琥珀蚕孵化酶基因AaHE的克隆及启动子活性分析  

Cloning and promoter activity analysis of the hatching enzyme gene AaHE of Antheraea assamensis(Lepidoptera:Saturniidae)

作　　者：陈安利[1,2] 董占鹏 唐顺明[3] 刘增虎[1] 李涛[1] 廖鹏飞[1] 李琼艳[1] CHEN An-Li;DONG Zhan-Peng;TANG Shun-Ming;LIU Zeng-Hu;LI Tao;LIAO Peng-Fei;LI Qiong-Yan(Sericultural and Apicultural Research Institute, Yunnan Academy of Agricultural Sciences, Mengzi, Yunnan 661101, China;Key Sericultural Laboratory of Shaanxi, Ankang University, Ankang, Shaanxi 725000, China;College of Biotechnology, Jiangsu University of Science and Technology, Zhenjiang, Jiangsu 212018, China)

机构地区：[1]云南省农业科学院蚕桑蜜蜂研究所,云南蒙自661101 [2]安康学院陕西省蚕桑重点实验室,陕西安康725000 [3]江苏科技大学生物技术学院,江苏镇江212003

出　　处：《昆虫学报》2021年第6期666-675,共10页Acta Entomologica Sinica

基　　金：云南省应用基础研究计划项目(2017FB069);陕西省教育厅重点科学研究计划项目(20JS001)。

摘　　要：【目的】琥珀蚕Antheraea assama具有典型的野蚕特征,蚕卵孵化不齐,严重影响琥珀蚕的室内规模化饲养。本研究旨在探究对琥珀蚕卵孵化起关键作用的孵化酶(hatching enzyme)基因及其启动子序列特征,为进一步选择合适的抑制剂或促进剂调节琥珀蚕卵的孵化奠定基础。【方法】采用RACE技术克隆琥珀蚕孵化酶基因的cDNA全长序列,对基因序列进行生物信息学分析;采用qRT-PCR检测琥珀蚕孵化酶基因在琥珀蚕不同发育天数卵中及5龄第3和4天幼虫不同组织(丝腺、马氏管、头、中肠、脂肪体、表皮、血液、精巢和卵巢)中的表达情况;采用染色体步移克隆琥珀蚕孵化酶基因的启动子序列,构建昆虫细胞重组表达载体转染家蚕Bombyx mori BmN细胞,检测琥珀蚕孵化酶基因启动子活性。【结果】获得了琥珀蚕孵化酶基因AaHE(GenBank登录号:KT336227.1)全长cDNA序列,长993 bp,编码294个氨基酸,预测蛋白质分子质量为33.7 kD,理论等电点为5.17。AaHE氨基酸序列含有一个信号肽和一个ZnMc结构域,AaHE是一种含有HExxH锌结合位点的锌依赖性蛋白水解酶,该类酶既是肽酶,同时又是一种消化酶。AaHE在琥珀蚕孵化前的卵及5龄幼虫中肠中特异性高表达,分别与AaHE的肽酶和消化酶的属性相吻合。AaHE启动子核心区存在多个转录因子结合位点,这可能与转录因子参与调节AaHE的表达有关。启动子活性分析表明,AaHE启动子在家蚕BmN细胞中能够启动EGFP基因的表达,具有明显的启动子活性。【结论】AaHE是锌依赖性蛋白水解酶,其启动子核心区存在多个转录因子结合位点。本研究为选择合适的抑制剂或促进剂调节琥珀蚕卵的孵化率提供了参考。【Aim】Antheraea assama has the typical characteristics of wild silkworms,with its eggs not uniformly hatched,which seriously affects its large-scale indoor rearing.This study aims to explore the characteristics of the hatching enzyme gene playing a key role in the egg hatching of A.assamensis and its promoter sequence,and to lay the foundation for further selection of appropriate inhibitors or accelerators to regulate the egg hatching of A.assamensis.【Methods】The full-length cDNA sequence of the hatching enzyme gene was cloned from A.assamensis by RACE,and the sequence was analyzed by bioinformatics method.qRT-PCR was used to detect the expression profiles of the hatching enzyme gene in eggs of different days after laying,and in different tissues(silk gland,Malpighian tubules,head,midgut,fat body,epidermis,blood,testis and ovary)of the day-3 and day-45th instar larvae of A.assamensis.The promoter sequence of the hatching enzyme gene was cloned from A.assamensis by chromosome walking,and the promoter activity was detected by constructing the insect cell recombinant expression vector and transfecting the BmN cells of Bombyx mori.【Results】The full-length cDNA sequence of the hatching enzyme gene AaHE(GenBank accession no.:KT336227.1)was obtained from A.assamensis.It is 993 bp in length,encoding 294 amino acids with the predicted molecular weight of 33.7 kD and the theoretical isoelectric point of 5.17.The amino acid sequence of AaHE contains a signal peptide and a ZnMc domain,and AaHE belongs to a group of zinc-dependent proteolytic enzymes with a HExxH zinc-binding site.The members of this enzyme group are both peptidase and digestive enzyme.AaHE was specifically and highly expressed in the eggs before hatching and the midgut of the 5th instar larva of A.assamensis,being consistent with the properties of peptidase and digestive enzyme.There are multiple transcription factor binding sites in the core region of AaHE promoter,which may be related to the transcription factor’s involvement in regulating the

关 键 词：琥珀蚕 孵化酶 RACE 表达谱 启动子活性 BmN细胞 

分 类 号：S885.9[农业科学—特种经济动物饲养] Q786[农业科学—畜牧兽医]