荧光素酶表达载体yy630的构建及鉴定  

Construction and Identification of Luciferase Expression Vector yy630

作　　者：李玉洁 山本義治 曹后男[1] 金美玉[1] 赵成日[1,2] Li Yujie;Yamamoto Yoshiharu Y.;Cao Hounan;Jin Meiyu;Zhao Chengri(Agricultural College,Yanbian University,Yanji,133002;Faculty of Applied Biological Sciences,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,应用生物科学部,岐阜,5011193日本

出　　处：《分子植物育种》2022年第2期479-485,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：使用Promega公司pNL1.1[Nluc]载体的Nluc’基因替换yy621载体中的LUC+基因,构建yy630载体。通过PCR扩增的方法从pNL1.1[Nluc]载体中得到带有BglⅡ和XbaⅠ限制性酶切位点的Nluc’基因片段,经BglⅡ和XbaⅠ消化后的片段插入到载体yy621的BglⅡ和XbaⅠ酶切位点之间,替换原有的LUC+基因。Nluc’基因是通过ATGGCTATGGCT序列替换pNL1.1[Nluc]载体Nluc基因的起始密码子ATG而形成的。经菌落筛选、PCR鉴定和测序验证,阳性菌落中的Nluc’基因序列与载体pNL1.1[Nluc]中的Nluc基因序列完全一致,载体630构建成功。本研究通过比较分析并筛选出适合植物体研究的荧光素酶载体对于研究植物基因调控机理有着重要的意义。The yy630 vector was constructed by replacing the LUC+gene in the yy621 vector with the Nluc’ gene of Promega pNL1.1[Nluc] vector. The Nluc’ gene fragment carrying the BglⅡ and XbaⅠrestriction endonuclease sites was obtained from the pNL1.1 [Nluc] vector by PCR amplification, and the fragment digested with BglⅡ and Xba Ⅰ restriction endonuclease was inserted between the Bgl Ⅱ and Xba Ⅰ restriction endonuclease sites of the vector yy621, replace the original LUC+gene. The Nluc’ gene is formed by replacing the initiation codon ATG of Nluc gene of pNL1.1 [Nluc] vector with the ATGGCTATGGCT sequence. After colony screening, PCR identification and sequencing verification, the Nluc’ gene sequence in the positive colony was completely consistent with the Nluc gene sequence in vector pNL1.1[Nluc], and the vector yy630 was successfully constructed. Comparative analysis and screening of luciferase vectors suitable for plant research has important implications for the study of plant gene regulation mechanisms.

关 键 词：荧光素酶 yy630载体 构建 

分 类 号：Q943.2[生物学—植物学]