凡纳滨对虾血蓝蛋白大亚基启动子的克隆及鉴定  

Cloning and Identification of the Promoter of Hemocyanin Large Subunit of Penaeus vannamei

作　　者：郭丽钒 黄贺楠 赵琼君 张涵 杨昊 杨培奎[1] GUO Li-fan;HUANG He-nan;ZHAO Qiong-jun;ZHANG Han;YANG Hao;YANG Pei-kui(College of Life Sciences and Food Engineering,Hanshan Normal University,Chaozhou,Guangdong,521041;Chaoan Guihu Weixing Freshwater Aquaculture Co.,Ltd.,Chaozhou,Guangdong,515653)

机构地区：[1]韩山师范学院生命科学与食品工程学院,广东潮州521041 [2]潮安归湖伟兴淡水养殖有限公司,广东潮州515653

出　　处：《韩山师范学院学报》2023年第6期40-48,共9页Journal of Hanshan Normal University

基　　金：广东省自然科学基金项目(项目编号:2019A1515110981);广东省普通高校特色创新类项目(项目编号:2019KTSCX101);广东省科技创新战略专项“攀登计划”(项目编号:pdjh2022b0333).

摘　　要：为了鉴定凡纳滨对虾血蓝蛋白大亚基基因启动子的核心区域和转录因子,探究该基因的转录调控机制.该研究首先通过PCR扩增凡纳滨对虾血蓝蛋白大亚基基因候选启动序列1969 bp并对其进行测序和生物信息学分析;其次,构建5个不同长度的缺失片段双荧光素酶报告载体,转染至293T细胞,利用双荧光素酶报告分析鉴定其核心区域.进一步通过生物信息学预测全长序列的转录起始位点和CpG岛,并对核心启动子区域的转录因子结合位点进行预测.结果表明,扩增得到的凡纳滨对虾蛋白大亚基启动子序列(HLP2)包含启动子元件;利用双荧光素酶报告载体分析发现HLP2的核心启动子区域(+87/+300 bp)位于起始密码子下游的外显子中,生物信息学分析发现该区域包括多种转录因子结合位点.实验结果为进一步探究对虾血蓝蛋白大亚基基因的转录调控机制提供理论依据.In order to identify the core region and transcription factors of the promoter of hemocyanin large subunit gene of Penaeus vannamei and explore the transcriptional regulation mechanism of the gene,this study first amplified the candidate promoter sequence 1969 bp of the hemocyanin large subunit gene of Penaeus vannamei by PCR,followed by sequencing and bioinformatics analysis.Secondly,dual luciferase reporter vectors with 5 different deletion fragments of the promoter were constructed and transfected into 293T cells.The core region of the hemocyanin large subunit gene was determined by detecting the activity of luciferase reporter vectors of different deletion fragments.Then,promoter element of the full-length sequence of promoter of hemocyanin large subunit gene were further predicted by bioinformatics,and the transcription factor binding sites of the core promoter region were predicted.The results showed that the promoter of hemocyanin large subunit(HLP2)contains the transcription start site(TSS)and CpG island.And dual-luciferase assay further confirmed that the core promoter was located in the+87/+300 bp region of exon,and bioinformatics analysis revealed that this core region included various transcription factor binding sites.The experimental results provide a theoretical basis for the transcription regulation mechanism of hemocyanin large subunit of Penaeus vannamei.

关 键 词：凡纳滨对虾 血蓝蛋白大亚基 启动子 克隆 

分 类 号：S917[农业科学—水产科学]