WRKY12基因在拟南芥响应镉胁迫中的功能分析

发布时间：2018-05-01 11:16

本文选题：WRKY12 + 拟南芥　；参考：《合肥工业大学》2017年硕士论文

【摘要】：土壤重金属镉污染状况日益严峻,已经成为全球面临的生态难题,解决这个问题的关键是如何将污染土壤修复再利用。已有研究表明利用超富集植物对土壤中的重金属进行吸收转运可以达到土壤净化的功效。研究植物对重金属信号的响应机制及重金属的吸收转运途径,对植物修复技术的应用提供更多理论依据。本课题主要是研究拟南芥转录因子WRKY12对镉胁迫信号的响应机制,初步探究WRKY12基因功能及其作用机理。获得了以下研究结果:1.用CdCl_2处理拟南芥WT幼苗发现,WRKY12基因可以被Cd诱导表达。拟南芥突变体wrky12-1、wrky12-2在镉胁迫的条件下与WT相比表现为明显的耐受性,说明WRKY12基因可能参与拟南芥镉胁迫应答机制,其功能缺失会导致拟南芥对镉的耐受。2.克隆拟南芥WRKY12基因,将其连接到含有强启动子的pART27载体上,构建WRKY12过表达载体,通过转基因技术,将重组载体转入拟南芥野生型体内,筛选获得WRKY12过表达阳性植株。3.WRKY12过表达植株在镉胁迫的条件下与WT相比表现出敏感表型,由此说明WRKY12基因在拟南芥响应镉胁迫应答中的功能可能是负调控作用。4.wrky12突变体和过表达植株根和茎的镉积累量数据显示,拟南芥幼苗在含有CdCl_2的培养基中生长一段时间后,突变体根部和茎部镉积累量明显高于WT;而WRKY12过表达植株根部和茎部镉积累量却显著低于WT。由此说明,拟南芥对镉的积累量增加可能由于WRKY12基因功能缺失导致。5.通过qRT-PCR技术分析参与GSH合成途径相关基因转录水平。在镉暴露的情况下,wrky12突变体体内GSH1、PCS2基因的表达量显著升高;而在WRKY12过表达植株中,这两种基因的表达量则呈现降低趋势。说明WRKY12转录因子可能参与GSH1、PCS2基因的转录调控。6.拟南芥GSH、PC含量测定发现,wrky12突变体Cd处理后的GSH含量高于WT,而WRKY12过表达植株的GSH含量与WT相比呈现降低趋势;wrky12突变体Cd处理后的PC含量明显高于WT,由此说明,WRKY12转录因子可能是通过调控GSH、PC的合成途径参与镉胁迫的响应机制。综上所述,WRKY12可能是参与拟南芥镉胁迫响应的负调控因子,通过调控GHS、PC合成途径相关基因的转录来调控GSH、PC的合成量,进而影响拟南芥对镉的耐受性。
[Abstract]:The pollution of heavy metals and cadmium in soil is becoming more and more serious, and it has become an ecological problem in the world. The key to solve this problem is how to repair and reuse the contaminated soil. The research shows that the use of hyperconcentration plants to absorb and transport heavy metals in soil can achieve the effect of soil purification. The response mechanism and the absorption and transport of heavy metals provide more theoretical basis for the application of phytoremediation technology. This topic is mainly to study the response mechanism of the Arabidopsis transcription factor WRKY12 to the cadmium stress signal and preliminarily explore the function and mechanism of WRKY12 gene. The following results are obtained: 1. the WT seedlings of Arabidopsis thaliana are treated by CdCl_2 It was found that the WRKY12 gene could be induced by Cd. The Arabidopsis mutants wrky12-1, wrky12-2 showed obvious tolerance compared with WT under the condition of cadmium stress, indicating that the WRKY12 gene might participate in the cadmium stress response mechanism of Arabidopsis, and its functional deletion could lead to the tolerance to cadmium in Arabidopsis by.2. cloning of the Arabidopsis WRKY12 gene of the Arabidopsis, and to connect it to containing the Arabidopsis thaliana. On the pART27 vector with strong promoter, WRKY12 overexpression vector was constructed. Through transgenic technology, the recombinant vector was transferred into the wild type of Arabidopsis thaliana, and the WRKY12 overexpressed plant.3.WRKY12 overexpressed plants showed a sensitive table compared with WT under the condition of cadmium stress. Thus the WRKY12 gene responds to the cadmium stress in Arabidopsis thaliana. The function of the forced response may be the negative regulatory effect of the.4.wrky12 mutant and the cadmium accumulation in the overexpressed plant roots and stems. The cadmium accumulation in the root and stem of the mutant was significantly higher than that of the WT after the growth of the Arabidopsis seedlings in the medium containing CdCl_2, while the cadmium accumulation in the roots and stems of the WRKY12 overexpressed plants was significantly lower. WT. suggests that the accumulation of cadmium in Arabidopsis may be due to the deletion of WRKY12 gene function resulting in the involvement of.5. in the GSH synthesis pathway related gene transcription level through qRT-PCR technology analysis. In the case of cadmium exposure, the expression of GSH1 and PCS2 genes in the wrky12 mutants increased significantly; and these two groups were in the WRKY12 overexpressed plants. The expression of the WRKY12 transcriptional factor may be reduced. It shows that the transcription factor of the PCS2 gene may participate in the GSH1, the transcriptional regulation of the PCS2 gene of.6. Arabidopsis thaliana, the content determination of PC found that the GSH content of the wrky12 mutant Cd is higher than that of WT, while the GSH content of the WRKY12 overexpressed plant is lower than that of the Cd. In WT, it is suggested that the WRKY12 transcription factor may be a response mechanism for cadmium stress by regulating the synthesis pathway of GSH and PC. To sum up, WRKY12 may be a negative regulator involved in the response of cadmium stress in Arabidopsis. By regulating the transcription of GHS, PC synthesis pathway related genes, the synthesis of GSH, PC and the tolerance to cadmium tolerance in Arabidopsis thaliana can be affected. Be subject to sex.

【学位授予单位】：合肥工业大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：Q943.2

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