利用CRISPR/Cas9系统研究REVOLUTA参与烟草叶芽发育的调控  

Regulation of Leaf Bud by REVOLUTA in Tobacco Based on CRISPR/Cas9 System

作　　者：王兵 赵会纳 余婧 陈杰 骆梅 雷波 WANG Bing;ZHAO Hui-na;YU Jing;CHEN Jie;LUO Mei;LEI Bo(Guizhou Academy of Tobacco Science,Molecular Genetics Key Laboratory of China Tobacco,Guiyang 550081;Guizhou Medical University,School of Biology and Engineering(School of Health Medicine Modern Industry),Guiyang 550025)

机构地区：[1]贵州省烟草科学研究院烟草行业分子遗传重点实验室,贵阳550081 [2]贵州医科大学生物与工程学院(健康医药现代产业学院),贵阳550025

出　　处：《生物技术通报》2023年第10期197-208,共12页Biotechnology Bulletin

基　　金：中国烟草总公司科技重大专项项目[110202101005(JY-05),110202201006(JY-06)];中国烟草总公司贵州省公司科技项目(2021XM03);贵州省烟草科学研究院科技项目(GZYKY2021-04)。

摘　　要：作物叶芽受到分生组织调控,调控叶芽是作物增产的有效措施之一。目前关于烟草分生组织调控的分子机理研究偏少,可用于株型改良的种质资源缺乏。本研究通过CRISPR/Cas9编辑系统靶向突变烟草REVOLUTA(REV)基因,分别构建两个不同REV单靶点序列C15NtREV和C16NtREV,通过农杆菌介导的叶盘转化方法获得再生苗,利用PCR测序鉴定转基因阳性单株,测序结果表明Ko-C15Ntrev突变体在NtREV氨基酸第26位置之后发生移码突变,而Ko-C16Ntrev突变体在NtREV氨基酸第60位置之后发生移码突变。此外,借助扫描电镜分别观测两个单靶点纯合突变体顶芽表型,结果表明烟草Ko-C15Ntrev双拷贝同源突变体出现顶芽缺失和叶片畸形的表型,而Ko-C16Ntrev单拷贝同源突变体未表现出顶芽缺失,但顶芽发育迟缓。相较于野生型烟草,Ko-C16Ntrev突变体自然株高较野生型增加3.76%,Ko-C16Ntrev突变体叶片数和腋芽鲜重分别较野生型减少21.47%和23.41%,且均达到极显著差异,说明NtREV参与烟草顶端分生组织发育,进而调节叶和腋芽发育。这些突变体为后续研究烟草的叶芽发育分子机理提供了重要研究材料。The leaf buds of crop are regulated by the meristem,which is one of the effective measures to increase crop yield.At present,there are few studies on the molecular mechanism of meristem regulation in tobacco,and there is a lack of germplasm resources that can be used for plant type improvement.In this study,two different REVOLUTA(REV)single-target sequences,C15NtREV and C16NtREV,were constructed respectively by targeting mutation of REV gene through CRISPR/Cas9 editing system,and regenerated seedlings were obtained by Agrobacterium-mediated leaf disc transformation method,and the transgenic plants were identified by PCR sequencing.The sequencing results showed that the Ko-C15Ntrev mutant had a shift mutation after amino acid position 26 of NtREV,while the Ko-C16Ntrev mutant had a shift mutation after amino acid position 60 of NtREV.In addition,the apical bud phenotypes of two single-target homozygous mutants were observed separately by scanning electron microscope,the results showed that the double-copy homologous mutants of Ko-C15Ntrev had apical bud deletion and leaf deformities,while the single-copy homologous mutants of Ko-C16Ntrev did not have the absence of apical buds,but the apical buds development delayed.Compared with wild type,the natural plant height of the Ko-C16Ntrev mutant increased by 3.76%,while the number of leaf blades and fresh axillary bud weight of the Ko-C16Ntrev mutant decreased by 21.47% and 23.41%,respectively,and both reached extremely significant differences,indicating that NtREV is involved in the development of shoot apical meristem in tobacco,which in turn regulates the development of leaf and axillary bud.These mutants provided important research materials for the subsequent study of the molecular mechanism of bud development in tobacco.

关 键 词：CRISPR/Cas9 REVOLUTA 叶芽 株型 侧生分生组织 烟草 

分 类 号：S572[农业科学—烟草工业]