Cloning and Analysis of Anthocyanidin Synthase Gene and Promoter in Nymphaea color ata
Anthocyanidins are responsible for the different colors of ornamental plants.Anthocyanidin synthase(ANS)plays an important role in catalyzing the last step of anthocyanidin biosynthesis pathway.In order to study the sequence characteristics and expression pattern of the ANS and its promoter,the gene sequence and coding region sequence of waterlily Nymphaea colorata ANS(NcANS)were cloned by PCR and RT-PCR from its leaf and petal,respectively.The length of NcANS was 1 277 bp,and there was a 200 bp intron between two exons.The coding region sequence contained a complete open reading frame(ORF),1 077 bp in length,encoding 358 amino acids,and included two conserved domains of DIOX-N and 2-ketoglutarate-Fe2+-dioxygenase,the latter which involved in active sites of 2-ketoglutarate and Fe2+.Multiple alignment and phylogenetic analysis showed that NcANS had the highest consistency with basal angiosperm Amborella trichopoda ANS in amino acid sequence,had the closest relationship with waterlily N.thermarum,and had the furthest relationship with mono-cotyledonous plant in evolution.The Real-time quantitative PCR result showed that the expression level of NcANS was the highest in anther,followed by the S3 stage of petal;the expression of NcANS in the five development stages of petal increased at first and then decreased sharply,reached the peak in the S3 stage.Additionally,the promoter sequence length of NcANS isolated by PCR was 1 382 bp,which contained binding sites of transcription factors such as MYB and MYC,and cis-acting elements involved in response to signals such as anaerobic,low temperature,light,methyl jasmonate,auxin,development,and metabolism.The results of this study provide a theoretical basis for further research on the function of NcANS gene and the molecular mechanism of the formation of N.colorata.
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