| 胡亚平,陈聪,吉前华,郭雁君,郭丽英,蒋惠,杨凤梅.砂糖橘几丁质酶orange1.1t05124基因克隆及原核表达[J].中国南方果树,2025,54(1): |
| 砂糖橘几丁质酶orange1.1t05124基因克隆及原核表达 |
| Cloning and prokaryotic expression of the orange1.1t05124 gene encoding chitinase from Citrus reticulata Blanco |
| 投稿时间：2024-08-14 修订日期：2024-10-21 |
| DOI： |
| 中文关键词: 砂糖橘 几丁质酶 原核表达 抗真菌蛋白 |
| 英文关键词:Citrus tachibana Chitinase Prokaryotic expression Antifungal Protein |
| 基金项目:2022SDZG“十四五”广东省农业科技创新十大主攻方向“揭榜挂帅‘项目 |
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| 中文摘要: |
| 柑橘的几丁质酶作为一种重要的抗真菌蛋白，可以降解真菌细胞壁中的几丁质。本研究利用砂糖橘的cDNA文库克隆了砂糖橘几丁质酶基因orange1.1t05124，它位于第8染色体末端，包含3个外显子，将基因克隆到pEASY-Blunt E1载体中，测序验证序列无误，随后转入大肠杆菌BL21（DE3）中，经IPTG诱导生成了目的蛋白， SDS-PAGE电泳验证成功表达，在产物的包涵体中纯化到了目标蛋白。通过生物信息学分析发现该蛋白具有信号肽，定位在细胞外，是单体的球状蛋白质，通过进化树分析发现该几丁质酶在植物中广泛存在同源蛋白，且都含有GH19几丁质酶催化结构域。对柑橘几丁质酶基因的克隆和表达为了解该基因家族的抗病机理，开发基于几丁质酶的新型生物农药、生物肥料提供了重要的基因资源。 |
| 英文摘要: |
| The chitinase of citrus, an essential antifungal protein, exerts its function by degrading chitin within the fungal cell wall. In this study, the citrus chitinase gene, designated as orange1.1t05124, was successfully amplified from the cDNA library of Shatangju mandarin (Citrus reticulata Blanco var. shatangju) using PCR. The gene was subsequently cloned into the pEASY-Blunt E1 vector, and the accuracy of the sequence was confirmed through sequencing. Following transformation into Escherichia coli BL21(DE3), the target protein was induced by IPTG and its successful expression was verified by SDS-PAGE electrophoresis. The desired protein was further purified from the inclusion bodies of the produced product. Bioinformatic analysis revealed the presence of a signal peptide in this protein, indicating its extracellular localization. Additionally, evolutionary tree analysis demonstrated that homologous proteins of this chitinase are widely distributed among plants and all contain the GH19 chitinase catalytic domain. The cloning and expression of the citrus chitinase gene not only contribute to understanding the disease resistance mechanisms within this gene family but also provide valuable genetic resources for the development of novel biopesticides and biofertilizers based on chitinase. |
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