Molecular Cloning And Characterization Of Lectin Gene From Zephyrathes Grandiflora

During the last few years important progress has been made in the study of plant anti-insect gene engineering, while a number of anti-insect genes were isolated and well documented. However, few genes resistant to homoptera (sap-sucking) insects were isolated. Recent studies have shown that some plant lectins from Amaryllidaceae species are toxic to sap-sucking insects in artificial diet assay, among which the lectin (GNA) from snowdrop (Galanthus nivalis) is the most toxic and has been most widely used in plant insect-resistant breeding. Transgenic plants expressing GNA showed significant insecticidal activity towards \\omoptera insects such as aphids and brown planthopper (BPH) in bioassay and feeding tests.In this paper a lectin gene was firstly isolated from leaves of Zephyranthes grandiflora, belonging to Amaryllidaceae, by the use of rapid amplification of cDNA ends (RACE) technique. Characterization, functional prediction and construction of plant and prokaryotic expression vector of this novel gene were studied. The analyzing results of the gene are very helpful to elucidate its molecular evolution, functional expression and potential application in insect resistance breeding. The major experiment results from this work were as follows:1.Molecular cloning and characterization of the novel lection gene. Primers were designed based on conserved sequences of other lectin genes from Amaryllidaceae. A novel lectin gene was isolated from Zephyrathes grandiflora. The full-length cDNA of Zephyrathes grandiflora lectin was 955bp, containing an open reading frame (576bp) encoding a peptide of 191 amino acids. The gene was named as zga (Zephyrathes grandiflora agglutinin), with its encoding protein (ZGA) showing 59% similarity to GNA. The results of signal cleavage site prediction, blocks' analysis and sugar-binding analysis showed that ZGA was very similar to those of other Amaryllidaceae species, which inferred that it possessed similar function to GNA.2.Construction of plant expression vector. Based on the cloning of Zephyrathes grandiflora agglutinin gene (zga), a plant expression vector p2300zga was successfully constructed and introduced into Agrobacterium tumefaciens EH A105.3.Construction of prokaryotic expression vector. According to the analysis of deduced amino acids of ZGA, prokaryotic expression vector pET32zga was successfully constructed and transformed into E.coli BL21.