利用CRISPR-Cas9基因编辑技术获得水稻OsMADS56基因突变体

Generation of OsMADS56 Mutants in Rice Using CRISPR/Cas9 Editing Approach

OsMADS56是水稻中长日照下的开花抑制基因,与拟南芥中同时影响开花时间和花发育的SOC1同源。为了深入探究OsMADS56基因对水稻开花时间的调控功能,本研究利用反向遗传学的方法通过CRISPR/Cas9基因编辑技术对OsMADS56基因设计了2个特异性敲除靶位点,构建了OsMADS56基因敲除载体,并通过农杆菌介导的转化方法成功获得了野生型粳稻9522背景的转基因苗。鉴定结果显示9棵OsMADS56转基因苗中包含了3种纯合突变类型,包括第一个外显子第45号碱基处缺失了一个G碱基、缺失了包含了第一个起始密码子的100个碱基对以及在第5个外显子第322号碱基处插入一个A碱基。通过蛋白序列比对分析,发现这三种类型的突变均造成移码突变和蛋白翻译提前终止引起保守结构域的缺失。本研究获得的水稻OsMADS56基因的三个突变体株系对今后进一步深入研究该基因的功能具有重要意义。

OsMADS56 is a flowering repressor under long day condition in rice,and is homologous to Arabidopsis SOC1,which affects both flowering time and flower development.To further understand the function of OsMADS56 in regulating rice flowering time,we designed two specific target sites for knocking out OsMADS56 gene through CRISPR/Cas9 gene editing technology.We constructed the knockout vector of OsMADS56 gene and successfully obtained transgenic seedlings of rice variety 9522 background by Agrobacterium-mediated transformation method.Identification results showed that there were three types of homozygous mutations among nine OsMADS56 transgenic seedlings,including the deletion of a G base in the 45 th-bit base of the first exon,the deletion of 100 base pairs containing the first starting codon,and the insertion of an A base in the 322 th-bit base of fifth exon.Through the analysis of protein sequences,we found that the three types of mutations all caused frame-shift mutation and premature termination of translation,leading to the loss of conserved domains.The resultant three mutant lines of rice OsMADS56 gene will be useful for further study of the function of this gene.