fsv1水稻胚珠发育过程中基因表达及不育机理研究

发布时间：2019-03-19 06:55

【摘要】：水稻是我国主要的粮食作物,其产量占全国粮食总产量的一半左右。水稻配子体育性如何对于水稻的产量具有至关重要的影响,因此对水稻配子体育性调控机理进行研究具有非常重要的意义。目前国内外关于水稻雄性不育机理的研究较多,雌性不育机理的研究还有所欠缺。本研究中,我们利用石蜡切片和子房整体透明技术对可育系(桂99)与其高度雌不育突变体fsv1)雌配子体形成过程进行了全面的观察并且采用高通量测序技术对胚珠发育的过程中桂99和fsv1胚珠中基因和miRNA的表达情况进行比较分析,以期为水稻雌配子体育性机理的研究提供分子基础。在本研究中,通过石蜡切片和子房整体透明技术,我们对桂99与fsvl雌配子体的形成过程进行观察,结果发现fsv1中雌配子体高度败育是由功能大孢子形成异常导致。发育异常的功能大孢子不能进行正常的有丝分裂,绝大多数fsv1胚珠中的没有形成可育的雌配子体,胚珠内部整个被珠心组织细胞所填满。通过利用高通量测序技术,我们比较分析了两个水稻品系胚珠三个发育时期(大孢子母细胞减数分裂时期,功能大孢子有丝分裂时期和胚囊成熟时期)中基因的表达变化情况。在水稻胚珠中一共检测到30,204个基因的表达。在同一水稻品系三个发育时期之间相比较,绝大部分基因在胚珠的三个发育时期中共同表达,每个时期又都存在其时期特异表达基因。在相同发育时期不同品系的胚珠之间相比较,绝大多数基因在桂99与fsvl胚珠中共同表达,但桂99与fsvl中也存在其品系特异表达基因。我们将在两个样本中共同表达,差异倍数≥2且FDR≤0.001基因定义为显著差异表达基因。结果发现在相同发育时期桂99与fsv1胚珠两两比较中,分别有45、495和932个基因在水稻胚珠三个发育时期显著差异表达。随着胚珠的发育,两个品系间差异表达的基因数目越来越多,说明桂99与fsv1胚珠分化差异越来越大。通过将这些差异表达基因进行GO功能注释分析、KEGG和MapMan代谢途径分析,我们发现绝大部分的差异表达基因与糖代谢、植物激素信号转导、蛋白质修饰与降解、氧化磷酸化等途径密切相关,这些途径中基因的表达变化很有可能影响了雌配子体的育性。另外我们还发现随着胚珠的发育,一些与生长素(例如TAA1和PIN1α)、转录因子(例如ARF6和KANADI4)和表观遗传(例如.JMJ709和AGO5)相关的基因在fsv1中表现出与桂99显著差异的表达量或完全相反的表达趋势。这些在雌不育突变体中异常表达的基因可能是导致雌配子体败育的关键因素,值得我们进行进一步的研究。MiRNA作为基因表达变化的重要调控因子之一,近年来受到研究者的广泛关注。miRNA能够通过调节靶基因的表达变化参与调控植物生长发育的多个阶段,包括生殖生长过程。在本研究中,我们应用高通量测序技术,对水稻桂99与fsv1两个品系胚珠发育过程中miRNA的表达变化进行分析,希望能够为揭示miRNA对水稻雌配子体育性调控的分子机理提供相关线索。根据测序结果,共有522个已知的miRNA和295个预测的miRNA在发育中的水稻胚珠中表达。其中100个已知的miRNA和217个预测的miRNA在桂99和fsv1之间显著差异表达。随着胚珠的发育,差异表达的已知miRNA和预测的miRNA的数目都呈下调的趋势,说明miRNA主要在胚珠发育早期对水稻雌配子体的育性进行调节。结合之前得到的转录组信息,我们对两个水稻品系之间显著差异表达的已知miRNA的靶基因进行分析,并将这些miRNA的靶基因进行GO功能注释分析。结果表明,差异表达的miRNA的靶基因主要与蛋白代谢、激素调节、转录调控及信号转导等过程相关。值得注意的是,许多靶向调控生长素基因(例如ARF8和ARF10)表达的miRNA和靶向作用于与花器官形成相关转录因子基因(例如SBP-box基因)表达的miRNA在fsv1中表现出与桂99显著的表达差异,这些miRNA可能对水稻雌配子体的育性发挥重要的调控作用。
[Abstract]:Rice is the main food crop in China, and its output is about half of the total grain output in China. It is of great significance to study the mechanism of rice gamete's physical and physical regulation. At present, there are many studies on the mechanism of male sterility in rice, and the mechanism of female infertility is still lacking. in this study, The formation of the female gametophyte of the fertile line (Guangxi 99) and its high female sterile mutant (fv1) was fully observed by using the paraffin section and the whole transparent technique of the ovary, and the expression of the genes and miRNAs in the ovule during the development of the ovule was carried out by high-throughput sequencing technology. The purpose of this study is to provide a molecular basis for the study of the mechanism of the female gamete of rice. In this study, we observe that formation of the female gametophyte in fv1 by the whole transparent technique of paraffin section and ovary. In most of the fv1 ovules, no fertile female gametophyte is formed, and the whole of the ovule is filled with the core of the nucellar tissue. By using the high-throughput sequencing technology, we compared the expression of the gene in the three developmental stages of the two rice lines (the meiosis of the megaspore, the mitosis of the function and the mature period of the embryo sac). The expression of 30,204 genes was detected in rice-ovule. During the three developmental stages of the same rice line, most of the genes were expressed in the three developmental stages of the ovule. Compared with the ovules of different strains in the same development period, most of the genes are expressed in the fvl ovules, but the specific expression genes of the strains are also present in the Gui 99 and fvl. We will be able to co-express in both samples, with a difference of 2 and the FDR-0.001 gene is defined as a significant difference expression gene. The results showed that there were 45,495 and 932 genes differentially expressed in the three developmental stages of rice-ovule during the same developmental period. With the development of the ovule, the number of genes between the two strains is increasing, and the difference of the differentiation between the two strains is more and more important. By carrying out the GO function annotation analysis, the KEGG and the MapMan metabolic pathway analysis of these differential expression genes, we found that most of the difference expression genes are closely related to the sugar metabolism, the plant hormone signal transduction, the protein modification and the degradation, the oxidative phosphorylation, and the like, The expression of the genes in these pathways is likely to affect the fertility of the female gametophyte. In addition, we have also found that with the development of ovule, some of the transcription factors (e.g., TA1 and PIN1), transcription factors (e.g., ARF6 and KANAD4), and epigenetic (e.g.,. The genes associated with JMJ709 and AG5) showed a significant difference in expression or a completely reverse expression trend in fv1. The abnormal expression of these genes in the female sterile mutant may be a key factor leading to female gametogenesis, and it is worth further study. MiRNA is one of the important regulators of gene expression, and has been widely concerned by the researchers in recent years. The miRNA can participate in a plurality of stages of regulating the growth and development of the plant by adjusting the expression change of the target gene, including the reproductive growth process. In this study, we applied the high-throughput sequencing technology to analyze the expression of miRNAs during the development of the ovule of rice-Gui 99 and fv1, and it is hoped that we can provide clues for revealing the molecular mechanism of miRNAs in the regulation of female gamete. According to the results of the sequencing,522 known miRNAs and 295 predicted miRNAs were expressed in the development of rice ovules. 100 of the known miRNAs and 217 of the predicted miRNAs were significantly differentially expressed between the Gui 99 and the fv1. With the development of the ovule, the number of the known miRNAs and the predicted miRNAs of the differentially expressed miRNAs is down-regulated, indicating that the miRNAs mainly regulate the fertility of the rice female gametophyte in the early stage of the development of the ovule. The target genes of known miRNAs that are differentially expressed between the two rice lines are analyzed and the target genes of these miRNAs are subjected to a GO function annotation analysis in combination with the previously obtained transcription group information. The results showed that the target genes of the differentially expressed miRNAs were mainly related to the processes of protein metabolism, hormone regulation, transcription regulation and signal transduction. It is to be noted that the miRNAs expressed by many of the targeted regulatory auxin genes (e.g., ARF8 and ARF10) and the miRNAs targeting the expression of the transcription factor gene (e.g., the SBP-box gene) associated with the floral organ formation exhibit a significant difference in expression with the Gui 99, These miRNAs may play an important regulatory role in the fertility of the rice female gametophyte.
【学位授予单位】：武汉大学
【学位级别】：博士
【学位授予年份】：2016
【分类号】：S511

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