Cloning And Functional Characterization Of BtSAMS1(S-adenosylmethionine Synthetase 1)in Balsas Teosinte

Aphid is an insect transmission vector that carries more than 110 types of plant viruses,and also a high egg-laying-rate insect which shows an outstanding reproductive performance and therefore,a severe threat to crop production.Maize is an important grain and feed crop,seriously threatened by pests.Balsas teosinte,the direct wild ancestor of modern maize,presented higher insect resistance.Screening insect-resistant genes from Balsas teosinte and applying them to modern maize is a novel idea to improve pest-resistance of cultivated maize.S-Adenosylmethionine synthetase(SAMS)catalyzes the conversion of methionine and ATP into S-adenosylmethionine(SAM),which is the only way to synthesize SAM in plant found so far.SAMS genes play important roles in plants growth and development,metabolism,and adversity stress response.In the present study,we analyzed the SAMS gene expression in the leaves of Balsas teosinte and maize before and after aphid infestation,and found the relative expression of SAMS increased 12.17-fold in teosinte after aphid infestation,while it increased 3.47-fold in maize.So,we speculate that Bt SAMS1 gene is positively involved in the insect-resistant defense response of plant.We have cloned BtSAMS1 gene from Balsas teosinte,and the gene sequences wer e analyzed by bioinformatics analysis.The Bt SAMS1 gene encodes S-adenosylmethionin e(SAM)synthetase,which is 1185 bp in length,encoding 394 amino acid.Bt SAMS1 is a stable hydrophilic protein located in cytoplasm.The secondary structure of Bt SAMS1 is mainly formed by unordered structures and their proportion is more than 50%.Based on the tertiary structure model,Bt SAMS1 is a tetramer which is composed of three d omains,N-terminal domain,intermediate domain,and C-terminal domain.The three do mains are symmetrically folded into a pseudo-triple structure.According to the homolo gy analysis and the phylogenetic tree,the Bt SAMS1 and Zm SAMS1 belong to the sa me evolutionary branch.The amino acid sequence of Bt SAMS1 shows no less than 85% sequence homology with other species,which indicates the peptide sequence of th e SAMS protein is highly conserved.To verify the resistance of BtSAMS1 gene to insects,we constructed a plant overexpression vector 35S::Bt SAMS1,and obtained transgenic Arabidopsis seedlings integrated with the Bt SAMS1 gene.The resistance of transgenic Arabidopsis with Bt SAMS1 gene to Myzus persicae was analyzed by observing the feeding trajectory,colonization performance and reproductive rate of aphids.The results showed that transgenic Arabidopsis seedlings presented higher resistance to peach aphids than untransformed Arabidopsis plants.Aphids preferred the untransformed plants and the colonization number was one-fold lower on transgenic plants compared with the untransfomed control.We proposed three possible pathways that might account for the aphid resistance of Bt SAMS1 gene,the biosynthesis of ethylene and polyamines,and the DNA methylation.The study suggests that Bt SAMS1 could be a candidate aphid-resistant gene and apply to pest-resistance improvement.