基于CRISPR/Cas9系统的番茄LeMYB330突变体制作及突变位点分析

Construction of Tomato LeMYB330 Mutant and Analysis of Mutation Site based on CRISPR/Cas9 System

植物MYB转录因子在植物防卫反应过程中发挥重要作用,番茄LeMYB330是与根结线虫效应蛋白MiPDCD6互作的靶标蛋白。本研究利用在线软件CRISPR-GE (http://skl.scau.edu.cn/)设计了番茄LeMYB330基因编辑双靶点,在番茄LeMYB330基因第3外显子区域找到彼此相距124 bp的2个靶点,分别为TG1:5’-ATGCAACAGTGGTACAACTGAGG-3’和TG2:5’-GGTGGTCAAGAAGTTGTATGAGG-3’。将靶点T1和靶点T2的sg RNA表达盒进行Golden Gate克隆连接到pYLCRISPR/Cas9Pubi-H双元表达载体,成功获得CRISPR/Cas9重组双元载体(pCas9-MYB330T1T2)。利用农杆菌介导的遗传转化将重组载体转入‘新金丰一号’番茄愈伤组织,经潮霉素抗性筛选获得共计50株CRISPR/Cas9突变番茄植株。番茄转基因后代测序结果表明,有3株阳性植株发生突变,由此成功构建了番茄LeMYB330突变体。试验结果可为加快番茄LeMYB330功能基因资源的开发利用提供有力的理论与方法支持。

Plant MYB transcription factor plays an important role in the process of plant defense response. Tomato LeMYB330 is a target protein interacting with root knot nematode effector protein MiPDCD6. In this study, the online software CRISPR-GE(http://skl.scau.edu.cn/) was used to design the editing double targets of tomato LeMYB330 gene. Two targets were found 124 bp apart from each other on the third exon region of LeMYB330, which met the target design requirements, named TG1: 5’-ATGCAACAGTGGTACAACTGAGG-3’ and TG2: 5’-GGTG GTCAAGAAGTTGTATGAGG-3’. Golden Gate clones of sg RNA expression vector of target T1 and target T2were cloned and connected to the dual expression vector pYLCRISPR/Cas9Pub-H, and the CRISPR/Cas9 recombinant dual vector(PCAS9-MYB330T1T2) was successfully obtained. Using agrobacterium-mediated genetic transformation, the recombinant vector was transferred into the tomato callus of ’Xinjinfeng No. 1’, and 50 T0 tomato mutant plants were obtained by hygromycin resistance screening, including 3 CRISPR/Cas9 mutant tomato plants.The sequencing results of transgenic progeny of tomato showed that one positive plant was mutated, so the tomato LeMYB330 mutant was successfully constructed. The results could provide powerful theoretical and methodological support for accelerating the development and utilization of tomato LeMYB330 functional gene resources.