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

To elucidate the effects of anthocyanidin synthase(ANS;EC 1.14.11.19) gene on anthocyanin accumulation in underground yam tubers,in the present 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.) ,and characterized with both the related softwares and the techniques as Northern 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 comprising a 1 077 bp largest open reading frame(ORF) ,a 9 bp 5′ noncoding region and a 301 bp 3′ noncoding region and having the typical characteristics of 5′-and 3′-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 largest ORF of DaANS1,in which had the 2-oxoglutarate-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 complete 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 of middle phase with the highest and thereafter slightly raised at the harvesting time,the profiles of which were paralleled with the change trends of both ANS enzyme activity and anthocyanin content. These results indicate that DaANS1 as a member of plant ANS genes can evaluate the genetic relationship between genus or species plants,and control anthocyanin accumulations of underground tubers based on its regulation at transcription level.