Expression Analysis, Construction Of Expression Vectors And Genetic Transformation Of A Pollen Specific Gene OsPSG076 In Rice(oryza Sativa L. )

Rice(Oryza sativa L.) is not only the second important crops in the world, but also a model monocot species for molecular genetic study. The completion of “The International Rice Genome Project” laid a solid foundation for cloning and functional investigation of genes encoding important agronomic traits.Pollen is the male gametophyte of flowering plants, and the normal development of pollen is of vital importance for successful fertilization. While the molecular mechanisms underlying pollen development remains unclear at present. Based on previous work in this laboratory on OsPSG076, a homologous of wheat pollen specific gene PSG076, the expression pattern of OsPSG076 in different rice organs was analyzed by RT-PCR to check if it is also a pollen specific expressed gene. Then, subcellular localization of OsPSG076-GFP fusion protein was examined by transient expression in onion epidermal cells. Finally, overexpression and RNA interference vectors were constructed and were used to transform rice. The main results are as follows:(1) Semi-quantitative RT-PCR was used to analyze OsPSG076 expression pattern using RNAs extracted from different organs. Results showed that high level expression of Os PSG076 was detected in anther/pollens, and very weak expression was also detected in roots, but there were no expression in all other tissues examined, indicating that Os PSG076 is anther /pollen dominantly expressed gene.(2) OsPSG076-GFP fusion protein expression vector pBI121-OsPSG076-GFP was constructed and was transient expressed in onion epidermal cells using biolistic bombardment technique. Microscopic observation result showed that like that of GFP control, OsPSG076-GFP fusion protein distributed throughout the whole cell.(3) OsPSG076 overexpression vector( pCAMBIA1304-OsPSG076-OX) and RNA interference vector( pCAMBIA1304-Os PSG076-RNAi) were successfully constructed,and these two vectors were transferred into rice variety Nipponbare, respectively. The gus and hyg genes were used to screen for T0 positive transformants by PCR method, and as a result, three vacant vector control transgenic lines, five overexpression transgenic lines and twelve RNAi transgenic lines were obtained.The above results provided useful materials for further studying the function of Os PSG076 gene during pollen development process.