玉米ZmHAL3基因功能研究

发布时间：2018-06-20 00:12

  本文选题：玉米 + ZmHAL3　； 参考：《沈阳农业大学》2017年硕士论文

【摘要】：HAL3蛋白是一种存在于真菌,植物和动物中的高度保守的黄素蛋白,它在拟南芥、水稻、酵母和烟草中的过表达都改善了转基因株系对盐离子的耐受性,并且这些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蛋白磷酸酶抑制剂活性,在拟南芥和水稻中具有4'-磷酸泛酰半胱氨酸脱羧酶(PPCDC)活性,玉米中ZmHAL3蛋白的功能尚不清楚。本文对ZmHAL3蛋白的氨基酸序列进行序列分析,发现该氨基酸序列与大肠杆菌Dfp的N端序列、拟南芥中AtHAL3a蛋白以及水稻OsHAL3蛋白都具有高度同源性。我们克隆了玉米ZmHAL3基因并构建质粒载体,利用PPCDC缺陷的大肠杆菌△dfp突变体菌株进行功能互补实验。结果表明:△dfp突变体菌株的转化子在42℃的LB以及30℃的VB培养基上都能正常生长,这表明ZmHAL3具有PPCDC功能。定点突变实验表明 ZmHAL3 蛋白中的 His91、Trp97、Asn143、Met146、Cys176、Met184和SANT106-109等氨基酸是该蛋白具有PPCDC活性的关键位点;而Pro56、Asp178和Gly180的突变对ZmHAL3蛋白的PPCDC活性基本没有影响。为了研究ZmHAL3蛋白中具有PPCDC功能的关键区域,我们对ZmHAL3基因进行了截段实验,结果表明ZmHAL3蛋白18-190位氨基酸是PPCDC功能的关键区域。为了进一步研究ZmHAL3蛋白的功能,我们采用实时荧光定量PCR在NaCl胁迫条件下检测玉米根和幼苗中ZmHAL3基因的转录水平。结果表明玉米根在50 mM和150 mMNaCl胁迫下,ZmHAL3基因的转录丰度变化同样都在两倍之间,没有显著变化。而在100mMNaCl胁迫4h下,ZmHAL3的表达量显著上调。并且在根和幼苗中的表达量变化相似。
[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，

本文编号：2041983