摘要
利用CRISPR-Cas9基因编辑技术对拟南芥真核生物释放因子1-1(eukaryotic release factor 1-1,ERF 1-1)基因进行高效率的靶向突变以获得该基因突变体,观察突变体表型并进一步探索ERF1-1基因在拟南芥中生长发育中的功能.根据原间隔序列邻近模块序列(protospacer adjacent motif,PAM)的特异性,选择1对位于ERF1-1基因内部的靶点序列,并设计1对向导核糖核酸(guide ribonucleic acid,gRNA),将与这对gRNA序列碱基互补配对的脱氧核糖核酸(deoxyribonucleic acid,DNA)序列构建到pHEE401载体中.将构建好的重组质粒转化到农杆菌GV3101菌株,接着利用含有重组质粒的农杆菌对拟南芥花序进行滴花法侵染.用含有潮霉素的培养平板筛选转基因T1代阳性苗并传代培养,通过测序检测每一代植株靶点附近是否发生编辑.采用目前最有效的CRISPR/Cas9基因靶向编辑技术突变拟南芥ERF1-1基因,成功获得了一系列不同形式的ERF1-1基因突变植株,在正常生长条件下ERF1-1突变植株与野生型相比具有轻微的生长发育缺陷表型.根据erf1-1突变植株的表型推测ERF1基因家族的成员ERF1-1、ERF1-2和ERF1-3可能在功能上冗余.研究成果可为后续拟南芥ERF1基因功能研究提供遗传学方面的数据.
The CRISPR-Cas9 gene editing technology is used to edit eukaryotic release factor 1-1(ERF1-1)gene in Arabidopsis thaliana.After obtaining erf1-1 mutants,the phenotypes of which are observed and the function of ERF1-1 gene is explored in Arabidopsis thaliana.A pair of target sequences inside the ERF1-1 gene coding region,with the protospacer adjacent motif(PAM)sequence flanking the 3′end,is chosen on a CRISPR target screening platform website and a pair of guide ribonucleic acids(gRNAs)is designed accordingly.The target segments are amplified by PCR and cloned into pHEE401 vector.The recombinant plasmid is transformed into Agrobacterium tumefaciens strain GV3101 and then the Arabidopsis inflorescence is infected with Agrobacterium tumefaciens using the floral dip method.The putative transgenic plants are screened in hygromycin-containing culture medium.DNAs from each generation of the transgenic plants are extracted and sequenced.Results show that the ERF1-1 gene in Arabidopsis is successfully mutated using the CRISPR-Cas9 gene editing technology and several different alleles of erf1-1 mutants are obtained.Under normal growth conditions,erf1-1 mutant plants show a slight growth and development phenotype compared with the wild type plants.It is speculated that functional redundancies may exist within homologous ERF1 genes.This work will provide genetic basis for the functional studies of ERF1 genes in Arabidopsis.
作者
熊伟
朱成新
王玉婷
郑媛
刘琳
莫蓓莘
XIONG Wei;ZHU Chengxin;WANG Yuting;ZHENG Yuan;LIU Lin;MO Beixin(College of Life Sciences and Oceanography,Guangdong Provincial Key Laboratory for Plant Epigenetics,Shenzhen University,Shenzhen 518071,Guangdong Province,P.R.China;Longhua Bioindustry and Innovation Research Institute,Shenzhen University,Shenzhen 518110,Guangdong Province,P.R.China;College of Physics and Optoelectronic Engineering,Shenzhen University,Key Laboratory of Optoelectronic Devices and Systems of Ministry of Education and Guangdong Province,Shenzhen 518060,Guangdong Province,P.R.China)
出处
《深圳大学学报（理工版）》
CAS
CSCD
北大核心
2021年第5期504-509,共6页
Journal of Shenzhen University(Science and Engineering)
基金
国家自然科学基金资助项目(31870287)
深圳市基础研究计划资助项目(JCYJ20190808115005598)
