白背飞虱海藻糖合成酶基因的表达特性及其在糖代谢调控中的作用  

Expression characteristics of trehalose-6-phosphate synthase genes and their roles in the regulation of carbohydrate metabolism in Sogatella furcifera (Hemiptera: Delphacidae)

作　　者：张道伟[1] 邱玲玉 康奎 余亚娅 曾伯平[1] 陈静 唐斌 ZHANG Dao-Wei;QIU Ling-Yu;KANG Kui;YU Ya-Ya;ZENG Bo-Ping;CHEN Jing;TANG Bin(College of Biology and Agriculture, Zunyi Normal University, Zunyi, Guizhou 563006, China;College of Life and Environmental Science, Hangzhou Normal University, Hangzhou 310036, China;College of Basic Medical Science, Zunyi Medical University, Zunyi, Guizhou 563006, China)

机构地区：[1]遵义师范学院生物与农业科技学院,贵州遵义563006 [2]杭州师范大学生命与环境科学学院,杭州310036 [3]遵义医科大学基础医学院,贵州遵义563006

出　　处：《昆虫学报》2019年第10期1117-1128,共12页Acta Entomologica Sinica

基　　金：国家自然科学基金项目(31672081);贵州省科学技术基金(黔科合JZ字[2014]2014号)

摘　　要：【目的】昆虫中海藻糖主要通过海藻糖合成酶(trehalose-6-phosphate synthase, TPS)在脂肪体中合成,当昆虫受极端环境胁迫时TPS能够诱导海藻糖累积从而起到保护作用。本研究旨在分析白背飞虱Sogatella furcifera 两个TPS基因的发育和组织表达模式及其对糖类物质代谢调控功能,探究TPS基因在白背飞虱生长发育中的具体作用。【方法】基于实验室前期获得的两个海藻糖合成酶基因SfTPS1和SfTPS2片段序列,在本实验中也进行了基因克隆和测序筛选,比对两者确定了白背飞虱两个TPS基因序列。并通过MEGA 7.0软件构建基于氨基酸序列的白背飞虱与其他昆虫TPS的系统发育树。利用qRT-PCR技术检测这两个基因在白背飞虱不同发育阶段(4龄第1天若虫至3日龄成虫)和成虫不同组织(头、足、翅、中肠、脂肪体、表皮和马氏管)中的表达情况。合成这两个基因的dsRNA,并注射到白背飞虱5龄第1天若虫中进行RNAi。在RNAi 48 和72 h后检测白背飞虱海藻糖酶基因TRE1-1, TRE1-2和TRE2的表达变化,海藻糖、葡萄糖和总糖原含量以及海藻糖酶活性。【结果】克隆获得白背飞虱SfTPS1和SfTPS2,ORF分别为2 424和2 115 bp,编码氨基酸数目分别为807个和704个,预测蛋白质分子量分别为90.37和80.56 kD,等电点分别为6.08 和6.10。而且白背飞虱2个TPS氨基酸序列与褐飞虱Nilaparvata lugens TPS1和TPS2的一致性最高。发育阶段表达模式表明,白背飞虱TPS基因SfTPS1和SfTPS2在4龄若虫到成虫阶段都有表达;组织表达模式表明,SfTPS1和SfTPS2在成虫马氏管、中肠和表皮中的表达较为显著。当SfTPS1被RNAi后,TRE1-1和TRE2的表达水平与对照组(dsGFP注射组)相比分别为略有上升和显著升高,TRE1-2的相对表达水平在SfTPS1被RNAi 48 h后显著上升而在72 h后显著下降;可溶性海藻糖酶活性无显著变化,膜结合型海藻糖酶活性显著增加;白背飞虱5龄若虫体内海藻糖、葡萄�【Aim】 Trehalose in insects is mainly synthesized in the fat body by trehalose-6-phosphate synthase (TPS), which can induce trehalose accumulation and thus plays a protective role when insects are under extreme environmental stress. This study aims to analyze the developmental and tissue expression patterns of two TPS genes in Sogatella furcifera, and their roles in the regulation of carbohydrate metabolism, so as to explore the specific roles of TPS genes in the growth and development of S. furcifera. 【Methods】 Based on the partial sequences of two trehalose synthase genes, SfTPS1 and SfTPS2,obtained in the early stage of the laboratory, gene cloning and sequencing were also carried out in this experiment. The two TPS gene sequences of the S. furcifera were determined by comparison. The phylogenetic tree based on the amino acid sequences of SfTPS and TPS proteins from other insect species was constructed by MEGA 7.0 software. The expression profiles of these two TPS genes in different developmental stages (from the day 1 4th instar nymph to 3 day-old adult), and different adult tissues (head, leg, wing, midgut, fat body, epidermis and Malpighian tubules) of S. furcifera were detected by qRT-PCR. The double-stranded RNA (dsRNA) of the two genes were synthesized and injected into the day 1 5th instar nymphs of S. furcifera for the RNAi. At 48 and 72 h after RNAi, the changes in expression levels of trehalase genes TRE1-1, TRE1-2 and TRE2, the contents of trehalose, glucose and total glycogen, and the trehalase activity were determined. 【Results】 The ORFs of SfTPS1 and SfTPS2 are 2 424 and 2 115 bp in length, respectively, and the numbers of encoded amino acids are 807 and 704, respectively. The predicted protein molecular weights are 90.37 and 80.56 kD, respectively, and the isoelectric points are 6.08 and 6.10, respectively. Moreover, the two TPS amino acid sequences of S. furcifera show the highest identities with Nilaparvata lugens TPS1 and TPS2. Developmental expression profiles revealed that SfTPS1

关 键 词：白背飞虱 海藻糖合成酶 海藻糖代谢 RNA干扰 QRT-PCR 

分 类 号：Q966[生物学—昆虫学]