连翘属抗寒相关基因GPAT的克

发布时间：2018-01-22 10:41

本文关键词： 耐冷连翘 GPAT 分子克隆体外表达　出处：《沈阳农业大学》2017年硕士论文　论文类型：学位论文

【摘要】：低温是限制林木、农作物分布及生长发育的主要因素,气温骤降对林木的损害巨大,造成极大的经济损失。连翘(Forsythia)声为木犀科连翘属植物为落叶灌木,连翘主要分为东北连翘(Forsythiamandshurica)和金钟连翘(Forsythia suspense)。高等植物质膜中脂肪酸不饱和度和冷敏感关系密切。甘油-3-磷酸酰基转移酶(GPAT)是磷脂酰甘油(Phosph-atidglglycerol,PG)生物合成过程中的第一个酰基酯化酶,直接决定植物膜PG的不饱和度。在抗冷植物中,GPAT优先选择18:1作为底物;在冷敏感植物中,GPAT优先选择16:0作为底物。由此导致二者生物膜的饱和度及其在低温下的流动性不同,进而影响其耐冷性。本研究以连翘属的两个材料,金钟连翘和东北连翘为研究材料。主要研究成果如下:(1)克隆了连翘属GPAT基因,分别命名为FsGPAT和FmGPAT。序列分析表明两个基因序列长度均为1347bp,编码448个氨基酸。连翘属GPAT基因具有GPAT基因家族的保守结构域GPAT_N结构域与LPLAT结构域。(2)连翘属GPAT与拟南芥10个GPAT基因家族对比发现,其与拟南芥的ATS7(at1g32200)基因序列的亲源性较高,而ATSl是拟南芥GPAT基因家族中唯一一个定位于叶绿体的。本研究表明连翘属GPAT基因相对表达量在富含叶绿体的绿色组织中相对较高,符合预测结果。连翘属GPAT与芝麻(Sesamum indicum)和牵牛花(Ipomoea nil)亲缘最近均为74%。(3)将FsGPAT基因编码区与表达载体pCAMBIA1302连接,构建真核表达载体pCAMBIA1302-FsGPAT,转入农杆菌LBA4404中,对烟草愈伤组织用转入表达载体的农杆菌侵染,生根移栽后得到转FsGP4T基因的烟草植株,对转基因烟草的进行PCR扩增检测,验证转基因烟草的基因组中已包含FsGPAT基因。(4)通过对连翘GPAT在不同组织表达量分析表明随着温度的降低GPAT表达量升高,说明GPAT表达量与植物的抗寒能力具有相关性。本研究将为连翘抗寒分子机制的进一步明确提供重要参考,同时对于利用转基因手段提高农作物或林木抗寒力奠定理论基础。
[Abstract]:Low temperature is the main factors of forest, crop distribution and growth, damage temperatures on trees, causing great economic losses. Forsythia (Forsythia) sound Forsythia genus for deciduous shrub, forsythia is divided into the northeast of Forsythia suspensa (Forsythiamandshurica) and Jin Zhonglianqiao (Forsythia suspense). The higher plant plasma membrane fat acid saturation and cold sensitive relationship. -3- glycerol phosphate acyltransferase (GPAT) is phosphatidylglycerol (Phosph-atidglglycerol, PG) first acyl esterified enzyme in the biosynthesis of PG, directly determines the plant membrane unsaturation. In cold resistant plants, GPAT 18:1 was selected as the preferred substrate; in cold sensitive plants, GPAT select 16:0 as the substrate. Thus the two different biofilm saturation and fluidity at low temperature, thereby affecting its cold tolerance. In this study, forsythia Of the two materials, and the northeast of Admiralty Forsythia Forsythia materials. The main research results are as follows: (1) cloning of Forsythia GPAT gene, named FsGPAT and FmGPAT. sequence analysis showed that two genes were 1347bp in length, encoding 448 amino acids. Forsythia GPAT gene has GPAT gene family conserved domain the GPAT_N and LPLAT domain. (2) found that Forsythia GPAT and 10 Arabidopsis GPAT gene family and its contrast, the Arabidopsis ATS7 (at1g32200) gene sequence of the pro source is higher, while ATSl is the Arabidopsis GPAT gene family in only one location in the chloroplast. This study shows that the relative expression of relative Forsythia a higher amount of rich in green tissues of chloroplast GPAT gene, consistent with the predicted results. Forsythia GPAT (Sesamum indicum) and sesame seeds and flowers (Ipomoea Nil) were recently were 74%. (3) FsGPAT gene encoding region and The expression vector pCAMBIA1302, to construct the eukaryotic expression vector pCAMBIA1302-FsGPAT into Agrobacterium LBA4404, of tobacco callus by Agrobacterium infection into expression vector, rooting and transplanting after tobacco plants transformed with FsGP4T gene, transgenic tobacco by PCR amplification assay, FsGPAT gene has been verified containing transgenic tobacco genome. (4) by GPAT in the amount of Forsythia suspensa analysis showed that with the decrease of temperature increased the expression level of GPAT expression in different tissues, GPAT expression and correlation with the cold resistance of the plant. This study will further clarify the molecular mechanism of cold resistance of Forsythia provides an important reference, at the same time to lay a theoretical foundation for the use of transgenic methods to improve the cold hardiness of crops and trees.

【学位授予单位】：沈阳农业大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：S567.19;Q943.2

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