茶树脱落酸β-葡萄糖基转移酶基因CsAOG的克隆和表达分析

Cloning and expression analysis of the CsAOG encoding abscisic acid β-glucosyltransferase in Camellia sinensis

脱落酸(ABA)β-葡萄糖基转移酶(abscisate beta-glucosyltransferase)是ABA分解代谢途径中的关键酶,在维持植物体内ABA正常生理水平上发挥着重要作用。通过RACE法克隆了茶树中ABAβ-葡萄糖基转移酶基因CsAOG,NCBI登录号为MF370238。CsAOG全长为2 108 bp,ORF大小为1 443 bp,无内含子,该基因编码480个氨基酸,分子量约为53.26 kDa,等电点为5.58,亚细胞定位于细胞质。CsAOG蛋白具有葡萄糖基转移酶(UDPGT)保守结构域。通过MEME分析工具在CsAOG序列中获得了9个保守元件。由系统进化和蛋白互作分析可知,CsAOG位于UGT73分支,且UGT73B4与UGT73B5的互作最强。实时荧光定量结果表明,叶中CsAOG表达水平最高,根部次之,茎中最低;在越冬期间,乌牛早CsAOG表达量先上调后下调,最后维持在较低水平;舒茶早和黄山早芽CsAOG表达量呈下调趋势。赤霉素处理后,乌牛早CsAOG表达量上调;高温胁迫后CsAOG表达量呈先升后降的趋势,12 h时表达量最高,而低温胁迫时表达量变化不大。通过对CsAOG基因的研究,为理解ABA在调控茶树生长发育和抗逆机制中的作用提供了理论基础。

Abscisic acid （ABA）β-glucosyltransferase is a key enzyme in ABA catabolic pathway, which plays an important role in regulating the ABA normal physiological level in plants. In this study, the full length of CsAOG gene encoding ABA D-glucosyltransferase from tea plants was cloned by RACE method （NCBI accession number MF370238）. The CsAOG had a full length of 2 108 bp and an ORF of 1 443 bp, without introns, which encodes 480 amino acids with a molecular weight of 53.26 kDa and an isoelectric point of 5.58. The subcellular localization of CsAOG protein is in cytoplasmic. The CsAOG protein has a conserved domain of glucosyltrans-ferase （UDPGT）. Nine conserved elements were obtained in the CsAOG sequence by the MEME analysis tool. Systematic evolution and protein interaction analysis showed that CsAOG is located at the UGT73 branch, and UGT73B4 and UGT73B5 have the strongest interaction. Real-time fluorescence quantitative results showed that the CsAOG expression level was the highest in leaves, followed by roots, and the lowest in stems. During the overwintering period, the expression of CsAOG in Camellia sinensis cv.＇Wuniuzao＇ was firstly up-regulated and then down-regulated, and then remained at a lower level; the CsAOG in Camellia sinensis cv.＇Shuchazao＇ and ＇Huangshanzaoya＇ showed a downward trend. After gibberellin treatment, the expression of CsAOG was up-regulated. After high-temperature stress, the expression of CsAOG increased firstly and then decreased and the expression level was the highest at 12 h, while it was not changed under low temperature. The study of CsAOG gene provides a theoretical basis for tmderstanding the role of ABA in regulating the growth and development of tea plants and the mechanism of stress resistance.