花生PEPC家族基因分析及反义PEPC1基因遗传转化研究

发布时间：2018-05-05 04:19

本文选题：花生 + 油脂合成　；参考：《山东农业大学》2017年博士论文

【摘要】：植物油是人类日常生活所需要的主要营养物质,也是重要的工业原料。由于人口增长、经济发展、农产品消费结构变化等因素的影响,使得国内外植物油脂市场需求量骤增。目前我国植物油供需矛盾突出,自给率不足35%。提高油料作物的含油量是增加油脂产量的有效途径。花生是我国最重要的油料作物之一,与大豆、油菜等主要油料作物相比,花生单位面积产油量最高。花生种子含油量45-56%,具有进一步提高的潜力。近年来,随着花生基因组测序工作的开展、花生转基因技术的逐步完善,使得通过转基因、分子育种手段培育高油花生品种成为可能。油脂合成和蛋白质合成存在一定的竞争性,两个代谢过程均需要利用磷酸烯醇式丙酮酸(Phosphoenolpyruvate,PEP)做底物。乙酰辅酶A羧化酶(ACCase)催化丙酮酸合成乙酰-CoA,进入脂肪酸合成途径,而磷酸烯醇式丙酮酸羧化酶(Phosphoenolpyruvate carboxylase,PEPC)可催化丙酮酸合成草酰乙酸,进入蛋白质合成途径。PEPC在植物中具有多种生理功能,研究表明PEPC参与碳/氮吸收、脂肪酸调控并与盐和干旱胁迫反应有关,但对其功能认识仍然非常有限。目前,花生中PEPC基因功能深入研究未见报道。本研究分析了花生PEPC家族基因的表达特性;在此基础上,构建AhPEPC1基因的反向表达载体,转化花生,通过对转基因植株性状分析,研究该基因在提高花生油脂含量中的作用及其分子机制。研究结果对于进一步提高花生含油量具有潜在的应用价值。本研究取得以下结果:1、通过氨基酸序列比对、保守结构域分析、基因结构预测以及系统发育进化分析等生物信息学分析方法,将花生PEPC家族5个基因分为两类:AhPEPC1、AhPEPC2、AhPEPC3和AhPEPC4基因编码植物型PEPC,AhPEPC5基因编码细菌型PEPC。AhPEPC1、AhPEPC2、AhPEPC3和AhPEPC4基因结构由10个外显子和9个内含子组成;AhPEPC5基因结构由20个外显子和19个内含子组成。2、通过花生不同组织和不同发育时期的荧光定量PCR分析,发现AhPEPC1、AhPEPC2、AhPEPC3、AhPEPC4和AhPEPC5基因在根、茎、叶和种子中均有表达但表达模式不同。AhPEPC1基因在种子中的表达量最高,AhPEPC3基因在根中的表达量较高,AhPEPC4基因在叶中的表达量较高。3、将AhPEPC1基因片段反向连接植物双元表达载体pCAMBIA1301,构建获得反义表达载体pCAMBIA1301-PEPC,农杆菌介导法转化花生。经潮霉素筛选后,利用特异引物PCR检测获得阳性植株。表型观察发现转基因花生农艺性状未发生明显改变,与对照花生相比,转基因花生种子含油量提高了5.7%-10.3%,脂肪酸组分相对含量没有变化,但种子蛋白质含量降低了7.5%-17.0%,转基因花生萌发过程中对盐胁迫耐受性增强。4、为了进一步分析转基因花生中AhPEPC1基因的抑制表达是否影响其他基因的功能,对转基因植株进行了转录组测序分析。结果表明,转基因植株与野生型花生相比,筛选到110个差异表达基因,其中25个基因表达上调,85个基因表达下调,没有引起花生中太多基因的表达变化,这与转基因花生和野生型花生表型差异不明显的结果一致。差异表达基因进行了KEGG富集分析,有34个基因成功获得了KEGG注释,其中氨基酸的生物合成中有两个基因(Aradu.M0JX8、Aradu.FE0Z7)下调表达。这些数据与转基因花生种子蛋白含量降低是一致的。
[Abstract]:Vegetable oil is the main nutrient and important industrial raw material needed in human daily life. Due to the influence of the population growth, economic development, and the change of the consumption structure of the agricultural products, the demand of the domestic and foreign vegetable oil market is increasing rapidly. At present, the plant oil supply and demand spear shield is prominent and the self-sufficiency rate is less than 35%. to improve the oil crops. The amount of oil is an effective way to increase the oil production. The peanut is one of the most important oil crops in our country. Compared with the main oil crops such as soybean and rape, peanuts have the highest oil production per unit area. The oil content of peanut seeds is 45-56%, which has the potential to be further improved. In recent years, with the development of the genome sequencing work of the peanut, the transgenic technique of peanut was carried out. The gradual improvement of the operation makes it possible to cultivate high oil peanut varieties by genetically modified and molecular breeding. There is a certain competition in oil synthesis and protein synthesis. The two metabolic processes need to use Phosphoenolpyruvate (PEP) as the substrate. Acetyl coenzyme A carboxylase (ACCase) catalyzes the synthesis of acetyl pyruvate. Acyl -CoA, into the fatty acid synthesis pathway, and the phosphoenolpyruvate carboxylase (Phosphoenolpyruvate carboxylase, PEPC) can catalyze the synthesis of oxoacetic acid by pyruvic acid, and into the protein synthesis pathway,.PEPC has a variety of physiological functions in plants. Research shows that PEPC is involved in carbon / nitrogen absorption, fatty acids are regulated and reacted with salt and drought stress. However, the understanding of its function is still very limited. At present, the PEPC gene function in peanut is not reported in depth. This study analyzed the expression characteristics of the peanut PEPC family gene. On this basis, the reverse expression vector of the AhPEPC1 gene was constructed to transform the peanut, and the gene was studied to improve the peanut through the analysis of the transgenic plant characters. The effect and molecular mechanism of oil content and molecular mechanism. The results are of potential application value for further improving the oil content of peanut. The following results are obtained: 1, bioinformatics analysis methods such as amino acid sequence alignment, conservative domain analysis, gene structure prediction and phylogenetic evolution analysis, and so on, the peanut PEPC family 5 The genes are divided into two categories: AhPEPC1, AhPEPC2, AhPEPC3 and AhPEPC4 genes encoding plant type PEPC. The AhPEPC5 gene encodes bacterial PEPC.AhPEPC1, AhPEPC2, AhPEPC3 and AhPEPC4 gene structures consisting of 10 exons and 9 introns; AhPEPC5 gene structure consists of 20 exons and 19 introns. AhPEPC1, AhPEPC2, AhPEPC3, AhPEPC4 and AhPEPC5 genes expressed in roots, stems, leaves and seeds were expressed in the roots, stems, leaves and seeds, but the expression of the.AhPEPC1 gene in the seeds was the highest, the expression of the AhPEPC3 gene in the root was higher, the expression of the AhPEPC4 gene in the leaves was higher.3, and the AhPEPC1 gene fragment was found in the PCR analysis. The reverse link plant dual expression vector pCAMBIA1301 was constructed to construct an antisense expression vector, pCAMBIA1301-PEPC, and Agrobacterium tumefaciens mediated transformation of peanut. After screening by hygromycin, the positive plants were detected by the specific primer PCR. The phenotypic observation showed that the agronomic characters of transgenic peanuts did not change obviously, and the transgenic peanuts were compared with the control peanut. The seed oil content increased by 5.7%-10.3%, and the relative content of fatty acid components did not change, but the content of seed protein decreased by 7.5%-17.0%. The tolerance of transgenic peanuts to salt stress was enhanced by.4. In order to further analyze the effect of the inhibition expression of AhPEPC1 gene in transgenic peanuts, the effect of other genes on the function of the transgenic peanuts was analyzed. The results showed that 110 differentially expressed genes were screened in the transgenic plant compared with the wild type peanut, of which 25 genes were up-regulated and 85 genes were down regulated, which did not cause the changes in the expression of too many genes in peanut, which was in agreement with the results of the phenotypic differences of transgenic peanuts and wild type peanuts. The differentially expressed genes were enriched and analyzed by KEGG, and 34 genes successfully obtained the KEGG annotation, of which two genes (Aradu.M0JX8, Aradu.FE0Z7) were down regulated in the biosynthesis of amino acids. These data were consistent with the decrease in the seed protein content of transgenic peanuts.

【学位授予单位】：山东农业大学
【学位级别】：博士
【学位授予年份】：2017
【分类号】：S565.2

【参考文献】