藤茶黄烷酮3-羟化酶基因AgF3H的克隆及表达分析  

Cloning and Expression Analysis of a Flavanone 3-hydroxylase Gene from Ampelopsis grossedentata

作　　者：许明 伊恒杰 郭佳鑫 唐进兰 林世强 杨志坚 郑金贵 XU Ming;YI Hengjie;GUO Jiaxin;TANG Jinlan;LIN Shiqiang;YANG Zhijian;ZHENG Jingui(College of Agriculture, Fujian Agriculture and Forestry University, Key Laboratory of Crop Biotechnology in Fujian Province University, Key Laboratory of Ministry of Education for Genetics, Breeding and Multiple Utilization of Crops, Fuzhou 350002, China)

机构地区：[1]福建农林大学农学院,作物生物技术福建省高校重点实验室,作物遗传育种与综合利用教育部重点实验室,福州350002

出　　处：《西北植物学报》2020年第2期185-192,共8页Acta Botanica Boreali-Occidentalia Sinica

基　　金：国家科技支撑计划(2013BAD01B05);福建农林大学科技创新专项基金(KFA17424A)。

摘　　要：该研究以藤茶叶片为材料,利用RACE技术克隆得到1个黄烷酮-3-羟化酶(flavanone 3-hydroxylase,F3H)基因,命名为AgF3H(登录号为JX087441)。AgF3H基因的cDNA全长为1323 bp,其中开放阅读框长1092 bp,编码363个氨基酸,含有双加氧酶家族的2个特征性结构域和5个保守基序。序列比对与系统进化树分析显示,AgF3H与其他物种F3H的氨基酸序列具有较高的相似性,且与葡萄、山葡萄和圆叶葡萄的亲缘关系最近。qRT-PCR结果显示,AgF3H基因在藤茶不同组织部位都有表达,在芽中的表达量最高,嫩茎中最低,在叶片中的表达量随着成熟度的增加而逐渐降低;不同组织中的AgF3H基因表达量与藤茶类黄酮主要成分二氢杨梅素的含量呈显著正相关。构建AgF3H基因过表达载体并导入烟草中,获得5株转基因植株,其中4株烟草的叶片总黄酮含量比野生型对照有不同程度提高,最高株系提高了26.8%。研究表明,AgF3H基因可能在藤茶类黄酮的生物合成中起重要作用,研究结果为进一步研究藤茶高黄酮含量形成的分子机制奠定了基础。In this study,a full length cDNA of flavanone 3-hydroxylase(F3H)was cloned from the leaf of Ampelopsis grossedentata using RACE technique,and named AgF3H(accession number JX087441).The full length cDNA was 1323 bp,containing a 1092 bp open reading frame(ORF)encoding a protein consisting of 363 amino acids,with 2 typical functional domains and 5 conserved motifs of dioxygenase superfamily.Sequence comparison and phylogenetic tree analysis showed that the amino acid sequence of AgF3H had considerable similarity with those of other species,and had the closest evolutionary relationship with the F3H protein of Vitis vinifera,Vitis amurensis and Vitis rotifera.Real time PCR showed that the expression level of AgF3H gene decreased gradually during the maturity of leaves.The highest expression level emerged in buds while the lowest expression level emerged in young stems.Correlation analysis revealed positive correlation between AgF3H gene expression and the content of dihydromyricetin,the main flavonoid component in A.grossedentata.After the overexpression vector of AgF3H gene was constructed and transferred to tobacco,five transgenic plants were obtained.Among them,the content of total flavonoids in leaves of four tobacco plants increased to different degrees compared with wild type control,and the highest line increased by 26.8%.The study indicated that AgF3H gene may play an important role in the biosynthesis of flavonoids in A.grossedentata and the results lay a foundation for further exploring the molecular mechanism of the formation of high flavonoids content in A.grossedentata.

关 键 词：藤茶 类黄酮3′-羟化酶 克隆 表达 

分 类 号：Q785[生物学—分子生物学] Q786