Clone Of RSSG58 Gene Promoter And Primary Study On Structure Of RSSG8 Gene In Rice (Oryza Sativa L.)

Rice is an model plant for developmental and molecular biology. It has important economic value. Along with the accomplishment of Rice Genome Plan, the study on rice development goes deep into molecular level. Sperm cell that acts as the male gamete plays a very important role in double fertilization process of rice. A pair of sperm cells that come from one pollen are transferred into embryo sac and one fuses with egg cell to produce zygote that develops to embryo, the other fuses with central cell and develops to endosperm. So it is very important to study expression of rice gene about sperm development for both academic sense and applied value. RSSG8 gene and RSSG58 gene were cloned from the cDNA library of rice sperm cells by subtractive method. And the two are both sperm-predominant expressed genes in rice.The cDNA sequence of RSSG58 was blasted and oriented with the database of Oryza sativa (japonica cultivar-group) in NCBI. Promoter sequence of the gene's upstream was forecasted by bioinformatic method. Primers were designed according to that, then two deletion fragments JP58B(1093 bp) and JP58S(462 bp) were obtained by PCR using Oryza sativa (japonica cultivar-group) etiolated seedling total DNA as template. The sequencing result showed that they possessed conservative elements of most higher plants promoter. For further study, the two fragments were inserted into plant expression vector pBJ121, and the recombinant plasmid named as pBI121-JP58B and pBI121-JP58S. Result of transient expression showed that the two fragments both have obvious activation of promoter for the report gene GUS.RSSG8 gene' structure was analyzed by bioinformatic method. It existed in rice genome with only one copy and had many restriction sites. It was located on rice chromosome 12, but the fragment of 2046~3762 bp had no homologous sequence in the rice genome data bank of GenBank. The open reading frame(ORF) of RSSG8 encodes a putative protein, which is consists of 1161 amino acid residues. The putative protein of RSSG8 had relative solubility, but no remarkable hydrophobicity. It had a putative molecular weight of 129.7 kD and an isoelectric point of 8.305. It had no signal peptide in the N-end, but had abundant a-helixs, β-sheets and loops in its secondary structure. It included four kinds of homologous domains, those were ANK, RRM, UBQ and Threonine-rich region domain. It also had many kinds of protein decorated and activated sites, such as Protein kinase C phosphorylation site, N-glycosylation site, ATP/GTP-binding site motif A (P-loop), Amidation site, etc. These suggested that it may be regulated by many factors in vivo, and had potential biological functions of receiving and reacting to cell signals. The putative tertiary structure showed that this protein had four circumvolution surrounded a central axis with a symmetrical spatial conformation, and folded into a looser globular protein.The open reading frame of RSSG8 was inserted into expression vector pQE30. And E coli. of M15 was transfected by the recombinant plasmid of pQE30-RSSG8. Then the positive clones was selected by colony Western blot. But no target fusion protein expressed in these positive clones was detected from the SDS-PAGE test after induced by IPTG. So it is essential for us now that ameliorate expression system and condition according to characteristics of the putative protein, and go deep into the function of RSSG8 gene.