Molecular Cloning A Novel Lectin Gene From Arisaema Amuremse And Analyzing Its Resistance To Nilaparvata Lugens And Myzus Persicae Of The Lectin

Some plant lectins have been found to confer crops enhanced resistant to many insect pests belonging to different orders including homopteran insects and successful used in the genetic engineering to develop pest-resistant crops. In order to identify more potential insect resistance genes, we researched Arisaema amuremse agglutinin (ArAA). The major experiment results of this work as follows:(1) Molecular cloning and sequence analysis. Primers were designed based on conserved sequences of lectin genes from Arisaema. A noval lectin gene was isolated from Arisaema amuremse. The full-length cDNA of ArAA was 1059 bp, containing an open reading frame (780 bp) encoding a peptide of 259 amino acids. The homology, conserved domains, the secondary and three-dimensional structure and the sites combinaed with mannose were analyzed and forecasted by bioinfermatics software.The result indicated that ArAA was similar to those of other Arisaema species lectin, which inferred that it possessed similar function to reported Arisaema lectin.(2) Expression and purification of ArAA protein in prokaryotic. According to the coding sequence of araa, the prokaryotic expression vector pET28a(+)-araa was successfully constructed, The recombinant ArAA (rArAA) protein was induced by IPTG to express and purified in E. coli BL21(DE3).(3) Hemagglutination and inhabition effect of rArAA. The rArAA protein even low concentration as 15μg/mL could coagulate rabbit red blood cells. Higher concentration rArAA made rabbit red blood cells distort even stave. Through the assay of inhabition hemagglutination, we founnd D-mannose was the strongest inhibitor for rArAA hemagglutination activity.(4) The stability of rArAA protein. The rArAA had a high thermal stability and little affected by acid and alkali. In addition, divalent cations is not necessary to rArAA hemagglutinating activity.(5) The insect resistance analysis of rArAA. The artificial diet assay by feeding Myzus persicae (Sulzer) and Nilaparvata lugens (Stal) with artificial diet containing the purified 0.1%(w/v) rArAA protein showed that rArAA had obvious resistance to Myzus persicae and Nilaparvata lugens. The araa gene thus represents a promising candidate to be used in genetic engineering for enhanced insect resistance and may make a valuable contribution to crop protection.