甘薯IbTPS1基因的克隆与活性鉴定

Cloning and identification of IbTPS1 from Ipomoea batatas

[目的]发掘植物海藻糖合成途径关键酶基因,探究其编码蛋白TPS活性,为作物遗传改良抗多种胁迫提供优良的候选基因。[方法]本研究基于干旱条件下从甘薯转录组数据库中得到一条与拟南芥AtTPS1基因高度同源的Unigene序列,通过RT-PCR技术克隆了甘薯TPS基因。利用生物信息学软件分析序列特征,酵母互补试验鉴定编码蛋白TPS活性。[结果]IbTPS1基因cDNA序列全长2811bp,编码936个氨基酸,且具有典型的GT1-TPS和HAD-TPP结构域;生物信息学预测表明IbTPS1编码的蛋白是一个不稳定亲水性蛋白,无信号肽,定位于细胞质中;二级结构元件多以无规则卷曲和α-螺旋为主;酵母互补实验证明表达IbTPS1基因的TPS突变酵母菌株(tps1Δ)和TPS,TPP双突变酵母菌株(tps1Δtps2Δ),以葡萄糖为唯一碳源的尿嘧啶缺失培养基上可恢复生长。[结论]证实甘薯IbTPS1基因的编码蛋白具有生物活性。

[Objectives] Present study was conducted to explore key enzymatic genes which involved in plant trehalose synthesis, to determine the activity of trehalose-6-phosphate synthase gene, and to provide an excellent candidate gene for crop genetic improvement with resistance to multiple stresses.[Methods] A sweet potato trehalose-6-phosphate synthase gene ( IbTPS1) which has high homology with AtTPS1, was screened from the transcriptome database under drought stress conditions, and was amplified by RT-PCR.The bioinformatics software was used to analyze the sequence characteristics, and the yeast complementation assay was used to identify the TPS activity.[Results] The cDNA full-length of IbTPS1was 2 811 bp that encoded 936 amino acid. IbTPS1 protein contained a typical GT1- TPS structure domain and HAD-TPP structure domain. Biological information analysis showed that IbTPS1 belonged to the unstable and hydrophilic protein without signal peptide, and localized in the cytoplasm. The secondary structural elements were mostly composed of random coils and α-helices. Yeast complementation assay results demonstrated that yeast TPS mutant ( tps1Δ) and TPS / TPP mutant ( t ps1Δtps2 Δ) expressing IbTPS1gene could restore growth on the medium supplemented with glucose.[Conclusion] The result indicated that sweetpotao IbTPS1 protein is an activetive trehalose-6-phosphate synthase.