海岛棉单萜合酶基因克隆及其受黄萎病诱导的表达分析

Clone of monoterpene synthetase gene GbTPS from Gossypiumbar badense and its expression induced by Verticillium wilt

【目的】克隆海岛棉品种新海15中单萜合成酶基因（Gb TPS）,研究其受黄萎病诱导的表达情况,为进一步研究该基因在棉花抗黄萎病中的功能打下基础。【方法】克隆Gb TPS基因的开放阅读框（ORF）全长序列,对其蛋白序列进行生物信息学分析,并采用实时荧光定量PCR及病毒诱导的基因沉默对其抗病性进行分析。【结果】通过PCR扩增得到一个全长为1761 bp的ORF序列,命名为Gb TPS（Gen Bank登录号：KP247548）。生物信息学分析发现,该基因编码586个氨基酸,预测分子量为67.7 k D,等电点为5.79。系统进化树分析结果表明,Gb TPS属于Tps-g亚家族。Gb TPS基因经黄萎病菌诱导后,表达量在4~24 h内显著（P〈0.05）或极显著（P〈0.01）高于空白对照组。病毒诱导的基因沉默受黄萎病诱导后其病情指数为52.38,与对照组相比并无明显差异。【结论】Gb TPS基因仅在棉花抗黄萎病的早期发挥积极作用。

【Objective】A monoterpene synthetase gene Gb TPS was cloned from Gossypiumbar badense, and its expression induced by Verticillium wilt were analysed, in order to lay a foundation for studying resistance of cotton to Verticillium wilt. 【Method】The full-length open reading frame（ORF） of Gb TPS gene was cloned, and its encoded protein was analysed by bioinformatics, the real-time fluorescent quantitative PCR（q RT-PCR） and virus induced gene silencing was used to analyse resistance to Verticillium wilt. 【Result】Full-length ORF was obtained, with length of 1761 bp, and named Gb TPS（Genebank accession number： KP247548）. The bioinformatics analysis showed that, the protein composed of 586 amino acids was coded by Gb TPS gene, its predicted molecular weight was 67.7 k D, its isoelectric point was 5.79. Phylogenetic tree analysis showed that, Gb TPS belonged to Tps-g subfamily. The expression of Gb TPS gene was significant（P0.05） or extermely significant（P0.01） higher than that of blank control within 4-24 h after being induced by Verticillium dahlia. The disease index of virus induced gene silencing was 52.38, and had no significant difference with that of control.【Conclusion】Only at the early stage, the Gb TPS gene plays a positive role in resistance to cotton Verticillium wilt.