肌醇代谢调节农杆菌介导拟南芥根受体转化机制的研究

发布时间：2018-03-02 17:33

本文选题：农杆菌转化　切入点：肌醇　出处：《中国农业大学》2017年博士论文　论文类型：学位论文

【摘要】：本研究分别用外源肌醇(myo-inositol,MI)预处理拟南芥(Arabiddopsis thaliana)生态型Col-)植株的根系和MI代谢终端的植酸(IP6)合成酶基因AtIPk1突变体atiPK1的根做农杆菌(Agrobacterium tumefaciens,菌株A208)转化受体。采用形态观察、转录组测序、实时荧光定量检测、组织化学分析、高效离子色谱分析(HPIC)和遗传转化效率比较等方法探究外源MI和肌醇磷酸盐(IPs)代谢途径AtIPK1基因影响拟南芥根对农杆菌介导转化敏感性的作用机理。我们的研究结果主要包括以下几个方面:外源MI预处理的拟南芥根受体对农杆菌介导转化的敏感性存在显著影响。添加外源MI(MI+,100 mg/LMI)处理提高了拟南芥根的农杆菌转化效率。基因表达水平在转化前后存在时效性。添加MI有利于愈伤组织的生长和发育,植物防御基因和T-DNA整合基因表达差异显著。MI+处理的根和根愈伤组织中IP6含量高于MI-处理,Pi含量则相反。结果表明,外源添加MI调节和维持细胞的生长发育有重要作用,影响了植物细胞的物质代谢水平。通过转录组分析,在两个处理(MI-和MI+)样本中挖掘了至少184个新基因和183个差异表达基因(DEGs)。基因功能注释表明DEGs涉及植物细胞壁的合成、激素信号调节、胁迫响应(植物防御)、物质转运、染色体修饰和DNA修复、次生物质代谢等过程。这些结果表明外源MI处理在一定程度上影响了植物基因的转录水平,对植物细胞的分子代谢有影响。拟南芥突变体atipk1的转化效率显著低于与野生型Col-0。突变体atipk1过表达AtIPK1后能够回补野生型的表型。虽然突变体atipk2p能够阻碍部分IP6的合成,但是,突变体atipk2β的表型和转化效率与野生型差异不显著。AtIPK1对转化的敏感性的调节可能与Pi和IP6代谢相关,在基因调控方面也有作用。外源MI调节了T-DNA整合(AtKu70、AtKu80、AtXRCC4),植物防御(AtWRKY62、AtWRKY70、AtPR1、AtPR2),与植物根转化相关(AtCSLA9、AtAGP17),细胞壁多糖前体合成(AtMIOX2)等基因的表达。突变体atipk1和AtIPK1过表达株系根受体中这些基因的表达差异更加显著。这些结果表明,根受体对农杆菌转化的敏感性与受体内源基因的表达调控相关,外源MI和AtIPK1有利于农杆菌介导的拟南芥根受体转化。外源MI和AtIPK1调节植物体内Pi与IP6的代谢平衡,直接或间接影响受体内基因的表达和物质代谢,对受体转化敏感性有显著的作用。这种作用可能是多个因子协同工作的结果。
[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.
【学位授予单位】：中国农业大学
【学位级别】：博士
【学位授予年份】：2017
【分类号】：Q945

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