本文選題：柑橘 + miRNA��； 參考：《華中農(nóng)業(yè)大學(xué)》2017年博士論文

【摘要】：柑橘是世界上廣泛栽培且具有較高經(jīng)濟(jì)價(jià)值的重要果樹(shù)。其常規(guī)雜交育種因受到珠心胚等因素干擾導(dǎo)致進(jìn)程緩慢、效率不高;同時(shí),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(jī)理研究將為柑橘育種提供重要理論依據(jù)。microRNA(mi RNA)是植物生長(zhǎng)發(fā)育過(guò)程中的一類重要調(diào)控因子。本研究通過(guò)比較柑橘單胚與多胚品種胚珠miRNA表達(dá)水平差異,鑒定可能與珠心胚發(fā)生相關(guān)的miRNA,為深入探討mi RNA介導(dǎo)調(diào)控珠心胚起始的內(nèi)在機(jī)制奠定基礎(chǔ)。另一方面,生物技術(shù)也是柑橘育種的重要途徑,體細(xì)胞胚發(fā)生是獲得離體再生植株的常見(jiàn)方式,是柑橘離體種質(zhì)保存與生物技術(shù)育種的重要環(huán)節(jié);柑橘多數(shù)品種的愈傷組織體胚發(fā)生能力隨繼代時(shí)間延長(zhǎng)而逐漸減弱甚至喪失,一定程度上限制了柑橘生物技術(shù)與遺傳改良研究的開(kāi)展。基于前人研究基礎(chǔ),本研究以高度保守的miR156為候選關(guān)鍵miRNA,對(duì)其在柑橘體細(xì)胞胚發(fā)生過(guò)程中的潛在功能及相關(guān)調(diào)控機(jī)制展開(kāi)深入分析。主要研究結(jié)果如下:1.小RNA高通量測(cè)序挖掘柑橘珠心胚起始相關(guān)miRNA以一對(duì)橘與一對(duì)柚/葡萄柚單胚/多胚品種為材料,選擇珠心胚起始細(xì)胞出現(xiàn)前后兩個(gè)時(shí)期,取胚珠進(jìn)行小RNA深度測(cè)序。在橘胚珠中鑒定了91條已知的和60條新的miRNAs,預(yù)測(cè)的靶基因分別為695和163個(gè);柚/葡萄柚中共鑒定了93條已知的及66條新的miRNAs,分別對(duì)應(yīng)417和152個(gè)預(yù)測(cè)的靶基因。在一對(duì)橘品種的胚珠中兩個(gè)時(shí)期均得到13個(gè)差異表達(dá)miRNAs,而柚/葡萄柚分別獲得31和41個(gè),僅有2個(gè)差異表達(dá)miRNAs(保守的miR1446a和新的miRN23-5p)在這兩對(duì)材料的胚珠中都表現(xiàn)差異表達(dá)。實(shí)時(shí)定量PCR驗(yàn)證了其中6個(gè)差異表達(dá)的miRNAs,大部分與測(cè)序結(jié)果一致。基于前人的轉(zhuǎn)錄組測(cè)序數(shù)據(jù),對(duì)其進(jìn)行重新優(yōu)化分析,獲得305個(gè)共有的差異表達(dá)基因。Gene ontology(GO)分析表明逆境響應(yīng)過(guò)程在上調(diào)基因中顯著富集,進(jìn)一步發(fā)現(xiàn)在該生物過(guò)程中大部分基因與氧化逆境響應(yīng)相關(guān),這些基因在兩個(gè)多胚品種的胚珠顯著高于單胚品種胚珠。因此,推測(cè)氧化逆境響應(yīng)過(guò)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(gè)多胚品種6個(gè)胚珠發(fā)育時(shí)期的表達(dá)量均低于單胚品種,而其預(yù)測(cè)的2個(gè)靶基因表達(dá)模式恰好相反。煙草瞬時(shí)表達(dá)系統(tǒng)進(jìn)一步表明,miRN23-5p能夠剪切其中一個(gè)靶基因Cs9g06920,暗示miRN23-5p與Cs9g06920的互作可能參與珠心胚起始過(guò)程。2.csi-miR156a在體細(xì)胞胚發(fā)生過(guò)程中的功能驗(yàn)證及調(diào)控作用研究利用RACE技術(shù)獲得csi-miR156a基因編碼區(qū)CsMIR156A全長(zhǎng),擴(kuò)增結(jié)果表明該序列在8個(gè)柑橘品種間無(wú)明顯差異。將攜帶前體MIR156a的超量表達(dá)載體轉(zhuǎn)入弱胚性的山金柑愈傷組織,并成功獲得轉(zhuǎn)基因愈傷組織系。體胚發(fā)生能力分析表明,轉(zhuǎn)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(gè)靶SPL(CsSPL3和CsSPL14)在miR156超表達(dá)愈傷組織系中的表達(dá)相比野生型下調(diào),且這2個(gè)靶SPL基因的表達(dá)模式與柑橘不同品種的體胚發(fā)生能力負(fù)相關(guān),推測(cè)這兩個(gè)SPL可能負(fù)調(diào)控體胚發(fā)生能力。亞細(xì)胞定位分析發(fā)現(xiàn)這兩個(gè)SPL均定位于細(xì)胞核。轉(zhuǎn)錄激活實(shí)驗(yàn)結(jié)果表明CsSPL14有自激活現(xiàn)象,能夠激活下游報(bào)告基因的表達(dá);而CsSPL3沒(méi)有自激活。構(gòu)建CsSPL3和CsSPL14的RNAi載體,轉(zhuǎn)化弱胚性的山金柑愈傷組織;經(jīng)體胚誘導(dǎo)及體胚發(fā)生能力統(tǒng)計(jì)發(fā)現(xiàn),與csi-miR156a超量表達(dá)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(shù)字表達(dá)譜分析比較超量表達(dá)mi R156的山金柑愈傷組織系與野生型,發(fā)現(xiàn)參與逆境響應(yīng)及激素介導(dǎo)的信號(hào)轉(zhuǎn)導(dǎo)途徑等生物過(guò)程在上調(diào)基因顯著富集,而下調(diào)基因主要參與甲基化及細(xì)胞周期等相關(guān)過(guò)程。qRT-PCR檢測(cè)8個(gè)參與逆境響應(yīng)過(guò)程差異表達(dá)基因的表達(dá)情況,表明這些基因在miR156超量表達(dá)與2個(gè)SPL基因干涉系均表現(xiàn)相似的表達(dá)模式。利用酵母雙雜交篩庫(kù)及點(diǎn)對(duì)點(diǎn)驗(yàn)證獲得2個(gè)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(bǔ)技術(shù)再次驗(yàn)證了以上互作關(guān)系。亞細(xì)胞定位分析表明Cs AKIN10定位在細(xì)胞核。綜上所述,本研究鑒定了柑橘兩對(duì)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(guān)的“miRNA-靶基因”調(diào)控組合;驗(yàn)證了csi-miR156a及其靶基因SPLs在體細(xì)胞胚發(fā)生過(guò)程中的功能,篩選了其中一個(gè)SPL的互作蛋白,并對(duì)SPL與其互作蛋白的相互作用調(diào)控體細(xì)胞胚發(fā)生的內(nèi)在機(jī)制進(jìn)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(xué)位授予單位】：華中農(nóng)業(yè)大學(xué)
【學(xué)位級(jí)別】：博士
【學(xué)位授予年份】：2017
【分類號(hào)】：S666

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本文編號(hào)：1830161