【摘要】：高等植物主要通過絡(luò)合作用和區(qū)室化作用進(jìn)行重金屬解毒,而谷胱甘肽合酶和植物絡(luò)合素合酶是植物體重金屬解毒途徑的關(guān)鍵酶。本研究以煙草(Nicotiana tabacum)為材料,克隆煙草谷胱甘肽合酶基因(NtGSH2)和植物絡(luò)合素合酶基因(NtPCS1)。通過序列比對(duì),組織表達(dá)模式,重金屬誘導(dǎo)表達(dá)等方面分析NtGSH2和NtPCS1基因的基本分子特征。并通過異源表達(dá)和過量表達(dá)分析NtPCS1基因的生物學(xué)功能。結(jié)果顯示:(1)來自不同物種的GSH2蛋白的氨基酸序列高度保守,而PCS蛋白的N端結(jié)構(gòu)域序列高度保守。(2)NtGSH2和NtPCS1基因在煙草的根、莖、葉、花和種子均表達(dá),且均在花器官中的表達(dá)量最高。(3)NtGSH2和NtPCS1基因的轉(zhuǎn)錄表達(dá)均受到Cd~(2+)和Zn~(2+)的誘導(dǎo)而上調(diào)。(4)NtPCS1基因編碼植物絡(luò)合素合酶蛋白,且Ag~(2+)和Cd~(2+)能夠顯著增加植物絡(luò)合素合酶蛋白的活性。(5)NtPCS1基因的異源表達(dá)能夠增強(qiáng)擬南芥Atpcs1突變體對(duì)Cd~(2+)的耐受性(6)NtPCS1基因的過量表達(dá)導(dǎo)致煙草對(duì)Cd~(2+)超敏感。(7)NtPCS1基因的過量表達(dá)導(dǎo)致煙草體內(nèi)PCs含量的顯著增加。(8)外源GSH的添加能夠減弱過表達(dá)轉(zhuǎn)基因煙草對(duì)Cd~(2+)的超敏感。(9)外源GSH的添加導(dǎo)致過表達(dá)轉(zhuǎn)基因煙草體內(nèi)積累更多的Cd~(2+)和Zn~(2+)。
[Abstract]:High plants detoxify heavy metals mainly by complexation and compartmentalization, and glutathione synthase and plant complexin synthase are the key enzymes in detoxification pathway of heavy metals in plants. In this study, tobacco glutathione synthase (NtGSH2) gene and plant complex synthase gene (NtPCS1) were cloned from tobacco (Nicotiana tabacum). The basic molecular characteristics of NtGSH2 and NtPCS1 genes were analyzed by sequence alignment, tissue expression patterns and heavy metal induced expression. The biological function of NtPCS1 gene was analyzed by heterologous expression and overexpression. The results showed that: (1) the amino acid sequence of GSH2 protein from different species was highly conserved, while the N-terminal domain of PCS protein was highly conserved. (2) NtGSH2 and NtPCS1 genes were expressed in roots, stems, leaves, flowers and seeds of tobacco. (3) the transcription expression of NtGSH2 and NtPCS1 genes were up-regulated by Cd~ (2) and Zn~ (2). (4) NtPCS1 gene encodes plant complex synthase protein. Ag~ (2) and Cd~ (2) could significantly increase the activity of plant complexin synthase protein. (5) the heterologous expression of NtPCS1 gene could enhance the tolerance of Arabidopsis Atpcs1 mutants to Cd~ (2). (6) the overexpression of NtPCS1 gene resulted in tobacco being hypersensitive to Cd~ (2) NtPCS1 gene. (8) the addition of exogenous GSH could weaken the hypersensitivity of overexpression transgenic tobacco to Cd~ (2). (9) the addition of exogenous GSH resulted in the accumulation of more Cd~ (2) and Zn~ (2) in over-expressed transgenic tobacco.
【學(xué)位授予單位】：西南科技大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2017
【分類號(hào)】：Q943.2

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