GhFSN1轉(zhuǎn)基因棉花后代株系的遺傳與生化分析
發(fā)布時(shí)間：2018-08-19 17:50
【摘要】：棉花在中國(guó)乃至世界各國(guó)農(nóng)業(yè)經(jīng)濟(jì)中都處于重要地位,傳統(tǒng)棉花的品質(zhì)和產(chǎn)量已經(jīng)難以滿足現(xiàn)代社會(huì)的需求,為解決這個(gè)矛盾,轉(zhuǎn)基因技術(shù)在棉花上的運(yùn)用于是越來(lái)越廣泛,因此關(guān)于棉花基因功能的基礎(chǔ)性研究,也就顯得十分必要了。本實(shí)驗(yàn)室在棉花中克隆出一個(gè)NAC家族基因,命名為GhFSN1,它在棉纖維次生細(xì)胞壁合成期特異表達(dá),與擬南芥中參與次生細(xì)胞壁合成調(diào)控的基因NST1-3同源性較高,之前本實(shí)驗(yàn)已經(jīng)對(duì)該基因在棉花纖維次生細(xì)胞壁發(fā)育中的功能進(jìn)行了較為深入的研究,本文在之前工作的基礎(chǔ)上,對(duì)GhFSN1轉(zhuǎn)基因棉花后代株系的遺傳及生化等方面進(jìn)行一些探究,取得了如下研究結(jié)果:1、GhFSN1轉(zhuǎn)基因棉花后代表型分析為了解GhFSN1基因在棉花纖維發(fā)育中的調(diào)控功能,我們以GhFSN1轉(zhuǎn)基因棉花為材料,對(duì)T2代、T3代和T4代轉(zhuǎn)基因棉花株系進(jìn)行了遺傳表型分析。首先通過(guò)陽(yáng)性鑒定和表達(dá)分析,我們篩選出了表達(dá)量相對(duì)于野生型具有明顯差異的轉(zhuǎn)基因棉花株系。對(duì)T2代轉(zhuǎn)基因棉花的表型進(jìn)行觀察,我們發(fā)現(xiàn)GhFSN1 RNAi植株不論在植株高度、葉片形態(tài)、棉纖維長(zhǎng)度、種子大小,還是在棉纖維細(xì)胞壁厚度上,都與野生型無(wú)明顯差別;而GhFSN1過(guò)量表達(dá)植株,植株高度有所下降,葉片出現(xiàn)上翹,種子變小,棉纖維長(zhǎng)度明顯變短,而纖維細(xì)胞壁的厚度增加。以上表型與T1代植株一致。繼續(xù)對(duì)T3代和T4代轉(zhuǎn)基因棉花植株成熟纖維進(jìn)行長(zhǎng)度統(tǒng)計(jì)分析發(fā)現(xiàn),不論是RNAi轉(zhuǎn)基因棉花株系,還是過(guò)量表達(dá)轉(zhuǎn)基因棉花株系,其表型均能穩(wěn)定遺傳。上述結(jié)果暗示在棉花中存在與GhFSN1功能冗余基因,同時(shí)也表明GhFSN1在棉纖維發(fā)育中發(fā)揮著十分重要的作用。2、GhFSN1過(guò)表達(dá)轉(zhuǎn)基因棉花后代株系的纖維轉(zhuǎn)錄組分析采用轉(zhuǎn)錄組測(cè)序技術(shù)比較分析了GhFSN1過(guò)量表達(dá)轉(zhuǎn)基因棉花和野生型棉花開花后18天的纖維中基因轉(zhuǎn)錄本變化,篩查鑒定受GhFSN1調(diào)控的差異表達(dá)基因。結(jié)果發(fā)現(xiàn),與野生型相比較,在T2代GhFSN1過(guò)量表達(dá)轉(zhuǎn)基因棉花纖維中共有2857個(gè)基因發(fā)生了差異表達(dá),其中1486個(gè)基因上調(diào)表達(dá),1371個(gè)基因下調(diào)表達(dá)。上調(diào)表達(dá)的基因主要富集在次級(jí)代謝生物合成、苯丙氨酸代謝、類黃酮生物合成、氨基糖和核苷酸糖代謝與苯丙烷類生物合成過(guò)程等途徑中,而下調(diào)表達(dá)的基因主要富集在次級(jí)代謝物的生物合成、角質(zhì)、軟木脂和蠟質(zhì)的生物合成、不飽和脂肪酸及果膠的生物合成以及脂肪酸代謝和脂肪酸延伸過(guò)程等途徑中。進(jìn)一步對(duì)各差異表達(dá)基因進(jìn)行分析,我們發(fā)現(xiàn),不僅有各種轉(zhuǎn)錄因子(與擬南芥次生壁合成調(diào)控相關(guān)轉(zhuǎn)錄因子同源),還包括大量纖維素、木聚糖、果膠、脂肪酸、細(xì)胞骨架等合成的基因,這些基因都可能與次生細(xì)胞壁合成相關(guān)。對(duì)這些基因進(jìn)行qRT-PCR驗(yàn)證,結(jié)果與轉(zhuǎn)錄組分析一致。我們推測(cè)GhFSN1基因通過(guò)不同調(diào)控途徑促進(jìn)棉纖維次生細(xì)胞壁的合成。3、pull-down驗(yàn)證GhFSN1蛋白與GhE2蛋白的互作之前本實(shí)驗(yàn)室通過(guò)酵母雙雜交等技術(shù),驗(yàn)證了 GhFSN1蛋白與GhE2蛋白的互作,暗示GhFSN1蛋白通過(guò)泛素化途徑降解,本文通過(guò)pull-down技術(shù)驗(yàn)證這一結(jié)果。分別構(gòu)建了 pMAL-GhE2蛋白表達(dá)載體及pGEX-4T-1-GhFSNl蛋白表達(dá)載體,導(dǎo)入大腸桿菌進(jìn)行表達(dá),并對(duì)MBP-GhE2蛋白與GST-GhFSN1蛋白分別進(jìn)行了純化,然后進(jìn)行pull-down實(shí)驗(yàn)驗(yàn)證,結(jié)果顯示,GhFSN1蛋白確實(shí)能與GhE2蛋白的發(fā)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.
【學(xué)位授予單位】：華中師范大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2017
【分類號(hào)】：S562;Q943.2
，
本文編號(hào)：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.
【學(xué)位授予單位】：華中師范大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2017
【分類號(hào)】：S562;Q943.2
，
本文編號(hào)：2192378
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