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

Abstract

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)

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

Characteristics, Developmental Expression and RNAi Effect Analysis of a Novel Trehalose-6-Phosphate Synthase Gene in Nilaparvata lugens

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