LOB1等转录因子对番茄果实软化的调控

发布时间：2018-01-01 05:27

本文关键词：LOB1等转录因子对番茄果实软化的调控　出处：《浙江大学》2017年博士论文　论文类型：学位论文

更多相关文章：番茄软化 转录调控 LOB1 MYB1/2

【摘要】：果实软化伴随着果实成熟进程而发生,为不可逆的生理现象,直接影响了果实的贮藏物流和货架寿命。番茄作为模式果实被广泛用作研究试材,其软化靶向研究主要集中于细胞壁降解酶,近年来相关转录因子相继被分离并有研究报道,但仍十分有限,是否存在关键的转录因子转录调控果实软化还不清楚。本文研究了 SlLOB1和SlMYB1/2对果实质地细及胞壁组分的影响,明确了 SlLOB1是果实软化的重要调控因子,主要结果如下。1.明确了SlLOB 的生物学功能。通过转录组分析,筛选得到果实特异表达的SlLOB1,且在果肉组织表达早于果皮组织,并与果实成熟软化进程呈正相关。进一步研究发现LOB1RNAi果实的软化速率减缓,而过表达LOB1促进果实提前软化。此外,不同于rin、Cnr等突变体,LOB1RNAi和过表达果实的成熟进程完全正常,且LOB1 RNAi果实积累更多番茄红素。2.探索了 SlLOB1参与果实软化的调控靶标。开展了 LOB1 RNAi果实的转录组分析,发现一系列与细胞壁代谢相关的基因在RNAi果实表达被显著下调,包括细胞壁降解酶,如果胶裂解酶(PL1-27)、甘露聚糖酶(MAN)、内切葡聚糖酶(CEL2)、木糖苷酶(xY)、伸展蛋白(EXP1),以及其他一些细胞壁相关基因,如细胞壁结构蛋白(AGP2)、细胞壁修饰蛋白(TBL)、细胞壁信号转导候选基因(LRR)及一些细胞壁相关的转录因子。通过对其中10个细胞壁相关基因的Q-RT验证表明,其表达模式与RNA-seq表达数据一致。综上所述,认为LOB1是一个果实软化激活子,可通过转录调控下游细胞壁软化结构基因,控制果实软化进程。3.解析了 SlLOB1参与果实软化转录调控机制。根据RNA-seq结果,利用双荧光素酶体系进一步分析了 LOB1对靶标基因的调控效应。结果表明LOB1可诱导EXP1启动子活性,其倍数约为67倍,Q-RT检测表明EXP1在LOB1RNAi果实中几乎不表达,通过Western杂交发现EXP1蛋白丰度在RNAi果实不可被检测,同时EXP1在过表达果实和叶片均被上调,说明EXP1受LOB1蛋白直接调控。通过EXP1启动子删剪实验,确定了其启动子上顺式结合元件TC-rich repeats是LOB1调控的直接靶点。4.获得了调控纤维素合成的SlMYB1/2转录因子。分离得到两个番茄MYB基因SlMYB1/2,与拟南芥纤维素合成相关的MYB83/46的同源。分析了 SlMYB1/2和次生细胞壁纤维素合成酶编码基因SlCESA4/5/6在番茄植株不同组织的表达模式,明确了SlMYB1/2对SlCESA4/5/6启动子的转录激活功能,同时SlMYB1/2瞬时表达和永久异源过表达至烟草均可促进纤维素的积累。因此,SlMYB1/2是调控纤维素合成的番茄MYB转录因子,是调控果实质地的重要候选基因。
[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.
【学位授予单位】：浙江大学
【学位级别】：博士
【学位授予年份】：2017
【分类号】：S641.2

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