棉花体细胞胚发育组学分析及JA和ABA调控的研究

发布时间：2018-04-03 10:40

  本文选题：体细胞胚发育　切入点：蛋白组　出处：《中国农业大学》2016年博士论文

【摘要】：棉花体细胞胚发育作为体细胞胚胎发生的重要组成部分,对提高转基因棉花再生率有重要的影响。本研究以中棉所24为实验材料,利用iTRAQ和RNA-seq技术对棉花体细胞胚发育过程中的蛋白和基因动态表达谱进行了分析,分离得到与棉花体细胞胚发育相关的特异基因,在组学水平上揭示体细胞胚发育的分子机制,并初步揭示了JA和ABA调控棉花体细胞胚胎发生的分子机制。主要结果如下：1、分离鉴定与棉花体细胞胚发育相关的关键通路,分析激素对体细胞胚发育的影响通过对球形胚和子叶胚进行蛋白质组分析,获得显著差异表达的蛋白209个,107个蛋白在球形胚显著上调,102个蛋白在子叶胚显著上调。功能聚类分析发现,它们主要参与了压力响应、激素合成和信号转导、糖和能量代谢、蛋白和细胞壁代谢、光合作用、细胞和脂类物质转运。在体细胞胚发育过程中,JA合成基因AOS和ABA受体PYL/PYR显著上调,GA受体GID1显著下调,推测这三种激素在控制胚发育方面有重要的作用。通过外源添加激素处理球形胚,发现JA有利于次级体细胞胚的产生,ABA会显著增加次级子叶胚的数量；相反,GA处理会造成体细胞胚膨大畸形,但其抑制剂多效唑有利于次级体细胞胚和类胚性愈伤物质的产生。这些结果表明：激素的动态变化对体细胞胚发育有重要的影响,通过调控激素水平可以控制体细胞胚发育。2、筛选调控棉花体细胞胚SAM和RAM发育的关键基因对球形胚、鱼雷胚和子叶胚进行转录组分析,将差异表达的基因进行GO和KEGG分析,发现它们主要涉及到激素的动态平衡、压力响应、生长素转运、SAM和RAM发育、子叶原基和胚根发育。PIN1调控的生长素转运不仅可以控制SAM和RAM的干细胞的维持和分化,还有利于基顶轴的建立。MP和它的靶标TMO5参与了体细胞胚胚根的起始和发育,表达水平的持续降低可能阻止了胚根的发育。3、JA调控棉花体细胞胚胎发生机制的研究外源JA处理诱导了GhMYC3-like的上调表达,过表达GhMYC3-like抑制愈伤的生长和体细胞胚的产生；干涉GhMYC3-like促进愈伤的生长,说明JA信号转导在控制体细胞脱分化方面发挥着重要的作用,JA可能通过诱导GhMYC3-like的表达调控H202的动态平衡,进而调控棉花体细胞胚胎发生。4、ABA调控棉花体细胞胚胎发生机制的研究外源ABA处理诱导LEC1、LEC2、FUS3和WUSCHEL基因的表达,这些基因的表达调控ABA和GA的动态平衡促进棉花体细胞胚的产生及成熟。ABA诱导GhMYB44-1表达,过表达GhMYB44-1抑制了PP2Cs基因的表达,进而调控ABA的动态平衡来促进体细胞胚发育和再生苗的获得。5、利用Sfold软件预测VIGS效率利用Sfold软件分析siRNA的AGdisruption、DSSE和AIS来比较靶标片段的干涉效率。通过实验分析,发现具有低的AGdisruption,高的DSSE和高的AIS的siRNA可以提高VIGS的效率。因此,借助Sfold软件可以提前预测基因的靶标片段干涉效率的高低,从而有效的设计VIGS实验。
[Abstract]:As an important part of somatic embryogenesis, somatic embryogenesis plays an important role in improving the regeneration rate of transgenic cotton.In this study, the protein and gene dynamic expression profiles of cotton somatic embryogenesis were analyzed by iTRAQ and RNA-seq techniques, and specific genes related to cotton somatic embryogenesis were isolated.The molecular mechanism of somatic embryogenesis and the molecular mechanism of JA and ABA regulating somatic embryogenesis in cotton were revealed at the stratigraphic level.The main results were as follows: 1. The key pathways related to the development of somatic embryos in cotton were isolated and identified, and the effects of hormones on the development of somatic embryos were analyzed by proteomic analysis of globular embryos and cotyledon embryos.Among 209 proteins, 107 proteins were significantly up-regulated in globular embryos and 102 proteins in cotyledon embryos.Functional cluster analysis showed that they were involved in stress response, hormone synthesis and signal transduction, glucose and energy metabolism, protein and cell wall metabolism, photosynthesis, cell and lipid transport.During somatic embryogenesis, AOS and ABA receptor PYL/PYR significantly up-regulated GA-receptor GID1, suggesting that these three hormones play an important role in controlling embryo development.It was found that JA could significantly increase the number of secondary cotyledon embryos by adding exogenous hormones to the globular embryos, but on the contrary, GA treatment would cause somatic embryogenesis deformity.These results indicated that the dynamic changes of hormones had an important effect on somatic embryogenesis. The somatic embryogenesis of cotton could be controlled by regulating hormone level, and the key genes regulating the development of SAM and RAM in cotton somatic embryos could be screened from globular embryos.Transcriptome analysis of torpedo embryos and cotyledon embryos was performed. Go and KEGG analysis showed that the differentially expressed genes were mainly involved in hormone homeostasis, stress response, growth hormone transport and RAM development.Studies on the Mechanism of regulating somatic embryogenesis in Cotton by exogenous JA treatment induced up-regulation of GhMYC3-like expression, overexpression of GhMYC3-like inhibited callus growth and somatic embryogenesis;Interference GhMYC3-like promotes callus growth, suggesting that JA signal transduction plays an important role in controlling somatic dedifferentiation. JA may regulate the dynamic balance of H202 by inducing the expression of GhMYC3-like.Regulation of cotton somatic embryogenesis by ABA in cotton somatic embryogenesis; exogenous ABA treatment induced the expression of LEC1, LEC2, FUS3 and WUSCHEL genes.The expression of these genes regulated the dynamic balance of ABA and GA and promoted the production of somatic embryos and the induction of GhMYB44-1 expression by maturation. The overexpression of GhMYB44-1 inhibited the expression of PP2Cs gene.Furthermore, the dynamic balance of ABA was regulated to promote somatic embryogenesis and the acquisition of regenerated plantlets. The Sfold software was used to predict the efficiency of VIGS. The Sfold software was used to analyze siRNA's agglomeration and AIS to compare the interference efficiency of target fragments.Through experimental analysis, it is found that the efficiency of VIGS can be improved by siRNA with low AGD, high DSSE and high AIS.Therefore, the interference efficiency of target fragment can be predicted in advance by Sfold software, and the VIGS experiment can be designed effectively.
【学位授予单位】：中国农业大学
【学位级别】：博士
【学位授予年份】：2016
【分类号】：S562
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本文编号：1704843