Cloning And Expression Of Plant Defensin Gene SmD1 Of Stellaria Media

Plant defensins are one of the basic component of plant defense system.They are small,cationic and cysteine-rich peptides and have strong resistance against fungal.So plant defensinscan be used as novel germicides to protect plants from diseases.Also,they are expected to help develop food preservatives for the improvement of food safety.Defensin gene SmD1 was cloned into E.coli expression system for heterologous expression.In our study,we synthesized the gene of Stellaria media defensin SmD1 according to its published nucleotide sequence.The synthetic plant defensin gene SmD1 was used as the template.Primers were designed according to this gene and the SmD1 gene fragment with restriction sites was amplified by PCR.Primers were also designed according to the DNA sequence of plasmid pET32a(+),and nucleotide sequence at the other end of the primer was complementary to gene SmD1.So Thioredoxin(TRX)gene was amplified using vector pET32a(+)as a template.At last,we amplified TRX-SmD1 gene by overlap PCR using the primer both complementary with TRX and SmD1 gene.We inserted TRX-SmD1 gene into pET22b(+)vector before transformed into E.coli BL21(DE3).After induction of IPTG,E.coli BL21(DE3)with the recombinant vector expressed 22 kDa fusion protein in the form of soluble expression both intracellularly and in the periplasmic space.We obtained a single protein band in 200 mmol/L imidazol elute of Ni-IDA agarose resin column.The peptide had antifungal activity after formic acid treatment.Meanwhile,we chose the factors which have significant impact on the expression of fusion protein,namely induction time,OD,induction temperature and IPTG concentration.The experiment showed that the maximum antifungal activity was obtained,when IPTG was 0.6 mmol/L,temperature was 25?,induction time was 12 h and OD was 0.8.We also inserted TRX-Sm D1 gene into pET28a(+)vector before transformed into E.coli BL21(DE3).After induction of IPTG,BL21(DE3)efficiently expressed 22 kDa fusion protein TRX-SmD1 in the form of inclusion body.After renaturation of the inclusion body,we observed a single protein band in the eluent of 200 mmol/L imidazol in Ni-IDA agarose resin column.Antifungal activity was observed after formic acid treatment in 50?.We chose culture components and fermentation conditionsto perform one factor optimization.At last,the concentration of glucose,fermentation time and concentration of magnesium sulfate were made as factors in response surface experiment.As a result,the antifungal activity reached maximum when the fermentation time is 6.34 h,the concentration of magnesium sulfate is 1.22g/L,the concentration of glucose is 14.64 g/L,the maximum can reach to 1.97 cm.The minimum inhibitory concentration(MIC)of defensin SmD1 against Botrytis cinerea was 1 ?g/m L.In this study,Stellaria media defensin SmD1 gene was cloned into E.coli and we obtained both soluble protein and inclusion body with high antifungal activity.Our study laid a foundation for the efficient production and development of plant defensins.