發(fā)布時(shí)間：2018-05-05 04:19

  本文選題：花生 + 油脂合成　； 參考：《山東農(nóng)業(yè)大學(xué)》2017年博士論文

【摘要】：植物油是人類日常生活所需要的主要營養(yǎng)物質(zhì),也是重要的工業(yè)原料。由于人口增長、經(jīng)濟(jì)發(fā)展、農(nóng)產(chǎn)品消費(fèi)結(jié)構(gòu)變化等因素的影響,使得國內(nèi)外植物油脂市場需求量驟增。目前我國植物油供需矛盾突出,自給率不足35%。提高油料作物的含油量是增加油脂產(chǎn)量的有效途徑。花生是我國最重要的油料作物之一,與大豆、油菜等主要油料作物相比,花生單位面積產(chǎn)油量最高�；ㄉN子含油量45-56%,具有進(jìn)一步提高的潛力。近年來,隨著花生基因組測序工作的開展、花生轉(zhuǎn)基因技術(shù)的逐步完善,使得通過轉(zhuǎn)基因、分子育種手段培育高油花生品種成為可能。油脂合成和蛋白質(zhì)合成存在一定的競爭性,兩個(gè)代謝過程均需要利用磷酸烯醇式丙酮酸(Phosphoenolpyruvate,PEP)做底物。乙酰輔酶A羧化酶(ACCase)催化丙酮酸合成乙酰-CoA,進(jìn)入脂肪酸合成途徑,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,進(jìn)入蛋白質(zhì)合成途徑。PEPC在植物中具有多種生理功能,研究表明PEPC參與碳/氮吸收、脂肪酸調(diào)控并與鹽和干旱脅迫反應(yīng)有關(guān),但對其功能認(rèn)識仍然非常有限。目前,花生中PEPC基因功能深入研究未見報(bào)道。本研究分析了花生PEPC家族基因的表達(dá)特性;在此基礎(chǔ)上,構(gòu)建AhPEPC1基因的反向表達(dá)載體,轉(zhuǎn)化花生,通過對轉(zhuǎn)基因植株性狀分析,研究該基因在提高花生油脂含量中的作用及其分子機(jī)制。研究結(jié)果對于進(jìn)一步提高花生含油量具有潛在的應(yīng)用價(jià)值。本研究取得以下結(jié)果:1、通過氨基酸序列比對、保守結(jié)構(gòu)域分析、基因結(jié)構(gòu)預(yù)測以及系統(tǒng)發(fā)育進(jìn)化分析等生物信息學(xué)分析方法,將花生PEPC家族5個(gè)基因分為兩類:AhPEPC1、AhPEPC2、AhPEPC3和AhPEPC4基因編碼植物型PEPC,AhPEPC5基因編碼細(xì)菌型PEPC。AhPEPC1、AhPEPC2、AhPEPC3和AhPEPC4基因結(jié)構(gòu)由10個(gè)外顯子和9個(gè)內(nèi)含子組成;AhPEPC5基因結(jié)構(gòu)由20個(gè)外顯子和19個(gè)內(nèi)含子組成。2、通過花生不同組織和不同發(fā)育時(shí)期的熒光定量PCR分析,發(fā)現(xiàn)AhPEPC1、AhPEPC2、AhPEPC3、AhPEPC4和AhPEPC5基因在根、莖、葉和種子中均有表達(dá)但表達(dá)模式不同。AhPEPC1基因在種子中的表達(dá)量最高,AhPEPC3基因在根中的表達(dá)量較高,AhPEPC4基因在葉中的表達(dá)量較高。3、將AhPEPC1基因片段反向連接植物雙元表達(dá)載體pCAMBIA1301,構(gòu)建獲得反義表達(dá)載體pCAMBIA1301-PEPC,農(nóng)桿菌介導(dǎo)法轉(zhuǎn)化花生。經(jīng)潮霉素篩選后,利用特異引物PCR檢測獲得陽性植株。表型觀察發(fā)現(xiàn)轉(zhuǎn)基因花生農(nóng)藝性狀未發(fā)生明顯改變,與對照花生相比,轉(zhuǎn)基因花生種子含油量提高了5.7%-10.3%,脂肪酸組分相對含量沒有變化,但種子蛋白質(zhì)含量降低了7.5%-17.0%,轉(zhuǎn)基因花生萌發(fā)過程中對鹽脅迫耐受性增強(qiáng)。4、為了進(jìn)一步分析轉(zhuǎn)基因花生中AhPEPC1基因的抑制表達(dá)是否影響其他基因的功能,對轉(zhuǎn)基因植株進(jìn)行了轉(zhuǎn)錄組測序分析。結(jié)果表明,轉(zhuǎn)基因植株與野生型花生相比,篩選到110個(gè)差異表達(dá)基因,其中25個(gè)基因表達(dá)上調(diào),85個(gè)基因表達(dá)下調(diào),沒有引起花生中太多基因的表達(dá)變化,這與轉(zhuǎn)基因花生和野生型花生表型差異不明顯的結(jié)果一致。差異表達(dá)基因進(jìn)行了KEGG富集分析,有34個(gè)基因成功獲得了KEGG注釋,其中氨基酸的生物合成中有兩個(gè)基因(Aradu.M0JX8、Aradu.FE0Z7)下調(diào)表達(dá)。這些數(shù)據(jù)與轉(zhuǎn)基因花生種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.

【學(xué)位授予單位】：山東農(nóng)業(yè)大學(xué)
【學(xué)位級別】：博士
【學(xué)位授予年份】：2017
【分類號】：S565.2

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本文編號：1846170