摘要
我们用反向遗传学途径研究了R2R3 MYB基因在逆境胁迫中的功能。在本论文中,基于极其保守的MYB DNA结合结构域和很高的序列一致性,我们一起分析了AtMYB50和AtMYB61基因的功能特性。主要分析了AtMYB50和AtMYB61基因在重金属铅、低磷和渗透等非生物胁迫方面的作用。T-DNA插入导致的功能缺失性突变体atmyb50、atmyb61-Ⅰ和atmyb61-2较野生型均表现出不同程度的铅、低磷和渗透胁迫耐受性。
无论是短期诱导还是长期诱导,AtMYB50和AtMYB61基因的表达被铅强烈抑制。AtMYB50和AtMYB61基因的功能缺失性突变体较野生型有明显较低的铅含量,至少在一定程度上与AtPDR12基因的表达增强有关,AtPDR12被认为是一个位于细胞膜上的铅离子泵,负责将细胞质中的铅离子或铅复合物泵出到细胞外。然而,GSH1基因在突变体中的表达没有大的改变,其是一个谷胱甘肽合成基因,谷胱甘肽能够螯合细胞质中的重金属离子并将其隔离到非活跃的细胞器中。该实验表明AtMYB50和AtMYB61基因敲除突变体对铅的耐受性不依赖于谷胱甘肽途径。这说明,这两个R2R3 MYB基因对重金属的耐受性不依赖于GSH途径,但至少部分地与AtPDR12基因有关。鉴于此,我们构建了双突变体atpdr12atmyb50和atpdr12atmyb61,发现在铅胁迫下其表型和铅含量均与atpdr12单突变体相似,说明AtMYB50和AtMYB61蛋白可能作为上游调控的转录因子调节了植株对Pb的耐受性。与此同时,我们构建了atpdr 50atmyb61双突变体,发现其铅胁迫耐受性较atmyb50和atmyb61单突变体显著提高,且铅含量也明显下降,表明AtMYB50和AtMYB61基因在调节铅耐受性方面具有独立性。综上所述,AtMYB50和AtMYB61基因的敲除性突变,可以增强铅耐受性并降低植株体内的铅含量。这可能对生长在铅污染土壤中的农作物的基因改良具有实际意义。有意思的是,除了对铅的耐受性,atmyb50和atmyb61突变体对CdCl2,CuSO4, CaCl2, MnCl2, AgNO3, FeSO4, FeCl3, NiSO4, ZnSO4, CoCl2和H2O2均没有耐受性,说明突变体对铅的耐受性可能具有专一性。总之,从突变体的表型到其铅含量,AtPDR12和GSH1基因表达水平的鉴定,及双突变体atmyb50atpdrl2和atmyb61atpdr12的生物学特性,表明AtMYB50andAtMYB61转录因子可能参与了AtPDR12基因的表达调控,从而将细胞质中的铅离子或铅复合物泵到细胞外,但其对AtPDR12基因的调控是直接还是间接效应仍不能肯定。
AtMYB50和AtMYB61基因敲除突变体也表现出低磷不敏感性。实验结果表明突变体中的磷含量没有明显变化,但花青素的含量则较野生型明显下降。然而,虽然突变体的主根长度明显优于野生型,但野生型和突变体在低磷胁迫条件下的根毛数量却都显著增多。低磷胁迫相关基因表达分析表明AtPT1、AtACP5和AtPAP1磷转运子在突变体中有较大的变化。突变体对低磷的不敏感可能与植株体内激素合成能力的优化有关。
另外,我们发现atmyb50和atmyb61突变体表现出增强的渗透和干旱胁迫耐受性。在渗透胁迫方面,我们用NbCl、KCl、LiCl和甘露醇作为胁迫介质检测了突变体在其作用下的表型,结果表明所有的突变体都不同程度地对这些渗透物质耐受,无论是在发芽率还是植株体积的大小方面。最后,我们证实atmyb50和atmyb61突变体相对于野生型对干旱胁迫也有耐受性。因此,我们认为atmyb50和atmyb61对渗透胁迫和干旱的耐受性可能有一定的相互关系。
本论文主要创新点：
1.MYB转录因子家族是拟南芥中最大的一个基因家族,主要与植物生长发育和逆境胁迫响应有关。我们的实验选择了重金属(尤其是Pb)作为逆境胁迫因子,以此揭示MYB转录因子是否参与了重金属胁迫的调节。
2.我们选择了两个拟南芥R2R3 MYB基因AtMYB50和.AtMYB61,它们在整个MYB家族中具有最近的进化关系。实验表明这两个转录因子具有许多相似的功能,这可能与它们具有很高的序列一致性有关。
We have undertaken a systematic reverse genetic approach to study functions of R2R3-MYB genes in Arabidopsis, particularly in terms of defence response. Here, we describe the functional,characterization of AtMYB50 and AtMYB61 together based on the extended degree of sequence similarity especially within their highly conserved MYB DNA-binding domains. In this study, the focus is on the involvement of AtMYB50 and AtMYB61 genes in the abiotic stress of lead (Pb), low phosphorus (LP) and osmotic. The T-DNA insertion mutants atmyb50, atmyb61-1 and atmyb61-2 were confirmed to be function deficiency mutants, which showed enhanced tolerance to Pb(Ⅱ), LP and osmotic stress compared with the wild type.
AtMYB50 and AtMYB61 genes were strongly repressed by Pb(Ⅱ) treatment in both short-term and long-term inductions. Decreased Pb(Ⅱ) contents in AtMYB50 and AtMYB61 knockout plants relative to wild type were detected when subjected to Pb(II) treatment. It may be, at least in part, a result of increased expression level of AtPDR12 gene, which is considered as a pump located in the cell membrane have the ability to exclude Pb(Ⅱ) and/or Pb(Ⅱ)-containing toxic compounds from the cytoplasm. However, the expression level of GSH1, a gene involved in GSH synthesis to chelate heavy metals and sequester them into inactive organelles, was not changed, suggesting enhanced Pb(Ⅱ) resistance of AtMYB50 and AtMYB 61-knockout mutants was glutathione (GSH)-independent. This implied that these two R2R3 MYB genes regulated Pb(Ⅱ) tolerance through a GSH-independent but AtPDR12-mediated pathway. Also, atpdr12atmyb50 and atpdr12atmyb61 double mutants were costructed to further test this mechanism, appearance similarity coupled to alike Pb(Ⅱ) content with the atpdr12 mutant indicates that AtMYB50 and AtMYB61 proteins may act as up-stream TFs in regulating Pb response. Also, increased resistance of atpdr50atmyb61 double mutant from phenotype as well as decreased Pb content relative to atmyb50 and atmyb61 single mutant when subjected to Pb(Ⅱ) treatment suggests that these two proteins may work independently in Pb(Ⅱ) response. These results imply that knockout of AtMYB50 and AtMYB61 genes lead to enhanced Pb(Ⅱ) tolerance coupled with lower Pb(II) accumulation, which may be useful for the development of crop plants that grow in heavy metal contaminated soil. Interestingly, both atmyb50 and atmyb61 plants were neither CdCl2, CuSO4, CaCl2, MnCl2, AgNO3, FeSO4, FeCl3, NiSO4, ZnSO4, CoCl2 nor H2O2 resistant, which suggests that AtMYB50 and AtMYB61 may specificly implicated in Pb tolerance. Take together, the mutant appearance and their Pb(II) contents, as well as the identification of the AtPDR12 and GSH1 expression levels, and characterastics of the double mutants atmyb50atpdrl2 and atmyb61atpdr12, suggest that AtMYB50 and AtMYB61 transcription factors may participate in regulation of the AtPDR12 pump to exclude Pb(Ⅱ) and/or Pb(Ⅱ)-containing toxic compounds from cytoplasm. However, it is still unclear whether they directly or an indirectly regulates the expression of AtPDR12.
AtMYB50 and AtMYB61-knockout mutants also exhibit insensitive to low phosphorus stress. Results indicated that P contents in the mutants were not changed significantly, while anthocyanidin contents decreased obviously. However, both WT and mutant displayed increased root hair numbers in low phosphorus condition though the later showed much longer primary roots. LP-related gene expression analyse suggested that AtPTl, AtACP5 and AtPAPl were varied significantly in the mutant under LP condition. Enhanced low phosphuros resistance of AtMYB50 and AtMYB61-knockout mutants may be assaciated with optimized in vivo hormone synthesis.
In addition, we also found atmyb50 and atmyb61 mutants displayed enhanced osmotic and drought tolerance. In terms of osmotic stress, we utilized NaCl, KC1, LiCl and Mannitol to test the phenotype. Results indicated that all mutants showed increased resistance to these osmotic mediators, no matter in seed germination ratio or in seedling volumn. Finally, we confirmed the increased tolerance of atmyb50 and atmyb61 mutants to drought stress in realative to WT. Thus, we conferred that the enhanced resistance of atmyb50 and atmyb61 to drought stress may be closely related to osmotic stress.
The innovations of this thesis can be summarized as follows:
1. MYB TFs is the biggest gene family in Arabidopsis, which is maily involved in the regulation of plant growth and development and defence responses. However, little is known about their roles in the rugulation of heavey metal resistance. Our results suggest that AtMYB50 and AtMYB61 play important roles in heavy metals (particularly Pb) resistance.
2. We chosed two R2R3 MYB genes AtMYB50 and AtMYB61, which had the most close relationship in the whole MYB family. Our results indicated that these two factors really possess many similar functions, which may be a result of sequence similarity.
引文
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