安徽农业大学学报2024，Vol.51Issue(5)：732-739,8.DOI:10.13610/j.cnki.1672-352x.20241031.017
多星韭黄酮醇合成酶基因AwFLS1的克隆及功能验证
Cloning and functional verification of flavonol synthase gene AwFLS1 from Allium wallichii
摘要
Abstract
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.关键词
黄酮醇合成酶/基因克隆/生物信息学分析/载体构建/体外酶活鉴定/多星韭Key words
flavonol synthase/gene cloning/bioinformatics analysis/vector construction/enzyme activity verification in vitro/Allium wallichii分类
农业科技引用本文复制引用
陈瑶,张艳,黄菊,赵鑫,鞠志刚,龚记熠,孙威..多星韭黄酮醇合成酶基因AwFLS1的克隆及功能验证[J].安徽农业大学学报,2024,51(5):732-739,8.基金项目
贵州省"千层次"人才项目([ZQ2018004]),贵州省高等学校高山杜鹃病虫害绿色防控重点实验室(黔教技[2022]044号)和贵州师范大学学术新苗基金项目([2021]B04)共同资助. （[ZQ2018004]）
