猴樟CbP5CS1基因克隆及表达载体构建  

Cloning and Expression Vector Construction of CbP5CS1 Gene from Cinnamomun bodinieri

作　　者：韩浩章[1] 张丽华[1] 李素华[1] 赵荣 王芳[1] 王晓立[1] 蒋亚华[1] Han Haozhang;Zhang Lihua;Li Suhua;Zhao Rong;Wang Fang;Wang Xiaoli;Jiang Yahua(School of Architecture and Engineering,Suqian University,Suqian,223800)

机构地区：[1]宿迁学院建筑工程学院,宿迁223800

出　　处：《分子植物育种》2024年第16期5350-5356,共7页Molecular Plant Breeding

基　　金：江苏省自然科学基金项目(BK20201481);宿迁市科技计划项目(M202001;L202004);宿迁学院创新团队项目(2021td05)共同资助。

摘　　要：为研究猴樟CbP5CS1蛋白的序列结构和功能,以猴樟转录组数据为基础,采用PCR技术克隆猴樟CbP5CS1基因CDS序列,构建表达载体。结果表明,克隆得到的序列具有一个2163 bp完整的ORF框,编码720个氨基酸,与沉水樟中收录的RWR86140.1基因序列一致性达98.47%,GenBank登录号为MW813958;生物信息学分析结果表明,CbP5CS1蛋白化学分子式为C_(3435)H_(5609)N_(963)O_(1050)S_(22),相对分子量为77.91 kD,理论等电点为6.16,结构稳定,无明显无序化特征,为脂溶性和亲水性蛋白,磷酸化氨基酸位点有95个;CbP5CS1蛋白具有PLN02418家族结构域,具备δ-1-吡咯啉-5-羧酸合成酶的功能,在樟属植物中具有较高的保守性;CbP5CS1蛋白亚细胞定位预测为细胞质,为跨膜蛋白,但不存在信号肽;重组质粒用Xba I酶切后,获得两条带片段,与预期的2226和11516 bp的大小一致,表明目的基因已经成功插入并连接到植物表达载体pCAMBIA1301上。In order to study the structure and function of the CbP5CS1 protein sequence,based on the transcriptome data of Cinnamomun bodinieri,the CDS gene was cloned by PCR and its expression vector was constructed.The results showed that the sequence had a complete ORF frame of 2163 bp encoding 720 amino acids,which was 98.47%consistent with the sequence of RWR86140.1 gene from Cinnamomun micranthum,and the GenBank entry number was MW813958;bioinformatics analysis of CbP5CS1 protein showed that the molecular formula of CbP5CS1 protein was C_(3435)H_(5609)N_(963O1050)S_(22),the relative molecular weight was 77.91 kD,and the theoretical isoelectric point was 6.16.CbP5CS1 protein was fat soluble and hydrophilic without obvious disorder characteristics,and it had 95 phosphorylation amino acid sites;CbP5CS1 protein has the PLN02418 family domain and has the function ofΔ1-pyrrolin-5-carboxylate synthetase,which showed high conserved in Cinnamomum species;CbP5CS1 protein was predicted to be cytoplasmic and transmembrane protein by subcellular localization,but there was no signal peptide;after the recombinant plasmid was digested with XbaI enzyme,two bands were obtained,which were the same size as expected 2226 and 11516 bp,indicating that the target gene had been successfully inserted and at-tached to the plant expression vector pCAMBIA1301.

关 键 词：猴樟 CbP5CS1 基因克隆 表达载体 

分 类 号：S792.23[农业科学—林木遗传育种]