荧光素酶表达载体yy625的构建及鉴定  

Construction and Identification of Luciferase Expression Vector yy625

作　　者：李玉洁 霍嘉鋆 曹后男[1] 金美玉[1] 山本義治 赵成日[1,2] Li Yujie;Huo Jiajun;Cao Hounan;Jin Meiyu;Yamamoto Yoshiharu Y.;Zhao Chengri(Agricultural College,Yanbian University,Yanji,133002;Gifu University,Gifu,5011193,Japan)

机构地区：[1]延边大学农学院,延吉133002 [2]岐阜大学,日本岐阜5011193

出　　处：《分子植物育种》2024年第22期7446-7452,共7页Molecular Plant Breeding

基　　金：国家自然科学基金项目(31660319);吉林省科技发展计划项目(20200402088NC);日本科学技术振兴机构项目(JST-ALCA);日本创新领域植物感知科研项目(Grant-in-Aid,23120511,25120712);日本具有挑战性的探索性研究项目(Grant-in-Aid,24657029)共同资助。

摘　　要：本研究为了构建含不同LUC报告基因的表达载体,利用TOYOBO公司pELuc(PEST)-test载体的ELuc'(PEST)基因替换yy621载体中的LUC基因,构建了yy625载体。利用PCR从pELuc(PEST)-test载体中克隆获得携带BglⅡ和XbaⅠ酶切位点的ELuc'(PEST)片段,将经BglⅡ和XbaⅠ酶切后的片段插入到载体yy621的BglⅡ和XbaⅠ位点之间,替换LUC基因。经菌落筛选、PCR鉴定和测序,阳性菌落中的ELuc'(PEST)序列与载体pELuc(PEST)-test中的ELuc (PEST)序列一致,表明载体625构建成功。筛选出更加灵敏、信号强的LUC报告基因,对于研究启动子序列中包含的基因调控元件有着重要的意义。In this study, in order to construct expression vectors containing different LUC reporter genes, the LUC gene in yy621 vector was replaced by ELuc'(PEST) gene of pELuc(PEST)-test vector from TOYOBO company,and the yy625 vector was constructed. The ELuc'(PEST) gene fragment carrying Bgl Ⅱ and Xba Ⅰ restriction endonuclease sites was obtained from the pELuc(PEST)-test vector by PCR, and the fragments digested by BglⅡand XbaⅠ restriction endonuclease were inserted between BglⅡ and XbaⅠ restriction endonuclease sites of vector yy621 to replace the original LUC gene. The colony screening, PCR identification and sequencing confirmed that the ELuc'(PEST) gene sequence in the positive colonies was identical to the ELuc(PEST) gene sequence in the pELuc(PEST)-test vector, and the vector 625 was successfully constructed. Screening more sensitive LUC reporter genes with strong signal is of great significance for studying gene regulatory elements contained in promoter sequences.

关 键 词：荧光素酶 yy625载体 构建 

分 类 号：Q943.2[生物学—植物学]