Localization Of HL-sp1-coding Protein And Analysis Of Its Candidate Interaction Proteins In HL-CMS Rice

Cytoplasmic male sterility(CMS)is a maternally inherited inability to produce functional pollens,and is of a normal femal organ in some angiosperms.In previous study on Honglian CMS(HL-CMS)rice in our laboratory,a CMS associated mitochondrial gene,HL-sp1,was identified,and a orf of coding 216 amino acids was predicted.But by Western Blot with ORF216's antibody,a larger molecular weight protein than ORF216 was detected in HL-CMS line YTA.Moreover,by Pull-Down with ORF216 as a bait,there were three potential interaction proteins trapped.In this study,HL-sp1-coding protein and one of its candidate interaction proteins were further identified,and the main results were as follows:1.Based on sequence and the transcript of HL-sp1,a potential ORF encoding a putative protein with 316 amino acid was predicted.2.Two transient expression vectors of pM999-orf316 and p M999-orf216 were constructed respectively,and their subcellular localization in transformed rice protoplasts was viscualized by laser scanning confocal microscopy.The results showed that the ORF316 was targeted into mitochondria,but the ORF216 was located in cytoplasm.3.A rice mitochondria gene ef-tu was cloned by overlapping PCR,and its expression vector p GEX4T-1-ef-tu was constructed.It was then successfully expressed in E.coli and the optimal inducing temperature was 28?.4.A prokaryotic expression vector pGEX-4T1-orf316 was constructed,which could be successfully expressed in E.coli under the optimal inducing temperature 25?.The expression protein,ORF316,was then purified by Ni-column affirnity chromatography as well as Western blot identification.But it was not verified that ORF316 could interact with OsEF-Tu,previously trapped by ORF216,by GST Pull-Down assay in this study.