The Primary Functional Analysis On CpKTI From Chimonanthous Praecox (L.) Link

Wintersweet (Chimonanthus praecox Link) is an important species originating from China, where it is cultivated with a long history owing to its important characteristics, especially its stress resistance. Cloning the CpKTI gene, and analysis its insect-resistant features,and we reviews on the plant protease inhibitor of characteristics, classification, biological role and regulation of gene expression.The main results are as follows: 1. Bioinformatic analysis of CpKTIA protease inhibitor gene, designated as CpKTI (GenBank accession number:DW222682), was obtained by sequencing the randomly selected clones, on the basis of Chimonanthus praecox flower cDNA library construction and its ESTs analysis. This sequence exhibited homology to Populus tremula and Glycine max etc. The identity of the derived protein has the high homogeneous characteristics of 90%. The further analysis showed that CpKTI is a protease inhibitor gene from Ch. Praecox. The full-length cDNA of CpKTI is 979 bp, containing a 594 bp open reading frame(ORF) and encoding a predicted protein of 197 amino acids, no intron, the Theoretical molecular weight is 21.72KD,the Isolectric Point I is 4.867.The structure characteristics of CpKTI protein were analyzed with bioinformatic method. The results showed that it had a 20aa signal peptide sequence,secondary structure of CpKTI protein composed of Alpha helix(14.72%), extended strand(35.03%) and Random coil(43.65%), with good Hydrophobic, meanwhile,this protein contained many phosphorylation sites, we conjectured the protein was diversified modified after translation, related to the multiple resistance of chitinase. 2. The construction of plant expression vector of CpKTI and Investigation of insect-resistance activity of transgenic tobacco plantsThe PMD19-T-CpKTI plasmid was digested with XBa I and Sac I to release a fragment containing the coding sequence of CpKTI and latter was inserted into bacterial vector pCAMBIA2301.The recombinant plasmid had been PCR amplified and enzyme digested,it indicated that the CpKTI gene had successful inserted into the recombinant plant expression vector.Using the leaf disc transformation procedure mediated by Agrobacterium(LBA4404) tumefacien, recombinant plasmid were transformed into tobacco plants,transgenic tobacco plantlets(30) were obtained, in which plantlets were identified by kanamycin-resistant,histochemistry assay of GUS activity, PCR and RT-PCR.Imported cabbageworm (Pieris rapae) attacked situation of transgenic tobacco and non-transgenic tobacco plantlets in the field were investigated.The results showed the transgenic tobacco and non-transgenic tobacco were all no damaged by imported cabbageworm. 3. The construction of prokaryotic expression vector of CpKTI and induced expressionThe CpKTI gene was cloned by PCR and insected into PMD19-T vector. The PMD19-T-CpKTI plasmid was digested with BamHâ… and Hindâ…¢to release a fragment containing the coding sequence of CpKTI and latter was inserted into bacterial vector PET-32a(+).The recombinant plasmid had been PCR amplified and enzyme digested,it indicated that the CpKTI gene had successful inserted into the recombinant bacterial expression vector.CpKTI was inserted into an prokaryotic expression vector pET-32a(+) with Resection of N_terminal signal region.Then the expression vector was transformed and expressed Escherichia coli BL21. and then through 1mmol/L IPTG inducement expression.The expressed product was dectected by SDS-PAGE.The result indicated the CpKTI had expressed in E.coli.