褐飞虱一个新的海藻糖合成酶基因的特性、 发育表达及RNAi效果分析

doi:10.3864/j.issn.0578-1752.2019.03.007

摘要/Abstract

摘要：

背景昆虫海藻糖合成酶基因是昆虫海藻糖合成的主要基因,大多数昆虫中只拥有一个海藻糖-6-磷酸合成酶（trehalose-6-phosphate synthase,TPS）基因,部分昆虫存在一个海藻糖-6-磷酸酯酶（trehalose-6-phosphate phosphatase,TPP）基因。前期研究发现褐飞虱（Nilaparvata lugens）中拥有两个TPS,对其功能研究发现TPS不仅能够调控海藻糖代谢,还可介导海藻糖酶调控几丁质合成与降解途径,控制昆虫的蜕皮过程。目的通过对褐飞虱转录组测序分析获得了一个新的TPS,检测该基因在褐飞虱不同发育阶段的表达情况,探究该基因的功能与前期发现的两个TPS的区别。方法对获得的新TPS基因序列采用克隆技术获得全长cDNA序列,经验证正确后,对其蛋白的一级、二级、三级结构及与其他昆虫的TPS进行比对分析,最后采用实时荧光定量PCR（qRT-PCR）技术测定3个不同TPS在褐飞虱不同发育阶段的表达,并采用RNA干扰（RNAi）技术抑制TPS3的表达。结果在前期研究的基础上,克隆出一个新的TPS,并命名为TPS3。TPS3开放阅读框长度为2 352 bp,编码783个氨基酸,预测蛋白分子量为88.9 kD,等电点为5.47,具有亲水性结构。生物信息学分析表明,褐飞虱3个TPS蛋白具有较高的同源性,都具有TPS和TPP两个保守结构域及其他特征序列,并且α-螺旋、β-折叠和无规则卷曲所占的比例较为接近。褐飞虱不同发育阶段3条TPS的相对表达量不同,TPS1的相对表达量从4龄0 h开始逐渐上升,至成虫阶段达到最高,TPS2的相对表达量从4龄末期开始明显上升且在整个5龄阶段都有较高的表达,TPS3的相对表达量在5龄末期和成虫初期较高。单独干扰褐飞虱TPS3 48 h后被干扰基因的相对表达量下降,dsTPS3能有效抑制TPS3的表达。结论在褐飞虱中发现一个新的TPS（TPS3）,其与褐飞虱中已经报道的TPS1和TPS2具有较高的同源性。不同发育阶段表达结果表明,3个TPS在发育过程中行使的功能不同。RNAi能够有效抑制TPS3的表达并导致褐飞虱蜕皮障碍和翅发育畸形。

关键词: 褐飞虱, 海藻糖-6-磷酸合成酶, 序列分析, 蛋白结构, 实时荧光定量PCR

Abstract:

【Background】 Insect trehalose-6-phosphate synthase (TPS) is the main gene for insect trehalose synthesis. Most insects possess one trehalose-6-phosphate synthase gene, and some insects have one trehalose-6-phosphate phosphatase (TPP) gene. Previous studies have found that there are two TPS genes in the brown planthopper (Nilaparvata lugens) and their functional studies have shown that TPS can not only regulate trehalose metabolism, but also mediate trehalase regulation of chitin synthesis and degradation pathways, and control insect molting process.【Objective】 A novel TPS was obtained by the transcriptome sequencing of N. lugens. The objective of this study is to detect the expression of the gene in different developmental stages of N. lugens, and to explore the difference between the function of the gene and the two previously discovered TPS genes.【Method】 The full-length cDNA sequence of the novel TPS was obtained by cloning technique. After correct verification, the primary, secondary, and tertiary structures of the protein were obtained. The protein structure and alignment of TPS were analyzed with other insects. Finally, qRT-PCR was used to determine the expression of three different TPS genes at different development stages of N. lugens, and RNA interference (RNAi) was used to inhibit the expression of TPS3.【Result】 Based on the previous study, this novel TPS was cloned and named as TPS3. The open reading frame of TPS3 is 2 352 bp, encoding 783 amino acids. The predicted molecular weight of the protein is 88.9 kD and the isoelectric point is 5.47, with a hydrophilic structure. Bioinformatics analysis showed that the three TPS proteins of N. lugens had high homology, with two conserved domains of TPS and TPP and other characteristic sequences, and the proportion of α-helix, β-sheet, and random curl was similar. The relative expression levels of three TPS genes in different developmental stages of N. lugens were different. The relative expression level of TPS1 gradually increased from 0 h of 4th instar to the highest level at the adult stage. The relative expression level of TPS2 increased obviously from the end of 4th instar and was higher in the whole 5th instar. The expression of TPS3 was relatively higher at the end of the 5th instar and the early stage of adult. The relative expression level of the interfered gene significantly decreased after 48 hours of single interference with TPS3. The dsTPS3 could effectively inhibit the expression of TPS3.【Conclusion】A novel TPS (TPS3) was found in N. lugens, which has high homology with TPS1 and TPS2 reported in N. lugens. The results of expression at different developmental stages showed that the three TPS genes performed different functions during development. RNAi could effectively inhibit the expression of TPS3 and lead to molting and wing deformity.

Key words: Nilaparvata lugens, trehalose-6-phosphate synthase (TPS), sequence analysis, protein structure, quantitative real-time PCR (qRT-PCR)

唐斌,沈祺达,曾伯平,肖仲久,邱玲玉,潘碧莹,李昆,张道伟. 褐飞虱一个新的海藻糖合成酶基因的特性、发育表达及RNAi效果分析[J]. 中国农业科学, 2019, 52(3): 466-477.

TANG Bin,SHEN QiDa,ZENG BoPing,XIAO ZhongJiu,QIU LingYu,PAN BiYing,LI Kun,ZHANG DaoWei. Characteristics, Developmental Expression and RNAi Effect Analysis of a Novel Trehalose-6-Phosphate Synthase Gene in Nilaparvata lugens[J]. Scientia Agricultura Sinica, 2019, 52(3): 466-477.

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基因名称 Primer name	正向引物 Forward primer (5′-3′)	反向引物 Reverse primer (5′-3′)
QNl18S QNlTPS1 QNlTPS2 QNlTPS3	CGCTACTACCGATTGAA AAGACTGAGGCGAATGGT AGAGTGGACCGCAACAACA GTGATGCGTCGGTGGCTAT	GGAAACCTTGTTACGACTT AAGGTGGAAATGGAATGTG TCAACGCCGAGAATGACTT CCGTTCATCATTGGGCATAGT
dsNlTPS3	GAGTCTGACCTGATAGCCTTTA	ATCGGAGTCCATTTAGTTGT
dsNlTPS3-T7	GGATCCTAATACGACTCACTATAGGGAGTCTGACCTGATAGCCTTTA	GGATCCTAATACGACTCACTATAGGATCGGAGTCCATTTAGTTGT
dsNlGFP	AAGGGCGAGGAGCTGTTCACCG	CAGCAGGACCATGTGATCGCGC
dsNlGFP-T7	GGATCCTAATACGACTCACTATAGGAAGGGCGAGGAGCTGTTCACCG	GGATCCTAATACGACTCACTATAGGCAGCAGGACCATGTGATCGCGC

蛋白名称 Protein name	氨基酸数 Amino acid number	相对分子量 Relative molecular weight	等电点 Isoelectric point	亲水性 Hydrophily
TPS1	807	90536	6.14	-0.180
TPS2	820	93215	6.07	-0.190
TPS3	783	88909	5.47	-0.126

	α-螺旋 α-helix (%)	β-折叠 β-sheet (%)	无规则卷曲 Random coil (%)
TPS1	40.27	12.76	45.35
TPS2	36.59	13.29	49.39
TPS3	35.50	18.01	46.49