【摘要】：目的:體細(xì)胞胚胎發(fā)生(Somatic embryogenesis)是棉花(Gossypium hirsutum L.)通過(guò)農(nóng)桿菌介導(dǎo)的遺傳轉(zhuǎn)化最關(guān)鍵的步驟之一,通常包括非愈傷組織的誘導(dǎo)、胚性愈傷組織的分化、胚狀體的形成以及胚狀體發(fā)育成苗等過(guò)程。棉花體細(xì)胞胚胎發(fā)生存在的缺點(diǎn)是基因型依賴、培養(yǎng)周期長(zhǎng)、不正常胚產(chǎn)生效率高,嚴(yán)重制約了棉花基因功能的驗(yàn)證和轉(zhuǎn)基因育種工作。研究棉花體細(xì)胞胚胎發(fā)生的生理及分子機(jī)制可以為棉花組織培養(yǎng)及轉(zhuǎn)基因育種工作提供重要的理論依據(jù)和技術(shù)支撐。方法:(1)本研究使用de novo轉(zhuǎn)錄組測(cè)序和iTRAQ蛋白組測(cè)序的方法,測(cè)定了新疆陸地棉品種新陸早33號(hào)體細(xì)胞胚胎發(fā)生過(guò)程中非胚性愈傷組織、胚性愈傷組織以及胚狀體中的差異表達(dá)基因和蛋白,并對(duì)差異表達(dá)基因和蛋白進(jìn)行了鑒定及表達(dá)模式分析。(2)對(duì)差異表達(dá)基因和蛋白的相關(guān)通路進(jìn)行了注釋和分類,進(jìn)一步明確了參與調(diào)控棉花體細(xì)胞胚胎發(fā)生的通路。(3)利用qRT-PCR、內(nèi)源含量測(cè)定以及外源添加等實(shí)驗(yàn),明確相關(guān)基因、蛋白及通路與棉花體細(xì)胞胚胎發(fā)生的關(guān)系。(4)用HPLC法,提取分離鑒定了棉花內(nèi)源多胺的種類,建立棉花組織多胺提取測(cè)定的技術(shù)體系。(5)通過(guò)分析多胺代謝途徑相關(guān)生理生化指標(biāo)、基因表達(dá)、ELISA法測(cè)定相關(guān)酶活性、生理互補(bǔ)實(shí)驗(yàn)等,揭示多胺調(diào)控棉花體細(xì)胞胚胎發(fā)生的可能機(jī)制。結(jié)果與結(jié)論:(1)De novo轉(zhuǎn)錄組測(cè)序一共獲得了101,670個(gè)unigene,在胚性愈傷組織向胚狀體轉(zhuǎn)化過(guò)程中差異表達(dá)的基因多于非胚性愈傷組織向胚性愈傷組織轉(zhuǎn)化過(guò)程中的差異表達(dá)基因。大量的差異表達(dá)基因參與了植物激素合成及信號(hào)轉(zhuǎn)導(dǎo)、脅迫響應(yīng)反應(yīng)、ROS平衡以及多胺代謝等途徑。用qRT-PCR驗(yàn)證了相關(guān)差異基因的真實(shí)性和準(zhǔn)確性。內(nèi)源IAA和KT含量變化規(guī)律與其合成途徑相關(guān)基因的表達(dá)模式一致。外源IAA和KT能夠促進(jìn)胚性愈傷組織向胚狀體的轉(zhuǎn)化,不同濃度IBA的添加會(huì)對(duì)非胚性愈傷組織、胚性愈傷組織的誘導(dǎo)效率有影響,外源添加多胺和過(guò)氧化氫也能在很大程度上促進(jìn)胚狀體的形成。適當(dāng)?shù)拿{迫條件對(duì)胚性愈傷組織的增殖以及胚狀體形成都有促進(jìn)作用。此外,赤霉素、脫落酸、乙烯、油菜素內(nèi)酯、水楊酸、茉莉酸及脂肪酸途徑相關(guān)基因也呈現(xiàn)出差異表達(dá)的規(guī)律。轉(zhuǎn)錄組動(dòng)力學(xué)分析表明不同植物激素間的平衡調(diào)控,多胺代謝和脅迫響應(yīng)過(guò)程協(xié)同調(diào)控了棉花體細(xì)胞胚胎發(fā)生。(2)iTRAQ蛋白組測(cè)序發(fā)現(xiàn),三組樣品中一共得到了5892個(gè)差異表達(dá)蛋白,這些蛋白主要參與了催化活性、結(jié)合活性、轉(zhuǎn)運(yùn)活性以及結(jié)構(gòu)分子活性,其中93.4%的差異蛋白在分子功能水平參與了結(jié)構(gòu)分子活性。差異表達(dá)蛋白統(tǒng)計(jì)分析發(fā)現(xiàn),與非胚性愈傷組織相比,胚性愈傷組織中分別有572個(gè)上調(diào)表達(dá)蛋白和452個(gè)下調(diào)表達(dá)蛋白;與胚性愈傷組織相比,胚狀體中分別有211個(gè)上調(diào)表達(dá)蛋白和647個(gè)下調(diào)表達(dá)蛋白。KEGG分析顯示遺傳信息的轉(zhuǎn)運(yùn)、植物激素合成及信號(hào)轉(zhuǎn)導(dǎo)、糖酵解過(guò)程、脂肪酸合成和代謝以及半乳糖代謝等通路參與了棉花體細(xì)胞胚胎發(fā)生。通過(guò)qRT-PCR分析相關(guān)差異基因在三個(gè)時(shí)期的表達(dá)情況進(jìn)一步確定了蛋白組測(cè)序的真實(shí)性和準(zhǔn)確性。對(duì)轉(zhuǎn)錄組和蛋白組的測(cè)序數(shù)據(jù)進(jìn)行關(guān)聯(lián)分析發(fā)現(xiàn),所測(cè)得基因序列和蛋白序列的關(guān)聯(lián)系數(shù)為0.27,差異表達(dá)基因與差異表達(dá)蛋白的關(guān)聯(lián)系數(shù)高于0.6。(3)建立了棉花組織多胺提取分離及測(cè)定的高效液相色譜法,可將腐胺、亞精胺、精胺在15分鐘完全分離并定量測(cè)定,線性關(guān)系良好(r0.99),回收率高(96.8%~103.1%)。測(cè)定棉花體細(xì)胞胚胎發(fā)生各時(shí)期內(nèi)源多胺的含量,結(jié)果顯示在胚性愈傷組織以及體細(xì)胞胚形成初期,三種類型多胺含量顯著提高,腐胺的含量在胚狀體中有下降趨勢(shì),而亞精胺和精胺的含量在胚狀體中沒(méi)有變化。多胺合成相關(guān)基因的表達(dá)量分析發(fā)現(xiàn),棉花精氨酸合成酶GhADC在體細(xì)胞胚胎發(fā)生過(guò)程中的表達(dá)與多胺含量(尤其是腐胺含量)的變化規(guī)律非常一致。此外,多胺代謝產(chǎn)物H2O2的含量在胚性愈傷組織時(shí)期也有顯著的提高,與多胺氧化酶GhPAO1和GhPAO4的表達(dá)規(guī)律一致。ELISA酶活測(cè)定發(fā)現(xiàn),多胺氧化酶PAO在胚性愈傷組織中表現(xiàn)出較高的活性,抑制PAO活性則表現(xiàn)出體細(xì)胞胚胎形成受阻。多胺及其代謝產(chǎn)物H2O2都能夠促進(jìn)胚性愈傷組織的生長(zhǎng)及胚狀體的形成,同時(shí)也能夠降低多胺合成抑制劑D-arginine及多胺氧化酶抑制劑1,8-DO的抑制效果。多胺代謝途徑另一個(gè)重要的信號(hào)分子NO的含量在體細(xì)胞胚胎發(fā)生過(guò)程中變化不明顯,且對(duì)體細(xì)胞胚胎發(fā)生沒(méi)有顯著作用。綜合上面的結(jié)果,我們得出結(jié)論:多胺可能通過(guò)多胺氧化酶調(diào)節(jié)其代謝產(chǎn)物過(guò)氧化氫來(lái)調(diào)控棉花體細(xì)胞胚胎發(fā)生。
[Abstract]:Objective: Somatic embryonic genesis (somatic embryogenesis) is one of the most critical steps in the transformation of cotton (Gossypium hirsutum L.) through Agrobacterium-mediated transformation, usually including the induction of non-callus, the differentiation of the embryonic callus, the formation of the embryoid and the development of the embryoid. The defects of the somatic embryogenesis of the cotton are genotype-dependent, long culture period, high production efficiency of non-normal embryos, and severely restrict the verification of the function of the cotton gene and the work of the transgenic breeding. The study of the physiological and molecular mechanism of somatic embryogenesis in cotton can provide important theoretical basis and technical support for cotton tissue culture and transgenic breeding. Methods: (1) The present study used de novo transcriptome sequencing and iTRAQ proteome sequencing method to determine the difference expression gene and protein of the non-embryonic callus, the embryogenic callus and the embryoid in the somatic embryogenesis of the No.33 Somatic Embryogenesis of the Gossypium hirsutum L. in Xinjiang. The differential expression gene and protein were identified and analyzed. (2) The related pathways of differentially expressed genes and proteins are annotated and classified, and the pathways involved in the regulation and control of somatic embryogenesis of the cotton are further defined. (3) Using qRT-PCR, endogenous content determination and exogenous addition, the relationship between the related gene, protein and pathway and the somatic embryogenesis of cotton was determined. And (4) separating and identifying the species of the endogenous polyamines of the cotton by using the HPLC method, and establishing a technical system for extracting and measuring the polyamine from the cotton tissue. (5) The possible mechanism of polyamine regulation and control of somatic embryogenesis of cotton was revealed by analyzing the relevant physiological and biochemical indexes, gene expression, enzyme-linked immunosorbent assay (ELISA) method, and so on. Results and Conclusion: (1) There was a total of 1,670 unganene in the sequence of the De novo transcription group, and the expression of the differentially expressed genes in the process of transformation of the embryogenic callus to the embryoid was more than that of the non-embryogenic callus in the transformation of the embryogenic callus. A large number of differentially expressed genes involved in plant hormone synthesis and signal transduction, stress response, ROS and polyamine metabolism. The authenticity and accuracy of the related differential gene were verified by qRT-PCR. The variation of endogenous IAA and KT was consistent with the expression pattern of its synthetic pathway. The exogenous IAA and KT can promote the transformation of the embryogenic callus to the embryoid, and the addition of the IBA with different concentration can influence the induction efficiency of the non-embryonic callus and the embryonic callus, and the exogenous addition of the polyamine and the hydrogen peroxide can greatly promote the formation of the embryoid. The appropriate stress condition can promote the proliferation of the embryogenic callus and the shape of the embryoid. In addition, the related genes of gibberellin, abscisic acid, ethylene, brassinolide, salicylic acid, jasmonic acid and fatty acid pathway also show the law of differential expression. The dynamic analysis of the transcription group showed that the balance regulation, the polyamine metabolism and the stress response of different plant hormones have been used to control the somatic embryogenesis of the cotton. (2) The sequencing of iTRAQ proteome showed that 5892 differentially expressed proteins were obtained in three groups of samples, which were mainly involved in the catalytic activity, binding activity, transport activity and structural molecular activity, of which 93.4% of the differential protein was involved in the structural molecular activity at the molecular function level. The statistical analysis of differential expression protein showed that there were 572 up-regulated and 452 down-regulated expression proteins in the embryonic callus compared with the non-embryonic callus,211 up-regulated and 647 down-regulated expression proteins in the embryoid. KEGG analysis showed that genetic information transfer, phytohormone synthesis and signal transduction, glycolysis, fatty acid synthesis and metabolism, and galactose metabolism were involved in somatic embryogenesis of cotton. The authenticity and accuracy of the proteome sequencing were further determined by qRT-PCR. It was found that the correlation coefficient of the gene sequence and the protein sequence was 0.27, the correlation coefficient of the difference expression gene and the differential expression protein was higher than 0.6. (3) The high performance liquid chromatography of the extraction and separation of polyamine from the cotton tissue was established, and the putrescine, spermidine and spermine were completely separated and measured in 15 minutes, the linear relationship was good (r0.99), and the recovery rate was high (96.8% ~ 103.1%). The content of the endogenous polyamines in the somatic embryogenesis of the cotton was determined. The results showed that the contents of the three types of polyamines increased significantly in the early stage of the formation of the embryogenic callus and the somatic embryos, and the content of the putrescine was decreased in the embryoid. While the content of spermidine and spermine did not change in the embryoid. The analysis of the expression of the related genes of polyamine synthesis showed that the expression of GADC in the process of somatic embryogenesis was very consistent with the changes of the content of polyamines (especially the content of putrescine). In addition, the content of H2O2 in the polyamine metabolite was significantly increased in the period of embryogenic callus and was consistent with the expression of the polyamine oxidase GPAO1 and GPAO4. The activity of the enzyme-linked immunosorbent assay (ELISA) showed that the polyamine oxidase (PAO) exhibited high activity in the embryogenic callus, and the inhibition of PAO activity was hindered by the formation of somatic embryos. The polyamine and its metabolite H2O2 can promote the growth of the embryogenic callus and the formation of the embryoid, and can also reduce the inhibitory effect of the polyamine synthesis inhibitor D-arginine and the polyamine oxidase inhibitor 1,8-DO. The content of NO is not obvious in the process of somatic embryogenesis, and there is no significant effect on somatic embryogenesis. Based on the results above, we have concluded that polyamines may modulate the somatic embryogenesis of the cotton by adjusting its metabolic product hydrogen peroxide through the polyamine oxidase.
【學(xué)位授予單位】：石河子大學(xué)
【學(xué)位級(jí)別】：博士
【學(xué)位授予年份】：2016
【分類號(hào)】：S562

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