β-1,3-葡聚糖合成酶(β-1,3-glucan synthase, GLS)催化合成的细胞壁核心组分β-1,3-葡聚糖对真菌的生存和毒力至关重要,该酶是研发抗菌药物的重要作用靶点。为了了解β-1,3-葡聚糖合成酶基因(BcGLS)在灰葡萄孢菌生存和侵染进程中的功能,该研究通过克隆获得BcGLS全长序列,序列全长7 137 bp,编码1 932个氨基酸,生物信息学分析结果显示,其编码蛋白(BcGLSp)理论相对分子质量为220.05 kDa,理论等电点为7.97;亚细胞预测其定位于细胞膜,存在17个跨膜结构,不存在信号肽,为非分泌蛋白;含有176个磷酸化位点,为亲水性蛋白。保守结构域分析结果表明,BcGLSp含有一个β-1,3-葡聚糖合成酶结构域和一个β-1,3-葡聚糖合成酶亚基FKS1,属于葡聚糖合成酶超家族。系统进化分析表明,灰葡萄孢菌BcGLS与植物致病菌如山茶叶杯菌、景天白粉菌等的葡聚糖合成酶亲缘关系近。不同逆境胁迫条件(氯化钠、十二烷基硫酸钠、过氧化氢)显著抑制灰葡萄孢菌菌丝生长,实时定量PCR进一步分析显示BcGLS在不同非生物胁迫条件下均上调表达,表明BcGLS基因的转录表达与环境压力密切相关。该研究为深入探讨BcGLS在灰葡萄孢菌生存和侵染进程中的基因功能和灰葡萄孢菌的新型防治策略提供理论材料和实践依据。
β-1, 3-glucan synthase (GLS) catalyzes the synthesis of cell wall core component β-1,3-glucan which is essential for fungal survival and virulence, and is the important target for antifungal agent development.To explore its gene function in the survival and infection process of Botrytis cinerea, this study primarily cloned the full-length sequence of β-1, 3-glucan synthase coding gene (BcGLS).Its total length was 7 137 bp by encoding 1 932 amino acids.Bioinformatic analysis indicated that BcGLS protein (BcGLSp) had a theoretical relative molecular weight of 220.05 kDa and a theoretical isoelectric point of 7.97.Subcellular prediction of BcGLSp was located in the cell membrane with 17 transmembrane regions, it had no signal peptide and was a non-secreted protein.BcGLSp was a hydrophilic protein with 176 phosphorylation sites.The results of conservative domain analysis revealed that BcGLSp contains one β-1,3-glucan synthase component and one β-1,3-glucan synthase FKS1 domain, belonging to glucan-synthase superfamily.Phylogenetic analysis displayed that BcGLS had close genetic relationship with those from plant pathogens such as Ciborinia camelliae and Erysiphe neolycopersici.Different abiotic stresses (NaCl、SDS、H2O2) significantly reduced Botrytis mycelial growth rate, and real-time PCR further showed that the expression of BcGLS was up-regulated under these stresses, indicating that the transcriptional expression of BcGLS was closely associated to environmental stresses.This study provides theoretical materials and practical basis for further research on the function of BcGLS gene in the survival and infection process of B.cinerea and the novel control strategies of B.cinerea.
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