【作者】 石之光；

【导师】 方宣钧；

【作者基本信息】 贵州大学 ， 生物化学与分子生物学， 2009， 硕士

【摘要】 在本研究中,我们用生物信息学的方法分析了玉米等物种ADH的保守功能域、蛋白二级结构和进化关系。分析证实,植物ADH通常含有3个保守功能域,它们是具有GroES结构的ADH_N结构域,Rossmann折叠NAD(P)(+)结合蛋白和锌结合位点,这3个保守结构在各个物种中具有相当的保守性。二级结构的分析表明,在我们研究的物种中,每个物种的两个ADH同工酶折叠情况大体相同,物种间蛋白二级结构也趋向一致。通过构建系统进化树,分析了供试物种中ADH以及ADH两个同工酶间的进化关系。初步显示,在进化上,单、双子叶植物源自不同的ADH祖先。双子叶植物ADH在物种间具有差异；而在单子叶植物不同物种其ADH同工酶出现分化。我们对一个百脉根的匿名序列(序列编号为CAG3057 9.1)进行了保守结构域预测、二级结构以及进化关系分析,初步推定该匿名序列为百脉根乙醇脱氢酶基因的未知同源基因LcADH(t)。保守结构域预测LcADH(t)含有ADH_N、NADB_Rossmann和锌结合位点这3个典型的ADH结构域。LcADH(t)的二级结构显示出与其他ADH极高的相似性,尤其体现在3个保守结构区。同时,在核苷酸和氨基酸序列上,与LcADH1又存在着一定的差别,这意味着LcADH(t)与LcADH1并非同一基因,且这种差别也非同一基因的种间差别。从进化关系分析上可知LcADH(t)与LcADH1之间在同一类的两个不同分支,据此,我们推断,LcADH(t)可能发挥着LcADH2的功能,值得期待进一步的功能验证。我们通过对陆地棉的一个409bp的cDNA片段进行解析,in silico拼接出一条完整的开放阅读框(Open Reading Frame, ORF)。我们推定这条ORF在陆地棉中真实存在,并在阅读框的两端设计了扩增引物,PCR扩增正向引物为：5—CACCACTAGATCACAAGAATAATAATGG—3,反向引物为：5—AAAAGGAGAAGCAATTTACATTATCTC—3.以陆地棉总RNA进行反转录合成的cDNA为模板进行PCR扩增,获得全长基因序列。ORF分析表明,序列中包含一个1305bp的ORF,编码434个氨基酸。通过BLAST和多重序列比对,我们克隆的序列可能是一个锌结合脱氢酶家族蛋白的同源蛋白,将其命名为棉花锌结合脱氢酶蛋白(cotton zinc-binding dehydrogenase family protein),将该基因命名为GhZBDH。保守结构域分析表明,从氨基酸链的63位至433位,是进行能量制造与转化的第三类锌结合乙醇脱氢酶AdhC结构域,87至221位是ADH_N结构域,269至394位是NAD (P)结合蛋白结构域。结构域预测的结果进一步暗示,这个蛋白质在一级结构上具有与其他物种ADH类似的构成状况,可能发挥着ADH功能。我们成功的将棉花锌结合脱氢酶基因构建到PQE-30载体之中,重组载体命名为pQE30-GhV3。然后应用热激转化法将pQE30-GhV3重组载体转化入表达菌株M15中,进行诱导表达。表达研究证明,这个蛋白表达的最适合温度为37℃,最适IPTG浓度为0.1mmol/L,最适诱导时间为3小时。我们在37℃、0.1mmol/L浓度的IPTG条件下,诱导3小时,使用Ni-NTA凝胶亲和层析柱进行层析,电泳分离纯化产物。结果显示,GhZBDH表达蛋白量很大,且其大小与预测的完全一致,初步表明,我们获得了大量的且高纯度的变性蛋白,为下一步的蛋白复性以开展定性实验准备了条件。更多还原

【Abstract】 In this study, we used bioinformatics method analyzed the conservative function domains, protein secondary structure and evolutionary relationship of ADH in Maize and other species. The results confirmed that plant ADH usually contained three conservative domains, they were GroES structure ADH_N domain, Rossmann fold NAD(P)(+) binding protein and zinc site, and they are considerable conservation in all species. Secondary structure analysis showed that the folding of tow ADH Isozymes are similar in testes species. We analyzed evolutionary relationship between two ADH Isozymes in tested species by constructing phylogenetic trees. The results showed that ADH in Dicotyledon and Monocotyledon were derived from different ancestors. ADH in Dicotyledon were different. However, in Monocotyledon, the ADH isozyme were divided.We predicted conservative domain, secondary structure and analyzed the evolutionary relationship to a Lotus corniculatus anonymous sequence, and we presumed that this sequence was the homologous gene of ADH---LcADH(t). LcADH(t) contained ADH_N, NADB_Rossmann and zinc site structure. Secondary structure of LcADH(t) showed that it had high similarity with other ADH, especially in the conservative domains, but it was different in nucleotide with LcADH1.This indicated that they were not the same gene. Revolutionary relationship showed that LcADH(t) and LcADHl were in different branches, So, we inferred that LcADH(t) might play a same role with LcADH2. Worth looking forward to further functional verification.We analyzed a 409bp cDNA sequence fragment from upland cotton, in silico splicing a complete open reading frame(ORF). We presumed that this ORF was real in upland cotton, so we designed amplification primers in both ends of this ORF, the forward primer was 5—CACCACTAGATCACAAGAATAATAA TGG—3, and the reverse primer was 5—AAAAGGAGA AGCA ATTTACATTATCTC—3. Synthesize cDNA with total RNA, and PCR amplification with cDNA template. We obtained the full-length gene sequence. Sequence analyzed showed that this sequence contained a 1305bp ORF and encoded 434 amino acids. Blast and multiple sequence alignment showed that this gene might be a zinc-binding dehydrogenase family homolog. So, we named it as cotton zinc-binding dehydrogenase family protein and named this gene as GhZBDH. Domain analyzed showed that from the amino acid chain 63-433 position was the third type of zinc binding alcohol dehydrogenase domain ADH_C that manufactured and conversed energy,87-221 position was the ADH_N domain,269-394 position was the NAD(P) binding protein. Domain predict result further suggested that this protein was similar with other ADH in primary structure, and it might play ADH function.We constructed this gene into pQE-30 vector, and named this recombinant vector as pQE30-GhV3. And then we carried out the inducible expression by transformed pQE30-GhV3 into M15 strain. The result showed that the most suitable expression temperature was 37℃, the optimal concentration of IPTG was 0.1mmol/L and the optimal induction time were 3 hours. We induced this protein 3 hours at 37℃and 0.1mmol/L IPTG concentration, and used Ni-NTA affinity chromatography column gel chromatography total protein, and separation purification products by electrophoresis. The result showed that the expression amount of GhZBDH was very large, and the size is fully consistent with the projected. We have obtained a large number and high-purity denatured protein.更多还原