| 穿心莲糖苷水解酶基因ApGH的克隆、生物信息学分析及表达研究 |
| 投稿时间：2024-01-22 修订日期：2024-04-19 点此下载全文
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| 中文摘要:目的：对穿心莲糖苷水解酶（ApGH）基因进行克隆、生物信息学分析、原核表达和相对表达量分析。方法：取穿心莲新鲜叶片，提取RNA并反转录为互补脱氧核糖核酸（cDNA）作为模板，以穿心莲转录组中筛选到的糖苷水解酶ApGH基因开放阅读框（ORF）设计特异性引物，进行PCR扩增，经测序后获得的基因序列利用生物信息学软件预测基因编码蛋白特征，构建原核表达载体在大肠杆菌中诱导表达目的蛋白，并利用实时荧光定量PCR（real-time PCR）分析ApGH基因的表达模式。结果：克隆所得ApGH基因的开放阅读框（ORF）全长1734 bp，编码577个氨基酸（GenBank登录号OR887606）。该蛋白为稳定亲水蛋白，无信号肽和跨膜结构域，属于糖苷水解酶家族；系统进化分析表明，ApGH归属于GH1家族。将ApGH基因构建到原核表达载体HIS-MBP-pET28a上，在大肠杆菌Transetta（DE3）中成功表达出重组蛋白。实时荧光定量PCR检测结果表明，ApGH基因在叶中表达量最高，茎和根中表达量较低。结论：本研究首次克隆得到穿心莲糖苷水解酶ApGH基因并对其蛋白序列特征进行了系统分析，证明其在大肠杆菌中成功表达重组蛋白，且主要在穿心莲叶中表达。本研究为进一步探索穿心莲糖苷水解酶的催化功能提供了依据。 |
| 中文关键词:穿心莲 糖苷水解酶 基因克隆 生物信息学分析 基因表达 |
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| Cloning, Bioinformatics Analysis and Expression of a Glucoside Hydrolase gene ApGH from Andrographis paniculata |
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| Abstract:Objective: The glycoside hydrolase gene (ApGH) of A. paniculata was cloned, and its bioinformatics, prokaryotic expression, and relative expression were analyzed. Methods: RNA was extracted from the fresh leaves of A. paniculata and reverse transcribed into complementary deoxyribonucleic acid (cDNA) as templates. Specific primers were designed according to the open reading frame (ORF) of the ApGH gene screened from the transcriptome of A. paniculata, after which PCR amplification was performed. The gene sequence obtained after sequencing was used to predict the protein features encoded using bioinformatics software. Prokaryotic expression vectors were constructed to heterologously express recombinant proteins in Escherichia coli. The expression of ApGH was examined by real-time fluorescence quantitative PCR (real-time PCR). Results: The ORF of the ApGH gene was 1734 bp in length and encoded 577 amino acid residues (GenBank accession number OR887606). It was a stable hydrophilic protein without signal peptides or transmembrane domains and belonged to the glycoside hydrolase family. Phylogenetic analysis revealed that ApGH was grouped into the GH1 family. The ApGH gene was ligated to the prokaryotic expression vector HIS-MBP-pET28a and subsequently transformed into Escherichia coli Transetta (DE3) for the expression of recombinant protein. The analysis of realtime quantitative PCR revealed that the expression level of ApGH was the highest in leaves, but lower in stems and roots. Conclusion: In this study, ApGH, the glycoside hydrolase gene from A. paniculata, was cloned for the first time, and its protein sequence characteristics were systematically analyzed. The recombinant protein was successfully expressed in Escherichia coli, and real-time PCR analysis showed that ApGH gene predominantly expressed in leaves. This study provides a basis for further study of the function of glycoside hydrolases from A. paniculata. |
| keywords:Andrographis paniculata glycoside hydrolase gene cloning bioinformatics analysis gene expression |
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