【作者】 刘红霞；

【导师】 李镇宇； 张星耀；

【作者基本信息】 北京林业大学 ， 生态学， 2008， 博士

【摘要】 植物凝集素是一种含有非催化结构域并能可逆结合到特异单糖或寡糖上的植物（糖）蛋白。植物凝集素的诸多功能都与它最重要的性质糖结合专一性有关。由于植物凝集素广泛存在并且种类繁多,加之其特定的作用机理,使得植物凝集素在植物抗病虫基因工程研究中具有较大的应用潜力。本研究以合欢Albizia julibrissin Durazz种子为研究材料,分离了合欢种子凝集素,研究了其理化性质及凝集素对几种真菌孢子萌发和菌丝生长的影响;同时采用RT-PCR和RACE相结合的方法完成了合欢种子凝集素基因的分离克隆。在此基础上利用生物信息学方法对获得的合欢种子凝集素基因进行了结构功能预测分析。通过上述研究得到了以下结果:1.采用硫酸铵沉淀、分子筛层析并结合蛋白超敏可逆染色试剂盒的方法纯化得到了合欢种子凝集素（Albizia julibrissin seed lectin,AJLs）,用SDS-PAGE测定其相对分子量为25.4kDa,IEF-PAGE测定其等电点为7.96。蒽酮比色法测定其中性糖含量为2.65%,是一种糖蛋白;凝血活性测定表明,其最低凝集限度为1.95μg/ml,N-乙酰葡糖胺（GlcNAc,N-acetylgucosamine）是合欢种子凝集素的专一性抑制糖。2.对合欢种子凝集素的功能研究表明,凝集素对灰葡萄孢Botrytis cinerea和大丽轮枝菌Verticillium dahliae的孢子萌发具有较强的抑制作用;对细交链孢菌Alternaria alternata菌丝生长有一定的抑制作用,对尖孢镰刀菌Fusariumoxysporum菌丝生长的抑制作用不明显。3.首次克隆得到了合欢凝集素基因的cDNA全长序列。该cDNA全长序列由1096个核苷酸组成,开放阅读框为747个核苷酸,编码248个氨基酸。5’端和3’端各有82和267个核苷酸的非编码区（UTR）,3’端有polyA尾。4.利用相关软件及在线生物信息学分析站点对合欢种子凝集素基因编码蛋白的理化性质进行了分析。结果表明,目的蛋白的pI为8.14,预测分子量为26.8kDa。用ClustalX1.83软件进行了同源性比对,并构建了目的蛋白的系统进化树。合欢种子凝集素基因编码蛋白的氨基酸序列与含羞草亚科Mimosoideae球花豆属的Parkia Platycephala氨基酸序列同源性达85%。5.利用在线生物信息学分析服务平台对合欢种子凝集素cDNA基因编码蛋白的结构进行了预测。结果表明,目的蛋白在第12～34位氨基酸之间具有明显的疏水区和跨膜区域。二级结构预测说明目的蛋白的α螺旋的比例在30%以上,β折叠均在20%以下,是一种混合型蛋白。应用同源建模法构建了合欢种子凝集素基因编码蛋白的三级结构,并采用PROCHECK软件对模型的结构质量进行了评价,最优模型为EsyPreD方法构建的模型。6.通过网络服务器平台进行了合欢种子凝集素cDNA基因编码蛋白的功能预测。结果显示,合欢种子凝集素基因编码的蛋白是Glyco_hydro₁8 superfamily的新成员,具有Glyco_hydro₁8的结构功能域,活性位点位于第146～154之间（序列为LDGVDFDIE）,而且有多个磷酸化位点。信号肽区域位于1～27个氨基酸,即MGTKLRNPKIILVLQVCLIMMVSTAKA。跨膜片段位于第12～34位氨基酸残基。亚细胞定位分析表明目的蛋白是一种分泌性蛋白（胞外蛋白）。本论文对合欢种子凝集素的研究填补了我国在含羞草亚科植物凝集素方面的研究空白,并为今后开展木本植物凝集素基因的在抗病虫研究方面的应用奠定了一定的理论基础,具有一定的的理论价值和现实意义。更多还原

【Abstract】 Plant lectins were defined as all plant protein possessing at least one non-catalytic domain, which binds reversibly to a specific mono- or oligosaccharide. Various functions of plant lectins were related with their sugar-recognition mechanisms. Since plant lectins were wide-spread through the plant kingdom and so many different lectins have been found, also their different biochemical properties, plant lectins would have much more applications, especially on pest control.This study reports the purification and some properties of a new lectin extracted from seeds of Mimosoideae, Albizia julibrissin, and also its antifungal activities. Based on this present studies, the seed lectin gene was cloned by RT-PCR and RACE from A. julibrissin and its structure and function was analyzed by bioinformatics. The main research conclusions were showed as follows:1. A new lectin was purified from Albizia julibrissin seeds by extraction, precipitation with （NH4）SO4, ion-exchange chromatography on DEAE Sepharose Fast Flow and followed native-PAGE. The purified lectin had a molecular mass of 25.4kDa on SDS-PAGE. Its isoelectric point is 7.96. The lectin was a glycoprotein . N-acetylglucosamine is the specific inhabitation sugar of the lectin.2. The function of this lectin showed that it strongly inhibited spore germination of Botrytis cinerea and Verticillium dahliae, and also partially inhibited the hyphal growth of Alternaria alternate. But it was no effect on the hyphal development of Fusarium oxysporum.3. A new lectin gene was cloned from Albizia julibrissin seed by RT-PCR and RACE. The full-length cDNA was 1096bp, and contained a 747bp open reading frame encoding a 248 amino acid protein with a 27 signal peptide.4. Protein structure was predicted with different methods. The results of primary structure analysis showed that the protein pI was 8.14, molecular weight was 26.8kDa. Using CLUSTAL （1.83） multiple sequence alignment methods determined Albizia julibrissin gene encoding amino acid sequence and related sequence homology. The amino acid sequence was found to be homologous to Mimosoideae, Parkia paltycephala, up to 85%.5. Related characters of coding protein were predicted. The result showed hydrophilic and transmembrane region located 12 to 34 amino acid residue. Secondary structure analysis indicated that the protein was mixed protein. Predicted three-dimensional structure of the protein by homology method in different net server and model quality was evaluated by PROCHECK software. The MODEL EsyPreD was BEST.6. Function analysis of the gene deduced amino acid sequence showed it belonged to Glyco-hydro₁8 superfamily. It had a domain of Glyco-hydro₁8 superfamily and located from 146 to 154 amino acid residue. The sequence was LDGVDFDIE. The subcellular location indicated the protein was extracellular protein, and had a transmembrane segment, the sequence was LVLQVCLIMMVSTAKAGGIVVYW.This data are great importance considering the lack of information on Mimosoideae plants. And also the conclusions provide some information for pest resistance research further in forest trees.更多还原