Myosins, a large family of structurally diverse mechanoenzymes, which, upon interaction with actin filaments, convert energy from ATP hydrolysis into mechanical force, play an important role in male reproductive processes. In this study we report the rice ( Oryza sativa L.) RSSG58 gene, which was cloned from the cDNA library of rice sperm cells by using sperm cell mainly expression subtractive clone as probe. This gene encodes a putative 66.7 W polypeptide, which shows similarity to the myosin heavy chain of Arabidopsis thaliana, and consists of 579 amino acids with an isoelectric point (pI) of 4.885. RSSG58, which is a member of a divergent gene family, generates transcripts of 2 278 bp and 2 437 bp that differ only in their polyadenylation sites. Southern hybridization showed that RSSG58 has only one copy in rice genome and RSSG58 transcripts are most abundant in sperm cells, with two distinct signals. 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 detected 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 abundantly expressed in rice sperm cells.
A gene with complete sequence of 1 176 bp containing an open reading frame (ORF) of 281 amino acids, RSG6 (accession number in GenBank: AF442490) was cloned from rice (Oryza sativa L.) sperm cell cDNA library by using sperm cell higher expression clone BF475207 as probe. It is the first gene isolated directly from sperm cells of higher plants. The sequences of RSG6 gene and its deduced amino acid did not reveal remarkable similarity to that of any known ones in GenBank. Southern hybridization suggested that there may be introns. in this gene. Reverse transcriptase-polymerase chain reaction analysis (RT-PCR) showed that the RSG6 gene was expressed in all nice organs or cell types examined (including roots, leaves, two-cell stage pollen, mature pollen, pollinated ovary and sperm cells), but the level of expression in sperm cells was particularly higher than that in all the other organs or cells. The RSG6 gene was cloned into a vector pQE30 and expressed in Escherichia coli M15 (pREP4). High specific antibody with high titer was obtained from rabbits.
