荔枝蒂蛀虫海藻糖酶基因克隆及生物信息学分析

姚 琼 1; 梁展图 2; 段双刚 1; 董易之 1; 徐 淑 1; 李文景 1

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姚琼 1 ，梁展图 2 ，段双刚 1 ，董易之 1 ，徐淑 1 ，李文景 1.荔枝蒂蛀虫海藻糖酶基因克隆及生物信息学分析[J].广东农业科学,2024,51(6):13-21

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Cloning and Bioinformatics Analysis of Trehalase Genes in Conopomorpha sinensis Bradley

DOI：10.16768/j.issn.1004-874X.2024.06.002

中文关键词: 海藻糖酶基因克隆生物信息学分析荔枝蒂蛀虫表达模式

英文关键词: rehalase gene cloning bioinformatics analysis Conopomorpha sinensis Bradley expression pattern

基金项目:国家自然科学基金（31801800）；广东省自然科学基金（2020A151501960）；广东省农业科学院协同创新中心项目（XTXM202209）；国家荔枝龙眼产业技术体系专项（CARS-32）；广东省农业科学院乡村振兴战略专项（2024TS-2-2）；广州市科技计划项目（2024E04J1261）

作者	单位
姚琼 1 ，梁展图 2 ，段双刚 1 ，董易之 1 ，徐淑 1 ，李文景 1	（1. 广东省农业科学院植物保护研究所 / 农业农村部华南果蔬绿色防控重点实验室 / 广东省植物保护新技术重点实验室，广东广州 510640 2. 华南师范大学生命科学学院，广东广州 510631）

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中文摘要:

【目的】海藻糖酶（Trehalase，Tre）是昆虫体内海藻糖代谢的关键酶，通过专一性地将海藻糖分解为葡萄糖，在昆虫能量代谢和生长发育中发挥着重要的作用。旨在克隆荔枝蒂蛀虫（Conopomorpha sinensis Bradley）可溶型海藻糖酶基因（CsTre1）和膜结合型海藻糖酶基因（CsTre2），探讨其在荔枝蒂蛀虫不同发育阶段和不同组织中的表达模式，解析这 2 个基因及其酶蛋白的分子特征。【方法】利用荔枝蒂蛀虫转录组数据和 RACE 技术，克隆 CsTre1 和 CsTre2 的全长 cDNA 序列，并应用 ORF Finder、ProtParam、SignalP 4.1、ProtScale、 NetPhos2.0 Server 和 IQ-TREE 等软件对其进行生物信息学分析；采用实时荧光定量 PCR（RT-qPCR）分析 CsTre1 和 CsTre2 在荔枝蒂蛀虫不同发育阶段及成虫不同组织的 mRNA 表达模式。【结果】CsTre1 的开放阅读框（ORF）长 1 701 bp，编码 566 个氨基酸，蛋白分子量为 64.53 kD。CsTre2 的 ORF 长 1 821 bp，编码 606 个氨基酸，蛋白分子量为 69.08 kD。信号肽预测分析表明，CsTre1 和 CsTre2 前端均有 1 个信号肽，其位置分别为 1-16 和 1-17。蛋白二级结构分析结果显示，二者均主要由 α- 螺旋和无规则卷曲组成，CsTre1 有 24 个 Ser、15 个 Tyr、10 个 Thr 可能成为蛋白激酶的结合位点，而 CsTre2 有 27 个 Ser、10 个 Tyr、13 个 Thr 可能成为蛋白激酶的结合位点。RTqPCR 结果显示，CsTre 在荔枝蒂蛀虫的蛹和成虫期均有表达。在成虫期中，CsTre1 的表达水平远高于 CsTre2，且 CsTre1 在雄成虫第 2 d 和第 5 d 的表达水平陡然下降，在雌成虫中则保持稳定高表达。【结论】该研究成功克隆了荔枝蒂蛀虫的 2 个海藻糖酶基因，其分子特征及表达模式结果表明，CsTre1 可能是荔枝蒂蛀虫主要调控海藻糖代谢的基因。研究结果可为阐明海藻糖酶基因的功能提供重要线索，为开展害虫防治策略的研究奠定基础。

英文摘要:

【Objective】Trehalase (Tre) is a key enzyme in trehalose metabolism of Conopomorpha sinensis Bradley, which plays an important role in energy metabolism and growth development by specifically hydrolyzing trehalose into glucose. The study aims to clone two trehalase genes (CsTre1 and CsTre2) from Conopomorpha sinensis Bradley, to clarify its expression patterns in different developmental stages and tissues, and to analyze the molecular characteristics of the two genes and their enzyme proteins.【Method】Based on the transcriptome data of C. sinensis, the full-length cDNA sequences of CsTre1 and CsTre2 were cloned with the rapid amplification of cDNA ends (RACE)-PCR. Bioinformatics analysis was performed with software such as ORF Finder, ProtParam, SignalP 4.1, ProtScale, NetPhos2.0 Server and IQ TREE. The mRNA expression patterns of CsTre1 and CsTre2 in different developmental stages and tissues of C. sinensis were detected by using real-time quantitative PCR (RT-qPCR).【Result】The open reading frame of CsTre1 was 1 701 bp, encoding 566 amino acids, and the protein molecular weight was 64.53 kD. The open reading frame length of CsTre2 was 1 821 bp, encoding 606 amino acids, and the protein molecular weight was 69.08 kD. Signal peptide prediction analysis showed that both front-ends of CsTre1 and CsTre2 had a signal peptide, with positions of 1-16 and 1-17, respectively. The analysis on the secondary structure of the sequence showed that both CsTre1 and CsTre2 were mainly composed of α-helix and random coil, CsTre1 had 24 Sers, 15 Tyrs, and 10 Thrs that may serve as binding sites for protein kinases, while CsTre2 had 27 Sers, 10 Tyrs, and 13 Thrs that may serve as binding sites for protein kinases. The RT-qPCR results revealed that CsTre was expressed throughout all developmental stages of C. sinensis. In the expression pattern of adult stage, the expression level of CsTre1 was much higher than that of CsTre2, and the expression level of male adults of CsTre1 dropped sharply after the fourth day.【Conclusion】 The study successfully cloned two trehalose genes of Conopomorpha sinensis Bradley. According to the results of analysis on their molecular characteristics and expression patterns, CsTre1 may be the main gene regulating trehalose metabolism in Conopomorpha sinensis Bradley. The research results provide important clues for elucidating the function of trehalase genes, which lay a solid foundation for the development pest control strategies.

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