本文選題：農(nóng)桿菌轉(zhuǎn)化　切入點(diǎn)：肌醇　出處：《中國農(nóng)業(yè)大學(xué)》2017年博士論文　論文類型：學(xué)位論文

【摘要】：本研究分別用外源肌醇(myo-inositol,MI)預(yù)處理擬南芥(Arabiddopsis thaliana)生態(tài)型Col-)植株的根系和MI代謝終端的植酸(IP6)合成酶基因AtIPk1突變體atiPK1的根做農(nóng)桿菌(Agrobacterium tumefaciens,菌株A208)轉(zhuǎn)化受體。采用形態(tài)觀察、轉(zhuǎn)錄組測序、實(shí)時熒光定量檢測、組織化學(xué)分析、高效離子色譜分析(HPIC)和遺傳轉(zhuǎn)化效率比較等方法探究外源MI和肌醇磷酸鹽(IPs)代謝途徑AtIPK1基因影響擬南芥根對農(nóng)桿菌介導(dǎo)轉(zhuǎn)化敏感性的作用機(jī)理。我們的研究結(jié)果主要包括以下幾個方面:外源MI預(yù)處理的擬南芥根受體對農(nóng)桿菌介導(dǎo)轉(zhuǎn)化的敏感性存在顯著影響。添加外源MI(MI+,100 mg/LMI)處理提高了擬南芥根的農(nóng)桿菌轉(zhuǎn)化效率。基因表達(dá)水平在轉(zhuǎn)化前后存在時效性。添加MI有利于愈傷組織的生長和發(fā)育,植物防御基因和T-DNA整合基因表達(dá)差異顯著。MI+處理的根和根愈傷組織中IP6含量高于MI-處理,Pi含量則相反。結(jié)果表明,外源添加MI調(diào)節(jié)和維持細(xì)胞的生長發(fā)育有重要作用,影響了植物細(xì)胞的物質(zhì)代謝水平。通過轉(zhuǎn)錄組分析,在兩個處理(MI-和MI+)樣本中挖掘了至少184個新基因和183個差異表達(dá)基因(DEGs)。基因功能注釋表明DEGs涉及植物細(xì)胞壁的合成、激素信號調(diào)節(jié)、脅迫響應(yīng)(植物防御)、物質(zhì)轉(zhuǎn)運(yùn)、染色體修飾和DNA修復(fù)、次生物質(zhì)代謝等過程。這些結(jié)果表明外源MI處理在一定程度上影響了植物基因的轉(zhuǎn)錄水平,對植物細(xì)胞的分子代謝有影響。擬南芥突變體atipk1的轉(zhuǎn)化效率顯著低于與野生型Col-0。突變體atipk1過表達(dá)AtIPK1后能夠回補(bǔ)野生型的表型。雖然突變體atipk2p能夠阻礙部分IP6的合成,但是,突變體atipk2β的表型和轉(zhuǎn)化效率與野生型差異不顯著。AtIPK1對轉(zhuǎn)化的敏感性的調(diào)節(jié)可能與Pi和IP6代謝相關(guān),在基因調(diào)控方面也有作用。外源MI調(diào)節(jié)了T-DNA整合(AtKu70、AtKu80、AtXRCC4),植物防御(AtWRKY62、AtWRKY70、AtPR1、AtPR2),與植物根轉(zhuǎn)化相關(guān)(AtCSLA9、AtAGP17),細(xì)胞壁多糖前體合成(AtMIOX2)等基因的表達(dá)。突變體atipk1和AtIPK1過表達(dá)株系根受體中這些基因的表達(dá)差異更加顯著。這些結(jié)果表明,根受體對農(nóng)桿菌轉(zhuǎn)化的敏感性與受體內(nèi)源基因的表達(dá)調(diào)控相關(guān),外源MI和AtIPK1有利于農(nóng)桿菌介導(dǎo)的擬南芥根受體轉(zhuǎn)化。外源MI和AtIPK1調(diào)節(jié)植物體內(nèi)Pi與IP6的代謝平衡,直接或間接影響受體內(nèi)基因的表達(dá)和物質(zhì)代謝,對受體轉(zhuǎn)化敏感性有顯著的作用。這種作用可能是多個因子協(xié)同工作的結(jié)果。
[Abstract]:In this study, the roots of Arabiddopsis thaliana ecotypic Col-pretreated with exogenous inositol myo-inositolol (MIM) and the root of AtIPk1 mutant atiPK1 were used as Agrobacterium tumefaciens (strain A208) transformation receptors. The transcriptional sequence was analyzed by morphological observation and transcriptional sequencing. Real-time fluorescence quantitative detection, histochemical analysis, High performance Ion Chromatography (HPLC) and comparison of genetic Transformation efficiency to explore the mechanisms of exogenous MI and Inositol Phosphate (IPs) Metabolism Pathway AtIPK1 Gene affecting the sensitivity of Arabidopsis Root to Agrobacterium tumefaciens mediated Transformation. The effects of exogenous MI pretreatment on the sensitivity of Arabidopsis root receptors to Agrobacterium tumefaciens mediated transformation were significant, and the efficiency of Agrobacterium tumefaciens transformation was improved by adding exogenous MI(MI 100mg / L MI-treated Arabidopsis root. The expression level was time-dependent before and after transformation. The addition of MI was beneficial to the growth and development of callus. The difference between plant defense gene and T-DNA integration gene expression was significant. The content of IP6 in root and root callus treated with MI was higher than that in MI-treated callus. The results showed that exogenous MI played an important role in regulating and maintaining the growth and development of cells. Through transcriptome analysis, at least 184 new genes and 183 differentially expressed genes were excavated in two treatments (MI- and MI) samples. Gene functional annotation showed that DEGs was involved in the synthesis of plant cell walls. Hormone signal regulation, stress response (plant defense, substance transport, chromosome modification and DNA repair, secondary substance metabolism, etc.) these results suggest that exogenous MI treatment affects the transcription level of plant genes to some extent. The transformation efficiency of Arabidopsis thaliana mutant atipk1 was significantly lower than that of wild type Col-0. the mutant atipk1 could compensate for the wild-type phenotype after overexpression of AtIPK1. Although the mutant atipk2p could inhibit the synthesis of some IP6, the transformation efficiency of Arabidopsis mutants was significantly lower than that of wild type Col-0. The phenotype and transformation efficiency of mutant atipk2 尾 were not significantly different from those of wild type. The regulation of the sensitivity of AtIPK1 to transformation may be related to the metabolism of Pi and IP6. Exogenous MI regulates the expression of genes such as T-DNA integrator AtKu70, AtKu80, AtXRCC4, plant defenses AtWRKY62and AtWRKY70, AtWRKY70, AtWRKY70, AtWRKY70, AtPR2, ATCSLA9AGP17, and cell wall polysaccharide precursor synthesis, AtMIOX2. These genes are expressed in root receptors of mutant atipk1 and AtIPK1 overexpression lines. The difference in gene expression is even more significant. These results suggest that, The sensitivity of root receptor to Agrobacterium tumefaciens transformation was related to the regulation of endogenous gene expression. Exogenous MI and AtIPK1 were beneficial to Agrobacterium tumefaciens mediated root receptor transformation. Exogenous MI and AtIPK1 regulated the metabolic balance of Pi and IP6 in plants. Direct or indirect effects on gene expression and substance metabolism in the recipient may play a significant role in receptor transformation sensitivity, which may be the result of the synergistic work of multiple factors.
【學(xué)位授予單位】：中國農(nóng)業(yè)大學(xué)
【學(xué)位級別】：博士
【學(xué)位授予年份】：2017
【分類號】：Q945

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