Expression Study Of RSSG58 Gene Of Sperm Cells From Rice (Oryza Sativa L.)

Here we report the rice (Oryza sativa L.) RSSG58 gene, cloned from the cDNA library of rice sperm cells by using sperm cell dominantly_expression subtractive clone as probe. It encodes a putative 66.7 kDa polypeptide, which shows a similarity to the myosin heavy chain of Arabidopsis thaliana. The RSSG58 protein consists of 579 amino acids with an isoeletric point (PI) of 4.885. RSSG58, which is a member of a divergent gene family, generates transcripts of 2 278 and 2 437 bp that differ only in their polydenylation sites.The RT-PCR analysis indicated that the transcriptions of the RSSG58 gene were various in the different development stages and tissues. The greatest accumulation of RSSG58 mRNA was also observed in sperm cells while weaker expression was detected in leaves, microspore mother cells, unicellular microspore pollen stage, two-cell stage pollens, mature pollens and pollinated ovaries. These results suggest that RSSG58 is especially abundant expressed in rice sperm cells.The open reading frame of RSSG58 gene was inserted into expression vector pQE30. After induction with IPTG, recombinant protein was expressed in E.coli M15(pREP4) consisting of 6 X His fused to the N-terminal. The purified recombinant protein was used to immunize rabbits to get specific antiserum with high titer. Purified antiserum was used to investigate the immunohistochemistry patterns of the protein during the process of male gametogenesis. The strongest immunofluorescence hybridization signal were detected in both the isolated sperm cells and the sperm cells in maturated pollens, a weak signal was detected in thegenerative cells of bicellular pollen, and no signal was observed at microsporocyte, unicellular microspore and vegetative organs (root, stem and leaf). These results suggest that RSSG58 is an abundantly expressed gene in rice sperm cells.One pairs of specific primers were designed according to the pubilished genomic sequence of rice and RSSG58 gene sequence of rice sperm cells, 5' upstream sequence 960 bp was amplified by polymerase chain reaction(PCR) technique from DNA template of rice, named as Pr58 and this fragment has most of conservative elements for higher plants' promoter. In order to established a foundation for further study of Pr58's function in promoting alien gene specific expressions in plant, we get Pr58's three absence sequences Pr58 I (765bp), Pr58II ( 530bp) and Pr58 III ( 258bp ) by PCR and then insert these fragments into vector pMGFP4(wi\h GFP report gene) to take place of CaMVSSS (a promoter) to get three expression vectors pRGFP I , pRGFPE and pRGFPlIL.The antisense RNA riboprobes of RSSG58 were regenerated by In Vitro transcription. Localization of RSSG58 mRNA were investigated by In Situ hybridization. We found that no signal appeared in the vegetative organs (stems, leaves) During the process of male gametogenesis, no detectable signal was observed at microsporocyte,tetrad and a weak signal began to appear in the unicellular microspore stages. The hybridization signal was increased along with the development process, and a very strong signal was observed in the sperm cells of the mature pollen. We would expect the activation of RSSG58 mRNA appeared to occur at the late unicellular microspore stages and early generative cells and the expression increased during pollen maturation, reached the highest level at the mature pollen stage.To investigate the function of RSSG58 gene, A 5' fragment of 590bp of RSSG58 cDNA was obtained and cloned into pFGC5941 vector in a reverted direction. Thus an antisense RSSG58 gene is constructed. The antisense recombinant is transformed inlo Arabidopsis thaliana by Agrobacterium-mediated transformation. Morphological features of Antisense transgenic Arabidopsis thaliana show that pollons crimple, pollen viability decline and the plants have a few seeds comparableto sense control plants. Cytology features at different pollen developmental stages under light microscopy showed microspore development proceeded normally until the bicellular pollen stage,...