水稻类成花素家族成员OsFTL5和OsFTL6双突变体的创建与分析  

Creation and analysis of rice FT-like family members OsFTL5 and OsFTL6 double mutants

作　　者：刘晓男 宇文铭玥 郝宏姣 田瑶 王鑫 兰志鹏[1,2] 张泗举 梁闪闪[1,2] 马轩[1,2] 栾维江 LIU Xiaonan;YUWEN Mingyue;HAO Hongjiao;TIAN Yao;WANG Xin;LAN Zhipeng;ZHANG Siju;LIANG Shanshan;MA Xuan;LUAN Weijiang(College of Life Sciences,Tianjin Normal University,Tianjin 300387,China;Tianjin Key Laboratory of Animal and Plant Resistance,Tianjin Normal University,Tianjin 300387,China)

机构地区：[1]天津师范大学生命科学学院,天津300387 [2]天津师范大学天津市动植物抗性重点实验室,天津300387

出　　处：《天津师范大学学报（自然科学版）》2021年第2期56-64,共9页Journal of Tianjin Normal University：Natural Science Edition

基　　金：国家自然科学基金资助项目(31770343);天津市水稻产业技术体系研究计划资助项目(ITTRRS2018006);天津市自然科学基金重点资助项目(17JCYBJC30000).

摘　　要：为了揭示水稻中类成花素家族(FT-like)成员的生物学功能,利用CRISPR-Cas9技术对OsFTL5和OsFTL62个成员进行基因编辑,以期得到编辑突变体.在OsFTL5基因的编码区选择了2个靶位点5T1和5T2,与OsFTL6基因的编码区选择的靶位点6T1分别组合后,构建了2个融合sgRNA表达框(U6a::6T1-sgRNA-U6b::5T1-sgRNA和U6a::6T1-sgRNA-U6c::5T2-sgRNA).将这2个表达框分别装载到CRISRP-Cas9表达载体,通过农杆菌介导的遗传转化法导入水稻品种中花11中,共获得了97株T0代转基因植株,其中68株转基因植株发生了编辑,产生了不同类型的突变.通过鉴定获得了OsFTL5和OsFTL6同时发生编辑的3个纯合双突变体(ftl5ftl6-1、ftl5ftl6-2、ftl5ftl6-3)和2个纯合OsFTL6单独编辑的突变体(ftl6-1、ftl6-2).在3个双突变体中,OsFTL5基因有单碱基缺失和单碱基插入2种编辑类型;OsFTL6基因有单碱基插入、小片段缺失和大片段缺失3种编辑类型.这些突变均造成了氨基酸阅读框移码,导致蛋白质翻译的提前终止.总之,本研究成功地利用CRISPR-Cas9技术同时实现了对OsFTL5及OsFTL6基因的定向编辑,得到了ftl5ftl6双突变体和ftl6单突变体,并且获得了纯合突变植株,为OsFTL5和OsFTL6功能的研究提供了材料.In order to reveal the biological functions of Flowering locus T like(FT-like)members in rice,CRISPR-Cas9 technology was used to generate the edited mutants of OsFTL5 and OsFTL6.Two target sites in OsFTL5 coding region(5T1 and 5T2)were selected and combined with one target site in OsFTL6 coding region(6T1)to produce two fusion vectors(U6a::6T1-sgRNA-U6b::5T1-sgRNA and U6a::6T1-sgRNA-U6c::5T2-sgRNA),respectively.Two fusion expression cassettes were introduced into CRISPR-Cas9 expression vector respectively,and then transformed into Zhonghua11 by Agrobacterium-mediated transformation.Totally 97 T0 generation transgenic plants were obtained,in which 68 transgenic plants were edited and produced different types of mutants.Then,three homozygous double mutants(ftl5ftl6-1,ftl5ftl6-2,ftl5ftl6-3)and two homozygous single mutants(ftl6-1,ftl6-2)were identified and analyzed in detail.In the three double mutants,there were two types of mutation with single base deletion or insertion at OsFTL5 locus,and three types of mutation with single base deletion and deletion of small fragment of large fragment at OsFTL6 locus.All of these mutations resulted in amino acid reading frame shifting to produce the premature termination of translation.In conclusion,ftl5ftl6 double mutants and ftl6 single mutant were successfully obtained using CRISPR-Cas9 technology,which provides materials for further study of the functions of OsFTL5 and OsFTL6.

关 键 词：水稻 OsFTL5 OsFTL6 CRISPR-Cas9 基因编辑 

分 类 号：Q943[生物学—植物学]