| 姚琼,梁展图,段双刚,等.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,(7-8):- |
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荔枝蒂蛀虫海藻糖酶基因克隆及生物信息学分析 |
| Cloning and Bioinformatics Analysis of Trehalase Genes in Conopomorpha sinensis Bradley |
| 投稿时间：2024-05-27 修订日期：2024-06-20 |
| DOI： |
| 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: trehalase 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“农作物病虫害智能识别与监测技术研发与应用”(XTXM202209)；国家荔枝龙眼产业技术体系(CARS-32) ；荔枝加工品种高产高效栽培体系集成与示范（2024TS-2-2）；广州市科技计划项目（2024E04J1261） |
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| 摘要点击次数: 14 |
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| 中文摘要: |
| 【目的】海藻糖酶( trehalase,Tre) 是荔枝蒂蛀虫(Conopomorpha sinensis Bradley)体内海藻糖代谢的关键酶,通过专一性地分解一分子海藻糖生成两分子葡萄糖,在能量代谢和生长发育中发挥着重要的作用。本研究旨在克隆荔枝蒂蛀虫可溶型(Tre1)和膜结合型(Tre2)两种海藻糖酶基因,探讨它们在荔枝蒂蛀虫不同发育阶段和不同组织中的表达模式,解析这两种基因及其酶蛋白的分子特征。【方法】通过荔枝蒂蛀虫转录组数据结合RACE技术,克隆该虫CsTre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)解析海藻糖酶基因CsTre1和CsTre2 在荔枝蒂蛀虫不同发育阶段及成虫不同组织部位的mRNA表达模式。【结果】可溶型海藻糖酶CsTre1的开放阅读框长1701 bp,编码566个氨基酸,蛋白分子量为64.53 kDa。膜结合型海藻糖酶CsTre2的开放阅读框长1821 bp,编码606个氨基酸,蛋白分子量为69.08 kDa。信号肽预测分析表明,CsTre1和CsTre2前端都具有一个信号肽,其位置分别为1-16和1-17。序列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。RT-qPCR结果显示,CsTre在蛹和成虫期都有表达。在成虫期的表达模式中,CsTre1的表达水平远远高于CsTre2,且CsTre1的雄成虫在第四天后的表达水平发生了陡然下降的趋势。【结论】本研究成功克隆了荔枝蒂蛀虫的两种海藻糖酶基因,根据其分子特征以及表达模式结果,推荐二者在成虫初期的交配和生殖行为中有重要生理功能。研究结果为阐明海藻糖酶基因的功能提供了重要线索,为开展害虫防治策略的研究奠定了基础。 |
| 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 one trehalose molecule into two glucose molecules. This study aims to clone trehalase genes (CsTre1 and CsTre2) from the Conopomorpha sinensis Bradley, to clarify its expression patterns in different developmental stages and tissues, and to analyze the molecular characteristics of the two types of the gene and their products.【Method】Based on the transcriptome data of C. sinensis, the full-length cDNA sequences of CsTre1 and CsTre2 were cloned using the rapid amplification of cDNA ends (RACE)-PCR. Bioinformatics analysis was performed using softwares such as ORF Finder, ProtParam, SignalP 4.1, ProtScale, NetPhos2.0 Server, and IQ TREE. The mRNA expression levels 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 soluble trehalase gene (CsTre1) is 1701 bp, encoding 566 amino acids, and the protein has a molecular weight of 64.53 kDa. The open reading frame length of membrane bound trehalase gene (CsTre2) is 1821 bp, encoding 606 amino acids, and the protein molecular weight is 69.08 kDa. Signal peptide prediction analysis showed that both CsTre1 and CsTre2 have a signal peptide, with positions 1-16 and 1-17, respectively. The analysis of the secondary structure of the sequence showed that both CsTre1 and CsTre2 were mainly composed of α-helix and random coil, CsTre1 has 24 Sers, 15 Tyrs, and 10 Thrs that may serve as binding sites for protein kinases, while CsTre2 has 27 Sers, 10 Tyrs, and 13 Thrs that may serve as binding sites for protein kinases. The Phylogenetic analysis showed that CsTre1 and CsTre2 belonged to two distinct branches. 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 is much higher than that of CsTre2, and the expression level of male adults of CsTre1 drops sharply after the fourth day. 【Conclusion】This study successfully cloned two trehalose genes of C.sinensis and analyzed their molecular characteristics and expression patterns. According to the results, both CsTre1 and CsTre2 might have important physiological functions in mating and reproductive behavior in the early adult. The research results provide important clues for elucidating the function of trehalase genes, which lay a solid foundation for developing pest control strategies. |
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