樗蚕孵化酶基因的克隆与表达特征分析  

Gene Cloning and Spatio-temporal Expression Analysis of Hatching Enzyme from Philosamia cynthia cynthia

作　　者：浦月霞 刘龙山 龚美霞 冉艳萍 贾雪峰 黄景滩 宋宪军 安春梅 唐顺明 Pu Yuexia;Liu Longshan;Gong Meixia;Ran Yanping;Jia Xuefeng;Huang Jingtan;Song Xianjun;An Chunmei;Tang Shunming(Guangxi General Station for Sericulture Technology Popularization,Nanning 530007,China;College of Biotechnol- ogy,Jiangsu University of Science and Technology,Jiangsu Key Laboratory of Sericultural Biology and Biotechnology,Zhenjiang Jiangsu 212018,China;Key Laboratory of Silkworm and Mulberry Genetic Improvement,Ministry of Agricul- ture,Sericultural Research Institute,Chinese Academy of Agricultural Sciences,Zhenjiang Jiangsu 212018,China)

机构地区：[1]广西壮族自治区蚕业技术推广总站,南宁530007 [2]江苏科技大学生物技术学院,江苏省蚕桑生物学与生物技术重点实验室,江苏镇江212018 [3]中国农业科学院蚕业研究所、农业部蚕桑遗传改良重点实验室,江苏镇江212018

出　　处：《蚕业科学》2018年第3期382-389,共8页ACTA SERICOLOGICA SINICA

基　　金：国家自然科学基金项目(No.31372376);广西壮族自治区自然科学基金项目(No.2017GXNSFAA198085);江苏省自然科学基金项目(No.BK20141285)

摘　　要：樗蚕（Philosamia cynthia cynthia）是重要的野蚕资源。为从分子水平研究樗蚕的孵化机制,以催青第9天的樗蚕卵为材料提取总RNA并反转录合成c DNA,根据鳞翅目昆虫孵化酶基因的保守序列设计简并引物,通过RACE-PCR获得樗蚕孵化酶基因全长c DNA,命名为Pcc HE（Gen Bank登录号：MH119084）。Pcc HE基因c DNA全长为964 bp,由5＇-UTR、3＇-UTR和885bp的ORF组成,编码294个氨基酸;Pcc HE蛋白序列含有孵化酶特征序列锌指结合基序和Met-转角基序;基于Pcc HE氨基酸序列与16种动物孵化酶同源氨基酸序列构建的系统进化分析树显示,樗蚕与蓖麻蚕之间的亲缘关系最近。半定量RT-PCR检测Pcc HE在樗蚕胚胎发育早期的表达维持在较低水平,催青第3天小幅度增加,第9天时表达水平开始大幅上升,至孵化前达到最高水平;Pcc HE在5龄3 d幼虫中肠中高水平表达,并且在马氏管中也有表达,这是首次在昆虫马氏管中发现孵化酶基因的表达。上述结果表明,Pcc HE与樗蚕的胚胎发育和孵化密切相关,还可能参与幼虫期的食物消化、蛋白质代谢等生物学过程。Philosamia cynthia cynthia is an important resource for wild silkworm. To investigate the hatching mechanism of Philosamia cynthia cynthia at moleculae level,total RNA was isolated from newly laid eggs on the ninth day of incubation and the c DNA was obtained by reverse transcription method. Degenerate primers were designed according to the conserved sequence of hatching enzyme gene（ HE） in Lepidoptera insect. Full length c DNA of Philosamia cynthia cynthia hatching enzyme was amplified by RACE-PCR,and named Pcc HE（ Gen Bank accession No： MH119-084）. The full length c DNA of Pcc HE was 966 bp,which contained 5＇-UTR,3＇-UTR and an ORF sequence of 885 bp encoding 294 amino acids. This protein contains the characteristic sequences of hatching enzyme,zinc binding motif and Met-turn motif. Phylogenetic analysis based on amino acid sequence of hatching enzymes indicated that Philosamia cynthia cynthia and Philasamia cynthia ricini had the closest genetic relationship. Semi-quantitative RT-PCR analysis indicated that the expression of Pcc HE transcripts in embryos at early stage remained at a relative low level,increased slightly on the 3 rd day,began to rise sharply on the9 th day and reached to the maximal level before hatching. Moreover,Pcc HE had the highest expression level in midgut on the 3 rd day of 5 th instar larval stage. In addition,its expreesion was also observed in Malpighian tubules,which was the first report on expression of hatching enzyme gene in insect Malpighian tubules. The above results indicate that Pcc HE is involved in embryonic development and hatching process at embryonic stage,and it may also participate in the biological process of digestion and protein metabolism at larval stage.

关 键 词：樗蚕 孵化酶 基因克隆 表达特性 

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]