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

维生素E是一种人体必需,却不能自主合成的脂溶性维生素。尿黑酸植基转移酶(HPT)是维生素E生物合成途径的关键限速酶,直接影响植物体内维生素E的总量。本实验利用同源克隆的方法,根据截形苜蓿的序列从紫花苜蓿中克隆得到HPT基因的完整开放阅读框(ORF)。NCBI Blast分析结果表明,该基因编码412个氨基酸,属于异戊烯转移酶UbiA超家族。多序列比对结果表明,该序列与其他物种的HPT蛋白序列相似度高达80%,将其命名为MsHPT。进化树分析结果表明,MsHPT与截型苜蓿MtHPT亲缘关系最近,蛋白序列相似度为96.84%。通过染色体步移技术得到该基因的启动子序列,分析结果显示,该基因的启动子区域含有胁迫响应元件、激素响应元件(脱落酸、赤霉素和乙烯)以及大量的光响应元件。实时荧光定量PCR检测结果表明,MsHPT基因在紫花苜蓿各组织中均有表达,叶片中的表达量最高,根次之。经低温、NaCl、PEG、GA3和ABA诱导后该基因表达均上调,表明其可能参与了植物的抗逆性调控。扩增MsHPT基因的开放阅读框,对其进行双酶切后转入双元表达载体pBI121中,通过农杆菌介导的方式将该基因转入拟南芥。通过PCR鉴定,得到7株阳性苗。本研究为进一步探索MsHPT在紫花苜蓿维生素E的合成以及紫花苜蓿抗逆调控中的作用奠定了基础。

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 MtHPTfrom 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 Real-time PCR results revealed that MsHPT was expressed throughout the plant,mainly in the leaves.Expression levels of MsHPTincreased 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 MsHPTin vitamin E biosynthesis and stress tolerance of M.sativa.