【摘要】：高溫條件下,生物體普遍的反應(yīng)是產(chǎn)生熱激蛋白,它在針對(duì)高溫對(duì)農(nóng)作物減產(chǎn)和品質(zhì)下降時(shí)起著至關(guān)重要的作用。生物體在高于正常生長(zhǎng)溫度5°C時(shí)就會(huì)迅速合成一些熱激蛋白在體內(nèi),而調(diào)控這些熱激蛋白和其他熱激誘導(dǎo)因子表達(dá)的調(diào)控因子-熱激轉(zhuǎn)錄因子(Heat shock transcription factor,HSF),對(duì)提高作物耐熱性能具有重要作用。目前,在擬南芥、水稻、楊樹(shù)、大白菜、玉米和蘋(píng)果等模式植物中對(duì)部分的HSF基因進(jìn)行了研究和報(bào)道,這為我們分析楊樹(shù)、擬南芥、水稻、和葡萄四種被子植物HSF基因家族的分子進(jìn)化提供了條件。本研究利用生物信息學(xué)軟件和在線網(wǎng)站等全基因組數(shù)據(jù)庫(kù),對(duì)這四種被子植物的相關(guān)基因鑒定分析、系統(tǒng)進(jìn)化樹(shù)、基因結(jié)構(gòu)和保守基序、基因復(fù)制事件、種間/種內(nèi)的微共線性分析、復(fù)制時(shí)間和選擇壓力分析、表達(dá)譜分析以及表達(dá)模式分析等方面全面展開(kāi)研究,獲得主要結(jié)果如下:1.擬南芥和水稻的HSF基因分別在Arabidopsis Information Resource和Rice annotation database網(wǎng)站上下載,楊樹(shù)和葡萄用模式植物擬南芥標(biāo)準(zhǔn)HSF蛋白序列為搜索條件,在Phytozome網(wǎng)站中進(jìn)行搜索下載,隨后通過(guò)ClustalX比對(duì),Pfam和SMART篩選和驗(yàn)證,最終確定楊樹(shù)有30個(gè)HSF基因,葡萄有10個(gè)HSF基因。2.對(duì)來(lái)自四個(gè)物種中的87個(gè)HSF基因,基于OD結(jié)構(gòu)域不同,將其劃分為A、B和C三個(gè)亞族。其中,A亞族又可以劃分為A1-9九個(gè)小亞族,B亞族被劃分為B1-5五個(gè)小亞族。3.對(duì)這四種被子植物中20對(duì)同源基因?qū)Φ膬?nèi)含子/外顯子進(jìn)行分析,位于同一亞族的相鄰基因,由于其親緣關(guān)系較近,都有著相似的基因結(jié)構(gòu),即相同的內(nèi)含子數(shù)目和外顯子長(zhǎng)度。其中,只有兩對(duì)同源基因(OsHsf-08/-21 and PtHsf-16/-20)的結(jié)構(gòu)不同,可能是由于內(nèi)含子在長(zhǎng)期進(jìn)化過(guò)程中的獲得或丟失導(dǎo)致的。利用MEME在線網(wǎng)站對(duì)HSF蛋白的保守基序進(jìn)行分析,再結(jié)合Pfam和SMART對(duì)HSF的保守基序進(jìn)行分析�；�1是HSF最常見(jiàn)的核心基序。4.通過(guò)四種被子植物內(nèi)的微共線性分析,說(shuō)明了HSF基因家族的擴(kuò)張主要通過(guò)大規(guī)模復(fù)制事件,而并非串聯(lián)復(fù)制。18組(31個(gè))同源基因?qū)⑴c了片段復(fù)制事件。其中,13對(duì)來(lái)自楊樹(shù)基因組,4對(duì)來(lái)自水稻基因組,1對(duì)來(lái)自擬南芥基因組,而葡萄中并沒(méi)有旁系同源關(guān)系的HSF基因區(qū)段。5.擬南芥、水稻以及葡萄的基因組中包含的HSF基因區(qū)段與楊樹(shù)HSF基因的復(fù)制區(qū)段具有廣泛的共線性,共形成了15組直系同源基因組。可能楊樹(shù)在經(jīng)歷兩輪遠(yuǎn)古基因復(fù)制事件后,73.3%的遠(yuǎn)古復(fù)制基因被保留下來(lái)。水稻有32%的基因被保留,擬南芥僅有9%,而葡萄的復(fù)制基因全部丟失了。6.選擇壓力分析表明在被子植物進(jìn)化過(guò)程中,純化作用依舊發(fā)揮著主要作用,但在個(gè)別基因部分編碼區(qū)域依然存在著強(qiáng)烈的正向選擇,這些擁有正向選擇的基因可能導(dǎo)致物種功能的進(jìn)化。7.微陣列數(shù)據(jù)分析結(jié)果顯示,大多數(shù)HSF基因在不同組織中都有著明顯的特異性表達(dá)。其中,7組片段復(fù)制基因?qū)τ兄嗨频谋磉_(dá)模式,僅有兩組(PtHsf-19/-17和PtHsf-23/-27)復(fù)制基因?qū)Φ谋磉_(dá)模式略有不同。8.利用qRT-PCR技術(shù)對(duì)楊樹(shù)HSF的全基因家族進(jìn)行熱激處理、冷脅迫處理和氧化脅迫處理三種誘導(dǎo)表達(dá)模式進(jìn)行分析。結(jié)果表明,大部分HSF基因都受這三種脅迫的誘導(dǎo)。PtHsf-10受到強(qiáng)烈的熱激誘導(dǎo)表達(dá),為此我們克隆了PtHsf-10的全長(zhǎng)基因,構(gòu)建其表達(dá)載體轉(zhuǎn)化擬南芥,為進(jìn)一步開(kāi)展其功能的研究奠定基礎(chǔ)。
[Abstract]:At high temperatures, the general response of organisms is the production of heat shock proteins, which play a vital role in the reduction of crop yield and quality at high temperatures. When organisms grow above the normal temperature of 5 degrees C, they rapidly synthesize some heat shock proteins in vivo and regulate the expression of these heat shock proteins and other heat shock inducible factors. Heat shock transcription factor (HSF) plays an important role in improving the heat tolerance of crops. At present, some HSF genes have been studied and reported in Arabidopsis, rice, poplar, Chinese cabbage, maize and apple model plants, which can be used to analyze the four quilts of poplar, Arabidopsis, rice and grape. Molecular evolution of the HSF gene family in plants provides the basis for this study. Using bioinformatics software and genome-wide databases such as online websites, we have identified and analyzed genes related to the four angiosperms, phylogenetic trees, gene structures and conserved motifs, gene replication events, interspecific/intraspecific microcollinearity analysis, replication time and selection. The main results are as follows: 1. The HSF genes of Arabidopsis and rice were downloaded from Arabidopsis Information Resource and Rice annotation database websites respectively, and the standard HSF protein sequences of poplar and grape model plant Arabidopsis thaliana were used as search conditions. After searching and downloading on Phytozome, through ClustalX comparison, Pfam and SMART screening and validation, 30 HSF genes were identified in poplar and 10 HSF genes were identified in grape. 2. For 87 HSF genes from four species, they were divided into three subgroups A, B and C based on OD domain. The intron/exon of 20 pairs of homologous gene pairs in the four angiosperms were analyzed. The adjacent genes in the same subgroup had similar gene structure, i.e. the same intron number and exon length, because of their close genetic relationship. The structure of the source gene (OsHsf-08/-21 and PtHsf-16/-20) is different, which may be caused by the acquisition or loss of introns during long-term evolution. The conserved motifs of HSF protein were analyzed by MEME online website, and then the conserved motifs of HSF were analyzed by Pfam and SMART. Microcollinearity analysis in angiosperms showed that the expansion of HSF gene family was mainly through large-scale replication events, not tandem replication. 18 pairs (31) of homologous genes participated in fragment replication events. The HSF gene fragments in Arabidopsis, rice and grape genomes have extensive collinearity with the replication fragments of poplar HSF gene, forming 15 groups of direct homologous genomes. It is possible that 73.3% of the ancient replication genes in poplar have been preserved after two rounds of ancient gene replication events. Thirty-two percent of the genes in rice were retained, only 9 percent in Arabidopsis, and all the replication genes in grape were lost. 6. Selection stress analysis showed that purification still played a major role in angiosperm evolution, but there were still strong positive selections in some coding regions of individual genes, and these genes with positive selections were possible. The results of microarray analysis showed that most HSF genes were expressed specifically in different tissues. Seven groups of HSF gene pairs had similar expression patterns, and only two groups (PtHsf-19/-17 and PtHsf-23/-27) had slightly different expression patterns. The results showed that most of the HSF genes were induced by these three stresses. PtHsf-10 was strongly induced by heat shock. Therefore, we cloned the full-length gene of PtHsf-10 and constructed its expression vector. Arabidopsis thaliana lays the foundation for further research on its function.
【學(xué)位授予單位】：安徽農(nóng)業(yè)大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2016
【分類號(hào)】：Q943.2

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本文編號(hào)：2247588