中国农业科学 ›› 2020, Vol. 53 ›› Issue (23): 4802-4812.doi: 10.3864/j.issn.0578-1752.2020.23.007

• 植物保护 • 上一篇    下一篇

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

於卫东1,2(),潘碧莹1,邱玲玉1,黄镇2,周泰2,叶林2,唐斌1,王世贵1()   

  1. 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
  • 收稿日期:2020-07-18 接受日期:2020-09-03 出版日期:2020-12-01 发布日期:2020-12-09
  • 通讯作者: 王世贵
  • 作者简介:於卫东,E-mail: hzyuweidong@hotmail.com
  • 基金资助:
    国家自然科学基金(31672081);国家自然科学基金(31371996)

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

YU WeiDong1,2(),PAN BiYing1,QIU LingYu1,HUANG Zhen2,ZHOU Tai2,YE Lin2,TANG Bin1,WANG ShiGui1()   

  1. 1College of Life and Environmental Science, Hangzhou Normal University, Hangzhou 310036
    2Zhejiang Dingyi Biotechnology Co., LTD, Quzhou 324100, Zhejiang
  • Received:2020-07-18 Accepted:2020-09-03 Online:2020-12-01 Published:2020-12-09
  • Contact: ShiGui WANG

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 X1NlTret1-2 X1的开放阅读框长度分别为1 920和1 578 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 X1 48 h后褐飞虱体内TPS1TPS2TRE1-1TRE1-2TRE2的表达均极显著下调;而干扰NlTret1-2 X1 48 h后褐飞虱体内TPS1TPS2TRE1-1的表达虽也极显著下降,但TRE1-2TRE2极显著上调;在注射dsNlTret1-like X1后,试虫可溶性海藻糖酶和膜结合型海藻糖酶的活性均极显著降低,而在注射dsNlTret1-2 X1后无显著变化。【结论】褐飞虱的两个Tret1在不同组织间发挥着不同的功能,其中NlTret1-like X1在特异性转运海藻糖参与能量供应中起到更为显著的作用。研究结果有助于探索Tret1在昆虫或无脊椎动物中调控海藻糖代谢平衡的调节机制,可为将来通过调控血糖平衡来控制褐飞虱等害虫提供理论依据。

关键词: 褐飞虱, 海藻糖转运蛋白, 结构, 海藻糖代谢, RNA干扰

Abstract:

【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 1 920 and 1 578 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-like X1 or dsNlTret1-2 X1. Furthermore, the content of glycogen and glucose in N. lugens did not change significantly, but unlike the dsNlTret1-2 X1 group, the trehalose content of N. lugens was significantly increased with the injection of dsNlTret1-like X1. Meanwhile, the expression of the TPS1, TPS2, TRE1-1, TRE1-2 and TRE2 in N. lugens was significantly down-regulated after the NlTret1-like X1 knocked down for 48 h. The expression of TPS1, TPS2 and TRE1-1 in N. lugens also decreased significantly after injection with dsNlTret1-2 X1 for 48 h, while TRE1-2 and TRE2 showed a very significant upward trend. Moreover, after injection of dsNlTret1-like X1, the activities of soluble trehalase and membrane-bound trehalase were significantly reduced, but there was no significant change after injection with dsNlTret1-2 X1. 【Conclusion】These two Tret1s of N. lugens play different functions in different tissues, among which NlTret1-like X1 plays a more significant role in the specific transport of trehalose involved in energy supply. The results are helpful to explore the regulatory mechanism of Tret1 regulating the balance of trehalose metabolism in insects or invertebrates, and provide a theoretical basis for the future control of pests by regulating blood sugar balance, such as N. lugens.

Key words: brown planthopper (Nilaparvata lugens), trehalose transporter (Tret), structure, trehalose metabolism, RNAi