fsv1水稻胚珠發(fā)育過程中基因表達(dá)及不育機(jī)理研究
發(fā)布時間：2019-03-19 06:55
【摘要】：水稻是我國主要的糧食作物,其產(chǎn)量占全國糧食總產(chǎn)量的一半左右。水稻配子體育性如何對于水稻的產(chǎn)量具有至關(guān)重要的影響,因此對水稻配子體育性調(diào)控機(jī)理進(jìn)行研究具有非常重要的意義。目前國內(nèi)外關(guān)于水稻雄性不育機(jī)理的研究較多,雌性不育機(jī)理的研究還有所欠缺。本研究中,我們利用石蠟切片和子房整體透明技術(shù)對可育系(桂99)與其高度雌不育突變體fsv1)雌配子體形成過程進(jìn)行了全面的觀察并且采用高通量測序技術(shù)對胚珠發(fā)育的過程中桂99和fsv1胚珠中基因和miRNA的表達(dá)情況進(jìn)行比較分析,以期為水稻雌配子體育性機(jī)理的研究提供分子基礎(chǔ)。在本研究中,通過石蠟切片和子房整體透明技術(shù),我們對桂99與fsvl雌配子體的形成過程進(jìn)行觀察,結(jié)果發(fā)現(xiàn)fsv1中雌配子體高度敗育是由功能大孢子形成異常導(dǎo)致。發(fā)育異常的功能大孢子不能進(jìn)行正常的有絲分裂,絕大多數(shù)fsv1胚珠中的沒有形成可育的雌配子體,胚珠內(nèi)部整個被珠心組織細(xì)胞所填滿。通過利用高通量測序技術(shù),我們比較分析了兩個水稻品系胚珠三個發(fā)育時期(大孢子母細(xì)胞減數(shù)分裂時期,功能大孢子有絲分裂時期和胚囊成熟時期)中基因的表達(dá)變化情況。在水稻胚珠中一共檢測到30,204個基因的表達(dá)。在同一水稻品系三個發(fā)育時期之間相比較,絕大部分基因在胚珠的三個發(fā)育時期中共同表達(dá),每個時期又都存在其時期特異表達(dá)基因。在相同發(fā)育時期不同品系的胚珠之間相比較,絕大多數(shù)基因在桂99與fsvl胚珠中共同表達(dá),但桂99與fsvl中也存在其品系特異表達(dá)基因。我們將在兩個樣本中共同表達(dá),差異倍數(shù)≥2且FDR≤0.001基因定義為顯著差異表達(dá)基因。結(jié)果發(fā)現(xiàn)在相同發(fā)育時期桂99與fsv1胚珠兩兩比較中,分別有45、495和932個基因在水稻胚珠三個發(fā)育時期顯著差異表達(dá)。隨著胚珠的發(fā)育,兩個品系間差異表達(dá)的基因數(shù)目越來越多,說明桂99與fsv1胚珠分化差異越來越大。通過將這些差異表達(dá)基因進(jìn)行GO功能注釋分析、KEGG和MapMan代謝途徑分析,我們發(fā)現(xiàn)絕大部分的差異表達(dá)基因與糖代謝、植物激素信號轉(zhuǎn)導(dǎo)、蛋白質(zhì)修飾與降解、氧化磷酸化等途徑密切相關(guān),這些途徑中基因的表達(dá)變化很有可能影響了雌配子體的育性。另外我們還發(fā)現(xiàn)隨著胚珠的發(fā)育,一些與生長素(例如TAA1和PIN1α)、轉(zhuǎn)錄因子(例如ARF6和KANADI4)和表觀遺傳(例如.JMJ709和AGO5)相關(guān)的基因在fsv1中表現(xiàn)出與桂99顯著差異的表達(dá)量或完全相反的表達(dá)趨勢。這些在雌不育突變體中異常表達(dá)的基因可能是導(dǎo)致雌配子體敗育的關(guān)鍵因素,值得我們進(jìn)行進(jìn)一步的研究。MiRNA作為基因表達(dá)變化的重要調(diào)控因子之一,近年來受到研究者的廣泛關(guān)注。miRNA能夠通過調(diào)節(jié)靶基因的表達(dá)變化參與調(diào)控植物生長發(fā)育的多個階段,包括生殖生長過程。在本研究中,我們應(yīng)用高通量測序技術(shù),對水稻桂99與fsv1兩個品系胚珠發(fā)育過程中miRNA的表達(dá)變化進(jìn)行分析,希望能夠為揭示miRNA對水稻雌配子體育性調(diào)控的分子機(jī)理提供相關(guān)線索。根據(jù)測序結(jié)果,共有522個已知的miRNA和295個預(yù)測的miRNA在發(fā)育中的水稻胚珠中表達(dá)。其中100個已知的miRNA和217個預(yù)測的miRNA在桂99和fsv1之間顯著差異表達(dá)。隨著胚珠的發(fā)育,差異表達(dá)的已知miRNA和預(yù)測的miRNA的數(shù)目都呈下調(diào)的趨勢,說明miRNA主要在胚珠發(fā)育早期對水稻雌配子體的育性進(jìn)行調(diào)節(jié)。結(jié)合之前得到的轉(zhuǎn)錄組信息,我們對兩個水稻品系之間顯著差異表達(dá)的已知miRNA的靶基因進(jìn)行分析,并將這些miRNA的靶基因進(jìn)行GO功能注釋分析。結(jié)果表明,差異表達(dá)的miRNA的靶基因主要與蛋白代謝、激素調(diào)節(jié)、轉(zhuǎn)錄調(diào)控及信號轉(zhuǎn)導(dǎo)等過程相關(guān)。值得注意的是,許多靶向調(diào)控生長素基因(例如ARF8和ARF10)表達(dá)的miRNA和靶向作用于與花器官形成相關(guān)轉(zhuǎn)錄因子基因(例如SBP-box基因)表達(dá)的miRNA在fsv1中表現(xiàn)出與桂99顯著的表達(dá)差異,這些miRNA可能對水稻雌配子體的育性發(fā)揮重要的調(diào)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.
【學(xué)位授予單位】：武漢大學(xué)
【學(xué)位級別】：博士
【學(xué)位授予年份】：2016
【分類號】：S511
本文編號：2443285
[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.
【學(xué)位授予單位】：武漢大學(xué)
【學(xué)位級別】：博士
【學(xué)位授予年份】：2016
【分類號】：S511
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