卷柏StTPS基因克隆及其在复苏过程中的表达分析  

Cloning and expression analysis in resurrection process of trehalose-6-phosphate synthase gene from Selaginella tamariscina

作　　者：席彩彩 谷巍[1] 孙红梅[1] 田荣 刘琪[1] 王小浩 XI Cai-cai;GU Wei;SUN Hong-mei;TIAN Rong;LIU Qi;WANG Xiao-hao(College of Pharmacy,Nanjing University of Chinese Medicine,Nanjing 210023,China)

机构地区：[1]南京中医药大学药学院

出　　处：《中国中药杂志》2018年第24期4842-4849,共8页China Journal of Chinese Materia Medica

基　　金：国家自然科学基金项目(81102772);江苏省中药资源产业化过程协同创新中心项目(ZDXM-2-6,ZDXM-3-24);2017年中医药公共卫生服务补助专项(财社[2017]66号);江苏省中药优势学科Ⅱ期建设项目(PAPD-2014)

摘　　要：卷柏为典型极端耐旱的复苏药用植物,海藻糖在植物复苏过程中发挥重要作用,海藻糖-6-磷酸合成酶(TPS)是植物体内合成海藻糖的关键酶。该研究根据卷柏转录组数据拼接获得海藻糖-6-磷酸合成酶基因序列,以卷柏总RNA为模板合成c DNA,采用RT-PCR法克隆验证该序列,并进行生物信息学分析,结果显示该序列编码区长2 799 bp (St TPS,Gen Bank号MH155231),编码932个氨基酸,无信号肽,St TPS可能定位于叶绿体上且无跨膜结构,包含2个糖原磷酸化酶糖基转移酶(GPGTF)家族的保守结构域。在此基础上,模拟卷柏复苏(脱水和复水)过程,应用qRT-PCR进行其在复苏过程中的表达量分析,并同步采用高效液相色谱-蒸发光散射检测器(HPLC-ELSD)测定卷柏复苏过程中海藻糖含量变化。结果显示St TPS基因相对表达量及海藻糖含量均随着脱水时间的延长而增加,随着复水时间的延长而下降,且二者之间呈正相关,初步说明St TPS在卷柏复苏过程中发挥重要作用。Selaginella tamariscina is a typical resuscitation medicinal plant with extreme drought tolerance. Trehalose plays an important role in the resurrection process,and the trehalose-6-phosphate synthase( TPS) is the key enzyme to synthesize trehalose in plants. In this study,the sequence of TPS was obtained by splicing from the transcriptome data of S. tamariscina. After the synthesis of c DNA based on the template of total RNA,the sequence was cloned by RT-PCR for verification and then analyzed by bioinformatics methods. The results indicated that the full-length coding sequence of St TPS was 2 799 bp( Gen Bank accession no. MH155231),and the encoded protein contained 932 amino acids. St TPS could be located in the chloroplastid according to subcellular localization prediction. There were two conserved domains belonging to glycogen phosphorylase glycosyltransferase( GPGTF) family but no signal peptide or transmembrane domain in St TPS. The expression of St TPS was determined by qRT-PCR and the variation of trehalose content was measured by HPLC-ELSD during the resurrection process of S. tamariscina. Meanwhile,the correlation between them was analyzed.The results showed that both the expression level of St TPS and the trehalose content increased associated with the extension of dehydration time,and declined associated with the extension of rehydration time which proved a significant positive correlation between the St TPS expression level and the trehalose content. The results suggested that the St TPS probably plays a central role in recovery process in S. tamariscina.

关 键 词：卷柏 海藻糖 海藻糖-6-磷酸合成酶 QRT-PCR 基因表达 

分 类 号：S567.239[农业科学—中草药栽培]