玉米二酰甘油酰基转移酶基因的鉴定及表达研究

发布时间：2018-08-28 15:20

【摘要】：二酰甘油酰基转移酶(Acyl-CoA:diacylglycerol acyltransferase,DGAT)作为植物油脂三酰甘油(TAG)合成途径中唯一的限速酶,参与植物油脂合成及生长发育等重要过程。本研究利用生物信息学等手段对玉米(Zea mays L.)DGAT基因家族进行鉴定和功能研究。主要的研究内容包括:(1)在全基因组水平上对玉米DGAT基因家族进行筛选和鉴定,并对家族成员进行全面的生物信息学分析;(2)玉米DGAT基因家族响应非生物胁迫的表达分析;(3)玉米两个II型DGAT基因的全长克隆及其酶学功能验证。主要结果如下:1.本研究共鉴定分离5个玉米DGAT基因家族成员,其中包括两个I型DGAT:ZmDGAT1.1和ZmDGAT1.2;两个II型DGAT:ZmDGAT2.1和ZmDGAT2.2;以及一个III型DGAT:ZmDGAT3。玉米的DGAT1和DGAT2基因家族均含有多个跨膜结构域,而玉米DGAT3基因不包含跨膜结构域。2.系统进化分析结果显示:各个物种的DGAT1、DGAT2和DGAT3的蛋白序列分别与其他物种的I,II或III型DGAT聚类在同一个簇内;其中在不同的基因家族分枝之内,同家族的不同物种的DGAT蛋白根据高低等植物进行了进一步的划分,其中玉米被划分到单子叶植物分组内,玉米DGAT蛋白均与高粱表现出较近的亲缘关系。进化过程中,DGAT基因家族从藻类到单、双子叶植物的直系同源基因的内含子、外显子和功能区片段的长度和数目发生了不同程度的增减。3.蛋白保守功能区的研究表明:两个玉米DGAT1编码的蛋白均包含膜结合酰基转移酶类蛋白MBOAT功能域(PF03062,Membrane bound O-acyl transferase)以及与油脂积累相关的ARE1调控元件(alkane-responsive element one)。两个玉米DGAT2编码的蛋白均包含溶血磷脂酰基转移酶类基因家族所包含的LPAT(Lysophospholipid acyltransferase)保守结构域(又称MBOAT2),也是膜结合类蛋白功能域。DGAT1和DGAT2的功能域都集中在蛋白的N端,而玉米DGAT3编码的蛋白包含DGAT3中的细胞分裂类蛋白Zap A以及与铁氧还蛋白/硫氧还蛋白相同的铁氧还蛋白结构域。4.不同非生物胁迫条件下的表达分析结果表明,玉米根和叶片组织中的DGAT1和DGAT2基因在多种非生物胁迫条件下上调表达,表明玉米DGAT基因作为脂类代谢过程中的关键基因,参与了植物响应非生物逆境胁迫过程,而且玉米DGAT基因在不同时间点和不同组织位的表达有明显差异,表现出不同的趋势,说明玉米DGAT基因家族成员对多种非生物胁迫有响应,并且在响应非生物胁迫过程中可能发挥着不同的作用。5.本文对两个II型玉米DGAT2基因的编码区进行了全长克隆,分别连接到酵母表达载体pYES2.1中,经过验证的表达载体转化到酵母脂类合成突变体H1246菌株进行异源表达。结果表明两个玉米DGAT2基因的表达能够恢复酵母突变体形成脂类的表型,说明两个玉米DGAT2基因编码的蛋白具有合成脂类的酶学功能。
[Abstract]:Diacylglycerol transferase (Acyl-CoA:diacylglycerol acyltransferase,DGAT) is the only rate-limiting enzyme in triacylglycerol (TAG) synthesis pathway, which plays an important role in plant oil synthesis, growth and development. In this study, bioinformatics was used to identify and study the (Zea mays L.) DGAT gene family in maize. The main research contents are as follows: (1) screening and identification of maize DGAT gene family at the whole genome level. The family members were analyzed by bioinformatics; (2) the expression of maize DGAT gene family in response to abiotic stress; (3) the full-length cloning and enzymatic function verification of two II type DGAT genes in maize. The main results are as follows: 1. In this study, five members of maize DGAT gene family were identified and isolated, including two type I DGAT:ZmDGAT1.1 and two II types of DGAT:ZmDGAT2.1 and ZmDGAT2.2; and one III type DGAT:ZmDGAT3.. Both DGAT1 and DGAT2 gene families in maize contain multiple transmembrane domains, while DGAT3 genes do not contain transmembrane domains. Phylogenetic analysis showed that the protein sequences of DGAT1,DGAT2 and DGAT3 of each species were clustered in the same cluster as those of other species, and were in different branches of gene family. The DGAT proteins of different species in the same family were further divided according to the higher and lower plants, in which maize was divided into monocotyledonous groups, and the DGAT protein of maize showed a close relationship with sorghum. During evolution, the length and number of homologous gene introns, exons and functional regions of the lineal homologous genes in dicotyledonous plants increased or decreased in varying degrees from algae to monocotyledonous plants. The study of conserved functional regions of protein showed that the two proteins encoded by DGAT1 in maize contained MBOAT functional domain (PF03062,Membrane bound O-acyl transferase) of membrane-binding acyltransferases and ARE1 regulatory elements related to lipid accumulation (alkane-responsive element one). The two proteins encoded by DGAT2 in maize contain the conserved domain of LPAT (Lysophospholipid acyltransferase) (also called MBOAT2) contained in the family of lysophosphatidyl transferase genes, and the functional domains of membrane binding protein domain. DGAT1 and DGAT2 are both concentrated in the N-terminal of the protein. The protein encoded by maize DGAT3 contains the cell division protein Zap A in DGAT3 and the ferredoxin domain. 4, which is the same as ferredoxin / thioredoxin. The expression of DGAT1 and DGAT2 genes in maize roots and leaves were up-regulated under various abiotic stress conditions, indicating that DGAT gene was the key gene in lipid metabolism. The DGAT gene expression in maize was different at different time points and different tissue sites, which indicated that DGAT gene family members were responsive to various abiotic stresses, and the expression of DGAT gene in maize was different at different time points and different tissue sites, indicating that maize DGAT gene family members were responsive to various abiotic stresses. And may play a different role in response to abiotic stress. In this paper, two full-length coding regions of II type maize DGAT2 gene were cloned and ligated into yeast expression vector pYES2.1, respectively. The verified expression vector was transformed into yeast lipid synthesis mutant H1246 for heterologous expression. The results showed that the expression of two maize DGAT2 genes could restore the lipid-forming phenotype of yeast mutants, indicating that the proteins encoded by the two maize DGAT2 genes had the enzymatic function of lipid synthesis.
【学位授予单位】：黑龙江八一农垦大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：S513

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