Molecular Cloning, Ectopic Expression And Subcellular Localization Of A Soybean BURP Domain-containing Protein Gene

With the completion of soybean EST project,the functional genetic information of soybean EST in public database has been very abundant.The functional genetic information of soybean EST provided the conditions for soybean functional genomics research.Research shows that, BURP protein play an important role in the process of plant growth and development.A new gene containing BURP domain was cloned from soybean for further study of the BURP protein function and mechanism of action.In this paper,we used the EST Blast method to clone a BURP domain-containing protein gene from Glycine max.RT-PCR was adapted to detect the expression of this gene in different tissue.Moreover,the GmBURP1 gene was transformed to E. coli.1 Cloning and analysis of Glycine max GmBURP1 geneA novel Glycine max BURP protein family gene GmBURP1 full-length cDNA of 1189bp was cloned by Blasting the Glycine max EST database with the Arabidopsis thaliana homologous gene RD22 amino acid sequence as a querying probe.Searching the cDNA sequence for potential coding regions by ORF finder(NCBI),an entire open reading frame(ORF) of 349 amino acids was found with a MW of 39.SKDa and a pI of 6.11.It was detected with a potential start codon at the 55^th site and a stop codon at the 1104^th site of the sequence.To examine the accuracy of in silico cloning cDNA sequence,Glycine max cDNA were used as templates to amplify the coding regions of this gene.Single strongly amplified band with about 1200bp was obtained,and then the amplified products were ligated into a pGEM-T Easy vector(Promega) and sequenced by primer walking.1 complete code nucleotide sequences were obtained.It was named GmBURP1 and deposited in GenBank with the accession number from FJ649315.The deduced amino acid sequence had typical characters of BURP protein family.2 Expression of in E.coli and subcellular Localization of cloned geneA pair of PCR primers was designed to amplify GmBURP1 gene cloned.The amplified products were ligated into expression vector pET28a and then transformed into E.coli strain Rosetta.After induced by isopropyl-β-D-thiogalactoside(IPTG),cells were harvested,and then the expressed proteins were extracted and identified by SDS-PAGE.GmBURP1 gene was expressed successfully in E.coli.This indicated that this gene can express under the control of the functional promoter.Furthermore,the deduced amino acid sequence of mature proteins were identical to the accurate amino acid sequence determined by ESI-Q-TOF.This GmBURP1 gene was fused with GFP gene,and constructed into P1390.The plasmid (P1390--GmBURP1GFP) was transformed into onion epidermal cells by particle bombardment.Bright green fluorescence was visualized in the cell membrane only with bombardment of P1390- GmBURP1GFP,whereas GFP plasmid showed the whole cell distribution of GFP.The result suggested that GmBURP1 might function in the cell membrane.