茶树八氢番茄红素脱氢酶cDNA全长克隆与表达分析  

Cloning of a full-length cDNA of phytoene desaturase gene in tea plant( Camellia Sinensis) and its expression

作　　者：李娜娜[1] 邵文韵 刘畅[1] 陆建良[1] 梁月荣[1] 

机构地区：[1]浙江大学茶叶研究所,浙江杭州310058

出　　处：《茶叶》2014年第2期69-74,共6页Journal of Tea

基　　金：国家茶叶产业技术体系经费资助(CARS-23)

摘　　要：八氢番茄红素脱氢酶是类胡萝卜素生物合成途径的关键酶之一。本实验采用3'/5'RACE和RTPCR技术成功扩增出茶树八氢番茄红素脱氢酶基因(PDS)的3'端和5'端序列,序列拼接获得全长cDNA序列,命名为CsPDS,并将其登录至GenBank,登录号KF646537。经生物信息学分析,所得基因cDNA序列全长为2295 bp,开放阅读框(ORF)1749 bp,编码582个氨基酸,预测分子量约为64.86 kDa,理论等电点(PI)为6.77,属于亲水性蛋白。该基因编码的氨基酸序列与柿树PDS序列的同源性达到87%,多序列比对表明茶树PDS具有高度保守区域,基于邻接法的进化树显示与柿树、葡萄的亲缘关系最近。实时荧光定量PCR分析结果显示,CsPDS在‘黄金芽’体内表达没有受抑制,表明‘黄金芽’黄色白化可能不是在CsPDS基因转录水平异常而引起。Phytoene desaturase is one of the key enzymes involving in carotenoid biosynthetic pathway. 3＇ /5＇ RACE and RT-PCR methods were used to amplify the 3＇- and 5＇- end sequence of phytoene desaturase gene（ PDS） from tea plant. A full-length cDNA sequence of the gene was obtained after sequence splicing,which was named as CsPDS,with a GenBank accession number KF646537. Its bioinformatic characterization indicated that the full length cDNA of CsPDS was 2295 bp, which contained an open reading frame of 1749 bp and encoded 582 amino acids with a putative molecular mass of 64. 86 kDa and a theoretical isoelectric point of 6. 77. The encoded proteine was a hydrophilic protein.The deduced amino acid sequence was most closely to Diospyros kaki with 87% sequence homology.Multiple sequence alignment showed that the highly conserved regions of the CsPDS, the closest evolutionary relationship with Diospyros kaki and Vitis vinifera was displayed by phylogenetic analysis of neighbor-joining method. Quantitative real-time PCR analysis showed that the expression of CsPDS gene was not inhibited in an albino tea cultivar Huangjinya＇,compared to normal tea cultivar,suggesting that the albinism of Huangjinya＇ may not be caused by the block of CsPDS gene expression at the transcriptional level.

关 键 词：茶树 八氢番茄红素脱氢酶 cDNA末端快速克隆 序列分析 表达分析 

分 类 号：S571.1[农业科学—茶叶生产加工] Q812[农业科学—作物学]