广东虫草邻氨基苯甲酸合酶基因克隆及不同实验条件下的表达

【背景】邻氨基苯甲酸合酶(TrpE)广泛存在于植物和微生物中,参与色氨酸和生长素的生物合成,在生长发育及胁迫响应过程中具有重要的作用。广东虫草(Tolypocladium guangdongense)是华南地区特有的一种极具开发价值的虫草资源。【目的】明确TgtrpE基因特征,并探究该基因在不同发育时期和不同温度下的表达模式。【方法】从广东虫草中克隆TgtrpE编码区序列,并进行生物信息分析和系统发育分析,采用实时荧光定量PCR(RT-qPCR)分析TgtrpE在广东虫草中不同发育时期及温度胁迫下的表达模式。【结果】TgtrpE编码区序列长度为1613bp(DNA)和1563bp(cDNA),编码521个氨基酸,预测其蛋白分子量约为56.86kD。系统发育分析显示,TgTrpE蛋白与子囊菌中的TrpE蛋白聚为一个分支,与奇异弯颈霉(Tolypocladiumparadoxum)和头状弯颈霉(Tolypocladiumcapitatum)具有较高的相似性。RT-qPCR结果表明:在广东虫草原基和幼嫩子实体的发育过程中,TgtrpE显著上调,而在成熟子实体期该基因显著下调;在温度胁迫过程中,TgtrpE显著下调表达。【结论】TgtrpE可能在广东虫草原基形成、子实体发育及温度胁迫响应过程中具有重要调控作用。

Clone and expression of anthranilate synthase gene from Tolypocladium guangdongense under differential experimental conditions

Background] Anthranilate synthase, one common enzyme in plants and microbes, is involved in the biosynthesis of tryptophan and auxin, and plays an important role in growth and development, together with the response to multiple stresses. Tolypocladium guangdongense is a new cordyceps resource only found in South China, and has a great exploitation value. Objective] Dissect the gene features of TgtrpE, and explore the expression pattern of TgtrpE at differential developmental stages and temperature stress conditions in T. guangdongense. Methods] The coding sequences of TgtrpE were cloned from T. guangdongense and used for bioinformatic and phylogenetic analysis. Real-time fluorescence quantitative PCR (RT-qPCR) was performed to analyze the expression pattern of TgtrpE under differential developmental stages and temperature stresses. Results] The sizes of TgtrpE were 1 613 bp (DNA) and 1 563 bp (cDNA) in length, respectively. It encodes 521 amino acids with a molecular weight approximate of 56.86 kD. The phylogeny result showed that the TgTrpE protein of T. guangdongense was clustered into one clade with those of ascomycetes fungi, and had a higher similarity with Tolypocladium paradoxum and Tolypocladium capitatum. RT-qPCR results showed that compared to the mycelial stage, TgtrpE was significantly up-regulated at primordium and young fruiting body stages, whereas TgtrpE showed a clear downregulation expression at mature fruiting body stage and in the response process of cold and heat stresses. Conclusion] TgtrpE may play an important role in the processes of primordium formation, fruiting body development and temperature stresses.