Molecular Cloning And Prokaryotic Expression Of ADC Gene From Brassica Napus L.

Arginine decarboxylase belongs to a huge gene family which has lots of homologous members.They possess the existence of a broad variety of plants and play a important role in polyamine metabolism.So far,many related genes have been cloned from plants.A great deal of experiments demonstrate the crucial functions of ADC gene during many biological phases,such as development of plant flower and fruit,stretch of stem,cell division,response to stress,embryo growth.Therefore it will be worthy of cloning the ADC gene from Brassica napus and verifying the gene function.In this paper,we firstly amplified the conservative region of ADC gene using degenerated primer. Then the full length cDNA of ADC gene named BnADCl andBnADC2 was obtained with the race method,Meanwhile we construct the prokaryotic expression vector and detect the ADC protein using SDS-PAGE.The brief results are as follows:1. Conservative region cloning of Brassica napus ADC geneTwo kinds of sequences 1040bp,1052bp were amplified using degenerated primer.These sequences have high similar with other ADC genes publicized by NCBI.The analysis of blast showed that the sequence obtained is part of the ADC gene.2.The cloning of full length cDNA of ADC gene from Brassica napus L.With the conservative sequences known.the 5'and 3'cDNA end of ADC gene were amplified with gene specific primers,then two full length cDNA of ADC homologous gene were obtained through sequence assembly.The nucleotide sequence of BnADC1 is 2649bp long including 5'UTR 344bp,ORF 2079bp,3'UTR 225bp and it encodes a protein of 692 amino acids; The nucleotide sequence of BnADC2 is 2625bp long including 5'UTR 352bp,ORF 2064bp,3'UTR 209bp and it encodes a protein of 687 amino acids. ORF of BnADCl and BnADC2 were cloned using primers combination:Full-ATG1 and Full-TGA1. Full-ATG2 andFull-TGA2.The result of homology analysis in NCBI suggests that these two cDNA full length sequences should belong to ADC gene of Brassica napus L.3.Constuction of prokaryotic expression vectorDesign the primer with the EcoR1 and Bg1Ⅱsites, then clone the BnADCl gene through PCR.Digest the BnADCl and prokaryotic expression vector pET30a(+) by double restriction endonuclease EcoRl and BglⅡ, then recycle the target gene and link to expression vector pET30a(+). Digestion and PCR verification showed that the prokaryotic exprssion vector with the purpose of gene——pET30a(+)/BnADCl has been successfully constructed and the inserted ADC1 gene sequence is correct. 4. Prokaryotic expressin vector pET30a(+)/BnADC1 expressed in E.coli BL21(DE3)Under the propriate induce condition, we tranplanted the prokaryotic expression vector into E.coli to induced BnADC1 gene expressing in E.coli BL21(DE3),and then proved the expression of BnADCl through the SDS-PAGE detection on the recombinant protein. Meanw-hile inducing conditions were discussed in the experiment.The results showed that the specific protein expressed in the condition of 37℃,1mmol/L IPTG,5h.