Cloning And Expression Vector Construction Of AtDND1, AtDND2and AtDND1Promoter In Arabidopsis

In order to recognize and defend the invasion of pathogens, plant innate immunity is a complicated set of multi-level defense system through long-term struggle with pathogens that the plant itself forms. The cyclic nucleotide gated channel (cyclic nucleotide-of gated ion channel, CNGC) is of a cation channel protein family found in recent years widespread in animal and plant cells. Studies have shown that a couple of CNGC genes involved in plant resistant response and the Arabidopsis mutants Atcngc2/dndl (defense no death) and Atcngc4/dnd2involved in HR response.In order to explore the roles of DNDI and DND2in plant innate immunity, we generated the overexpression constructs of AtDND1and AtDND2and transformed into Arabidopsis. We also generated AtDNDl constructs containing GUS and GFP in order to do the tissue localization and subcellular localization of AtDND1in the future. The results are as following:(1)Through reverse transcription PCR, AtDND1(2181bp) and,AtDND2(2085bp) wered cloned from Arabidopsis thaliana. The sequencing results showed100%identity with the gene in NCBI.(2) Expression vectors pPZP221-DND1and pPZP221-DND2were generated which had CaMV35S as the promoters and T-nos as the terminators. Both vectors are transformed into Agrobacterium and further into Arabidopsis for enegy study.(3) Using Arabidopsis genomic DNA as template, DND1promoter was cloned. The sequencing results showed100%indentity with the gene in NCBI. Expression vector pR1-pDND1was generated which containing gusA gene and transformed into Agrobacterium for further study of tissue localization of DND1.(4) The DND1gene without stop codon was cloned in Arabidopsis. Sequencing results showed99%identity with the gene in NCBI. And the single base transition doesn’t change the amino acid sequence.(5) Expression vector pR3-pDND1-DND1was generated which contains DND1promoter, DND1cDNA sequence, and GFP sequence for further study of subcellular localization of DND1.