大别山野生油茶中茶氨酸检测与茶氨酸合成酶基因克隆

Determination of theanine and cloning of theanine synthetase gene in Dabieshan wild Camellia oleifera

茶氨酸是茶树叶片中最丰富的游离氨基酸,具有重要的生理药理功能,但迄今仅在蘑菇、蕈和一些山茶科(属)植物中检测到茶氨酸。茶氨酸因有一种独特的风味特色"umami鲜爽味"而被人类营养学广泛研究,并发现合成茶氨酸的植物不仅在植物分类上具有积极意义,而且对于植物资源的有效发掘有巨大经济价值;同时还可以间接去研究茶树中茶氨酸的代谢机理以及茶氨酸合成酶的分离纯化和TS基因的克隆表达。该文运用HPLC、LC-TOF/MS对大别山地区野生幼年与成年油茶根、叶中茶氨酸进行检测,并结合分子生物学手段对油茶中茶氨酸合成酶(theanine synthetase,TS)基因进行克隆与生物信息学分析。结果表明:在幼年的油茶根中检测到茶氨酸,含量为0.08mg·g-1(鲜重),而在幼年的油茶叶片和成年油茶根、叶中均未检测到茶氨酸;在幼年油茶根中克隆出一条长为1 071bp油茶TS基因开放阅读框,其基因序列与茶树谷氨酰胺合成酶(glutamine synthetase,GS,AB117934)基因和TS(DD410896)序列的同源性达到98%,氨基酸序列与茶树中GS(AB117934)和TS(DD410896)的相似性高达99%。经生物信息学分析,该序列编码的TS蛋白具有20个磷酸化位点,不存在信号肽序列与跨膜结构,含有卷曲螺旋结构的亲水性细胞质蛋白。该研究将为油茶新经济价值的发掘,为茶氨酸在油茶中合成代谢途径的研究提供一定的理论基础,同时也为进一步研究茶氨酸在茶树中代谢机理提供了新的研究思路。

Theanine was discovered as the most abundant free amino acid in Camellia sinensis leaves with many physiological and pharmacological function.However,only limited reports have subsequently been published about theanine in the plant kingdom,for instance,mushroom,Xerocomus badius,and some theaceae plants.Theanine has been extensively studied about human nutrition for tea unique taste characteristic＂umami＂.Moreover,synthesize theanine has not only positive significance in plant classification,but also great economic value for the effective exploration of the plant resources.Furthermore,the mechanism of theanine synthesis in C.sinensis,separation and purification of theanine synthetase and cloning and expression of TS gene were indirectly studied.And HPLC and LC-TOF/MS were applied to determine theanine in the roots and leaves in the young and mature Dabieshan wild C.oleiferaplants while molecular biology methods were used to clone theanine synthetase（TS）gene in C.oleifera;Bioinformatic analysis was carried out to analyze the gene sequence.The results revealed that the theanine content of roots in young C.oleifera plants was 0.08mg·g^-1（fresh weight）.However,theanine was not detected in leaves of young plants.It was also found there was no theanine in the roots and leaves in mature C.oleifera plants.One TS ORF（1 071bp）was acquired from the roots in young C.oleifera plants.The homology of the cloned TS with glutamine synthetase gene（GS,AB117934）and TS（DD410896）were up to 98%.Moreover,the cloned TSshowed a high similarity about 99%to GS（AB117934）and TS（DD410896）fromC.sinensis at the protein levels.We found that there were20 phosphorylation sites in the polypeptide chain after bioinformatic analysis.Bioinformatics prediction showed that the protein contained hydrophilic and winded helix domain.However,there was neither signal peptide nor transmembrane in the sequences,and the protein was non-secreted which functioned in the cell.This paper would provide theoretical basis and new approach for study of the synthesis,metabolic pathways and metabolic mechanism of theanine.Furthermore,it was beneficial to develop economical value of C.Oleifera.