Molecular Characterization And Fuctional Analysis Of Pollen Specific Genes In Gossypium Hirsutum

Pollen development is a highly programmed process, in which a lot of genes are involved. These genes can be classified into two groups by the gene-expression profile in anther development:early anther development gene and late anther development gene. It is generally believed that late anther development genes are involved in pollen maturation, pollen germination and pollen tube elongation. Our previous study isolated two genes from cotton flower cDNA library. In this study, the expression patterns and factions of these two genes have been characterized. The main results are as following:1. GhPSP231gene is pollen specific in cotton.Northern hybridization, Real time RT-PCR and in situ hybridization results indicated that the transcripts of GhPSP231are specifically accumulated in maturing pollen of cotton at late anther development and regulated by cotton anther development. Western blot and immunohistochemistry assay were performed to determine the protein expression profile of GhPSP231. The result showed that GhPSP231protein was also specifically detected in maturing pollen. These results indicated that GhPSP231is a late anther development gene, and may be involved in pollen maturation and/or pollen germination.2. GhPSP231promoter is active specifically in pollen.The promoter fragment upstream of the GhPSP231translation start codon was isolated by Genome walking PCR. GhPSP231promoter::GUS fusion expression vector was constructed and tansformed into tobacoo by Agrobacterium-mediated method. The expression of GUS gene driven by GhPSP231promoter was only detected in pollen at late anther development stage, and the GUS activities were significantly increased continually during anther development. It suggested that the GhPSP231promoter activity is limited in pollen and regulated by anther development.To indentify the regulatory elements of GhPSP231promoter, five vectors containing1067bp,863bp,650bp,446bp and241bp promoter sequence were constructed and named GhPSP231P1to GhPSP231P5, respectively. Then the vectors were tansformed into tobacco respectively. Histochemical GUS staing analysis showed that the446bp fragment upstream the coding region of GhPSP231gene was enough to drive GUS expression exclusively and specially in pollen. However, the241bp fragment upstream the coding region of GhPSP231gene did not drive GUS expression. Taken together, the pollen specific regulatory element of GhPSP231promoter was localized between-446bp to-241bp.3. Overexpression of GhPSP231in yeast affects cell cytokinesisIn order to investigate the fuction of GhPSP231protein, the coding sequence of GhPSP231gene was cloned into the yeast vector pERE5N, and introduced into yeast (Schizosaccharomyces pombe) cells. After20hours of inductive cultivating, morphological changes of yeast cell were detected under the optical microscope. The results indicated that the percentage of the cells with cell division plate in transformed cell lines overexpressing GhPSP231was higher than in control cells. However, the growth rate of transformed cell lines overexpressing GhPSP231was lower than control cells. In addition, decolorized aniline blue staining showed that expression of GhPSP231in fission yeast induced over or ectopic deposition of callose in primary septum or cell wall. These results indicated that GhPSP231affects cell cytokinesis in fission yeast.4. Overexpression of GhPSP231in tobacoo reduces pollen viability.To investigate the role of GhPSP231in plant anthers, we overexpressed GhPSP231driven by GhPSP231promoter in tobacco, and6transgenic lines were obtained. RT-PCR analysis showed that they were expressed in anthers of each transgenic line except line7. It was founded that the overexpression transgenic plants had some small and wrinkled pollen grains. Malformed pollen grains in GhPSP231overexpression transgenic tobaccos showed no viability seen by FDA/PI staining. To further test pollen viability and function, we measured pollen germination on artificial media. Germination of the pollen from GhPSP231overexpressed transgenic tobacco was considerably lower than germination of wild-type pollen. DAPI staining showed that the differentiation of vegetative cell and germ cell in GhPSP231overexpressed transgenic tobacco was abnormal.5. Suppression of GhPSP231expression in cotton leads to serious male sterilityRNA interference was utilized to reveal the biological function of GhPSP231in cotton. We constructed GhPSP231RNAi vector and then transformed into cotton.5transgenic lines were obtained, and each of them was sterility. The pollen grains in GhPSP231RNAi transgenic cotton were small and wrinkled. Neighter of them can germinate in stigma of wild type. Further analysis revealed that GhPSP231RNAi transgenic plants can form normal microspores and anther walls, but the microspores can not form mature pollen grains.6. Molecular characterization of GhMYB24and its roles in late anther developmentGhMYB24encoding R2R3-MYB-like protein with210amino acids was isolated in cotton. GhMYB24protein is localized in the nucleus and act as a transcriptional activator. Northern blot analysis revealed that the transcripts of GhMYB24gene were predominantly detected in anthers. It was further found that strong expression of GhMYB24was mainly detected in pollen and was regulated during anther development by in situ hybridization. Overexpression of GhMYB24in Arabidopsis caused flower malformation, shorter filaments, non-dehiscence anthers and less viable pollen grains. Further analysis revealed that the septum and stomium cells of anthers were not broken, and fewer fibrous bands were found in the endothecium cells in transgenic plants. RT-PCR analysis demonstrated that the expression levels of the genes involved in phenylpropanoid biosynthetic pathway and ROS (reactive oxygen species) homeostasis were altered in GhMYB24-overexpression transgenic plants. Furthermore, the genes involved in JA biosynthesis and its signaling pathway were up-regulated in the transgenic plants. Yeast two-hybrid assay indicated that GhMYB24interacted with GhJAZ1/2in cells. Taken together, our results suggested that GhMYB24may play an important role in normal anther/pollen development.