茶树GDP-D-甘露糖焦磷酸化酶基因cDNA全长的克隆与表达分析

摘要

抗坏血酸（Vc）是重要的抗氧化剂，在茶树体内的各种生理代谢及抵御非生物胁迫中起重要作用，Vc 含量关系到绿茶品质优劣。GDP-D-甘露糖焦磷酸化酶（GMP）是茶树 Vc 生物合成途径中的关键酶之一。本研究通过 RACE-PCR 方法从茶树中克隆了 GMP cDNA 全长序列，共计1510 bp，其中包含1086 bp 的开放阅读框（ORF），编码361个氨基酸，推测蛋白分子量为39.599 kDa。BLAST 分析表明，茶树 GMP 基因氨基酸序列与猕猴桃的亲缘性最高，达到96%。实时定量 PCR 分析表明，茶树新梢芽下第三叶中 GMP 基因表达量最高，嫩茎中最低，不同品种茶树新梢叶片中表达量存在明显差异。高温胁迫初期，GMP 基因表达量及抗坏血酸含量迅速升高，然后逐渐下降且均低于同期对照。%Ascorbic acid(Vitamin C, Vc) is an important antioxidant in tea plant, playing important roles in metabolism and responses to abiotic stress, and tea qualityis positive correlation with the content ofVc.GDP-D-mannosepyrophosphorylase(GMP) is an important enzyme in the synthesis of ascorbic acid.The full-length cDNA sequence ofGMPgene was isolated from the shoots ofCamellia sinensisby RT-PCR and RACE. The entire GMPcDNAwas 1510bp, containing a 1086bp complete open reading frame which encoding a protein with 361 amino acidsand a calculated molecular weight of39.599kDa.Blastanalysis showedthatGMPgene in Camellia sinensiswas most closely toActinidiawith 96% amino acids similarity.Quantitative real-time PCR analysis showed that the expression levels ofGMPgene in the third leaves were highest while the stem is the lowest, and the different varieties also existed an obvious differences. High temperature stress stimulate the expression of GMPgene and the accumulation ofVcin incipient stage, then reduced rapidly.