GhFSN1转基因棉花后代株系的遗传与生化分析
发布时间：2018-08-19 17:50
【摘要】：棉花在中国乃至世界各国农业经济中都处于重要地位,传统棉花的品质和产量已经难以满足现代社会的需求,为解决这个矛盾,转基因技术在棉花上的运用于是越来越广泛,因此关于棉花基因功能的基础性研究,也就显得十分必要了。本实验室在棉花中克隆出一个NAC家族基因,命名为GhFSN1,它在棉纤维次生细胞壁合成期特异表达,与拟南芥中参与次生细胞壁合成调控的基因NST1-3同源性较高,之前本实验已经对该基因在棉花纤维次生细胞壁发育中的功能进行了较为深入的研究,本文在之前工作的基础上,对GhFSN1转基因棉花后代株系的遗传及生化等方面进行一些探究,取得了如下研究结果:1、GhFSN1转基因棉花后代表型分析为了解GhFSN1基因在棉花纤维发育中的调控功能,我们以GhFSN1转基因棉花为材料,对T2代、T3代和T4代转基因棉花株系进行了遗传表型分析。首先通过阳性鉴定和表达分析,我们筛选出了表达量相对于野生型具有明显差异的转基因棉花株系。对T2代转基因棉花的表型进行观察,我们发现GhFSN1 RNAi植株不论在植株高度、叶片形态、棉纤维长度、种子大小,还是在棉纤维细胞壁厚度上,都与野生型无明显差别;而GhFSN1过量表达植株,植株高度有所下降,叶片出现上翘,种子变小,棉纤维长度明显变短,而纤维细胞壁的厚度增加。以上表型与T1代植株一致。继续对T3代和T4代转基因棉花植株成熟纤维进行长度统计分析发现,不论是RNAi转基因棉花株系,还是过量表达转基因棉花株系,其表型均能稳定遗传。上述结果暗示在棉花中存在与GhFSN1功能冗余基因,同时也表明GhFSN1在棉纤维发育中发挥着十分重要的作用。2、GhFSN1过表达转基因棉花后代株系的纤维转录组分析采用转录组测序技术比较分析了GhFSN1过量表达转基因棉花和野生型棉花开花后18天的纤维中基因转录本变化,筛查鉴定受GhFSN1调控的差异表达基因。结果发现,与野生型相比较,在T2代GhFSN1过量表达转基因棉花纤维中共有2857个基因发生了差异表达,其中1486个基因上调表达,1371个基因下调表达。上调表达的基因主要富集在次级代谢生物合成、苯丙氨酸代谢、类黄酮生物合成、氨基糖和核苷酸糖代谢与苯丙烷类生物合成过程等途径中,而下调表达的基因主要富集在次级代谢物的生物合成、角质、软木脂和蜡质的生物合成、不饱和脂肪酸及果胶的生物合成以及脂肪酸代谢和脂肪酸延伸过程等途径中。进一步对各差异表达基因进行分析,我们发现,不仅有各种转录因子(与拟南芥次生壁合成调控相关转录因子同源),还包括大量纤维素、木聚糖、果胶、脂肪酸、细胞骨架等合成的基因,这些基因都可能与次生细胞壁合成相关。对这些基因进行qRT-PCR验证,结果与转录组分析一致。我们推测GhFSN1基因通过不同调控途径促进棉纤维次生细胞壁的合成。3、pull-down验证GhFSN1蛋白与GhE2蛋白的互作之前本实验室通过酵母双杂交等技术,验证了 GhFSN1蛋白与GhE2蛋白的互作,暗示GhFSN1蛋白通过泛素化途径降解,本文通过pull-down技术验证这一结果。分别构建了 pMAL-GhE2蛋白表达载体及pGEX-4T-1-GhFSNl蛋白表达载体,导入大肠杆菌进行表达,并对MBP-GhE2蛋白与GST-GhFSN1蛋白分别进行了纯化,然后进行pull-down实验验证,结果显示,GhFSN1蛋白确实能与GhE2蛋白的发生相互作用。
[Abstract]:Cotton plays an important role in the agricultural economy of China and even the world. The quality and yield of traditional cotton can not meet the needs of modern society. In order to solve this contradiction, transgenic technology is more and more widely used in cotton. Therefore, it is necessary to study the basic function of cotton genes. A NAC family gene, named GhFSN1, has been cloned from cotton in our laboratory. It is specifically expressed in the secondary cell wall synthesis phase of cotton fiber and has high homology with the gene NST1-3 involved in the regulation of secondary cell wall synthesis in Arabidopsis. The function of this gene in the development of cotton fiber secondary cell wall has been studied in this experiment before. On the basis of previous work, the following results were obtained: 1. Phenotypic analysis of GhFSN1 transgenic cotton progeny in order to understand the regulatory function of GhFSN1 gene in cotton fiber development, we used GhFSN1 transgenic cotton as material. The genetic phenotype of transgenic cotton lines of T2 generation, T3 generation and T4 generation was analyzed. Firstly, through positive identification and expression analysis, we screened out transgenic cotton lines with significantly different expression levels compared with wild type. Leaf morphology, cotton fiber length, seed size, or cell wall thickness of cotton fiber were not significantly different from those of the wild type, but GhFSN1 overexpression decreased plant height, leaf warped, seed reduced, cotton fiber length significantly shortened, and fiber cell wall thickness increased. Statistical analysis of mature fiber length of transgenic cotton plants of T3 and T4 generations showed that the phenotype of transgenic cotton plants could be inherited steadily, whether they were RNAi transgenic cotton lines or overexpressed transgenic cotton lines. These results indicated that there were functional redundancy genes with GhFSN1 in cotton, and GhFSN1 played a role in cotton fiber development. 2. Fiber transcriptome analysis of GhFSN1 overexpression transgenic cotton progeny lines using transcriptome sequencing technology comparative analysis of GhFSN1 overexpression transgenic cotton and wild-type cotton 18 days after flowering changes in fiber gene transcripts, screening and identification of GhFSN1 regulated by the differential expression genes. Compared with type 2, 2857 genes were differentially expressed in transgenic cotton fibers overexpressing GhFSN1, of which 1486 genes were up-regulated and 1371 genes were down-regulated. The up-regulated genes were mainly concentrated in secondary metabolic biosynthesis, phenylalanine metabolism, flavonoid biosynthesis, aminoglycose and nucleotide metabolism. Phenylpropanoid biosynthesis and other pathways, the down-regulated genes are mainly concentrated in biosynthesis of secondary metabolites, biosynthesis of keratin, cork fat and wax, biosynthesis of unsaturated fatty acids and pectin, fatty acid metabolism and fatty acid elongation. We found that there are not only various transcription factors (homologous to transcription factors related to the regulation of secondary wall synthesis in Arabidopsis), but also a large number of genes synthesized by cellulose, xylan, pectin, fatty acids, cytoskeleton and so on. These genes may be related to secondary cell wall synthesis. These genes were verified by qRT-PCR and the results were analyzed by transcriptome. We speculate that GhFSN1 promotes the synthesis of cotton fiber secondary cell wall through different regulatory pathways. 3. Before pull-down validation of the interaction between GhFSN1 protein and GhE2 protein, our laboratory verified the interaction between GhFSN1 protein and GhE2 protein by yeast two-hybrid technology, suggesting that GhFSN1 protein is degraded by ubiquitination pathway. In this paper, we used pull-down method to verify the interaction between GhFSN1 protein and GhE2 protein. The expression vector of pMAL-GhE2 protein and the expression vector of pGEX-4T-1-GhFSNl protein were constructed and transfected into E.coli. The MBP-GhE2 protein and GST-GhFSN1 protein were purified respectively. The pull-down test showed that GhFSN1 protein could interact with GhE2 protein. Effect.
【学位授予单位】：华中师范大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：S562;Q943.2
，
本文编号：2192378
[Abstract]:Cotton plays an important role in the agricultural economy of China and even the world. The quality and yield of traditional cotton can not meet the needs of modern society. In order to solve this contradiction, transgenic technology is more and more widely used in cotton. Therefore, it is necessary to study the basic function of cotton genes. A NAC family gene, named GhFSN1, has been cloned from cotton in our laboratory. It is specifically expressed in the secondary cell wall synthesis phase of cotton fiber and has high homology with the gene NST1-3 involved in the regulation of secondary cell wall synthesis in Arabidopsis. The function of this gene in the development of cotton fiber secondary cell wall has been studied in this experiment before. On the basis of previous work, the following results were obtained: 1. Phenotypic analysis of GhFSN1 transgenic cotton progeny in order to understand the regulatory function of GhFSN1 gene in cotton fiber development, we used GhFSN1 transgenic cotton as material. The genetic phenotype of transgenic cotton lines of T2 generation, T3 generation and T4 generation was analyzed. Firstly, through positive identification and expression analysis, we screened out transgenic cotton lines with significantly different expression levels compared with wild type. Leaf morphology, cotton fiber length, seed size, or cell wall thickness of cotton fiber were not significantly different from those of the wild type, but GhFSN1 overexpression decreased plant height, leaf warped, seed reduced, cotton fiber length significantly shortened, and fiber cell wall thickness increased. Statistical analysis of mature fiber length of transgenic cotton plants of T3 and T4 generations showed that the phenotype of transgenic cotton plants could be inherited steadily, whether they were RNAi transgenic cotton lines or overexpressed transgenic cotton lines. These results indicated that there were functional redundancy genes with GhFSN1 in cotton, and GhFSN1 played a role in cotton fiber development. 2. Fiber transcriptome analysis of GhFSN1 overexpression transgenic cotton progeny lines using transcriptome sequencing technology comparative analysis of GhFSN1 overexpression transgenic cotton and wild-type cotton 18 days after flowering changes in fiber gene transcripts, screening and identification of GhFSN1 regulated by the differential expression genes. Compared with type 2, 2857 genes were differentially expressed in transgenic cotton fibers overexpressing GhFSN1, of which 1486 genes were up-regulated and 1371 genes were down-regulated. The up-regulated genes were mainly concentrated in secondary metabolic biosynthesis, phenylalanine metabolism, flavonoid biosynthesis, aminoglycose and nucleotide metabolism. Phenylpropanoid biosynthesis and other pathways, the down-regulated genes are mainly concentrated in biosynthesis of secondary metabolites, biosynthesis of keratin, cork fat and wax, biosynthesis of unsaturated fatty acids and pectin, fatty acid metabolism and fatty acid elongation. We found that there are not only various transcription factors (homologous to transcription factors related to the regulation of secondary wall synthesis in Arabidopsis), but also a large number of genes synthesized by cellulose, xylan, pectin, fatty acids, cytoskeleton and so on. These genes may be related to secondary cell wall synthesis. These genes were verified by qRT-PCR and the results were analyzed by transcriptome. We speculate that GhFSN1 promotes the synthesis of cotton fiber secondary cell wall through different regulatory pathways. 3. Before pull-down validation of the interaction between GhFSN1 protein and GhE2 protein, our laboratory verified the interaction between GhFSN1 protein and GhE2 protein by yeast two-hybrid technology, suggesting that GhFSN1 protein is degraded by ubiquitination pathway. In this paper, we used pull-down method to verify the interaction between GhFSN1 protein and GhE2 protein. The expression vector of pMAL-GhE2 protein and the expression vector of pGEX-4T-1-GhFSNl protein were constructed and transfected into E.coli. The MBP-GhE2 protein and GST-GhFSN1 protein were purified respectively. The pull-down test showed that GhFSN1 protein could interact with GhE2 protein. Effect.
【学位授予单位】：华中师范大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：S562;Q943.2
，
本文编号：2192378
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