Isolation and Detection of PLC? (Sperm Factor Protein) in Testes of Rattus rattus diardii Using One-Step RT-PCR

摘要

The fertilization process in many species studied involves induction of Ca2+ oscillations in eggs. Prolonged time period of calcium oscillations is essential for regulating various kinds of events that will occur after fertilization. PLC zeta (PLCζ) has been discovered as a novel isoenzyme of PLC which is responsible in promoting intracellular Ca2+ oscillation in oocytes. Until now, PLCζ has been identified in many mammalian species. Although PLCζ has been identified in the rodent family, such as the laboratory mice, rats and hamsters, no research has been carried out to identify PLCζ in house rats. This study was carried out in order to identify the existence of PLCζ in testes of house rats (Rattus rattus Diardii), one of the major pests in houses. Testes from ten male rats were subjected to RNA extraction using easy-BLUETM Total RNA Extraction Kit. One-step RT-PCR technique was chosen to identify PLCζ using forward and reverse primers from published sequence of PLC-zeta. PCR amplicons were extracted from Agarose gel, purified and further subjected to sequencing. Sequences obtained were aligned using NCBI Blast search at www.ncbi.nlm.nih.gov/blast to check for identification from published PLC-zeta sequences. Results from this study showed that PLCζ from the testes of house rat was successfully amplified by using One-step RT-PCR technique. The sequence also showed strong homology to published sequence of Rattus norvegicus PLCζ. We hope that with the understanding of molecular properties of PLCζ in house rats could lead to development of contraceptive vaccines, an alternative approach to control the rat population.