Cloning And Expression Analysis Of DzDWF1 Gene From Dioscorea Zingiberensis

Dioscorea zingiberensis is the most important source of steroidal saponins in the world.Diosgenin,the second metabolite of Dioscorea zingiberensis,is most concerned for its medicinal value.Diosgenin is a compound extracted from rhizome,many researchs had pointed out that it has many important functionds in human body,such as protect cardiovascular system,anti-tumor.The synthetic pathways of diosgenin has become a hot research area in rescent years,in which MVA pathway,MEP pathway,many enzymes and genes in the middle and upper reach had been excavated deeply.DWF1(dwarf and fragile)is a downstream gene in the synthetic pathway,it is a oxidordeuctase that catalyze 24-methylenecholesterol and isofucosterol to campesterol and stigmasterol,respectively.But at present,the key genes and enzymes in the process of DWF1 reduction have not been excavated and analyzed.In this article,RACE Technique and Nest PCR were used to clone the full length of the c DNA of DWF1,its function had been analyzed.The results are as following:(1)The full length of the cDNA of DWF1 is 2057 bp,the bioinformatics analysis result revealed that the length of the Open Reading Frame is 1686 bp,which encodes 561 amino acids.Homology alignment showed that DzDWF1 has high homology with other DWF1.Phylogenetic analysis shows that it has the highest affinity with DWF1 of Arabidopsis thaliana,next with monocotyledons and farthest with dicotyledon.There is a conversed FAD binding domain of typical oxidordeuctase in the sequence of DWF1,signal P prediction indicated that no signal peptide was found.The result of subcellular localization implied that DzDWF1 is located in Endoplasmic reticulum,consistent with the subcellular localization prediction.(2)By constructing the prokaryotic expression vector p ET32a-DzDWF1,the plasmid of the recombinant vector was transferred into E.coli BL21 competent cells.IPTG was used to induce the expression of the target protein.High protein solubility was found under low temperature conditions by protein solubility analysis.Target protein was purfied by His-Nickel column,then elaste protein with imidazole.Western blot analisis result showed that the band is the target protein.(3)qRT-PCR technique was applied to explore the expression pattern of DzDWF1 in rhizome,stem and leaves,the consequence indicated that DzDWF1 expressed in all organs,but the highest expression was found in young leaves,the lowest in rhizome.Exogenous application of methyl jasmonate was also carried.After 0 h,6 h,12 h,2 4h,48 h,72 h of treatment,detect differences in expression levels of DzDWF1.The consequence suggested that after 48 h of treatment,the highest expression level was measured.