本文選題：玉米 + ZmHAL3�。� 參考：《沈陽(yáng)農(nóng)業(yè)大學(xué)》2017年碩士論文

【摘要】：HAL3蛋白是一種存在于真菌,植物和動(dòng)物中的高度保守的黃素蛋白,它在擬南芥、水稻、酵母和煙草中的過表達(dá)都改善了轉(zhuǎn)基因株系對(duì)鹽離子的耐受性,并且這些HAL3蛋白在不同真核生物中具有不同的功能,如:在酵母中具有Ppzl蛋白磷酸酶抑制劑活性,在擬南芥和水稻中具有4'-磷酸泛酰半胱氨酸脫羧酶(PPCDC)活性,玉米中ZmHAL3蛋白的功能尚不清楚。本文對(duì)ZmHAL3蛋白的氨基酸序列進(jìn)行序列分析,發(fā)現(xiàn)該氨基酸序列與大腸桿菌Dfp的N端序列、擬南芥中AtHAL3a蛋白以及水稻OsHAL3蛋白都具有高度同源性。我們克隆了玉米ZmHAL3基因并構(gòu)建質(zhì)粒載體,利用PPCDC缺陷的大腸桿菌△dfp突變體菌株進(jìn)行功能互補(bǔ)實(shí)驗(yàn)。結(jié)果表明:△dfp突變體菌株的轉(zhuǎn)化子在42℃的LB以及30℃的VB培養(yǎng)基上都能正常生長(zhǎng),這表明ZmHAL3具有PPCDC功能。定點(diǎn)突變實(shí)驗(yàn)表明 ZmHAL3 蛋白中的 His91、Trp97、Asn143、Met146、Cys176、Met184和SANT106-109等氨基酸是該蛋白具有PPCDC活性的關(guān)鍵位點(diǎn);而Pro56、Asp178和Gly180的突變對(duì)ZmHAL3蛋白的PPCDC活性基本沒有影響。為了研究ZmHAL3蛋白中具有PPCDC功能的關(guān)鍵區(qū)域,我們對(duì)ZmHAL3基因進(jìn)行了截段實(shí)驗(yàn),結(jié)果表明ZmHAL3蛋白18-190位氨基酸是PPCDC功能的關(guān)鍵區(qū)域。為了進(jìn)一步研究ZmHAL3蛋白的功能,我們采用實(shí)時(shí)熒光定量PCR在NaCl脅迫條件下檢測(cè)玉米根和幼苗中ZmHAL3基因的轉(zhuǎn)錄水平。結(jié)果表明玉米根在50 mM和150 mMNaCl脅迫下,ZmHAL3基因的轉(zhuǎn)錄豐度變化同樣都在兩倍之間,沒有顯著變化。而在100mMNaCl脅迫4h下,ZmHAL3的表達(dá)量顯著上調(diào)。并且在根和幼苗中的表達(dá)量變化相似。
[Abstract]:HAL3 is a highly conserved flavin protein found in fungi, plants and animals, and its overexpression in Arabidopsis, rice, yeast and tobacco improves the tolerance of transgenic lines to salt ions. These HAL3 proteins have different functions in different eukaryotes, such as Ppzl protein phosphatase inhibitor activity in yeast and PPCDCactivity in Arabidopsis and rice. The function of ZmHAL3 protein in maize is unclear. The amino acid sequence of ZmHAL3 protein was analyzed in this paper. It was found that the amino acid sequence was highly homologous with the N-terminal sequence of Escherichia coli DFP, AtHAL3a protein in Arabidopsis thaliana and OsHAL3 protein in rice. The maize ZmHAL3 gene was cloned and the plasmid vector was constructed. The PPCDC deficient Escherichia coli dfp mutants were used for functional complementation test. The results showed that the transformants of dfp mutants could grow normally on LB at 42 鈩，

本文編號(hào)：2041983