马铃薯野生种 SpSBP1 基因的克隆及CRISPR/Cas9载体构建  

Cloning and CRISPR/Cas9 Vector Construction of SpSBP1 Gene in Potato

作　　者：陈娜 邵勤 李晓鹏 高阳 卢其能[1] CHEN Na;SHAO Qin;LI Xiaopeng;GAO Yang;LU Qineng(College of Life Science and Resources and Environment,Yichun University,Yichun 336000,China)

机构地区：[1]宜春学院生命科学与资源环境学院,江西宜春336000

出　　处：《华北农学报》2022年第6期23-33,共11页Acta Agriculturae Boreali-Sinica

基　　金：江西省作物生长发育调控重点实验室开放基金项目(2210819005);江西省教育厅科学技术项目(GJJ201618);江西现代农业科研协同创新专项课题(JXXTCX201704-05)。

摘　　要：为了探究SBP1基因在植物自交不亲和方面的重要作用,以二倍体马铃薯野生种为材料,利用RT-PCR技术克隆获得马铃薯野生种SpSBP1(登录号:MZ803088)的cDNA全长序列,并对其进行生物信息学分析及CRISPR/Cas9载体构建。结果显示,SpSBP1基因的cDNA全长为1176 bp,包含92 bp的5′非编码区和163 bp的3′非编码区,其最大开放阅读框为921 bp,编码306个氨基酸。蛋白结构域分析显示,SpSBP1蛋白包括Smc超家族结构域以及RING finger和Zinc finger结构域。蛋白同源序列比对及系统进化树分析显示,马铃薯野生种SpSBP1与马铃薯野生种ScSBP1的同源性最高,其次为花烟草。生物信息学分析显示,SpSBP1分子质量为34.73144 ku,理论等电点pI为5.10,推测得出该蛋白为酸性不稳定亲水性蛋白。二级结构预测该蛋白主要由α螺旋和无规则卷曲所构成。同时该蛋白属于非分泌蛋白且不存在跨膜结构。从二倍体马铃薯野生种中克隆出了SpBP1基因,同时成功构建了CRISPR/Cas9载体,并进行了遗传转化试验。In order to explore the important role of SBP1(S-RNase-binding protein 1)gene in plant self-incompatibility,a diploid wild potato was used as the material to obtain the full-length cDNA sequence of potato SpSBP1(GenBank:MZ803088)by using RT-PCR cloning technique,and performed bioinformatics analysis and CRISPR/Cas9 vector construction for SpSBP1 gene.The results showed that the full length cDNA of SpSBP1 gene was 1176 bp,containing 92 bp 5′non-coding region and 163 bp 3′non-coding region.The maximum open reading frame(ORF)of SpSBP1 gene was 921 bp,which encoding 306 amino acids.Protein domain analysis showed that SpSPB1 protein included Smc superfamily domain,RING finger domain and Zinc finger domain.The homologous sequence alignment and phylogenetic tree analysis showed that SpSBP1 had the highest homology with S.chacoense,followed by Nicotiana alata.Bioinformatics analysis showed that the molecular weight of SpSBP1 was 34.73144 ku,with a theoretical isoelectric point of 5.10,and it was speculated that SpSBP1 was an acid unstable hydrophilic protein.The secondary structure predicted that the protein was mainly composed ofαhelix and random curling.And the protein was a non-secretory protein and had no transmembrane structure.SpSBP1 gene was cloned from wild diploid potato,and CRISPR/Cas9 vector was successfully constructed,and also the genetic transformation was carried out.

关 键 词：马铃薯野生种 自交不亲和 SpSBP1 克隆 生物信息学分析 CRISPR/Cas9载体构建 

分 类 号：S532.03[农业科学—作物学] Q78[生物学—分子生物学]