柑橘珠心胚起始相关miRNA挖掘与csi-miR156a调控体细胞胚发生作用机理

发布时间：2018-05-01 16:21

本文选题：柑橘 + miRNA　；参考：《华中农业大学》2017年博士论文

【摘要】：柑橘是世界上广泛栽培且具有较高经济价值的重要果树。其常规杂交育种因受到珠心胚等因素干扰导致进程缓慢、效率不高;同时,珠心胚因含有母本全部遗传信息而具有固定优良性状的潜能。因此,珠心胚发生的分子机理研究将为柑橘育种提供重要理论依据。microRNA(mi RNA)是植物生长发育过程中的一类重要调控因子。本研究通过比较柑橘单胚与多胚品种胚珠miRNA表达水平差异,鉴定可能与珠心胚发生相关的miRNA,为深入探讨mi RNA介导调控珠心胚起始的内在机制奠定基础。另一方面,生物技术也是柑橘育种的重要途径,体细胞胚发生是获得离体再生植株的常见方式,是柑橘离体种质保存与生物技术育种的重要环节;柑橘多数品种的愈伤组织体胚发生能力随继代时间延长而逐渐减弱甚至丧失,一定程度上限制了柑橘生物技术与遗传改良研究的开展。基于前人研究基础,本研究以高度保守的miR156为候选关键miRNA,对其在柑橘体细胞胚发生过程中的潜在功能及相关调控机制展开深入分析。主要研究结果如下:1.小RNA高通量测序挖掘柑橘珠心胚起始相关miRNA以一对橘与一对柚/葡萄柚单胚/多胚品种为材料,选择珠心胚起始细胞出现前后两个时期,取胚珠进行小RNA深度测序。在橘胚珠中鉴定了91条已知的和60条新的miRNAs,预测的靶基因分别为695和163个;柚/葡萄柚中共鉴定了93条已知的及66条新的miRNAs,分别对应417和152个预测的靶基因。在一对橘品种的胚珠中两个时期均得到13个差异表达miRNAs,而柚/葡萄柚分别获得31和41个,仅有2个差异表达miRNAs(保守的miR1446a和新的miRN23-5p)在这两对材料的胚珠中都表现差异表达。实时定量PCR验证了其中6个差异表达的miRNAs,大部分与测序结果一致。基于前人的转录组测序数据,对其进行重新优化分析,获得305个共有的差异表达基因。Gene ontology(GO)分析表明逆境响应过程在上调基因中显著富集,进一步发现在该生物过程中大部分基因与氧化逆境响应相关,这些基因在两个多胚品种的胚珠显著高于单胚品种胚珠。因此,推测氧化逆境响应过程可能是影响柑橘珠心胚起始的重要因素。qRT-PCR分析表明,miRN23-5p在2个多胚品种6个胚珠发育时期的表达量均低于单胚品种,而其预测的2个靶基因表达模式恰好相反。烟草瞬时表达系统进一步表明,miRN23-5p能够剪切其中一个靶基因Cs9g06920,暗示miRN23-5p与Cs9g06920的互作可能参与珠心胚起始过程。2.csi-miR156a在体细胞胚发生过程中的功能验证及调控作用研究利用RACE技术获得csi-miR156a基因编码区CsMIR156A全长,扩增结果表明该序列在8个柑橘品种间无明显差异。将携带前体MIR156a的超量表达载体转入弱胚性的山金柑愈伤组织,并成功获得转基因愈伤组织系。体胚发生能力分析表明,转基因愈伤系的体胚发生能力明显高于野生型。qRT-PCR分析表明,2个靶SPL(CsSPL3和CsSPL14)在miR156超表达愈伤组织系中的表达相比野生型下调,且这2个靶SPL基因的表达模式与柑橘不同品种的体胚发生能力负相关,推测这两个SPL可能负调控体胚发生能力。亚细胞定位分析发现这两个SPL均定位于细胞核。转录激活实验结果表明CsSPL14有自激活现象,能够激活下游报告基因的表达;而CsSPL3没有自激活。构建CsSPL3和CsSPL14的RNAi载体,转化弱胚性的山金柑愈伤组织;经体胚诱导及体胚发生能力统计发现,与csi-miR156a超量表达愈伤组织系表型相似,山金柑CsSPL3和CsSPL14RNAi愈伤组织系的体胚发生能力均显著提高。利用数字表达谱分析比较超量表达mi R156的山金柑愈伤组织系与野生型,发现参与逆境响应及激素介导的信号转导途径等生物过程在上调基因显著富集,而下调基因主要参与甲基化及细胞周期等相关过程。qRT-PCR检测8个参与逆境响应过程差异表达基因的表达情况,表明这些基因在miR156超量表达与2个SPL基因干涉系均表现相似的表达模式。利用酵母双杂交筛库及点对点验证获得2个与CsSPL14潜在的互作蛋白,分别为CsARK1和SnRK催化亚基KIN10(CsAKIN10)。随后利用双分子荧光互补技术再次验证了以上互作关系。亚细胞定位分析表明Cs AKIN10定位在细胞核。综上所述,本研究鉴定了柑橘两对单胚/多胚品种胚珠中已知与新的miRNA,并发掘了可能与珠心胚起始相关的“miRNA-靶基因”调控组合;验证了csi-miR156a及其靶基因SPLs在体细胞胚发生过程中的功能,筛选了其中一个SPL的互作蛋白,并对SPL与其互作蛋白的相互作用调控体细胞胚发生的内在机制进行了讨论。
[Abstract]:Citrus is an important fruit tree widely cultivated in the world and has high economic value. Its conventional cross breeding is slow and inefficient because of the disturbance of the pearl embryo and other factors. At the same time, the embryo of the heart of the heart has the potential of fixed excellent characters with all the genetic information of the mother. .microRNA (MI RNA) is an important regulatory factor in the process of plant growth and development. By comparing the difference in the expression level of miRNA in the ovules of the citrus single embryo and multi embryo, this study identifies the miRNA that may be related to the embryogenesis of the ovule, which lays the foundation for the study of the internal mechanism of the MI RNA mediated control of the beginning of the embryo of the heart. On the other hand, biological technology is also an important way of citrus breeding. Somatic embryogenesis is a common way to obtain regenerated plants in vitro. It is an important link in the preservation of germplasm in vitro and biotechnology breeding of citrus, and the callus embryogenesis ability of most citrus cultivars gradually weakened or even lost with the subculture time. On the basis of previous research, the potential function and related regulatory mechanism of miR156 in the process of citrus somatic embryogenesis were analyzed in this study based on previous research. The main research results are as follows: 1. small RNA high throughput sequencing excavation. A pair of citrus and grapefruit / Grapefruit single embryo / multi embryo varieties were selected as the starting related miRNA of Citrus pomp embryo, and the two stages of the embryo start cells were selected before and after the emergence of the ovule. 91 known and 60 new miRNAs were identified in the orange ovules, and 695 and 163 target genes were predicted, and the pomelo / Grapefruit was found in the orange pomelo. A total of 93 known and 66 new miRNAs were identified for 417 and 152 target genes. 13 differentially expressed miRNAs were obtained in two stages of the ovule of a pair of orange varieties, while pomelo / Grapefruit was 31 and 41 respectively, and only 2 differentially expressed miRNAs (conservative miR1446a and new miRN23-5p) in the ovules of two pairs of materials. The real time quantitative PCR verified 6 of the differentially expressed miRNAs, most of which were consistent with the sequencing results. Based on the previous transcriptional sequence data, it was re optimized and analyzed, and 305 common differentially expressed genes.Gene Ontology (GO) analysis showed that the adverse response process was significantly enriched in the up-regulated gene. One step found that most of the genes were associated with the response to oxidative stress in the biological process. These genes were significantly higher than the single embryo ovules in two multi embryo varieties. Therefore, it is suggested that the response process of oxidative stress may be an important factor affecting the initiation of citrus somatic embryo..qRT-PCR analysis shows that miRN23-5p is in 6 ovules in 2 multi embryo varieties. The expression of the 2 target gene expression patterns was exactly the opposite. The tobacco transient expression system further indicated that miRN23-5p could cut one of the target genes Cs9g06920, suggesting that the interaction between miRN23-5p and Cs9g06920 might participate in the process of somatic embryogenesis of.2.csi-miR156a in the beginning of the embryo of the Pearl. RACE technology was used to obtain the full length of CsMIR156A in the csi-miR156a gene coding region. The amplification results showed that there was no significant difference between the 8 citrus varieties. The overexpression vector carrying the precursor MIR156a was transferred into the callus of the weakly embryogenic citrus, and the transgenic callus system was successfully obtained. The ability analysis showed that the somatic embryogenesis of the transgenic callus was significantly higher than the wild type.QRT-PCR analysis, indicating that the expression of 2 target SPL (CsSPL3 and CsSPL14) in the miR156 overexpressed callus system was lower than that of the wild type, and the expression pattern of the 2 target SPL genes was negatively correlated with the somatic embryogenesis of different citrus varieties. The two SPL may negatively regulate somatic embryogenesis. The subcellular localization analysis found that the two SPL were all located in the nucleus. The transcriptional activation experiment showed that CsSPL14 had self activation and could activate the expression of the downstream reporter gene; while CsSPL3 did not activate itself. The RNAi vector of CsSPL3 and CsSPL14 was constructed and the weakly embryogenic orange callus was transformed. Somatic embryogenesis and somatic embryogenesis were found to be similar to the phenotype of csi-miR156a overexpressed callus, and the somatic embryogenesis of Kumquat CsSPL3 and CsSPL14RNAi callus lines increased significantly. Using digital expression spectrum analysis, the callus and wild type of citrus, which overexpressed mi R156, was found to be involved in adversity. The biological processes, such as response and hormone mediated signal transduction pathway, were significantly enriched in the up-regulated gene, while the down regulated genes were mainly involved in methylation and cell cycle related processes, such as.QRT-PCR detection of 8 differentially expressed genes involved in adverse response processes, indicating that these genes were overexpressed in miR156 and the average of the 2 SPL gene interference lines. 2 potential interacting proteins with CsSPL14, CsARK1 and SnRK catalytic subunit KIN10 (CsAKIN10), were obtained by yeast two hybrid sieves and point to point verification. Subsequently, the above interaction was verified by the double molecular fluorescence complementary technique. The subcellular localization analysis showed that Cs AKIN10 was located in the nucleus. In this study, we identified the known and new miRNA in the ovules of two Citrus single embryo / multi embryo varieties, and explored the regulation combination of "miRNA- target gene" that may be related to the beginning of the embryo of the pearly embryo. The function of csi-miR156a and its target gene SPLs in the process of somatic embryogenesis was verified, and one of the SPL interaction proteins was screened and SPL and their interaction with each other were screened. The intrinsic mechanism of protein interaction regulating somatic embryogenesis is discussed.

【学位授予单位】：华中农业大学
【学位级别】：博士
【学位授予年份】：2017
【分类号】：S666

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