Cloning Of Disease Resistance Of Gene From Polygonum Sibiricum And Construction Of Plant Expression Vector With Double Genes For Genetics Transformation

A partial sequence of a Thionins gene(PsTHI1),Chitinase gene(PsCHIl) and Glucanase gene(PsGLUC1) were obtained from a random clone in Polygonum sibiricum SSH library and the PsTHI was cloned by using rapid amplification of cDNA end(RACE) technology.Bioinformatics analysis indicated that The PsTHI1 was found to be highly homologous with that from other dicotyledon.All of them possessed a conserved plant THI signature sequence.As result,it was possibly involved in antimicrobial activity and was identified as a typical plant THI.The PsCHI1 possessed a conserved Chitinases family 191 signature(PS00773) sequence.The deduced amino acid sequence shows high identity to classâ…£chitinases,therefore,it was identified as a typical plant classâ…£CHI.It was predicted using Bio-software PsCHI1 exits in secretory pathway,without transmembrane domain,A homology model based on the structure of a class I chitinase from rice(Oryza Sativa L.Japonica) was used as an aid to interpreting the available data attacking against pathogen cell walls.On the basis of required structure and function of plant chitinases,It was concluded that PsCHIl was possibly involved in antimicrobial activity.The PsGLUCl was a negatively charged acidic protein.This protein had a highly sequence similarity with endo-1,3;1,4-beta-D-glucanase of Oryza sativa L.japonica,thus can be preliminary predicated as a member of glucanase family.By real-time PCR analysis,the differential expression of PsGLUC1 and PsCHI1 in various tiussue and stress stage were investigated under salt and alkaline stress.The results showed that the expression of PsCHI1 has a significantly higher transcript amount in leaves than in stem,no matter in NaHCO₃ stress or without stress condition,demonstrating a tissue-specific expression property.However,the PsGLUCl exhibited a higher expression level in both leaves and stems under NaHCO₃ stress compared with nontreatment.These results indicated that both genes were induced by salt and alkaline stress,and their overexpression probably have correlation with the stress resistance of Polygonum sibiricum.In order to introduce two exogenous genes simultaneously in one transformation manipulation.A plant bi-expression system containing both the PsGLUC1 and PsCHI1 genes, based on the PsGLUC1/pROKâ…¡and PsCHI1/pROKâ…¡vector,was successfully constructed,by the "restriction-site-substitution method of the intermediate vector pGEM-T easy" and "35S-&Tnos-primer-PCR "method.The "35S-&Tnos-primer-PCR method" had relatively higher efficient than "the restriction-site-substitution of the intermediate vector pGEM-T easy" method,since it can avoid such complicated steps as replacement of restriction sites,ligation, and transformation concerning in "the restriction-site-substitution of the intermediate vector pGEM-T easy" method.These attempts provide valuable theoretical and practical experiences in overcoimg the difficulties of introducing multiple genes in disease-resistant breeding field.Seventeen independent strains of transgenic tobaccos overexpressing both the PsGLUC1 and PsCHI1 genes were obtained using Agrobacteria-mediated transformation method.Six of them were subjected to PCR analysis and all of them a present positive result.And the RT-PCR detection results demonstrated that the two exogeneous genes can be expressed in transcriptional level.This establish basis for further application of cultivation of disease-resistant-forest-tree-species by bi-expression of disease resistant genes.