家蚕脂肪酶-1基因(Bmlipase-1)的启动子活性分析  

Promoter Activity Analysis of Bombyx mori lipase-1 Gene

作　　者：夏定国[1,2] 张婷婷[1] 赵巧玲[1,2] 张国政[1,2] 裘智勇[1,2] 张业顺[1,2] Xia Dingguo Zhang Tingting Zhao Qiaoling Zhang Guozheng Qiu Zhiyong Zhang Yeshun(College of Biotechnology, Jiangsu University of Science and Technology, Zhenjiang Jiangsu 212018, China The Ser- icultural Research Institute, Chinese Academy of Agricultural Sciences, Zhenjiang Jiangsu 212018, China)

机构地区：[1]江苏科技大学生物技术学院,江苏镇江212018 [2]中国农业科学院蚕业研究所,江苏镇江212018

出　　处：《蚕业科学》2017年第3期395-401,共7页ACTA SERICOLOGICA SINICA

基　　金：国家自然科学基金项目(No.31372377);江苏科技大学科技计划项目(No.635181302)

摘　　要：家蚕脂肪酶-1(Bmlipase-1)在蚕体中肠组织中特异性表达,具有抵抗家蚕核型多角体病毒(Bm NPV)侵染的活性。用PCR方法扩增了Bmlipase-1启动子的8个不同区域片段,以绿色荧光蛋白基因(EGFP)为报告基因,分别构建重组家蚕杆状病毒并注射感染家蚕幼虫后,通过荧光观察、荧光定量PCR和ELISA方法,检测报告基因在家蚕中肠中的表达量,以明确对Bmlipase-1具有转录调控活性的启动子片段。结果表明:Bmlipase-1启动子-1 453~192 bp区域的活性最强;在-1 453^-679 bp区域有与Bmlipase-1在中肠组织特异性表达相关的启动子保守序列及1个CAAT box和大量的GATA序列等正调控元件,推测该区域可能与Bmlipase-1的组织特异性表达有关;-81~493 bp区域涵盖核心启动子区、第1外显子区和第1内含子区,含有大量Pbx-1转录因子结合位点、OTC序列和GATA序列,推测该区域发挥对Bmlipase-1转录的基础调控作用;-1 980^-1 452 bp区域存在负调控元件。研究结果有助于进一步揭示Bmlipase-1的转录调控机制。Bombyx mori lipase-1 gene（Bmlipase-1）,which is specifically expressed in midgut tissue,has strong antiviral activity against B.mori nucleopolyhedrovirus（Bm NPV）.We cloned eight different fragments of Bmlipase-1 promoter using PCR,and constructed recombinant baculovirus carrying enhanced green fluorescent protein reporter gene（EGFP）for each promoter fragment.In order to determine the promoter fragment with transcriptional regulation activity,after infecting silkworm larvae using the constructed recombinant baculovirus,expression level of reporter gene in silkworm midgut tissue was determined by fluorescence observation,real-time fluorescent quantitative PCR（qRT-PCR）and enzyme linked immunosorbent assay（ELISA）.The results showed that-1 453 to 192 bp fragment of Bmlipase-1promoter had the strongest activity.The-1 453 to-679 bp region contains a conserved promoter sequence related to tissue-specific expression of Bmlipase-1 in midgut and many positive regulatary elements including a CAAT box and a large number of GATA sequences,suggesting that this region is involved in regulation of tissue-specific expression of Bmlipase-1.The-81 to 493 bp region covers the core promoter,the first exon and the first intron,contains a large number of Pbx-1 transcription factor binding sites,OTC sequences and GATA sequences,suggesting that it functions basically in regulation of Bmlipase-1transcription.The-1 980 to-1 452 bp region contains negative regulation elements.These results are favorab le to further exploration of regulatory mechanism of Bmlipase-1 transcription.

关 键 词：家蚕 Bmlipase-1基因 启动子 活性区域 杆状病毒表达系统 

分 类 号：Q782[生物学—分子生物学] S881.2[农业科学—特种经济动物饲养]