本文選題：陸地棉 + GhFLP1��； 參考：《西北農(nóng)林科技大學(xué)》2016年碩士論文

【摘要】：棉花作為主要的紡織纖維和油料種子作物,是一種重要的經(jīng)濟(jì)作物。我國(guó)當(dāng)前土地資源緊張,棉花種植面積銳減,培育短季棉品種,實(shí)現(xiàn)麥棉兩熟是解決這一問(wèn)題的重要途徑。開(kāi)花是植物從營(yíng)養(yǎng)生長(zhǎng)進(jìn)入生殖生長(zhǎng)的重要轉(zhuǎn)變過(guò)程,作物的早熟性與開(kāi)花密切相關(guān),所以了解和掌握作物開(kāi)花規(guī)律從而調(diào)控這一過(guò)程對(duì)于提高棉花的早熟性有重要意義。FLP1(flowering-promoting factor 1-like protein 1)基因,是擬南芥中開(kāi)花促進(jìn)因子基因FPF1(flowering-promoting factor-1)的同源基因,目前在擬南芥中并沒(méi)有對(duì)FLP1基因進(jìn)行深入的功能驗(yàn)證。本研究從陸地棉中克隆得到了FLP1的同源基因GhFLP1,對(duì)其進(jìn)行生物信息學(xué)分析,表達(dá)模式分析以及轉(zhuǎn)擬南芥等試驗(yàn)對(duì)其功能進(jìn)行了深入研究。根據(jù)實(shí)驗(yàn)室原有研究結(jié)果,對(duì)轉(zhuǎn)GhFPF1基因棉花進(jìn)行了功能研究,初步明確了其在棉花中的作用,試驗(yàn)結(jié)果如下:1、經(jīng)克隆測(cè)序得到GhFLP1基因長(zhǎng)537 bp,無(wú)內(nèi)含子,包含339 bp開(kāi)放閱讀框,編碼112個(gè)氨基酸。其蛋白質(zhì)相對(duì)分子質(zhì)量為12.176 kDa,理論等電點(diǎn)為9.13。GhFLP1與其他物種FLP1蛋白序列存在明顯保守區(qū)域,與雷蒙德氏棉D(zhuǎn)基因組中的FLP1序列相似性高達(dá)99%。2、外源激素處理試驗(yàn)證實(shí)GhFLP1能夠響應(yīng)GA和SA的處理,表明GhFLP1可能參與植物開(kāi)花和逆境脅迫調(diào)控。3、在不同品種中表達(dá)模式分析發(fā)現(xiàn)GhFLP1呈現(xiàn)出比較強(qiáng)烈的組織表達(dá)特異性,在花蕾中的表達(dá)量遠(yuǎn)遠(yuǎn)高于其他組織。結(jié)果還表明,該基因在早熟品種中棉所36、中棉所74花蕾中的表達(dá)量明顯高于晚熟品種中棉所60、魯棉研28。4、在擬南芥中過(guò)表達(dá)GhFLP1,轉(zhuǎn)基因擬南芥開(kāi)花時(shí)間提前,蓮座葉數(shù)目減少,轉(zhuǎn)基因擬南芥中一些開(kāi)花時(shí)間促進(jìn)基因如AtFT、AtAP1、AtLFY、AtSOC1表達(dá)量均有升高,抑制開(kāi)花基因AtFLC表達(dá)量明顯下調(diào)。5、構(gòu)建了棉花VIGS載體pCLCrVA-GhFLP1,對(duì)棉花進(jìn)行接種。發(fā)病植株的GhFLP1表達(dá)量下調(diào),目前植株表型并沒(méi)有觀察到明顯變化。6、對(duì)轉(zhuǎn)GhFPF1基因棉花進(jìn)行表型觀察,發(fā)現(xiàn)轉(zhuǎn)基因株系與野生型相比花期延遲,植株變高。水培結(jié)果表明,轉(zhuǎn)基因株系的根系變得更加發(fā)達(dá)。qRT-PCR結(jié)果表明轉(zhuǎn)基因株系中,GhFPF1在根和莖中的表達(dá)量遠(yuǎn)遠(yuǎn)高于其他組織。
[Abstract]:As the main seed crop of textile fiber and oil, cotton is an important cash crop. At present, the land resources in our country are tight, the planting area of cotton is decreasing sharply, the important way to solve this problem is to cultivate short season cotton varieties and to realize wheat and cotton double cropping. Flowering is an important process of plant transformation from vegetative growth to reproductive growth, and the early maturity of crops is closely related to flowering. Therefore, understanding and mastering the rules of crop flowering to regulate this process is of great significance for improving the precocity of cotton. FLP1flowering-promoting factor 1-like protein 1) gene is the homologous gene of FPF1(flowering-promoting factor-1) in Arabidopsis thaliana. At present, there is no in-depth functional verification of FLP1 gene in Arabidopsis thaliana. In this study, the homologous gene GhFLP1 of FLP1 was cloned from upland cotton, and its function was studied by bioinformatics analysis, expression pattern analysis and transgenic Arabidopsis thaliana test. According to the original results of laboratory studies, the function of transgenic cotton with GhFPF1 gene was studied and its function in cotton was preliminarily determined. The results were as follows: 1. The length of GhFLP1 gene was 537 BP, and the length of GhFLP1 gene was 537 BP, which contained 339bp open reading frame. Encode 112 amino acids. The relative molecular weight of its protein is 12.176 kDa.The theoretical isoelectric point (9.13.GhFLP1) is conserved with other species' FLP1 protein sequence. The similarity of the FLP1 sequence with the D genome of Raymond's cotton is as high as 99.2. The exogenous hormone treatment test proves that GhFLP1 can respond to the treatment of GA and SA. The results showed that GhFLP1 might be involved in plant flowering and stress regulation. The expression patterns of GhFLP1 in different cultivars showed strong tissue expression specificity, and the expression level in flower buds was much higher than that in other tissues. The results also showed that the expression of this gene in the buds of early maturing varieties Zhongmiansuo 36 and Zhongmiaojiao74 was significantly higher than that of late varieties Zhongmiansuo 60 and Lumiyan 28.4.The expression of GhFLP1 in Arabidopsis thaliana was overexpressed. The flowering time of transgenic Arabidopsis thaliana was earlier and the number of rosette leaves was decreased. Some flowering time promoting genes in transgenic Arabidopsis thaliana, such as AtFTFT-AtAP1, AtLFYYN, AtSOC1, were all increased, and the expression of inhibiting flowering gene AtFLC was significantly down-regulated. The cotton VIGS vector pCLCrVA-GhFLP1 was constructed to inoculate cotton. The expression of GhFLP1 in the infected plants was down-regulated and no obvious phenotypic changes were observed at present. The phenotypic changes of transgenic cotton with GhFPF1 gene were observed. It was found that the flowering period of transgenic lines was longer than that of wild-type plants and the plants were higher than those of wild-type plants. The results of hydroponic culture showed that the root system of transgenic lines became more developed. QRT-PCR results showed that the expression of GhFPF1 in roots and stems of transgenic lines was much higher than that in other tissues.
【學(xué)位授予單位】：西北農(nóng)林科技大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2016
【分類(lèi)號(hào)】：S562

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本文編號(hào)：1962307