2A肽介导的割手密ScNAC1、ScDREB融合基因表达载体构建  

Construction of Co-expression Vector Carrying ScNAC1 and ScDREB by Using 2A Peptide-linked

作　　者：徐荣[1] 吴清莲 孟玉 陈疏影[1,2] 王先宏 何丽莲[2] 李富生[1] XU Rong;WU Qing-lian;MENG Yu;CHEN Shu-ying;WANG Xian-hong;HE Li-lian;LI Fu-sheng(College of Agriculture and Biotechnology, Yunnan Agricultural University, Yunnan Kunming 650201, China;Sugarcane Research Institute,Yunnan Agricultural University, Yunnan Kunming 650201, China)

机构地区：[1]云南农业大学农学与生物技术学院,云南昆明650201 [2]云南农业大学甘蔗研究所,云南昆明650201

出　　处：《西南农业学报》2019年第7期1492-1497,共6页Southwest China Journal of Agricultural Sciences

基　　金：国家自然科学基金(31560417);国家重点研发计划项目(2018YFD1000503);云南省现代农业甘蔗产业技术体系建设专项(2009-2019)

摘　　要：【目的】本研究旨在构建适合甘蔗遗传转化的双基因表达载体,用于提高甘蔗栽培品种的抗旱能力,并探讨基因表达规律及互作效应。【方法】本研究以前期课题组在割手密中克隆到的具有抗旱功能ScNAC1和ScDREB为目的基因,以对单子叶植物高效、专一的Ubiquitin为启动子,以适于单子叶植物遗传转化的表达载体pCAMBIA1300-nu为基础载体,利用改造后的FMDV2A多肽进行连接,采用重叠延伸PCR(简称SOE PCR)将ScNAC1和ScDREB基因相融合,利用同源重组的方法连接表达载体。【结果】经PCR验证获得约2000 bp的目的条带,利用BamH I与Sac I双酶切,切出了载体的1条大片段和连接目的基因的3条小片段,测序结果验证扩增产物的正确性,融合基因在扩增过程中无突变发生。【结论】本研究成功构建了符合需要的融合基因表达载体,为后期甘蔗的遗传转化研究奠定了基础。【Objective】The purpose of this study was to construct a dual gene expression vector suitable for genetic transformation of sugarcane, which will be used to improve the drought resistance of sugarcane cultivars and explore gene expression patterns and interaction effects. 【Method】In this study, the ScNAC1 and ScDREB genes with drought resistance cloned from S. spontaneum L. in our previous research were as the target genes. And Ubiquitin, a highly efficient and specific promoter to monocotyledonous plant, was used as a promoter for genetic transformation of monocots. The expression vector pCAMBIA1300 nu was a basic vector. The modified FMDV2 A polypeptide was used for the linkage, and the ScNAC1 & ScDREB genes were fused by a gene splicing by overlap extension PCR(SOE PCR), then the expression vectors were linked by homologous recombination.【Result】The target band of about 2000 bp was obtained and verified by PCR. A large fragment of the vector and three small fragments of the target gene were obtained by double digestion of BamH I and Sac I. Sequencing results showed that there was no mutation in the amplification process of the fusion gene.【Conclusion】A fusion gene expression vector that met our needs were successfully constructed, which lay a good foundation for the later genetic transformation of sugarcane.

关 键 词：ScNAC1 ScDREB 融合基因 2A肽 pCAMBIA1300nu 

分 类 号：S566[农业科学—作物学]