| RSG6 gene was cloned from the cDNA library of rice sperm cells by using sperm cell predominant expression subtractive clone as probe. The result of bioinformatics analysis showed that it had no remarkable homology with the known genes in GenBank. And the RSG6 gene is localized on the chromosome 8 and 10 of Oryza sativa L. with one copy for each; its homologous fragments exist also on the chromosome 4 and 12 of rice.By using DIG RNA labeling kit and by the way of in vitro transcript, a RNA probe was synthesized, which was a fragment of the cDNA of RSG6 gene and was 258bp in length. By the method of in situ hybridization, the distribution of mRNA of RSG6 was detected. The result showed that the transcript activity began at the late unicellular stage microspore, increased at the bicellular pollen, and was most vigorous at the two sperm cells in mature pollen. On the other hand, using the purified antiserum of RSG6 protein, the expressed RSG6 protein was localized by Western-blot and immunohistochemistry in the sections of different tissues and organs of rice. The results revealed that the expression of RSG6 appeared to begin at the bicellular microspore, and kept activity in the sperm cells of mature pollens. This result was coincided with that of the in situ hybridization of RNA. The immunofluorescence result of isolated sperm cells even showed that RSG6 protein was present close to or attached to the surface of sperm cells. Thus, it is suggested that RSG6 might be very important in the sexual reproduction of rice, especially in signal conducting and recognizing of the procession of sperm-egg fusion.In order to investigating the function of RSG6, anti-expression vector, pBIN19-Fan, was constructed by using the 258bp fragment of the cDNA ofRSG6, the CaMV35S promoter in pBI221 and the expression vector of plant, pBIN19. In addition, a reporter gene, GUS, was attached to the anti-fragment and another re-combinant plasmid, pBIN^-Fan-GL/S1, was constructed. The two re-combinant plasmids were transformed to the Agrobacterium tumefaciens EFIA105, and the later was used to infect callus of rice. 27% and 21% of the callus transformed by pBIN19-Fan and pBIN19-Fan-GC/5 respectively survived in the selective medium. Tested by polymerase chain reaction (PCR) amplification of the fragment of GUS gene, the callus transformed by pBIN19-Fan-GUS was positive in 42.86%. Meanwhile, the sense expression vector, pBI12l-RSG6, was constructed by using the open-reading-fragment of RSG6 and the expression vector, pBI121. All these work laid foundation for the further study on the function of RSG6 gene.Specific primers were designed according to the published genomic sequence of rice in GenBank and the sequence of RSG6 gene. Then 5'upstream fragments of RSG6 were amplified by PCR from DNA template of rice. Bioinformatics analysis showed that there were plenty of conservative elements of higher plants' promoters in the amplified sequences. Four deletion fragments, PS1, PS2, PB1 and PB2 were produced by PCR. By using GUS gene from pBI221 as the reporter gene and the expression vector of plant, pBIN19, four re-combinant plasmid, pBIN19-G?SSl, pBIN19-GUSS2, pBIN19-GWSBl, and pBIN19-GC/5B2were then constructed. In addition, the CaMV35S promoter was used as the positive control and the re-combinant plasmid, pBIN19-GUS, was also constructed. Re-combinant plasmids were transformed into Agrobacterium tumefaciens EH105, and the later were used to infect leaves and mature pollen of tobacco and callus of rice. GUS transient expression demonstrated that all of the four deletion fragments could work as promoter, but their efficiency were different from each other and in various tissues. By the way of leaf dish transformation, transgenic tobacco plants were obtained. Tested by PCR, transgenic plants of pBESF19-GUSS1, pBIN19-GUSS2, pBIN19-GWSBl, pBIN19-GWSB2 and pBIN19-GUS were positive in 44.98%,36.94%, 42.13%, 30.26%, and 34.26% respectively. Our present work have laid a solid foundation for the further study on...
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