山药ANS基因的克隆和分子特性及其与花青素积累的关系

 为了阐明花青素合成酶(ANS; EC 1114111119) 基因在山药地下块茎花青素积累过程中的功能, 利用RT-PCR和RACE技术从田薯(Dioscorea alata L. ) 地下块茎中分离到一个ANS 基因(DaANS1) ,并运用相关软件、Norhtern杂交和原核表达等技术分析了该基因; 同时测定了ANS酶活性和花青素含量。结果显示, 该基因序列大小为1 387 bp, 最大开放阅读框(ORF) 、5′端和3′端的非翻译区分别由1 077 bp、9 bp和301 bp组成, 且有真核生物基因5′- 和3′- 末端序列的典型特征, 是一个完整的全长cDNA序列;DaANS1最大ORF可编码358个氨基酸, 其分子量和等电点分别为40.4 kD和5.26, 并含有依赖于2 - 酮戊二酸和Fe²⁺氧化的保守结构域, 其中包括与2 - 酮戊二酸特异结合的精氨酸2个(Arg295、304) 及与Fe²⁺结合的保守组氨酸5个(His238、243、249、276、294) 和天冬氨酸3个(Asp240、260、279); DaANS1与所选被子植物ANS基因的同源性均远高于与裸子植物的同源性, 而且与被子植物中双子叶旋花科植物ANS基因的亲缘关系最近, 但仅能将属、种间植物合理分类; DaANS1 表达丰度从初前期增至最强的盛前期后一直降至最低的后中期, 到收获时又稍增强, 其表达模式与ANS酶活性和花青素含量的变化具协同性。这些结果表明, DaANS1 为植物ANS基因的一员, 可评价属、种间植物的亲缘关系, 在转录水平上受到调控而影响田薯地下块茎花青素形成。

Cloning and Molecular Characteristics of ANS Gene and Its Correlations with Anthocyanin Accumulation in Yam

To elucidate the effects of anthocyanidin synthase (ANS; EC 1114111119) gene on anthocyanin accumulation in underground yam tubers, in the p resent paper, one of ANS gene (DaANS1 ) was not only isolated by RT-PCR and RACE techniques from the underground tubers of yam (Dioscorea alata L. ) , andcharacterized with both the related softwares and the techniques asNorthern hybridization and gene expression in E.coli, but also both ANS enzyme activity and anthocyanin concentration were determined. The results shows, that DaANS1 was a full-length cDNA sequence with 1 387 bp in size in terms of comp rising a 1 077 bp largest open reading frame (ORF) , a 9 bp 5' noncoding region and a 301 bp 3′noncoding region and havingthe typical characteristics of 5′2 and 3′2 ends of eukaryotic gene cDNA sequence; that a polypeptide of 358 amino acids with a 40.4 kD molecular weight and a theoretical pI of 5.26 was putatively encoded by the largestORF of DaANS1 , in which had the 22oxoglutarate2 and Fe²⁺ -dependent conserved oxidation regions containing two of conserved Arginine (Arg295, 304) related to the combination of 2-oxoglutarate and the Fe²⁺combination-related amino acid residues as both five of conserved Histidine (His238, 243, 249, 276, 294) and three of Aspartate (Asp240, 260, 279) ; that DaANS1 shared the higher similarities with ANS genes of the selected angiosperm species than gymnosperm ANS genes at the levels of comp lete cDNA sequences, coding regions and its deduced amino acid sequences, and had the closest genetic relationship with ANS genes of convolvulaceae plants in dicots of angiosperms, but was used to reasonably sort only between genus or species plants; DaANS1 expression abundances were straightly reduced to the lowest at the part of later stage of the underground tuber bulking after increased from the initiation of early stage to the initiation ofmiddle phase with the highest and thereafter slightly raised at the harvesting time, the profiles of which were paralleled with thechange trends of both ANS enzyme activity and anthocyanin content. These results indicate thatDaANS1 as a member of plant ANS genes can evaluate the genetic relationship between genus or species plants, and controlanthocyanin accumulations of underground tubers based on its regulation at transcrip tion level.