Cloning And Characterization Of Resistance-related CDNA From Tobacco Induced By Oligochitosan

Oligochitosan is is an effective elicitor for inducing plant resistance. To understand the induced resistance mechanism of plant by oligochitosan, the technique of mRNA differential display has been used to isolate genes from Nicotiana tabacum var.Samsun NN and 96 differential displayed bands were obtained.To screen genes related to resistance, eleven cDNA fragments were re-amplified and cloned. Sequencing date and homology analysis show that one of these cDNA fragments has high identity with Nicotiana benthamiana SKP1 gene. Because the SKP1 protein is relevant to plant resistance to virus, so the 5'end and full-length of SKP1 cDNA from Nicotiana tabacum var. samsun NN (NtSKP1) was cloned using the SMART-RACE technique. Sequence analysis shows that there are a 5′UTR Py-rich stretch site (function: cis-acting element conferring high transcription levels), a HSE site (function: cis-acting element involved in heat stress responsiveness) and a CATT-motif (function part of a light responsive element) in the 5'untranslated areas of NtSKP1. Southern blot analysis shows that there is one gene copy in genome.To obtain the SKP1 protein of tobacco with high purity at a large scale, the recombinant plasmid pET23b-SKP1 was constructed and transformed into E. coli BL21 (DE3) for expression. The results of SDS-PAGE and HPLC-MS analysis indicate that a protein with relative molecular mass 18 KD was expressed in E.coliand was purified by NTA Resin.Using GFP as a reporter, the NtSKP1 was localized in the cytoplasm or nucleus in the different cell phases, the location of NtSKP1 is in accordance with 26 S proteasome which interacts with SKP1 in vivo.Further more, The RNAi vector pART27-SKP1a-SKP1s and pART27-pHANNIBAL were constructed. Nicotiana tabacum var. sam sun NN was transformed by leaf disc method using Agrobacterium tumefaciens LBA4404 containing pART27-SKP1a-SKP1s or pART27-pHANNIBAL. The transgenic tobacco containing pART27-pHANNIBAL has been selected, while the transgenic tobacco containing pART27-SKP1a-SKP1s is still under selection.