Cloning And Characterization Of A Differentially Expressed Gene Of Sperm Cells From Rice(Oryza Sativa L.)

Sperm cell plays a very important role in double fertilization of angiosperms. Apair of sperm cells that come from one pollen are transferred into embryo sac andone fuses with egg cell to produce zygote that develops to embryo, the other fuseswith central cell and develops to endosperm. The previous studies had investigatedextensively the process using cytological and embryological observations. It ispossible to understand gene expression in sexual cells with the help of newtechnologies and methods. A lot of pollen-specific expressed genes relating to pollendevelopment have been isolated. Several genes have been isolated from egg cell andzygote of Zea mays too. But little is known about the gene expression in sperm cellsof higher plants. Rice is the major source of food energy and a monocotyledonousplant with small genome size (430Mb), which has become a model system fordevelopmental and molecular biology. We identified 127 clones expressed only insperm cells or had higher expression level in sperm cells. In this dissertation, one ofclones was identified and characterized. Some results were obtained as following:1. One 1176bp gene containing an ORF of 281 amino acids, RSG6 (Accessionnumber in GenBank: AF442490) was cloned from rice sperm cell cDNA libraryby using sperm cell higher expression clone BF475207 as probe. Sequence ofRSG6 and the deduced amino acid sequence did not reveal remarkable similarityto any known sequences in GenBank, showing that may be a new gene.2. Southern hybridization showed 2-6 bands. No signal in all organs or cells except in sperm cell cDNA was detected by Northern hybridization. Reverse transcriptase-polymerase chain reaction analysis showed that RSG6 gene expressed in all rice organs or cell types examined (including roots, leaves, two-cell stage pollen, mature pollen and sperm cells), but the level of expression in sperm cells was higher particularly, which suggested that RSG6 is a specially abundant expressed gene of rice sperm cells.3. The open reading frame of RSG6 gene was cloned into expression vector pQESO. After induction with IPTG, RSG6 gene was expressed in E.coli M15 (pREP4) consisting of 6 X His fused to the N-terminal. The protein product accounted for 9.9% of the bacterial total protein. The expression product was purified by Ni-NTA resins with more than 90% purity.4. The purified recombinant protein was used to immunize rabbits to prepare antiserum. The liter of antiserum was 1:102400. Western blot analysis confirmed antiserum only recognized His6-RSG6 recombinant protein.This work isolated a differentially expressed gene directly from sperm cells of higher plants and investigated its expression pattern. This gene was expressed in E.coli, and high specific antibody was raised against the purified recombinant protein. All the results provide a basis to study its function at the protein level.