Cloning And Expression Analysis Of DaANS Gene In Dioscorea Alata.L

Anthocyanidin synthase, as the key enzyme in anthocyanin biosynthetic pathway, can catalyze leuco anthocyanins dehydration and oxidation to form colored anthocyanins. At present, research on the molecular level of anthocyanidin synthase gene in Dioscorea alata.L has not been reported. By studying the function and regulation mechanism of DaANS gene, and elucidating the relationship between DaANS gene and anthocyanin accumulation, to lay the foundation for the study of molecular mechanism of the formation of yam tuber anthocyanins. In the present paper, the full cDNA of ANS gene was isolated by RT-PCR and RACE techniques from the tuber of Dioscorea alata. L, and the upstream promoter sequence was amplified, the expression pattern of DaANS was analysed, the bioinformatics of DaANS gene and its promoter was also analysed. The main results of the research are as follows:1:The full cDNA of ANS gene was isolated by RT-PCR and RACE techniques from the tuber of Dioscorea alata L. The full cDNA of ANS was named as DaANS (accession number:KP729182) containing an open reading frame (1071bp) and encoding a protein of356amino acids, size of about40kDa, isoelectric point of5.81. The full-length of DNA sequence is1534bp, containing one intron (113bp).2:By using Genome walking, the promoter upstream of DaANS gene, size of about2Kb was cloned. The analysis found that in addition to including basic promoter element TATA-box and CAAT-box, also has many other elements such as the regulation of gibberellin responsive elements and light reaction of cis regulatory elements.3:The relative expression of DaANS was determined by real-time quantitative PCR in six different organizations and different developmental stages of tubers.The results showed that:the expression of DaANS gene has obvious characters of space and time.4:The of DaANS gene was expressioned in E.coli, The gene is found under the condition of4h25℃,1mM IPTG, get the highest expression level, can be purified to soluble protein.5:Construction of DaANS expression vector PCXSN-35S-DaANS, and using the leaf disc transformation into tobacco, and detected3positive transgenic plants.