本文選題：WRKY12 + 擬南芥�。� 參考：《合肥工業(yè)大學》2017年碩士論文

【摘要】：土壤重金屬鎘污染狀況日益嚴峻,已經(jīng)成為全球面臨的生態(tài)難題,解決這個問題的關(guān)鍵是如何將污染土壤修復再利用。已有研究表明利用超富集植物對土壤中的重金屬進行吸收轉(zhuǎn)運可以達到土壤凈化的功效。研究植物對重金屬信號的響應機制及重金屬的吸收轉(zhuǎn)運途徑,對植物修復技術(shù)的應用提供更多理論依據(jù)。本課題主要是研究擬南芥轉(zhuǎn)錄因子WRKY12對鎘脅迫信號的響應機制,初步探究WRKY12基因功能及其作用機理。獲得了以下研究結(jié)果:1.用CdCl_2處理擬南芥WT幼苗發(fā)現(xiàn),WRKY12基因可以被Cd誘導表達。擬南芥突變體wrky12-1、wrky12-2在鎘脅迫的條件下與WT相比表現(xiàn)為明顯的耐受性,說明WRKY12基因可能參與擬南芥鎘脅迫應答機制,其功能缺失會導致擬南芥對鎘的耐受。2.克隆擬南芥WRKY12基因,將其連接到含有強啟動子的pART27載體上,構(gòu)建WRKY12過表達載體,通過轉(zhuǎn)基因技術(shù),將重組載體轉(zhuǎn)入擬南芥野生型體內(nèi),篩選獲得WRKY12過表達陽性植株。3.WRKY12過表達植株在鎘脅迫的條件下與WT相比表現(xiàn)出敏感表型,由此說明WRKY12基因在擬南芥響應鎘脅迫應答中的功能可能是負調(diào)控作用。4.wrky12突變體和過表達植株根和莖的鎘積累量數(shù)據(jù)顯示,擬南芥幼苗在含有CdCl_2的培養(yǎng)基中生長一段時間后,突變體根部和莖部鎘積累量明顯高于WT;而WRKY12過表達植株根部和莖部鎘積累量卻顯著低于WT。由此說明,擬南芥對鎘的積累量增加可能由于WRKY12基因功能缺失導致。5.通過qRT-PCR技術(shù)分析參與GSH合成途徑相關(guān)基因轉(zhuǎn)錄水平。在鎘暴露的情況下,wrky12突變體體內(nèi)GSH1、PCS2基因的表達量顯著升高;而在WRKY12過表達植株中,這兩種基因的表達量則呈現(xiàn)降低趨勢。說明WRKY12轉(zhuǎn)錄因子可能參與GSH1、PCS2基因的轉(zhuǎn)錄調(diào)控。6.擬南芥GSH、PC含量測定發(fā)現(xiàn),wrky12突變體Cd處理后的GSH含量高于WT,而WRKY12過表達植株的GSH含量與WT相比呈現(xiàn)降低趨勢;wrky12突變體Cd處理后的PC含量明顯高于WT,由此說明,WRKY12轉(zhuǎn)錄因子可能是通過調(diào)控GSH、PC的合成途徑參與鎘脅迫的響應機制。綜上所述,WRKY12可能是參與擬南芥鎘脅迫響應的負調(diào)控因子,通過調(diào)控GHS、PC合成途徑相關(guān)基因的轉(zhuǎn)錄來調(diào)控GSH、PC的合成量,進而影響擬南芥對鎘的耐受性。
[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.

【學位授予單位】：合肥工業(yè)大學
【學位級別】：碩士
【學位授予年份】：2017
【分類號】：Q943.2

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