茶树病程相关蛋白基因CsPR5的克隆及表达分析

Cloning and expression analysis of the gene encoding pathogenesis-related protein CsPR5 in the tea plant

为探究茶树中病程相关蛋白5(pathogenesis-related protein 5,PR5)在茶树中的功能,采用cDNA末端快速扩增(rapid amplification of cDNA end,RACE)技术克隆了PR5基因的全长;通过荧光定量PCR方法检测了CsPR5在龙井43茶树不同组织部位、植物激素(茉莉酸、乙烯利和水杨酸)处理、茶炭疽病菌Colletotrichum camelliae侵染和小贯小绿叶蝉Empoasca onukii为害后的表达特征。结果表明,CsPR5基因开放阅读框为843 bp,编码281个氨基酸。CsPR5基因在茶树根、茎、嫩叶、花和种子5个组织中的相对表达量分别为0.187、3.093、0.928、0.045和0.012,且五者之间差异显著,主要在茎和叶中表达,其分别为根中相对表达量的16.54倍与4.96倍。水杨酸处理6 h及乙烯利处理6、12 h后,茶树CsPR5基因的相对表达量分别为1.622、2.344和1.845,分别比对照显著上调2.20倍、3.18倍和2.35倍;但是外施茉莉酸对茶树CsPR5基因的相对表达量无影响。茶炭疽病菌侵染显著诱导了茶树CsPR5基因的相对表达量,处理后的相对表达量为1.977,是对照的1.90倍;小贯小绿叶蝉取食显著抑制了其为害部位CsPR5的相对表达量,为7.273,仅为对照的38.80%,但该诱导不具系统性。

To explore the functions of pathogenesis-related protein 5（PR5） in the tea plant, the fulllength cDNA of PR5 was cloned by using RACE（rapid amplification of cDNA end）. The expression patterns of CsPR5 in different tissues of Longjing 43 and Longjing 43 treated with exogenous plant hormones（salicylic acid, ethephon and jasmonic acid）, infected by Colletotrichum camelliae and infested with Empoasca onukii were analyzed by RT-qPCR. The open reading frame（ORF） of CsPR5 was 843 bp and encoded 281 amino acids residues, which contained a signal peptide in the N-terminus region, 16 conservative cysteine residues, multiple phosphorylation sites and two potential N-glycosylation sites.RT-qPCR analysis results showed that the expression patterns of CsPR5 in the roots, stems, young leaves, flowers and seeds of tea plants were 0.187, 3.093, 0.928, 0.045 and 0.012, respectively, and the measured five tissues were significantly different from each other. CsPR5 mostly expressed in the stemsand leaves, which accounted for nearly 16.54 and 4.96 times higher than that in the root, respectively.After salicylic acid treatment for six hours and ethephon treatment for six hours and 12 h, the relative expression levels of CsPR5 gene in tea plants were 1.622, 2.344 and 1.845, respectively, which were significantly up-regulated by 2.20, 3.18 and 2.35 times. However, exogenous application of jasmonic acid showed no effect on m RNA accumulation of CsPR5. Infection of C. camelliae with tea anthracnose significantly induced higher relative expression of CsPR5 gene in tea plants. The relative expression of CsPR5 gene was 1.977, which was 1.90 times that of the control. The infestation by E. onukii downregulated the expression level of CsPR5 at feeding site, and the expression level was 7.273, which was only38.80% of the control, but the induction was not systematic.