本文關(guān)鍵詞：LOB1等轉(zhuǎn)錄因子對(duì)番茄果實(shí)軟化的調(diào)控　出處：《浙江大學(xué)》2017年博士論文　論文類型：學(xué)位論文

  更多相關(guān)文章： 番茄 軟化 轉(zhuǎn)錄調(diào)控 LOB1 MYB1/2

【摘要】：果實(shí)軟化伴隨著果實(shí)成熟進(jìn)程而發(fā)生,為不可逆的生理現(xiàn)象,直接影響了果實(shí)的貯藏物流和貨架壽命。番茄作為模式果實(shí)被廣泛用作研究試材,其軟化靶向研究主要集中于細(xì)胞壁降解酶,近年來相關(guān)轉(zhuǎn)錄因子相繼被分離并有研究報(bào)道,但仍十分有限,是否存在關(guān)鍵的轉(zhuǎn)錄因子轉(zhuǎn)錄調(diào)控果實(shí)軟化還不清楚。本文研究了 SlLOB1和SlMYB1/2對(duì)果實(shí)質(zhì)地細(xì)及胞壁組分的影響,明確了 SlLOB1是果實(shí)軟化的重要調(diào)控因子,主要結(jié)果如下。1.明確了SlLOB 的生物學(xué)功能。通過轉(zhuǎn)錄組分析,篩選得到果實(shí)特異表達(dá)的SlLOB1,且在果肉組織表達(dá)早于果皮組織,并與果實(shí)成熟軟化進(jìn)程呈正相關(guān)。進(jìn)一步研究發(fā)現(xiàn)LOB1RNAi果實(shí)的軟化速率減緩,而過表達(dá)LOB1促進(jìn)果實(shí)提前軟化。此外,不同于rin、Cnr等突變體,LOB1RNAi和過表達(dá)果實(shí)的成熟進(jìn)程完全正常,且LOB1 RNAi果實(shí)積累更多番茄紅素。2.探索了 SlLOB1參與果實(shí)軟化的調(diào)控靶標(biāo)。開展了 LOB1 RNAi果實(shí)的轉(zhuǎn)錄組分析,發(fā)現(xiàn)一系列與細(xì)胞壁代謝相關(guān)的基因在RNAi果實(shí)表達(dá)被顯著下調(diào),包括細(xì)胞壁降解酶,如果膠裂解酶(PL1-27)、甘露聚糖酶(MAN)、內(nèi)切葡聚糖酶(CEL2)、木糖苷酶(xY)、伸展蛋白(EXP1),以及其他一些細(xì)胞壁相關(guān)基因,如細(xì)胞壁結(jié)構(gòu)蛋白(AGP2)、細(xì)胞壁修飾蛋白(TBL)、細(xì)胞壁信號(hào)轉(zhuǎn)導(dǎo)候選基因(LRR)及一些細(xì)胞壁相關(guān)的轉(zhuǎn)錄因子。通過對(duì)其中10個(gè)細(xì)胞壁相關(guān)基因的Q-RT驗(yàn)證表明,其表達(dá)模式與RNA-seq表達(dá)數(shù)據(jù)一致。綜上所述,認(rèn)為LOB1是一個(gè)果實(shí)軟化激活子,可通過轉(zhuǎn)錄調(diào)控下游細(xì)胞壁軟化結(jié)構(gòu)基因,控制果實(shí)軟化進(jìn)程。3.解析了 SlLOB1參與果實(shí)軟化轉(zhuǎn)錄調(diào)控機(jī)制。根據(jù)RNA-seq結(jié)果,利用雙熒光素酶體系進(jìn)一步分析了 LOB1對(duì)靶標(biāo)基因的調(diào)控效應(yīng)。結(jié)果表明LOB1可誘導(dǎo)EXP1啟動(dòng)子活性,其倍數(shù)約為67倍,Q-RT檢測(cè)表明EXP1在LOB1RNAi果實(shí)中幾乎不表達(dá),通過Western雜交發(fā)現(xiàn)EXP1蛋白豐度在RNAi果實(shí)不可被檢測(cè),同時(shí)EXP1在過表達(dá)果實(shí)和葉片均被上調(diào),說明EXP1受LOB1蛋白直接調(diào)控。通過EXP1啟動(dòng)子刪剪實(shí)驗(yàn),確定了其啟動(dòng)子上順式結(jié)合元件TC-rich repeats是LOB1調(diào)控的直接靶點(diǎn)。4.獲得了調(diào)控纖維素合成的SlMYB1/2轉(zhuǎn)錄因子。分離得到兩個(gè)番茄MYB基因SlMYB1/2,與擬南芥纖維素合成相關(guān)的MYB83/46的同源。分析了 SlMYB1/2和次生細(xì)胞壁纖維素合成酶編碼基因SlCESA4/5/6在番茄植株不同組織的表達(dá)模式,明確了SlMYB1/2對(duì)SlCESA4/5/6啟動(dòng)子的轉(zhuǎn)錄激活功能,同時(shí)SlMYB1/2瞬時(shí)表達(dá)和永久異源過表達(dá)至煙草均可促進(jìn)纖維素的積累。因此,SlMYB1/2是調(diào)控纖維素合成的番茄MYB轉(zhuǎn)錄因子,是調(diào)控果實(shí)質(zhì)地的重要候選基因。
[Abstract]:Fruit softening occurs with the fruit ripening process, which is an irreversible physiological phenomenon, which directly affects the storage logistics and shelf life of the fruit. As a model fruit, tomato is widely used as a research material. In recent years, the related transcription factors have been isolated and reported, but they are still very limited. The effects of SlLOB1 and SlMYB1/2 on fruit texture and cell wall composition were studied. SlLOB1 is an important regulatory factor for fruit softening. The main results are as follows. 1. The biological function of SlLOB is clarified. Transcriptional analysis is carried out. The specific expression of SlLOB1 in fruit was obtained, and the expression was earlier in pulp tissue than in pericarp tissue. Further study found that the softening rate of LOB1RNAi fruit slowed down, while over-expression of LOB1 promoted the fruit softening ahead of time. In addition, it was different from rin. The maturation process of Cnr and other mutant Lob1 RNAi and over-expressed fruit was completely normal. And the fruit of LOB1 RNAi accumulated more lycopene. 2. The regulatory target of SlLOB1 involved in fruit softening was explored. Transcriptome analysis of LOB1 RNAi fruit was carried out. It was found that a series of genes related to cell wall metabolism were significantly down-regulated in RNAi fruits, including cell wall degrading enzymes, such as PL1-27, mannanase (MAN). Endoglucanase (CEL2), xylosidase (XY), extensin (EXP1), and other cell wall related genes, such as cell wall structural protein (AGP2). Cell wall modified protein (TBLN), a cell wall signal transduction candidate gene (LRRRR), and some cell wall related transcription factors. Q-RT analysis of 10 of the cell wall related genes was carried out. In conclusion, LOB1 is a fruit softening activator, which can regulate the downstream cell wall softening structure gene by transcription. The mechanism of SlLOB1 involved in the transcription regulation of fruit softening was analyzed. According to the results of RNA-seq. Double luciferase system was used to further analyze the regulatory effect of LOB1 on target gene. The results showed that LOB1 could induce the activity of EXP1 promoter, and the multiple was about 67 times. Q-RT analysis showed that EXP1 was almost not expressed in LOB1RNAi fruit, and EXP1 protein abundance could not be detected in RNAi fruit by Western hybridization. At the same time, EXP1 expression was up-regulated in both fruit and leaf, indicating that EXP1 was directly regulated by LOB1 protein. EXP1 promoter was used to prune. The cis-binding element TC-rich on its promoter was determined. Repeats is the direct target of LOB1 regulation. 4. SlMYB1/2 transcription factors regulating cellulose synthesis were obtained. Two tomato MYB gene SlMYB1/2 were isolated. The homology of MYB83/46 related to cellulose synthesis in Arabidopsis thaliana was analyzed. Expression patterns of SlMYB1/2 and Cellulose Synthase coding Gene SlCESA4/5/6 in different tissues of Tomato. The transcriptional activation of SlCESA4/5/6 promoter by SlMYB1/2 was clarified. At the same time, transient expression of SlMYB1/2 and permanent heterogenous overexpression to tobacco could promote the accumulation of cellulose. Therefore, SlMYB 1 / 2 is a MYB transcription factor regulating cellulose synthesis in tomato. It is an important candidate gene to regulate fruit texture.
【學(xué)位授予單位】：浙江大學(xué)
【學(xué)位級(jí)別】：博士
【學(xué)位授予年份】：2017
【分類號(hào)】：S641.2

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