Cloning And Prokaryotic Expression Of Soybean PR-5Protein GmOLPa

Plant produces Pathogenesis-related protein(PRP/PRs) by biological or abiological stresses. The Thaumatin-like protein (TLP) belongs to Pathogenesis-related protein(PRP/PRs) family which is related to plant defense. GmOLPa protein which is one kind of Thaumatin-like proteins (TLP) is reverse osmosis protein.Total RNA was extracted from the salt-induced soybean root and reverse transcribed to cDNA. GmOLPa was cloned by RT-PCR. Then GmOLPa was constructed into prokaryotic and eukaryotic expression vectors. A more simple and effective system was established by agrobacterium rhizogenes infection.The GmOLPa gene cloned from soybean is1050bp, and sequencing analysis by DNAMAN software showed GmOLPa had99%similarity to published GmOLPa (Gene bank ID:AB116251). This gene had a revered domain (25-224aa) which belong to GH64-TLP-SF super-family. Phylogenetic analysis between GmOLPa protein and partly members of Thaumatin-like family using DNAMAN software proved that GmOLPa protein was a separate branch.Two interference fragments with different restriction enzyme sites had been cloned from pMD18-T-Gm plasmid. pHANNIBAL-Gm-RNAi vector was constructed using pHANNIBAL skeleton. The functional domain of this expression vector (35s promoter, sense strand, anti-sense strand, intron and stop codon) was inserted into pCAMBIA1301(the plasmid with reporter gene GUS). So, pHANNIBAL-Gm-RNAi was constructed and would be used in functional analysis of the GmOLPa.Objective fragment which was700bp cloned from pMD18-T-Gm plasmid and inserted into pET-28a(+). Recombinant protein was induced in E.coil. The best expression system was optimized which is0.1mmol/L IPTG and induced4h. The weight of target protein molecular is about30KDa.It will be a good experimental basis on the protein purification, antibody preparation, the large-scale application of the protein and further study of function of the protein.The high-efficient system is optimized through agrobacterium rhizogenic C58C1mediated transformation with pCAMBIA1301plasmid infecting soybean by the not tissue culture.Detecting the GUS gene of DNA by PCR in the root and observing the results of the chemical tissue dyeing in the leaves of GUS which invaded10min,15min,20min,25min and30min. The optimal conditions is invaded25min that means which is the optimized time for bacterium infecting soybean.