)Target region sequencing showed that M2 and M8 plants had the 177 bp and 7 bp deletion of SlMLO1, respectively, M7 had the 12 bp deletion of SlMLO6, and M9 had a single base T insertion of SlMLO6. Except for four single gene homozygous mutants above, the other mutations were heterozygous.(3)RT-qPCR showed that compared with the wild type plant, SlMLO1/6 gene expression of the mutants was significantly decreased, especially M2, M7, and M8 plants.(4)Phenotypic identification indicated that SlMLO1/6 might be tomato bacterial wilt susceptibility genes. In conclusion, the knockout vector is successfully constructed for resistance MLO genes and tomato transformation is also achieved, homozygous mutants acquire resistance to bacterial wilt. Amino acid deletion and frameshift mutation may be the crucial reasons for the gene function change of Slmlo1/6 in resistance. The results provide a theoretical reference and genetic engineering materials for the gene function study in resistance to bacterial wilt and disease resistance breeding application of tomato.

Key words: tomato, Slmlo1/6, gene editing, genetic transformation, mutant