本文選題：柑橘 + miRNA�。� 參考：《華中農業(yè)大學》2017年博士論文

【摘要】：柑橘是世界上廣泛栽培且具有較高經濟價值的重要果樹。其常規(guī)雜交育種因受到珠心胚等因素干擾導致進程緩慢、效率不高;同時,珠心胚因含有母本全部遺傳信息而具有固定優(yōu)良性狀的潛能。因此,珠心胚發(fā)生的分子機理研究將為柑橘育種提供重要理論依據。microRNA(mi RNA)是植物生長發(fā)育過程中的一類重要調控因子。本研究通過比較柑橘單胚與多胚品種胚珠miRNA表達水平差異,鑒定可能與珠心胚發(fā)生相關的miRNA,為深入探討mi RNA介導調控珠心胚起始的內在機制奠定基礎。另一方面,生物技術也是柑橘育種的重要途徑,體細胞胚發(fā)生是獲得離體再生植株的常見方式,是柑橘離體種質保存與生物技術育種的重要環(huán)節(jié);柑橘多數品種的愈傷組織體胚發(fā)生能力隨繼代時間延長而逐漸減弱甚至喪失,一定程度上限制了柑橘生物技術與遺傳改良研究的開展�；谇叭搜芯炕A,本研究以高度保守的miR156為候選關鍵miRNA,對其在柑橘體細胞胚發(fā)生過程中的潛在功能及相關調控機制展開深入分析。主要研究結果如下:1.小RNA高通量測序挖掘柑橘珠心胚起始相關miRNA以一對橘與一對柚/葡萄柚單胚/多胚品種為材料,選擇珠心胚起始細胞出現前后兩個時期,取胚珠進行小RNA深度測序。在橘胚珠中鑒定了91條已知的和60條新的miRNAs,預測的靶基因分別為695和163個;柚/葡萄柚中共鑒定了93條已知的及66條新的miRNAs,分別對應417和152個預測的靶基因。在一對橘品種的胚珠中兩個時期均得到13個差異表達miRNAs,而柚/葡萄柚分別獲得31和41個,僅有2個差異表達miRNAs(保守的miR1446a和新的miRN23-5p)在這兩對材料的胚珠中都表現差異表達。實時定量PCR驗證了其中6個差異表達的miRNAs,大部分與測序結果一致�；谇叭说霓D錄組測序數據,對其進行重新優(yōu)化分析,獲得305個共有的差異表達基因。Gene ontology(GO)分析表明逆境響應過程在上調基因中顯著富集,進一步發(fā)現在該生物過程中大部分基因與氧化逆境響應相關,這些基因在兩個多胚品種的胚珠顯著高于單胚品種胚珠。因此,推測氧化逆境響應過程可能是影響柑橘珠心胚起始的重要因素。qRT-PCR分析表明,miRN23-5p在2個多胚品種6個胚珠發(fā)育時期的表達量均低于單胚品種,而其預測的2個靶基因表達模式恰好相反。煙草瞬時表達系統進一步表明,miRN23-5p能夠剪切其中一個靶基因Cs9g06920,暗示miRN23-5p與Cs9g06920的互作可能參與珠心胚起始過程。2.csi-miR156a在體細胞胚發(fā)生過程中的功能驗證及調控作用研究利用RACE技術獲得csi-miR156a基因編碼區(qū)CsMIR156A全長,擴增結果表明該序列在8個柑橘品種間無明顯差異。將攜帶前體MIR156a的超量表達載體轉入弱胚性的山金柑愈傷組織,并成功獲得轉基因愈傷組織系。體胚發(fā)生能力分析表明,轉基因愈傷系的體胚發(fā)生能力明顯高于野生型。qRT-PCR分析表明,2個靶SPL(CsSPL3和CsSPL14)在miR156超表達愈傷組織系中的表達相比野生型下調,且這2個靶SPL基因的表達模式與柑橘不同品種的體胚發(fā)生能力負相關,推測這兩個SPL可能負調控體胚發(fā)生能力。亞細胞定位分析發(fā)現這兩個SPL均定位于細胞核。轉錄激活實驗結果表明CsSPL14有自激活現象,能夠激活下游報告基因的表達;而CsSPL3沒有自激活。構建CsSPL3和CsSPL14的RNAi載體,轉化弱胚性的山金柑愈傷組織;經體胚誘導及體胚發(fā)生能力統計發(fā)現,與csi-miR156a超量表達愈傷組織系表型相似,山金柑CsSPL3和CsSPL14RNAi愈傷組織系的體胚發(fā)生能力均顯著提高。利用數字表達譜分析比較超量表達mi R156的山金柑愈傷組織系與野生型,發(fā)現參與逆境響應及激素介導的信號轉導途徑等生物過程在上調基因顯著富集,而下調基因主要參與甲基化及細胞周期等相關過程。qRT-PCR檢測8個參與逆境響應過程差異表達基因的表達情況,表明這些基因在miR156超量表達與2個SPL基因干涉系均表現相似的表達模式。利用酵母雙雜交篩庫及點對點驗證獲得2個與CsSPL14潛在的互作蛋白,分別為CsARK1和SnRK催化亞基KIN10(CsAKIN10)。隨后利用雙分子熒光互補技術再次驗證了以上互作關系。亞細胞定位分析表明Cs AKIN10定位在細胞核。綜上所述,本研究鑒定了柑橘兩對單胚/多胚品種胚珠中已知與新的miRNA,并發(fā)掘了可能與珠心胚起始相關的“miRNA-靶基因”調控組合;驗證了csi-miR156a及其靶基因SPLs在體細胞胚發(fā)生過程中的功能,篩選了其中一個SPL的互作蛋白,并對SPL與其互作蛋白的相互作用調控體細胞胚發(fā)生的內在機制進行了討論。
[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.

【學位授予單位】：華中農業(yè)大學
【學位級別】：博士
【學位授予年份】：2017
【分類號】：S666

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