發(fā)布時(shí)間：2018-05-15 00:19

  本文選題：番茄 + 花青素��； 參考：《中國(guó)農(nóng)業(yè)科學(xué)院》2016年博士論文

【摘要】：花青素是植物體內(nèi)一類重要的次生代謝物質(zhì),不僅能吸引昆蟲傳粉和協(xié)助種子傳播,還能提高植物對(duì)生物和非生物脅迫的抵抗能力。近年來(lái),在擬南芥、玉米和矮牽牛中已克隆了大量的參與花青素合成調(diào)控的轉(zhuǎn)錄因子,但番茄中研究相對(duì)滯后,且都集中在R2R3-MYB型轉(zhuǎn)錄因子。為此,本研究通過(guò)圖位克隆的方法在番茄中克隆了第一個(gè)參與花青素合成調(diào)控的b HLH型轉(zhuǎn)錄因子AH,并結(jié)合低溫誘導(dǎo)、低溫冷害試驗(yàn)以及轉(zhuǎn)錄組試驗(yàn)研究了AH基因的功能及其對(duì)提高番茄幼苗耐低溫的作用,旨在為番茄花青素合成調(diào)控機(jī)理和番茄耐低溫育種研究提供參考依據(jù)。主要結(jié)果如下:1.FMTT271中花青素缺失表型受一個(gè)單隱性基因控制,且與AH基因?yàn)榈任换��?因此,將FMTT271中花青素缺失基因命名為ah。應(yīng)用圖位克隆的方法,我們將ah基因定位于番茄9號(hào)染色體長(zhǎng)臂CAPS標(biāo)記CAPS2和CAPS4之間,其物理距離約為130kb,根據(jù)番茄參考基因組注釋信息(ITAG2.4),該區(qū)間共有5個(gè)ORFs。2.根據(jù)同源分析結(jié)果,結(jié)合基因表達(dá)分析,推測(cè)ORF5(Solyc09g065100)為AH基因。c DNA全長(zhǎng)測(cè)序結(jié)果表明,ah突變體中Solyc09g065100基因的第6個(gè)外顯子上一個(gè)堿基(G到T)的突變,使Gly突變成終止密碼子,導(dǎo)致Solyc09g065100基因翻譯提前終止。將Solyc09g065100基因在ah突變體FMTT271中過(guò)量表達(dá),轉(zhuǎn)基因番茄T1均能夠互補(bǔ)FMTT271的表型,且葉片和果實(shí)中均大量積累花青素。以上試驗(yàn)結(jié)果表明Solyc09g065100即為AH。結(jié)合q RT-PCR分析結(jié)果及轉(zhuǎn)錄激活試驗(yàn),我們推測(cè)AH可能通過(guò)上調(diào)花青素合成途徑中晚期結(jié)構(gòu)基因的表達(dá)促進(jìn)花青素的合成。3.AH基因受發(fā)育調(diào)控,進(jìn)而下調(diào)花青素合成途徑中結(jié)構(gòu)基因的表達(dá),抑制花青素的合成。此外,低溫可能通過(guò)激活A(yù)H的表達(dá)進(jìn)而促進(jìn)番茄葉片中花青素的積累。二氨基聯(lián)苯胺(DAB)和臺(tái)盼藍(lán)染色試驗(yàn)表明,AH基因能夠提高番茄幼苗對(duì)低溫的抵抗能力。4.RNA-seq分析結(jié)果發(fā)現(xiàn),NIL-PH和NIL-GH下胚軸中共含有551差異表達(dá)基因(DEGs)。相比于NIL-GH,在NIL-PH中285個(gè)表現(xiàn)為上調(diào),266個(gè)表現(xiàn)為下調(diào)。GO terms分析表明PH/GH上調(diào)基因顯著的富集于“響應(yīng)非生物脅迫刺激”、“響應(yīng)激素刺激”和“類黃酮物質(zhì)合成”,而下調(diào)基因顯著的富集于“響應(yīng)非生物脅迫刺激”、“氧化還原反應(yīng)”和“節(jié)律過(guò)程”。此外,根據(jù)低溫誘導(dǎo)番茄葉片RNA-seq分析結(jié)果發(fā)現(xiàn),39個(gè)基因的表達(dá)最有可能受AH的調(diào)控,其中10個(gè)在16-PL中表現(xiàn)為上調(diào),包括花青素合成基因F3’5’H和DFR。GO term分析表明上調(diào)基因主要富集于“苯丙烷生物合成”、“細(xì)胞內(nèi)氨基酸衍生物物質(zhì)合成”和“芳香類物質(zhì)合成”,而下調(diào)基因主要富集于“響應(yīng)碳水化合物的刺激”。5.基因表達(dá)模式分析結(jié)果表明,在6個(gè)RNA-seq樣品中,Sl AN2和Sl ANT1-like的表達(dá)模式與AH相一致(相關(guān)系數(shù)分別為0.938和0.818),推測(cè)AH可能與Sl AN2和Sl ANT1-like相互作用,共同調(diào)控花青素的合成。根據(jù)轉(zhuǎn)基因試驗(yàn)表達(dá)分析以及RNA-seq分析結(jié)果,我們推測(cè)AH可能直接作用于F3’5’H和DFR。順式作用元件分析結(jié)果表明,在F3’5’H和DFR兩個(gè)基因的轉(zhuǎn)錄位點(diǎn)上游(-2000bp和-1500bp)均含有大量的b HLH結(jié)合位點(diǎn)E-box(CANNTG)。
[Abstract]:Anthocyanins are an important secondary metabolite in plants, which not only attract insect pollination and assist seed transmission, but also enhance resistance to biological and abiotic stresses. In recent years, a large number of transcription factors involved in the synthesis and regulation of anthocyanins have been cloned in Arabidopsis, corn and Petunia, but in tomato research phase In this study, we cloned the first B HLH type transcription factor AH, which was involved in the regulation of anthocyanin synthesis in tomato, and combined with low temperature induction, low temperature cold injury test and transcriptional test to study the function of AH gene and to improve the low temperature tolerance of tomato seedlings. The main results are as follows: the anthocyanin deletion phenotype in 1.FMTT271 is controlled by a single recessive gene, and the AH gene is the allele. Therefore, the anthocyanin deletion gene in FMTT271 is named as the ah. application map cloning method, We locate the ah gene between the CAPS marker CAPS2 and CAPS4 of the long arm of the tomato chromosome 9. The physical distance is about 130kb. According to the reference genomic information of tomato (ITAG2.4), there are 5 ORFs.2. according to the results of homologous analysis and the analysis of gene expression. It is concluded that ORF5 (Solyc09g065100) is the.C DNA whole sequence of AH gene. The mutation of one base (G to T) of the sixth exons of the Solyc09g065100 gene in the mutant of the H mutant causes Gly to turn into a terminating codon and lead to the early termination of the Solyc09g065100 gene translation. The Solyc09g065100 gene is overexpressed in the ah mutant FMTT271, and the transgenic tomato T1 can both complement the FMTT271 phenotype and have a large number of leaves and fruits. The above results show that Solyc09g065100 is AH. binding Q RT-PCR analysis and transcriptional activation test. We speculate that AH may promote the development regulation of anthocyanin synthesis.3.AH gene by up regulation of the expression of late structure gene in the anthocyanin synthesis pathway, and then down down the table of structural genes in the anthocyanin synthesis pathway. Two amino diphenyl amine (DAB) and trypan blue staining test showed that the AH gene could improve the resistance of tomato seedlings to low temperature by.4.RNA-seq analysis. The results showed that the NIL-PH and NIL-GH hypocotyls were 551 poor in the NIL-PH and NIL-GH hypocotyls. Different expression genes (DEGs). Compared with NIL-GH, 285 up regulation in NIL-PH and 266 down regulated.GO terms analysis showed that PH/GH up regulation genes were significantly enriched in "response to abiotic stress stimulation", "response to hormone stimulation" and "flavonoid synthesis", while the down regulated genes were significantly enriched in "response to abiotic stress spines." In addition, according to the RNA-seq analysis of tomato leaves induced by low temperature, the expression of 39 genes was most likely to be regulated by AH, and 10 of them were up-regulated in 16-PL, including the anthocyanin synthesis gene F3 '5' H and DFR.GO term analysis indicating that the up-regulated genes were mainly enriched in benzene. Propane biosynthesis, "synthesis of amino acid derivatives in cells" and "synthesis of aromatic substances", and the down regulated genes are mainly enriched in the "response to carbohydrate stimulus".5. gene expression pattern analysis results show that in 6 RNA-seq samples, the expression patterns of Sl AN2 and Sl ANT1-like are in accordance with AH (correlation coefficient, respectively) For 0.938 and 0.818), it is presumed that AH may interact with Sl AN2 and Sl ANT1-like to jointly regulate the synthesis of anthocyanins. According to the expression analysis of the transgenic test and the results of RNA-seq analysis, we speculate that AH may directly act on F3 '5' H and DFR. cis acting elements, indicating that the F3 '5' H and the transcriptional sites of two genes are on the F3 '5. Both -2000bp and -1500bp contain a large number of B HLH binding sites E-box (CANNTG).

【學(xué)位授予單位】：中國(guó)農(nóng)業(yè)科學(xué)院
【學(xué)位級(jí)別】：博士
【學(xué)位授予年份】：2016
【分類號(hào)】：S641.2

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相關(guān)期刊論文 前1條

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本文編號(hào)：1890132