两个褐飞虱海藻糖转运蛋白基因的结构及调控海藻糖代谢功能  

The Structure Characteristics and Biological Functions on Regulating Trehalose Metabolism of Two NlTret1s in Nilaparvata lugens

作　　者：於卫东[1,2] 潘碧莹 邱玲玉 黄镇 周泰 叶林 唐斌 王世贵[1] YU WeiDong;PAN BiYing;QIU LingYu;HUANG Zhen;ZHOU Tai;YE Lin;TANG Bin;WANG ShiGui(College of Life and Environmental Science,Hangzhou Normal University,Hangzhou 310036;Zhejiang Dingyi Biotechnology Co.,LTD,Quzhou 324100,Zhejiang)

机构地区：[1]杭州师范大学生命与环境科学学院,杭州310036 [2]浙江鼎益生物科技有限公司,浙江衢州324100

出　　处：《中国农业科学》2020年第23期4802-4812,共11页Scientia Agricultura Sinica

基　　金：国家自然科学基金(31672081,31371996)。

摘　　要：【目的】海藻糖是昆虫中的主要血糖物质,在昆虫的发育及生理活动中发挥重要功能。其中,海藻糖转运蛋白(Tret)在将海藻糖从其生成组织(例如脂肪体)运输到其消耗组织的过程中起着重要作用。本研究通过分析褐飞虱(Nilaparvata lugens)两条Tret1的序列结构,进一步抑制NlTret1的表达,探讨这两个NlTret1在褐飞虱体内的生物学功能。【方法】以褐飞虱两条Tret1序列为研究对象,利用生物信息学技术分析其蛋白结构以及与其他昆虫之间的同源性。采用RNAi(RNA interference)技术将合成的外源dsRNA(double-stranded RNA)注射到实验室饲养褐飞虱种群体内,抑制其体内NlTret1的表达,分别在注射48 h后取材,抽取总RNA并用反转录试剂盒合成第一链cDNA,采用qRT-PCR(quantitative real-time PCR)技术检测dsNlTret1的干扰效果以及RNAi后褐飞虱体内海藻糖代谢通路中相关基因的表达,最后测定葡萄糖、海藻糖和糖原含量以及海藻糖酶活性。【结果】生物信息学分析表明,NlTret1-like X1和NlTret1-2 X1的开放阅读框长度分别为1920和1578 bp,分别编码639和525个氨基酸,预测蛋白分子量分别为69.29和58.71 kD,等电点分别为8.32和8.36;NlTret1-like X1和NlTret1-2 X1的二级结构主要包含螺旋和卷曲;保守结构域分析显示它们均属于MFS家族。进化分析结果显示,不同昆虫的Tret1蛋白具有较高的同源性,且褐飞虱与其他半翅目昆虫具有较近的亲缘关系;与注射dsGFP组相比,注射靶标基因的dsRNA后均能够显著沉默本基因的表达;褐飞虱体内糖原、葡萄糖在注射dsNlTret1-like X1和dsNlTret1-2 X1后,其含量均无显著变化,然而不同于注射dsNlTret1-2 X1组,在注射dsNlTret1-like X1后褐飞虱体内海藻糖含量极显著升高;干扰NlTret1-like X148 h后褐飞虱体内TPS1、TPS2、TRE1-1、TRE1-2和TRE2的表达均极显著下调;而干扰NlTret1-2 X148 h后褐飞虱体内TPS1、TPS2和TRE1-1的表达虽也极显著下降【Objective】Trehalose is the main blood sugar substance in insects and plays an important role in insect development and physiological activities.Among them,trehalose transporter(Tret)plays a key role in the transportation of trehalose from trehalose-producing tissues(such as fat body)to trehalose-consuming tissues.The objective of this study is to explore the biological functions of these two NlTret1s in brown planthopper(Nilaparvata lugens)by analyzing the sequence structure of two NlTret1s and further suppressing the expression of NlTret1s.【Method】Taking these two NlTret1 sequences as the research object,the protein structure and homology with other insects were analyzed by bioinformatics technology.The RNA interference(RNAi)technology was used to inject the synthetic exogenous dsRNA(double-stranded RNA)into the laboratory feeding population of N.lugens,to inhibit the expression of the NlTret1s.The total RNA was extracted to synthesize the first-strand cDNA using reverse transcription Kit,qRT-PCR(quantitative real-time PCR)technology was used to detect the RNAi effect of dsNlTret1s and the expression of related genes in the trehalose metabolism pathway in N.lugens after RNAi,and finally glucose,trehalose and glycogen content as well as trehalase enzyme activity were determined.【Result】Bioinformatics analysis showed that the open reading frames of NlTret1-like X1 and NlTret1-2 X1 are 1920 and 1578 bp in length,encoding 639 and 525 amino acids,respectively.The predicted protein molecular weights are 69.29 and 58.71 kD,and the isoelectric points are 8.32 and 8.36,respectively.The secondary structure of NLTret1-like X1 and NLTret1-2 X1 is mainly composed of helix and coil.Conservative domain analysis showed that they all belong to the MFS family.The results of evolutionary analysis showed that Tret1 of different insects had high homology,and N.lugens was closely related to other hemiptera insects.Compared with the dsGFP group,the target gene was inhibited significantly after injection with dsNlTret1-lik

关 键 词：褐飞虱 海藻糖转运蛋白 结构 海藻糖代谢 RNA干扰 

分 类 号：S435.112.3[农业科学—农业昆虫与害虫防治]