本文選題：出芽短梗霉　切入點(diǎn)：ApHAL3　出處：《沈陽農(nóng)業(yè)大學(xué)》2017年碩士論文

【摘要】：HAL3蛋白家族是高度保守的黃素蛋白家族,酵母中的HAL3蛋白是一種Ppz蛋白磷酸酶的抑制因子,而擬南芥、水稻中的HAL3蛋白與大腸桿菌Dfp蛋白的N端具有非常相似的功能,即:4'-磷酸泛酰半胱氨酸脫羧酶(PPCDC)功能。PPCDC為輔酶A合成過程的關(guān)鍵酶,它可以催化4'-磷酸泛酰半胱氨酸(PPC)脫羧生成4'-磷酸泛酰巰基乙胺(PP)。HAL3蛋白還和生物的耐鹽性高度相關(guān),該基因的過表達(dá)能夠提高植物的轉(zhuǎn)基因株系對(duì)滲透脅迫、鹽脅迫的耐性。出芽短梗霉是一種耐鹽性較強(qiáng)的真菌,出芽短梗霉中ApHAL3基因的功能目前尚未有人報(bào)道。本文對(duì)出芽短梗霉ApHAL3基因的功能及功能位點(diǎn)進(jìn)行了研究。對(duì)出芽短梗霉ApHAL3基因編碼的氨基酸序列進(jìn)行分析,發(fā)現(xiàn)該氨基酸序列與大腸桿菌中編碼PPCDC的Dfp蛋白N端序列、擬南芥AtHAL3a、釀酒酵母ScHAL3以及水稻OsHAL3蛋白序列有較高的一致性。ApHAL3蛋白也具有寡聚黃素蛋白半胱氨酸脫羧酶蛋白家族的四個(gè)保守結(jié)構(gòu)域:(Ⅰ)PXMNXXMW結(jié)構(gòu)域(Ⅱ)組氨酸結(jié)構(gòu)域(Ⅲ)底物結(jié)合結(jié)構(gòu)域和(Ⅳ)底物識(shí)別夾結(jié)構(gòu)域。為了研究ApHAL3基因的功能,本文利用RT-PCR技術(shù)克隆得到ApHAL3蛋白的cDNA序列并構(gòu)建質(zhì)粒載體,利用PPCDC缺陷的大腸桿菌ΔdZp突變體菌株進(jìn)行功能互補(bǔ)實(shí)驗(yàn)。通過溫度敏感表型和營養(yǎng)缺陷實(shí)驗(yàn)分析可知:APHAL3可以功能互補(bǔ)大腸桿菌PPCDC缺陷的突變體菌株Δdfp。該結(jié)果表明ApHAL3蛋白具有PPCDC的功能。構(gòu)建多個(gè)攜帶點(diǎn)突變ApHAL3蛋白的載體,利用互補(bǔ)實(shí)驗(yàn)發(fā)現(xiàn):ApHAL3中保守的 Leu40、Trp106、Met120、Asn172、Cys212、Met220 以及 Ser116Ala117Asn118Ser119 是 PPCDC活性的關(guān)鍵位點(diǎn);而His100和Met175這兩個(gè)單點(diǎn)突變可以降低ApHAL3蛋白的PPCDC活性,表現(xiàn)為大腸桿菌Δdfp的轉(zhuǎn)化子在42℃培養(yǎng)時(shí)的生長速率下降;Va120、Arg105、Ala170、Leu202、Ala211、Asp214、Gly216 以及 Asp235 單點(diǎn)發(fā)生突變不影響 ApHAL3 蛋白的互補(bǔ)功能。分別用 1 mol/L、1.5 mol/L 和 2 mol/LNaCl 處理出芽短梗霉 4 h、8 h、12 h、20 h,利用qRT-PCR檢測出芽短梗霉ApHAL3基因在鹽脅迫下的轉(zhuǎn)錄水平。結(jié)果表明:NaCl濃度為1 mol/L、1.5 mol/L和2 mol/L時(shí),在鹽脅迫12 h時(shí),ApHAL3基因的表達(dá)量顯著下調(diào);NaCl濃度為1 mol/L和1.5 mol/L時(shí),在鹽脅迫20h,ApHAL3基因的表達(dá)量稍微上調(diào),但不顯著。
[Abstract]:The HAL3 protein family is a highly conserved family of flavin proteins. The HAL3 protein in yeast is an inhibitor of Ppz protein phosphatase. In Arabidopsis thaliana and rice, the HAL3 protein has very similar function to the N-terminal of Dfp protein in Escherichia coli. That is, the function of PPCDC.PPCDC is the key enzyme in the synthesis of coenzyme A. it can catalyze the decarboxylation of PPC4 to produce PPN. HAL3 protein is also highly related to the salt tolerance of organisms. Overexpression of this gene can improve the tolerance of transgenic plants to osmotic stress and salt stress. The function of ApHAL3 gene was not reported. The function and function site of ApHAL3 gene were studied in this paper. The amino acid sequence of ApHAL3 gene was analyzed. The amino acid sequence and the N-terminal sequence of Dfp protein encoding PPCDC in Escherichia coli were found. Arabidopsis thaliana AtHAL3a, Saccharomyces cerevisiae ScHAL3 and Rice OsHAL3 protein sequence highly consistent. ApHAL3 protein also has four conserved domains of oligoflavin protein cysteine decarboxylase protein family (鈪，

本文編號(hào)：1666904