Cloning And Functional Study Of Soybean Lectin Gene LEC-S Using Transenic Tobacco

Plant lectins can be defined as plant proteins that possess at least one noncatalytic domain that binds reversibly to a specific mono-or oligosaccharide. Lectins have been isolated from a variety of plants, animals, bacteria, viruses and fungi. Legume lectins are the best-studied group of lectins and hundreds of these proteins have been isolated and extensively characterized. One possible physiological function that has emerged is the defensive role of these carbohydrate-binding proteins against phytopathogenic microorganisms, phytophagous insects and plant-eating animals. So far, many plant lectin genes have been used for conferring pathogen and pest resistance in plants by genetic engineering, such as Galanthus nivalis lectin (GNA) gene, pea lectin (PNA) gene, Phaseolus vulgaris lectin (PHA) gene, Amaranthus hypochondriacus agglutinin (AHA) gene, and type II ribosome-inactivating proteins (RIPs) gene.In our previous work,46cDNA fragments concerned soybean resistance to soybean frogeye leaf spot disease were obtained, among which the fragment DN40M shows highly homologous with soybean lectin. The entire sequences of the corresponding gene was gotten using RACE (rapid amplification of cDNA ends) technique, and was renamed lec-s (Accession number:DQ235094). This research was aimed at transgenic tobacco with lec-s gene encoding soybean lectin, whose resistance and output may be meliorated.In this study, lec-s gene was amplified by RT-PCR from soybean strain Hefeng29and proper restriction enzyme sites were added at each end by PCR. It was cloned into pBI121to make recombinant pBI121::lec-5, which was proved to be constructed successfully by restriction enzyme analysis. Then, this transgenic construct was transferred into Agribacterium tumefaciens EHA105by the freeze-thaw method. The positive clones were determined by PCR and restriction enzyme digestion.The tobacco discs were infected and transformed by A. tumefaciens strain EHA105/pBI121::lec-s, and transgenic tobacco plants were obtained by screening with kanamycin. Genomic integration and expression of the transferred genes were determined by PCR, Southern blot hybridization and reverse transcriptase PCR. Resistance bioassays revealed that the resistance of transgenic plants to TMV increased, numbers of lesions on leaves caused by TMV were markedly reduced in transgenic plants compared with the controls. The resistance of transgenic plants to Phytophthora nicotianae, also increased. Quantitative RT-PCR analysis indicated that four defense-related genes, PR-1a, GST1, Pal, and hsr515, were up-regulated in transgenic lines inoculated with TMV. The up-regulations of these defense-relate genes were not observed in leaves of control plant after inoculation. The results suggested that lec-s gene may play a role in the regulation of plant defense responses through the induction of downstream defense-related genes after the recognition of microbial pathogens, thus the transformation of tobacco with lec-s gene could enhance the capability of TMV resistance. Insect bioassays using detached leaves from transgenic tobacco plants demonstrated that the expressed soybean lectin significantly reduced the weight gain of larvae of the beet armyworm, Spodoptera exigua (Lepidoptera:Noctuidae). Further on, the lectins retarded the development of the larvae and their metamorphosis. These findings suggested that soybean lectins has potential as a protective agent against pathogens and insect pests through a transgenic approach.