石半夏凝集素基因的克隆及甘露糖特异结合凝集素的抗真菌研究

【作者】 林玲；

【导师】 唐克轩；

【作者基本信息】 复旦大学 ， 遗传学， 2007， 博士

【摘要】 凝集素是一类含有至少一个非催化结构域并能可逆结合到特异单糖或寡糖上的蛋白质。单子叶植物甘露糖结合型凝集素(monocot mannose-bindingagglutinins)是其中比较大的一个家族,其中以雪花莲凝集素为代表的一个亚类显示出良好的抗同翅目昆虫活性,以天麻抗真菌蛋白为代表的另一亚类则具有突出的抗真菌活性,它们在植物抗虫和抗病基因工程中均具有很大的应用潜力。真菌对农作物的危害呈现间歇爆发的特征,并存在一定的普遍性。病菌除导致植物叶片病斑从而影响光合作用外,还可侵染茎秆和果实,影响品质,造成经济损失。为了为植物抗病基因工程提供更多的候选基因,本文对植物凝集素基因进行了研究。以石半夏(Pinellia cordata)为材料,利用RACE-PCR技术,成功地克隆出了石半夏凝集素基因,并对其抗真菌功能进行了研究。此外,本文还对已经克隆的马蹄莲(Zantedeschia aethiopica)、文殊兰(Crinum asiaticum)、石斛(Dendrobium officinale)和姜(Zingiber officinale)凝集素分别进行了抗真菌功能的初步研究,得到如下结果:1.首次从天南星科植物石半夏中克隆了石半夏凝集素基因pcl。pcl的全长cDNA为1182bp,含有一个768bp的开放阅读框,编码一个由256个氨基酸组成的前体蛋白。对PCL和其他单子叶甘露糖结合凝集素的序列进行了同源性比对,预测了该蛋白的二级和三级结构,结果表明,PCL可能是一个有凝集素功能的蛋白。半定量RT-PCR分析表明,pcl在石半夏植物的各个部位都有转录,其中在珠芽中的转录水平最高。2.分别构建了含pcl、zaa(马蹄莲凝集素基因)和caa(文殊兰凝集素基因)的原核表达载体,并成功地实现了这三个基因在E.coli中的表达,对表达的重组蛋白进行了纯化,并从植物中提取了天然的PCL,作为对照进行了凝血活性实验。结果表明重组PCL具有和天然蛋白一样的凝血活性。抑菌实验表明,重组ZAA能抑制叶霉病菌(Fulvia fulva)的生长,重组CAA蛋白和重组PCL蛋白抑菌效果不明显。3.分别构建了含pcl、doa(石斛凝集素基因)和zoa(姜凝集素基因)的植物表达载体,并导入到根癌农杆菌C58C1中,对拟南芥进行了转化。分别获得了含doa基因和zoa基因的转基因拟南芥植株及其后代,研究表明,外源基因在转基因植株后代中的分离比符合孟德尔遗传。初步抑菌实验表明,转姜凝集素基因的拟南芥对叶霉病菌有一定抗性作用。本研究为利用基因工程技术改良农作物抗病性提供了更多的候选基因。更多还原

【Abstract】 Agglutinins are proteins possessing at least one noncatalytic domain, which binds reversibly to specific mono- or oligo- saccharides. Among them a subfamily represented by Galanthus nivalis agglutinin (GNA) is characterized with effective resistance activity against Homopteran insects, while another subfamily represented by gastrodianin is more effective against pathogenic fungi. Both of the subfamilies show great potentials in genetics engineering of plants to improve their resistance against harmful insects or fungal pathogens.To seek more effective genes with insect resistance or anti-fungal activities, agglutinin genes pcl was successfully isolated from Pinellia cordata by using rapid amplification of cDNA ends (RACE)-PCR method respectively, and its function was also studied. Otherwise the anti-fungal analysis of Zantedeschia aethiopica agglutinin, Crinum asiaticum agglutinin, Dendrobium officinale agglutinin and Zingiber officinale agglutinin was studied in order to find more lectins against pathogenic fungi. The results are as follows:1. The full-length cDNA of pcl gene was cloned for the first time from Pinellia cordata, which belongs to Araceae family. The full-length cDNA of pcl was 1182bp with an opening reading frame of 768bp encoding a protein with 256 amino acids. Sequence homology analysis was carried out by aligning PCL with other monocot mannose-binding agglutinins. The secondary structure and third-dimensional structure of PCL were predicted. Semi-quantitative RT-PCR analysis revealed that pcl mRNA transcription could be detected in all the tested tissues ,with the highest transcription in bulbil.2. The ORFs of Pinellia cordata agglutinin (PCL), Zantedeschia aethiopica agglutinin (ZAA) and Crinum asiaticum agglutinin (CAA) were constructed into the bacterial expression vector. The recombinant proteins in the resulting vectors were successfully expressed and purified from E.coli. The recombinant PCL displayed the similar agglutination activities towards rabbit red cells as the natural PCL isolated from the plants. Anti-fungal assay revealed that the recombinant ZAA could inhibit the growth of leaf mould.3. Three kinds of univalent plant expression vectors were constructed based onpHB, with constitutive expression 35S promoter activating the genes. Vector 1: pHB35S::PCL, vector 2: pHB35S::DOA , and vector 3: pHB35S::ZOA. The recombinant vectors were introduced into Agrobacterium tumefaciens C58C1 with freezing-thawing method. The pollens of Arabidopsis plants were infected by A. tumefaciens and the transgenic plants were selected with hygromycin. PCR test and western blot analysis of the transgenic plants were carried out to confirm the transgenic events. Functional analysis indicated that the expression of ZOA improved the transgenic Arabidopsis tolerance against pathogenic fungi.The cloning of pcl provides a basis for further investigating its insect and disease-resistance functions. The recombinant ZAA could inhibit the growth of leaf mould and the expression of ZOA could improve the transgenic Arabidopsis tolerance against pathogenic fungi. This study is of potential importance not only for the obtaining of intellectual properties of more anti-insect and disease genes but also for the improvement of crops for insect and disease resistances by genetic engineering.更多还原