本文關(guān)鍵詞：鹽脅迫下鹽地堿蓬甘油-3-磷酸�；D(zhuǎn)移酶基因的功能分析　出處：《山東師范大學》2016年碩士論文　論文類型：學位論文

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【摘要】：高等植物體內(nèi)的多不飽和脂肪酸合成途徑分為真核途徑和原核途徑。原核途徑是指在葉綠體中存在的途徑,真核途徑是指內(nèi)質(zhì)網(wǎng)中甘油脂的合成以及向葉綠體的轉(zhuǎn)運。在這兩種途徑中,由飽和脂肪酸形成磷酸酯(PA)這一過程中都需要甘油-3-磷酸�；D(zhuǎn)移酶(Glycerol-3-phosphate acyltransferase,GPAT)的催化作用,進而由磷酸酯(PA)合成磷脂酰甘油(PG),即甘油-3-磷酸�；D(zhuǎn)移酶(GPAT)是磷脂酰甘油合成過程中的第一個�；セ�。本研究從鹽地堿蓬葉片中分離得到葉綠體甘油-3-磷酸�；D(zhuǎn)移酶基因,并對該基因的表達和功能進行了分析。同時研究了鹽脅迫對擬南芥GPAT缺失突變體的影響。主要研究結(jié)果如下:1.利用擬南芥、番茄、菠菜、甜菜等植物中已知的甘油-3-磷酸�；D(zhuǎn)移酶基因設(shè)計一對簡并引物。通過PCR的方法從鹽地堿蓬葉片中克隆甘油-3-磷酸�；D(zhuǎn)移酶基因的中間片段,再通過5'RACE和3'RACE技術(shù)克隆得到5'片段和3'片段,拼接全長后設(shè)計兩端特異引物進行全長克隆。該基因編碼區(qū)全長1167bp,編碼388個氨基酸,分子量約為43 kDa。經(jīng)過同源序列比對后發(fā)現(xiàn),鹽地堿蓬葉綠體甘油-3-磷酸�；D(zhuǎn)移酶基因與菠菜的甘油-3-磷酸�；D(zhuǎn)移酶基因同源性最高,達到87%。2.將獲得的鹽地堿蓬甘油-3-磷酸�；D(zhuǎn)移酶基因與pB7WG2D載體重組,構(gòu)建正義表達載體,利用花序侵染法獲得擬南芥過表達株系。以葉片DNA為模板,根據(jù)表達載體上的35S啟動子序列設(shè)計引物與基因3'端引物進行PCR擴增,檢測過表達株系。3.在鹽脅迫下,野生型擬南芥與過表達株系的萌發(fā)率、主根長度都受到了抑制,但是過表達株系受到的抑制程度較低,說明在萌發(fā)期過表達株系的抗鹽能力更好;用RT-PCR檢測過表達株系中鹽地堿蓬甘油-3-磷酸�；D(zhuǎn)移酶基因的表達量,發(fā)現(xiàn)在100 mM NaCl條件下,表達量最高。用0和100 mM NaCl處理幼苗期的植株,測定葉片中的葉綠素含量、光合熒光參數(shù)和光系統(tǒng)I活性,結(jié)果發(fā)現(xiàn),在鹽處理下,過表達株系的葉綠素含量、ΦPSII、Fv/Fm及PSI活性顯著高于野生型擬南芥,Fo、1-qP和NPQ顯著低于野生型擬南芥。這說明與野生型相比,過表達株系受到鹽脅迫的抑制作用更小,光合能力更強。通過測定PG脂肪酸組成發(fā)現(xiàn),過表達株系PG中不飽和脂肪酸的含量顯著高于野生型擬南芥。4.選取SALK_136675C(ATGPAT6),SALK_060056(ATGPAT2),SALK_037660C(ATGPAT1),SALK_045942(ATGPAT4)這四個突變體進行實驗,結(jié)果發(fā)現(xiàn),SALK_136675C和SALK_060056這兩個突變體的抗鹽能力顯著低于野生型。鹽處理下,SALK_136675C和SALK_060056的萌發(fā)率及主根長度顯著低于野生型擬南芥。測定了幼苗期100 mM NaCl條件下SALK_136675C,SALK_060056和野生型擬南芥的葉綠素含量、各葉綠素熒光參數(shù)和光系統(tǒng)I活性參數(shù),結(jié)果發(fā)現(xiàn),在鹽處理下,SALK_136675C和SALK_060056葉片中的葉綠素含量、ΦPSII、Fv/Fm及PSI活性顯著低于野生型植株,Fo、1-qP和NPQ顯著高于野生型植株。這些結(jié)果說明,SALK_136675C和SALK_060056的光系統(tǒng)受到鹽脅迫的傷害更大。與野生型擬南芥相比,SALK_136675C和SALK_060056葉片中PG中不飽和脂肪酸的含量明顯減少。上述結(jié)果表明,鹽地堿蓬葉綠體甘油-3-磷酸�；D(zhuǎn)移酶基因能夠通過提高PG不飽和脂肪酸的含量來增強擬南芥的抗鹽能力。
[Abstract]:In higher plants the biosynthesis of polyunsaturated fatty acids into prokaryotic and eukaryotic channels. The prokaryotic pathway is a pathway existed in chloroplast, the eukaryotic pathway refers to the synthesis of glycerides in the endoplasmic reticulum and transport to the chloroplast. In this two ways, the formation of phosphate by saturated fatty acid (PA) this process needs to -3- glycerol phosphate acyltransferase (Glycerol-3-phosphate acyltransferase, GPAT) catalyzed by phosphate (PA) and the synthesis of phosphatidylglycerol (PG), namely -3- glycerol phosphate acyltransferase (GPAT) is the first enzyme acyl acyl glycerol phosphate esterification synthesis process. This study isolated the chloroplast -3- glycerol phosphate acyltransferase gene from Suaeda salsa leaves, and the expression and function of the gene was analyzed. At the same time, study the effects of salt stress on Arabidopsis GPAT mutants. The main research The results are as follows: 1. using Arabidopsis, tomato, spinach, -3- glycerol phosphate acyltransferase gene known beet and other plants in the degenerate primers were designed. The middle fragment -3- glycerol phosphate acyltransferase gene from Suaeda salsa leaves by PCR method, then 5'and 3' fragments were obtained by 5'RACE and 3'RACE cloning technology after splicing specific primers design two full-length cloning. The gene encoding the full-length 1167bp encoding 388 amino acids with a molecular weight of 43 kDa. by homologous sequence alignment showed that -3- glycerol phosphate acyltransferase gene of Suaeda -3- chloroplast glycerol phosphate acyltransferase gene and spinach salt highest homology to 87%.2. will get the salt Suaeda -3- glycerol phosphate acyltransferase gene and recombinant vector pB7WG2D, construct expression vector, obtain the Arabidopsis inflorescence infection method using table As with leaf strains. DNA as template, according to the expression vector of 35S promoter sequence primers and 3'gene primer for PCR amplification, detection of.3. overexpression strains under salt stress, the germination rate of wild type Arabidopsis and overexpression lines and root length were inhibited, but the expression is the degree of inhibition by low strain, indicated that the over expression lines of salt resistance in better germination; detected by RT-PCR over expression strains of Suaeda salsa -3- glycerol phosphate acyltransferase gene, found in 100 mM under the condition of NaCl, the highest expression level, with 0 and 100 mM NaCl treatment at seedling stage determination of plant chlorophyll content, photosynthetic fluorescence parameters and photosystem I activity, the results showed that under salt stress, the chlorophyll content, the over expression lines PSII, Fv/Fm and PSI were significantly higher than that of wild type Arabidopsis, Fo, 1-qP and NPQ were significantly lower than those of the wild Born in Arabidopsis. This shows that compared with the wild type, overexpression strains inhibited less salt stress, photosynthetic ability. Through the determination of PG fatty acid composition showed that over expression strain PG content of unsaturated fatty acid was significantly higher than that of wild type Arabidopsis.4. selected SALK_136675C (ATGPAT6) (ATGPAT2), SALK_060056 SALK_037660C, (ATGPAT1), SALK_045942 (ATGPAT4) four mutants of this experiment, results showed that SALK_136675C and SALK_060056 of the two mutants of salt resistance were significantly lower than the wild type. Under salt stress, the germination rate and root length of SALK_136675C and SALK_060056 were significantly lower than the wild type Arabidopsis seedlings. 100 mM under NaCl SALK_136675C determination of the content of chlorophyll SALK_060056 and wild type Arabidopsis, found the I activity parameters, chlorophyll fluorescence parameters and optical system, under salt treatment, SALK_136675C and SALK_0600 The content of chlorophyll in leaves, 56 PSII, Fv/Fm and PSI activity was significantly lower than that of wild type plants, Fo, 1-qP and NPQ were significantly higher than that of wild type plants. These results demonstrate that the optical system SALK_136675C and SALK_060056 by salt stress. Compared with wild type Arabidopsis, SALK_136675C and SALK_060056 in the leaves of PG in unsaturated the fatty acid content decreased significantly. The results showed that the suaedasalsa chloroplast -3- glycerol phosphate acyltransferase gene can enhance Arabidopsis salt resistance by increasing PG content of unsaturated fatty acids.

【學位授予單位】：山東師范大學
【學位級別】：碩士
【學位授予年份】：2016
【分類號】：Q943.2
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本文編號：1402444