草业学报2017，Vol.26Issue(3)：82-90,9.DOI:10.11686/cyxb2016007
紫花苜蓿尿黑酸植基转移酶的克隆、表达分析以及遗传转化研究
Cloning and expression analysis of homogentisate phytyltransferase from Medicago sativa and its genetic transformation in Arabidopsis thaliana
摘要
Abstract
Vitamin E is an essential nutrient for humans,but we cannot synthesize it by ourselves.Homogentisate phytyltransferase (HPT) is a key enzyme that determines vitamin E content.In this study,according to the sequence of MtHPT from Medicago truncatula,we cloned the Opening Reading Frame (ORF) of HPT from Medicago sativa by homology cloning strategy and termed MsHPT.NCBI blast results showed that it encoded a protein of 412 amino acid and belonged to the PT_UbiA superfamily.Multiple sequence alignment results showed that the similarity between the MsHPT protein and other HPT was 80％.In order to further study the gene,promoter sequences were cloned using genome walking technology.Analysis of the MsHPT promoter showed that defense and stress responsiveness cis elements,hormone (MeJA,ABA,GA and ethylene) responsiveness cis elements and light responsiveness elements presented in the promoter.Quantitative Re-al-time PCR results revealed that MsHPT was expressed throughout the plant,mainly in the leaves.Expression levels of MsHPT increased significantly in response to cold,NaCl,PEG,GA3 and ABA treatments.The ORF of MsHPT was amplified with restriction enzyme recognition sites added,inserted into vector pBI121 and transformed into Arabidopsis thaliana by Agrobacterium tumefaciens-mediated transformation.According to the PCR results,seven positive lines were obtained.This study provides the basic information for further studies of the function of MsHPT in vitamin E biosynthesis and stress tolerance of M.sativa.关键词
紫花苜蓿/尿黑酸植基转移酶/维生素E/抗逆性/遗传转化Key words
Medicago sativa/homogentisate phytyltransferase/vitamin E/stress tolerance/genetic transformation引用本文复制引用
姜霁珊,方志红,陈敏,王运琦,吴欣明,贾慧丽,武语迪,高洪文,王学敏..紫花苜蓿尿黑酸植基转移酶的克隆、表达分析以及遗传转化研究[J].草业学报,2017,26(3):82-90,9.基金项目
国家重点基础研究发展计划(973)(2014CB138703),物种资源保护项目(2130135)和中国农业科学院科技创新工程(ASTIP-IAS10)资助. （973）
