荧光素酶表达载体yy621的构建及鉴定  

Construction and Identification of Luciferase Expression Vector yy621

作　　者：李玉洁 南奇延[2] 金美玉[1] 曹后男[1] 山本義治 赵成日[1] Li Yujie;Nan Qiyan;Jin Meiyu;Cao Hounan;Yamamoto Yoshiharu Y.;Zhao Chengri(Agricultural college,Yanbian University,Yanji,133002;Yanbian University Hospital,Yanji,133000;Gifu University,Gifu,5011193,Japan)

机构地区：[1]延边大学农学院,延吉133002 [2]延边大学附属医院,延吉133000 [3]岐阜大学,日本岐阜5011193

出　　处：《分子植物育种》2021年第14期4672-4680,共9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)共同资助。

摘　　要：在载体yy449的CaMV 35S minimal启动子序列与LUC基因序列之间增加BglⅡ限制性内切酶位点,构建yy621载体。用PCR的方法分别扩增CaMV 35S minimal启动子序列与LUC基因。引物设计要满足如下条件:CaMV 35S minimal启动子序列扩增片段的上游应包括BamHⅠ、下游应包括BglⅡ限制性内切酶位点;LUC基因序列扩增片段的上游应包括BglⅡ、下游应包括XbaⅠ限制性内切酶位点。以上两个片段先用BglⅡ限制性内切酶消化后进行连接,得到CaMV 35S minimal启动子序列与LUC基因序列之间含BglⅡ限制性内切酶的识别位点的片段,然后再用BamHⅠ和XbaⅠ限制性内切酶进行消化,插入到载体yy449的BamHⅠ和XbaⅠ酶切位点之间,替换原有的CaMV 35S minimal启动子序列与LUC基因序列。经菌落筛选、PCR鉴定和测序验证,阳性菌落中的CaMV 35S minimal启动子序列与LUC基因序列之间包含BglⅡ限制性内切酶的识别位点,载体yy621构建成功。本实验中构建的荧光素酶表达载体yy621,适于今后用抗性相关基因替换LUC基因,测定合成启动子对抗性相关基因的具体调控与赋予植物对不良环境的具体抗性表现,或者用全长启动子序列替换CaMV 35S minimal启动子序列(-46~+1),对进一步观察LUC基因的表达调控具有重要的意义。The yy621 vector was constructed by adding a BglⅡrestriction endonuclease site between the CaMV 35S minimal promoter sequence and the LUC gene sequence of vector yy449.The CaMV 35S minimal promoter sequence and the LUC gene were amplified by PCR,respectively.The primers should be designed to meet the following conditions:the upstream of the amplified fragment of the CaMV 35S minimal promoter sequence should include BamHⅠ,and the downstream should include the BglⅡrestriction endonuclease site;the upstream of the LUC gene sequence amplification fragment shall include BglⅡand downstream shall include the XbaⅠrestriction endonuclease site.The two fragments were digested by BglⅡrestriction endonuclease and then ligated to obtain the fragment containing the recognition site of BglⅡrestriction enzyme between the CaMV 35S minimal promoter sequence and the LUC gene sequence,and digested with BamHⅠand XbaⅠrestriction endonucleases,they were inserted between BamHⅠand XbaⅠsites of vector yy449 to replace the original CaMV 35S minimal promoter sequence and LUC gene sequence.Colony screening,PCR identification and sequencing confirmed that the recognition site of BglⅡrestriction endonuclease was included between the CaMV 35S minimal promoter sequence and the LUC gene sequence in the positive colony,and the vector yy621 was successfully constructed.The luciferase expression vector yy621 constructed in this experiment is suitable for replacing the LUC gene with a resistance-related gene in the future,and determining the specific regulation of the synthetic promoter-related gene and the specific resistance of the plant to the adverse environment,or replace the CaMV 35S minimal promoter sequence(-46~+1)with full-length promoter to observe the expression and regulation of LUC gene.

关 键 词：荧光素酶 yy621载体 构建 

分 类 号：Q943.2[生物学—植物学]