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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 a kind of fat-soluble vitamins that human body can not synthesize independently. Homocysteine transferase (HPT) is the key rate-limiting enzyme in the biosynthetic pathway of vitamin E and directly affects the total amount of vitamin E in plants. In this experiment, the homologous cloning method was used to clone the complete open reading frame (ORF) of HPT gene from alfalfa according to the sequence of truncated alfalfa. NCBI Blast analysis showed that the gene encodes 412 amino acids and belongs to the prenyltransferase UbiA superfamily. Multiple sequence alignment showed that this sequence shared 80% identity with other HPT proteins and named MsHPT. The results of phylogenetic tree analysis showed that MsHPT had the closest genetic relationship with MtHPT, and the similarity of the protein sequence was 96.84%. The promoter sequence of this gene was obtained by chromosome walking technique. The results showed that the promoter region of the gene contained stress response elements, hormone response elements (abscisic acid, gibberellin and ethylene) and a large number of light-responsive elements. The results of real-time PCR showed that MsHPT gene was expressed in all tissues of alfalfa and the highest expression level in leaves. The gene expression was up-regulated after being induced by low temperature, NaCl, PEG, GA3 and ABA, indicating that it may be involved in plant stress resistance regulation. The open reading frame (ORF) of MsHPT gene was amplified and double-digested. The double-stranded vector was transformed into binary vector pBI121 and transformed into Arabidopsis by Agrobacterium-mediated transformation. By PCR identification, seven positive seedlings were obtained. This study laid the foundation for further exploring the role of MsHPT in the synthesis of alfalfa vitamin E and alfalfa stress resistance.