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

作　　者：陈瑶 张艳 黄菊 赵鑫 鞠志刚 龚记熠[1,3] 孙威 CHEN Yao;ZHANG Yan;HUANG Ju;ZHAO Xin;JU Zhigang;GONG Jiyi;SUN Wei(Key Laboratory of Plant Physiology and Development Regulation,School of Life Science,Guizhou Normal University,Guiyang 550025;Pharmacy College,Guizhou University of Traditional Chinese Medicine,Guiyang 550025;Key Laboratory of State Forestry Administration on Biodiversity Conservation in Karst Mountain Area of Southwest of China,Guiyang 550025)

机构地区：[1]贵州师范大学生命科学学院,植物生理与发育调控重点实验室,贵阳550025 [2]贵州中医药大学药学院,贵阳550025 [3]西南喀斯特山地生物多样性保护重点实验室,贵阳550025

出　　处：《安徽农业大学学报》2024年第5期732-739,共8页Journal of Anhui Agricultural University

基　　金：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);贵州师范大学学术新苗基金项目([2021]B04)共同资助。

摘　　要：黄酮醇合成酶(Flavonol synthase,FLS)可以催化3种二氢黄酮醇形成相对应的黄酮醇,是调控黄酮醇合成的关键酶。以多星韭(Allium wallichii)盛开期花朵为材料,提取RNA并通过反转录获得cDNA,利用PCR技术对AwFLS1基因进行克隆并完成生物信息学分析;利用qRT-PCR技术分析AwFLS1基因在多星韭花朵不同发育时期、不同组织器官的表达,初步推测其功能;构建原核表达载体BL21-pET32a(+)-AwFLS1,制备可溶性重组蛋白并进行体外酶活性鉴定。结果表明,多星韭AwFLS1基因CDS全长为1008 bp,编码335个氨基酸,推测形成一种不稳定且不定位于膜上的非分泌亲水性蛋白,分子量约为38.22 kDa,具有保守的2-酮戊二酸/FeⅡ依赖型双加氧酶结构域。AwFLS1基因在多星韭花朵不同发育时期表达量呈逐渐降低趋势,在小苞期最高;在不同组织器官中,表达量存在显著差异,在叶中表达量最高,根中表达量最低。AwFLS1重组蛋白可在体外催化3种二氢黄酮醇底物形成对应的黄酮醇,底物偏好性有待进一步验证。研究结果证实了多星韭AwFLS1在类黄酮代谢途径中的功能,为解析多星韭黄酮醇合成机制奠定了理论基础,为利用基因工程技术提高药用植物活性成分含量提供了候选的基因资源。Flavonol synthase(FLS)plays a key role on flavonol synthesis and can catalyze three kinds dihy-droflavonols to form corresponding flavonols.RNA was extracted from blooming flowers of Allium wallichii and reverse 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 development 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 assay in vitro was also prepared.The results showed that CDS of AwFLS1 was 1008 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 contained the conserved 2-ketoglutaric acid/FeⅡ-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 further verification.In this study,the function of AwFLS1 involved in flavonoid synthesis was verified,which lay a theoretical 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 

分 类 号：S682[农业科学—观赏园艺]