利用CRISPR/Cas9基因编辑技术创建水稻OsPUX2突变体

CRISPR/Cas9 Targeted Editing of OsPUX2 in Rice

[目的]U-box蛋白是一类决定靶标蛋白特异性的E3泛素连接酶(少部分属于泛素链聚集因子-E4),在植物抗病、抗逆和生长发育各阶段都发挥着重要作用。为揭示U-box蛋白家族基因OsPUX2在水稻防御反应中的具体生物学功能,利用CRISPR/Cas9编辑技术对OsPUX2基因进行编辑。[方法]在OsPUX2第1外显子设计1个20 bp的编辑靶点,构建了OsPUX2基因敲除载体pRGEB32-OsPUX2-gRNA载体,并通过农杆菌介导的转化方法侵染水稻Pik-H4 NIL愈伤组织,经潮霉素检测获得转基因阳性植株,并对T 0代转基因植株进行靶点区域序列进行PCR和测序分析,分析ospux2的突变类型。[结果]成功获得了ospux2的敲除突变体材料,对突变类型的分析发现,转基因编辑后代共存在7种突变类型,以缺失突变为主,其中一种纯合突变类型在OsPUX2第一个外显子的第115号碱基处缺失了一个C碱基,导致蛋白的翻译在第84个氨基酸处提前终止。[结论]该研究获得ospux2突变株系对进一步研究该基因的功能具有重要意义。

[Objective]U-box proteins are a class of E3 ubiquitin ligases that determine the specificity of target proteins,play an important role in plant disease resistance,stress resistance and all stages of growth and development.In order to reveal the specific biological function of the U-box protein family gene OsPUX2 in rice defense response,the OsPUX2 gene was edited by CRISPR/Cas9 editing technology.[Method]A 20-bp editing target was designed in the first exon of OsPUX2 and the pRGEB32-OsPUX2-gRNA vector was constructed.Rice Pik-H4 NIL callus was infected by Agrobacterium-mediated transformation and tested with hygromycin.The T0 generation transgenic plants were subjected to PCR and sequencing analysis of the target region sequence to analyze the mutation type of ospux2.[Result]The knockout mutant of ospux2 was successfully obtained.There were a total of 7 mutation types in the transgenic edited progeny,which are mainly deletion mutations.One of the homozygous mutation types with a C base deletion at the+115 bp of the first exon of OsPUX2 gene leads to termination of protein translation at 84 aa.[Conclusion]The ospux2 mutant strain obtained in this study is of great significance to further study the function of this gene.