水稻OsFTL12和OsFTL13基因双突变体的创建及新型编辑类型的鉴定

Construction of rice OsFTL12 and OsFTL13 genes double mutants and identification of new editing type

为了研究水稻成花素家族基因的功能,在OsFTL12基因的编码区选择2个靶点,OsFTL13基因的编码区选择1个靶点,构建各个靶点的融合表达盒,将其连接到CRISPR-Cas9载体中,获得包含OsFTL12和OsFTL13基因靶点的CRISPR-Cas9重组载体.以粳稻品种中花11为材料,利用农杆菌介导的遗传转化法,创建水稻OsFTL12和OsFTL13两个基因的双突变体,对获得的阳性转基因植株进行测序鉴定.结果显示:阳性转基因植株中包括3种不同类型的编辑突变体,其中,OsFTL13基因仅有1种编辑类型,为1 bp缺失;而OsFTL12基因有3种编辑类型,分别为纯合1 bp缺失、纯合19 bp缺失和双等位突变.进一步分析双等位突变,发现该突变体的一条同源染色体含有1 bp缺失,而另一条同源染色体上有376 bp的CRISPR-Cas9载体序列插入,这在CRISPR-Cas9编辑系统中鲜见.通过自交获得了纯合的CRISPR-Cas9载体序列插入突变体.

In order to reveal the functions of rice florigen genes,two target sites in OsFTL12 coding region and one target site in OsFTL13 coding region were selected to construct the fusion expression cassettes of each target,which was connected to the CRISPR-Cas9 vector.The recombinant CRISPR-Cas9 vector containing OsFTL12 and OsFTL13 gene targets was obtained.Oryza sativa L.spp.Japonica cv.Zhonghua11 was adopted as material,and double mutants of OsFTL12 and OsFTL13 were created by Aturobacterium-mediated genetic transformation method.The positive transgenic plants obtained were sequenced and identified.The results showed that there were three different editing mutants in the positive transgenic plants.OsFTL13 gene only has one type of mutation which is 1 bp deletion,while OsFTL12 gene has three editing types,which are homozygous 1 bp deletion,homozygous 19 bp deletion and bi-allelic mutation.The bi-allelic mutation in OsFTL12 gene was analyzed in detail and found that one homologous chromosome contained 1 bp deletion and the other homologous chromosome contained 376 bp insertion of CRISPR-Cas9 vector sequence in the bi-allelic mutation of OsFTL12 gene.This is rare in the CRISPR-Cas9 editing system.Then homozygous CRISPR-Cas9 vector sequence insertion mutants were obtained by self-fertilization.