Stone Pta Gene Cloning And Mannose Specific Binding Lectin, Anti-fungal Research,

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.