盐胁迫下盐地碱蓬甘油-3-磷酸酰基转移酶基因的功能分析

发布时间：2018-01-09 20:07

  本文关键词：盐胁迫下盐地碱蓬甘油-3-磷酸酰基转移酶基因的功能分析　出处：《山东师范大学》2016年硕士论文　论文类型：学位论文

  更多相关文章： 盐地碱蓬 叶绿体甘油-3-磷酸酰基转移酶 盐胁迫 PG 不饱和脂肪酸

【摘要】：高等植物体内的多不饱和脂肪酸合成途径分为真核途径和原核途径。原核途径是指在叶绿体中存在的途径,真核途径是指内质网中甘油脂的合成以及向叶绿体的转运。在这两种途径中,由饱和脂肪酸形成磷酸酯(PA)这一过程中都需要甘油-3-磷酸酰基转移酶(Glycerol-3-phosphate acyltransferase,GPAT)的催化作用,进而由磷酸酯(PA)合成磷脂酰甘油(PG),即甘油-3-磷酸酰基转移酶(GPAT)是磷脂酰甘油合成过程中的第一个酰基酯化酶。本研究从盐地碱蓬叶片中分离得到叶绿体甘油-3-磷酸酰基转移酶基因,并对该基因的表达和功能进行了分析。同时研究了盐胁迫对拟南芥GPAT缺失突变体的影响。主要研究结果如下:1.利用拟南芥、番茄、菠菜、甜菜等植物中已知的甘油-3-磷酸酰基转移酶基因设计一对简并引物。通过PCR的方法从盐地碱蓬叶片中克隆甘油-3-磷酸酰基转移酶基因的中间片段,再通过5'RACE和3'RACE技术克隆得到5'片段和3'片段,拼接全长后设计两端特异引物进行全长克隆。该基因编码区全长1167bp,编码388个氨基酸,分子量约为43 kDa。经过同源序列比对后发现,盐地碱蓬叶绿体甘油-3-磷酸酰基转移酶基因与菠菜的甘油-3-磷酸酰基转移酶基因同源性最高,达到87%。2.将获得的盐地碱蓬甘油-3-磷酸酰基转移酶基因与pB7WG2D载体重组,构建正义表达载体,利用花序侵染法获得拟南芥过表达株系。以叶片DNA为模板,根据表达载体上的35S启动子序列设计引物与基因3'端引物进行PCR扩增,检测过表达株系。3.在盐胁迫下,野生型拟南芥与过表达株系的萌发率、主根长度都受到了抑制,但是过表达株系受到的抑制程度较低,说明在萌发期过表达株系的抗盐能力更好;用RT-PCR检测过表达株系中盐地碱蓬甘油-3-磷酸酰基转移酶基因的表达量,发现在100 mM NaCl条件下,表达量最高。用0和100 mM NaCl处理幼苗期的植株,测定叶片中的叶绿素含量、光合荧光参数和光系统I活性,结果发现,在盐处理下,过表达株系的叶绿素含量、ΦPSII、Fv/Fm及PSI活性显著高于野生型拟南芥,Fo、1-qP和NPQ显著低于野生型拟南芥。这说明与野生型相比,过表达株系受到盐胁迫的抑制作用更小,光合能力更强。通过测定PG脂肪酸组成发现,过表达株系PG中不饱和脂肪酸的含量显著高于野生型拟南芥。4.选取SALK_136675C(ATGPAT6),SALK_060056(ATGPAT2),SALK_037660C(ATGPAT1),SALK_045942(ATGPAT4)这四个突变体进行实验,结果发现,SALK_136675C和SALK_060056这两个突变体的抗盐能力显著低于野生型。盐处理下,SALK_136675C和SALK_060056的萌发率及主根长度显著低于野生型拟南芥。测定了幼苗期100 mM NaCl条件下SALK_136675C,SALK_060056和野生型拟南芥的叶绿素含量、各叶绿素荧光参数和光系统I活性参数,结果发现,在盐处理下,SALK_136675C和SALK_060056叶片中的叶绿素含量、ΦPSII、Fv/Fm及PSI活性显著低于野生型植株,Fo、1-qP和NPQ显著高于野生型植株。这些结果说明,SALK_136675C和SALK_060056的光系统受到盐胁迫的伤害更大。与野生型拟南芥相比,SALK_136675C和SALK_060056叶片中PG中不饱和脂肪酸的含量明显减少。上述结果表明,盐地碱蓬叶绿体甘油-3-磷酸酰基转移酶基因能够通过提高PG不饱和脂肪酸的含量来增强拟南芥的抗盐能力。
[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.

【学位授予单位】：山东师范大学
【学位级别】：硕士
【学位授予年份】：2016
【分类号】：Q943.2
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本文编号：1402444