本文關(guān)鍵詞： 耐冷 連翹 GPAT 分子克隆 體外表達(dá)　出處：《沈陽農(nóng)業(yè)大學(xué)》2017年碩士論文　論文類型：學(xué)位論文

【摘要】：低溫是限制林木、農(nóng)作物分布及生長(zhǎng)發(fā)育的主要因素,氣溫驟降對(duì)林木的損害巨大,造成極大的經(jīng)濟(jì)損失。連翹(Forsythia)聲為木犀科連翹屬植物為落葉灌木,連翹主要分為東北連翹(Forsythiamandshurica)和金鐘連翹(Forsythia suspense)。高等植物質(zhì)膜中脂肪酸不飽和度和冷敏感關(guān)系密切。甘油-3-磷酸�；D(zhuǎn)移酶(GPAT)是磷脂酰甘油(Phosph-atidglglycerol,PG)生物合成過程中的第一個(gè)�；セ�,直接決定植物膜PG的不飽和度。在抗冷植物中,GPAT優(yōu)先選擇18:1作為底物;在冷敏感植物中,GPAT優(yōu)先選擇16:0作為底物。由此導(dǎo)致二者生物膜的飽和度及其在低溫下的流動(dòng)性不同,進(jìn)而影響其耐冷性。本研究以連翹屬的兩個(gè)材料,金鐘連翹和東北連翹為研究材料。主要研究成果如下:(1)克隆了連翹屬GPAT基因,分別命名為FsGPAT和FmGPAT。序列分析表明兩個(gè)基因序列長(zhǎng)度均為1347bp,編碼448個(gè)氨基酸。連翹屬GPAT基因具有GPAT基因家族的保守結(jié)構(gòu)域GPAT_N結(jié)構(gòu)域與LPLAT結(jié)構(gòu)域。(2)連翹屬GPAT與擬南芥10個(gè)GPAT基因家族對(duì)比發(fā)現(xiàn),其與擬南芥的ATS7(at1g32200)基因序列的親源性較高,而ATSl是擬南芥GPAT基因家族中唯一一個(gè)定位于葉綠體的。本研究表明連翹屬GPAT基因相對(duì)表達(dá)量在富含葉綠體的綠色組織中相對(duì)較高,符合預(yù)測(cè)結(jié)果。連翹屬GPAT與芝麻(Sesamum indicum)和牽�；�(Ipomoea nil)親緣最近均為74%。(3)將FsGPAT基因編碼區(qū)與表達(dá)載體pCAMBIA1302連接,構(gòu)建真核表達(dá)載體pCAMBIA1302-FsGPAT,轉(zhuǎn)入農(nóng)桿菌LBA4404中,對(duì)煙草愈傷組織用轉(zhuǎn)入表達(dá)載體的農(nóng)桿菌侵染,生根移栽后得到轉(zhuǎn)FsGP4T基因的煙草植株,對(duì)轉(zhuǎn)基因煙草的進(jìn)行PCR擴(kuò)增檢測(cè),驗(yàn)證轉(zhuǎn)基因煙草的基因組中已包含F(xiàn)sGPAT基因。(4)通過對(duì)連翹GPAT在不同組織表達(dá)量分析表明隨著溫度的降低GPAT表達(dá)量升高,說明GPAT表達(dá)量與植物的抗寒能力具有相關(guān)性。本研究將為連翹抗寒分子機(jī)制的進(jìn)一步明確提供重要參考,同時(shí)對(duì)于利用轉(zhuǎn)基因手段提高農(nóng)作物或林木抗寒力奠定理論基礎(chǔ)。
[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.

【學(xué)位授予單位】：沈陽農(nóng)業(yè)大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2017
【分類號(hào)】：S567.19;Q943.2

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本文編號(hào)：1454440