罗雨嘉,邱玲玉,刘永康,庞晓青,姚琼,王世贵,唐斌,徐彩娣,2022,褐飞虱两个糖转运蛋白的功能分析及调控海藻糖代谢的影响[J].环境昆虫学报,(4):935-945
褐飞虱两个糖转运蛋白的功能分析及调控海藻糖代谢的影响
Functional analysis of two sugar transporters of the brown planthopper (Nilaparvata lugens) and their effects on regulating trehalose metabolism
  
DOI:
中文关键词:  褐飞虱  海藻糖代谢  海藻糖转运蛋白  RNA干扰
英文关键词:Nilaparvata lugens  trehalose metabolism  trehalose transporter  RNA interference
基金项目:广东省农业科学院协同创新中心项目(XTXM202202);杭州市科技计划引导项目(20201231Y072)
作者单位
罗雨嘉,邱玲玉,刘永康,庞晓青,姚琼,王世贵,唐斌,徐彩娣 1.杭州师范大学生命与环境科学学院杭州 3111212. 广东省农业科学院植物保护研究所/广东省植物保护新技术重点试验室 广州 5106403. 杭州师范大学经亨颐教育学院杭州 311121 
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中文摘要:
      海藻糖转运蛋白(Trehalose transporter,Tret)可将昆虫“血糖”——海藻糖由脂肪体转运到血淋巴中,是维持昆虫体内海藻糖平衡的重要转运蛋白。本研究通过对褐飞虱Nilaparvata lugens两条糖转运蛋白序列(NlTret1、NlTret1 X1)进行生物信息学分析,并利用RNAi技术沉默NlTret1与NlTret1 X1基因,探讨其对褐飞虱调控海藻糖代谢的生物学功能。生物信息学分析表明,NlTret1与NlTret1 X1分别有1 353 bp和1 488 bp的开放阅读框,编码具有450和495个氨基酸残基,蛋白分子量大小为49.984 kDa和53.059 kDa,理论等电点pI为6.53和7.46;保守结构域分析NlTret1和NlTret1 X1分别包含10个和12个跨膜结构域,属于MFS超家族;二级结构以及三级结构预测NlTret1和NlTret1 X1主要包含无规卷曲和α螺旋结构。进化树分析显示NlTret1和NlTret1 X1皆与同为半翅目昆虫的Tret1蛋白亲缘关系接近。与注射dsGFP相比RNAi后显著抑制了靶标基因的表达量。荧光定量检测海藻糖代谢通路TRE和TPS基因,结果显示注射dsNlTret1 48 h后NlTRE1-1、NlTPS2基因表达量极显著下调,NlTRE1-2、NlTRE2与NlTPS1、NlTPS3基因表达量极显著上调;而注射dsNlTret1 X1则为NlTRE1-1、NlTRE2与NlTPS1、NlTPS2基因表达量极显著降低,NlTRE1-2和NlTPS3基因表达量极显著增高。注射dsNlTret1与dsNlTret1 X1后海藻糖酶活性均显著性降低,同时NlTret1的沉默显著抑制了糖原含量和海藻糖含量,NlTret1 X1的沉默仅使葡萄糖含量显著增高。褐飞虱两条糖转运蛋白序列经初步分析确定为海藻糖转运蛋白,这两个海藻糖转运蛋白在转运糖类物质中发挥着不同的作用,其中NlTret1 X1可能参与葡萄糖运输功能,而NlTret1则更可能参与海藻糖特异性转运。研究结果有利于探究海藻糖转运蛋白Tret调控海藻糖代谢的作用机制,为将来通过其调控昆虫海藻糖代谢平衡治理褐飞虱等害虫提供理论依据。
英文摘要:
      Trehalose transporter (Tret) transport trehalose-the “blood sugar” of insects, from fat body to hemolymph, and is an important transporter to maintain trehalose balance in insects. In this study, bioinformatics analysis was conducted on two sugar transporter sequences (NlTret1 and NlTret1 X1) of Nilaparvata lugens, and NlTret1 and NlTret1 X1 genes were silencing by RNAi technology to explore their function on regulation of trehalose metabolism. Bioinformatics analysis showed that NlTret1 and NlTret1 X1 had an open reading frame of 1 353 bp and 1 488 bp, and encoded 450 and 495 amino acid residues, respectively. The molecular weights of the proteins were 49.984 kDa and 53.059 kDa, and the theoretical isoelectric point was 6.53 and 7.46, respectively. Conserved domain analysis showed that NlTret1 and NlTret1 X1 contained 10 and 12 transmembrane domains, respectively, which belonged to the MFS superfamily. Secondary and tertiary structure prediction showed that NlTret1 and NlTret1 X1 mainly contained coils and helix structures. The phylogenetic tree analysis showed that both NlTret1 and NlTret1 X1 were closely related to Tret1 proteins of Hemiptera. Compared with dsGFP injection, RNAi significantly inhibited the expression of the target gene. QRT-PCR detection of trehalose metabolic pathway TRE and TPS genes showed that the expression levels of NlTRE1-1 and NlTPS2 were significantly down-regulated at 48 h after dsNlTret1 injection, and the expression levels of NlTRE1-2, NlTRE2, NlTPS1 and NlTPS3 increased significantly. However, the expression levels of NlTRE1-1, NlTRE2, NlTPS1 and NlTPS2 were significantly decreased after injecting dsNlTret1 X1, and the expression levels of NlTRE1-2 and NlTPS3 were extremely significantly increased. Trehalase activity was significantly decreased after dsNlTret1 and dsNlTret1 X1 injection. Meanwhile, silencing of NlTret1 significantly decreased glycogen and trehalose content, while silencing of NlTret1 X1 only significantly increased glucose content. To sum up, two trehalose transporters were identified as trehalose transporters by preliminary analysis. These two trehalose transporters played different roles in the sugars transport, among which NlTret1 X1 might be involved in glucose transport, while NlTret1 was more likely to be involved in transporting trehalose specifically. These results were conducive to exploring the mechanism of Tret regulating trehalose metabolism, and provided a theoretical basis for pest management such as brown planthopper by regulating trehalose metabolism balance in the future.
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