本文選題：體細胞胚發(fā)育　切入點：蛋白組　出處：《中國農(nóng)業(yè)大學》2016年博士論文

【摘要】：棉花體細胞胚發(fā)育作為體細胞胚胎發(fā)生的重要組成部分,對提高轉(zhuǎn)基因棉花再生率有重要的影響。本研究以中棉所24為實驗材料,利用iTRAQ和RNA-seq技術(shù)對棉花體細胞胚發(fā)育過程中的蛋白和基因動態(tài)表達譜進行了分析,分離得到與棉花體細胞胚發(fā)育相關(guān)的特異基因,在組學水平上揭示體細胞胚發(fā)育的分子機制,并初步揭示了JA和ABA調(diào)控棉花體細胞胚胎發(fā)生的分子機制。主要結(jié)果如下：1、分離鑒定與棉花體細胞胚發(fā)育相關(guān)的關(guān)鍵通路,分析激素對體細胞胚發(fā)育的影響通過對球形胚和子葉胚進行蛋白質(zhì)組分析,獲得顯著差異表達的蛋白209個,107個蛋白在球形胚顯著上調(diào),102個蛋白在子葉胚顯著上調(diào)。功能聚類分析發(fā)現(xiàn),它們主要參與了壓力響應(yīng)、激素合成和信號轉(zhuǎn)導、糖和能量代謝、蛋白和細胞壁代謝、光合作用、細胞和脂類物質(zhì)轉(zhuǎn)運。在體細胞胚發(fā)育過程中,JA合成基因AOS和ABA受體PYL/PYR顯著上調(diào),GA受體GID1顯著下調(diào),推測這三種激素在控制胚發(fā)育方面有重要的作用。通過外源添加激素處理球形胚,發(fā)現(xiàn)JA有利于次級體細胞胚的產(chǎn)生,ABA會顯著增加次級子葉胚的數(shù)量；相反,GA處理會造成體細胞胚膨大畸形,但其抑制劑多效唑有利于次級體細胞胚和類胚性愈傷物質(zhì)的產(chǎn)生。這些結(jié)果表明：激素的動態(tài)變化對體細胞胚發(fā)育有重要的影響,通過調(diào)控激素水平可以控制體細胞胚發(fā)育。2、篩選調(diào)控棉花體細胞胚SAM和RAM發(fā)育的關(guān)鍵基因?qū)η蛐闻�、魚雷胚和子葉胚進行轉(zhuǎn)錄組分析,將差異表達的基因進行GO和KEGG分析,發(fā)現(xiàn)它們主要涉及到激素的動態(tài)平衡、壓力響應(yīng)、生長素轉(zhuǎn)運、SAM和RAM發(fā)育、子葉原基和胚根發(fā)育。PIN1調(diào)控的生長素轉(zhuǎn)運不僅可以控制SAM和RAM的干細胞的維持和分化,還有利于基頂軸的建立。MP和它的靶標TMO5參與了體細胞胚胚根的起始和發(fā)育,表達水平的持續(xù)降低可能阻止了胚根的發(fā)育。3、JA調(diào)控棉花體細胞胚胎發(fā)生機制的研究外源JA處理誘導了GhMYC3-like的上調(diào)表達,過表達GhMYC3-like抑制愈傷的生長和體細胞胚的產(chǎn)生；干涉GhMYC3-like促進愈傷的生長,說明JA信號轉(zhuǎn)導在控制體細胞脫分化方面發(fā)揮著重要的作用,JA可能通過誘導GhMYC3-like的表達調(diào)控H202的動態(tài)平衡,進而調(diào)控棉花體細胞胚胎發(fā)生。4、ABA調(diào)控棉花體細胞胚胎發(fā)生機制的研究外源ABA處理誘導LEC1、LEC2、FUS3和WUSCHEL基因的表達,這些基因的表達調(diào)控ABA和GA的動態(tài)平衡促進棉花體細胞胚的產(chǎn)生及成熟。ABA誘導GhMYB44-1表達,過表達GhMYB44-1抑制了PP2Cs基因的表達,進而調(diào)控ABA的動態(tài)平衡來促進體細胞胚發(fā)育和再生苗的獲得。5、利用Sfold軟件預(yù)測VIGS效率利用Sfold軟件分析siRNA的AGdisruption、DSSE和AIS來比較靶標片段的干涉效率。通過實驗分析,發(fā)現(xiàn)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軟件可以提前預(yù)測基因的靶標片段干涉效率的高低,從而有效的設(shè)計VIGS實驗。
[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.
【學位授予單位】：中國農(nóng)業(yè)大學
【學位級別】：博士
【學位授予年份】：2016
【分類號】：S562
，

本文編號：1704843