铁观音茶树RING型E3泛素连接酶基因克隆及其干旱胁迫下的表达分析

Cloning and Expression Analysis of Ring-type E3 Ubiquitin Ligase Gene in Camellia sinensis cv. Tieguanyin Under Drought Stress

以铁观音茶树为试材,对其中E3泛素连接酶基因进行克隆和生物信息学分析,并采用qPCR进行不同干旱条件下的定量表达分析。研究结果表明,该序列全长为1 138bp,开放阅读框(ORF)为810bp,编码269个氨基酸(GenBank登录号KR819177)。生物信息学分析发现该铁观音茶树E3泛素连接酶基因不含跨膜结构以及信号肽,具有多个磷酸化位点,亚细胞定位于叶绿体中。经BLAST比对,该基因编码的氨基酸序列与烟草、亚麻荠、葡萄、醉蝶花、芜菁中的E3泛素连接酶基因编码的氨基酸序列分别有51%、50%、50%、50%和49%的同源性,且相关保守功能结构域翻译的蛋白质序列具有RING-finger结构,初步确定该基因为铁观音茶树的E3泛素连接酶基因。qPCR分析结果显示在不同干旱胁迫处理下铁观音茶树的E3泛素连接酶基因的表达量,与对照组相比显著增加。本研究认为铁观音茶树RING型E3泛素连接酶基因参与茶树抗旱响应机制。

The E3 ubiquitin ligase gene fromCamellia sinensis cv.Tieguanyin tea plants were cloned and subjected to a bioinformatics analysis.Under varied drought conditions on the plants,the quantitative expressions of the gene was determined using qPCR.The gene sequence（GenBank accession number,KR819177）was found to be 1 138 bp long containing an open reading frame（ORF）of 810 bp and encoding 269 amino acids.The bioinformatics revealed that the ligase gene did not have a transmembrane or signal peptide but multiple phosphorylation sites,and that its subcellular cells located in the chloroplasts.Its nucleotide and deduced amino acid sequences showed 51%,50%,50%,50%,and 49% homology with the E3 ubiquitin ligase genes from Nicotiana sylvestris,Camelina sativa,Vitis vinifera,Tarenaya hassleriana,and Brassica rapa,respectively;while its conserved functional domains related to the translated protein sequences had a RING-finger structure.Consequently,it was preliminarily identified as the E3 ubiquitin ligase gene of C.sinensis cv.Tieguanyin.Subsequently,the qPCR analysis indicated that the transcript of this gene,when the tea plant was under varied drought stresses,was significantly more up-regulated than control.Hence,it was concluded that the previously identified RING-type E3 ubiquitin ligase gene found in C.sinensis cv.Tieguanyin indeed involved in the drought response mechanism of the plant.