棉花（Gossypium hirsutum L.）体细胞胚胎发生的生理及分子机制研究
发布时间：2019-06-04 08:20
【摘要】：目的:体细胞胚胎发生(Somatic embryogenesis)是棉花(Gossypium hirsutum L.)通过农杆菌介导的遗传转化最关键的步骤之一,通常包括非愈伤组织的诱导、胚性愈伤组织的分化、胚状体的形成以及胚状体发育成苗等过程。棉花体细胞胚胎发生存在的缺点是基因型依赖、培养周期长、不正常胚产生效率高,严重制约了棉花基因功能的验证和转基因育种工作。研究棉花体细胞胚胎发生的生理及分子机制可以为棉花组织培养及转基因育种工作提供重要的理论依据和技术支撑。方法:(1)本研究使用de novo转录组测序和iTRAQ蛋白组测序的方法,测定了新疆陆地棉品种新陆早33号体细胞胚胎发生过程中非胚性愈伤组织、胚性愈伤组织以及胚状体中的差异表达基因和蛋白,并对差异表达基因和蛋白进行了鉴定及表达模式分析。(2)对差异表达基因和蛋白的相关通路进行了注释和分类,进一步明确了参与调控棉花体细胞胚胎发生的通路。(3)利用qRT-PCR、内源含量测定以及外源添加等实验,明确相关基因、蛋白及通路与棉花体细胞胚胎发生的关系。(4)用HPLC法,提取分离鉴定了棉花内源多胺的种类,建立棉花组织多胺提取测定的技术体系。(5)通过分析多胺代谢途径相关生理生化指标、基因表达、ELISA法测定相关酶活性、生理互补实验等,揭示多胺调控棉花体细胞胚胎发生的可能机制。结果与结论:(1)De novo转录组测序一共获得了101,670个unigene,在胚性愈伤组织向胚状体转化过程中差异表达的基因多于非胚性愈伤组织向胚性愈伤组织转化过程中的差异表达基因。大量的差异表达基因参与了植物激素合成及信号转导、胁迫响应反应、ROS平衡以及多胺代谢等途径。用qRT-PCR验证了相关差异基因的真实性和准确性。内源IAA和KT含量变化规律与其合成途径相关基因的表达模式一致。外源IAA和KT能够促进胚性愈伤组织向胚状体的转化,不同浓度IBA的添加会对非胚性愈伤组织、胚性愈伤组织的诱导效率有影响,外源添加多胺和过氧化氢也能在很大程度上促进胚状体的形成。适当的胁迫条件对胚性愈伤组织的增殖以及胚状体形成都有促进作用。此外,赤霉素、脱落酸、乙烯、油菜素内酯、水杨酸、茉莉酸及脂肪酸途径相关基因也呈现出差异表达的规律。转录组动力学分析表明不同植物激素间的平衡调控,多胺代谢和胁迫响应过程协同调控了棉花体细胞胚胎发生。(2)iTRAQ蛋白组测序发现,三组样品中一共得到了5892个差异表达蛋白,这些蛋白主要参与了催化活性、结合活性、转运活性以及结构分子活性,其中93.4%的差异蛋白在分子功能水平参与了结构分子活性。差异表达蛋白统计分析发现,与非胚性愈伤组织相比,胚性愈伤组织中分别有572个上调表达蛋白和452个下调表达蛋白;与胚性愈伤组织相比,胚状体中分别有211个上调表达蛋白和647个下调表达蛋白。KEGG分析显示遗传信息的转运、植物激素合成及信号转导、糖酵解过程、脂肪酸合成和代谢以及半乳糖代谢等通路参与了棉花体细胞胚胎发生。通过qRT-PCR分析相关差异基因在三个时期的表达情况进一步确定了蛋白组测序的真实性和准确性。对转录组和蛋白组的测序数据进行关联分析发现,所测得基因序列和蛋白序列的关联系数为0.27,差异表达基因与差异表达蛋白的关联系数高于0.6。(3)建立了棉花组织多胺提取分离及测定的高效液相色谱法,可将腐胺、亚精胺、精胺在15分钟完全分离并定量测定,线性关系良好(r0.99),回收率高(96.8%~103.1%)。测定棉花体细胞胚胎发生各时期内源多胺的含量,结果显示在胚性愈伤组织以及体细胞胚形成初期,三种类型多胺含量显著提高,腐胺的含量在胚状体中有下降趋势,而亚精胺和精胺的含量在胚状体中没有变化。多胺合成相关基因的表达量分析发现,棉花精氨酸合成酶GhADC在体细胞胚胎发生过程中的表达与多胺含量(尤其是腐胺含量)的变化规律非常一致。此外,多胺代谢产物H2O2的含量在胚性愈伤组织时期也有显著的提高,与多胺氧化酶GhPAO1和GhPAO4的表达规律一致。ELISA酶活测定发现,多胺氧化酶PAO在胚性愈伤组织中表现出较高的活性,抑制PAO活性则表现出体细胞胚胎形成受阻。多胺及其代谢产物H2O2都能够促进胚性愈伤组织的生长及胚状体的形成,同时也能够降低多胺合成抑制剂D-arginine及多胺氧化酶抑制剂1,8-DO的抑制效果。多胺代谢途径另一个重要的信号分子NO的含量在体细胞胚胎发生过程中变化不明显,且对体细胞胚胎发生没有显著作用。综合上面的结果,我们得出结论:多胺可能通过多胺氧化酶调节其代谢产物过氧化氢来调控棉花体细胞胚胎发生。
[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.
【学位授予单位】：石河子大学
【学位级别】：博士
【学位授予年份】：2016
【分类号】：S562
本文编号：2492589
[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.
【学位授予单位】：石河子大学
【学位级别】：博士
【学位授予年份】：2016
【分类号】：S562
【参考文献】
相关期刊论文 前1条
1 罗明,肖月华,侯磊,罗小英,李德谋,裴炎;棉花LIM结构域基因(GhLIM1)的克隆和表达分析[J];遗传学报;2003年02期
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