多星韭黄酮醇合成酶基因AwFLS1的克隆及功能验证OACSTPCD

Flavonol synthase(FLS)plays a key role on flavonol synthesis and can catalyze three kinds dihydro-flavonols to form corresponding flavonols.RNA was extracted from blooming flowers of Allium wallichii and re-verse transcripted to cDNA,and AwFLS1 gene was cloned by PCR and analyzed through bioinformatics methods.For preliminarily predicting the function of AwFLS1,its expression analysis was performed during flower develop-ment stages and in different tissues through qRT-PCR method.Subsequently,prokaryotic expression vector BL21-pET32a(+)-AwFLS1 was successfully constructed and soluble recombinant protein identified via enzyme as-say in vitro was also prepared.The results showed that CDS of AwFLS1 was 1 008 bp,encoding 335 amino acids,which constituted an unstable non-secreted and non-hydrophilic protein with a molecular weight of about 38.22 kDa and con-tained the conserved 2-ketoglutaric acid/Fe Ⅱ-dependent dioxygenase domain.AwFLS1 expression decreased gradually at different developmental stages,and it was highest at the bracteole stage.In various tissues,AwFLS1 transcript levels were significant different with the highest in the leaves and the lowest in the roots.Enzyme assay revealed that AwFLS1 could catalyze three dihydroflavonols to produce corresponding flavonols in vitro,and its substrate preference needs fur-ther verification.In this study,the function of AwFLS1 involved in flavonoid synthesis was verified,which lay a theoreti-cal foundation for the analysis of flavonol synthesis mechanism in A.wallichii and provide alternative genetic resources for improving active ingredients of medicinal plants through using genetic engineering technology.