Expression Analyses Of Two Cotton GhPRP Genes And Transgenic Cotton (Gossipium Hirsutum)

Cotton fibers which are derived from the epidermis of the ovule are single-cell trichomes.Fiber development consists of four overlapping stages:initiation,elongation, secondary cell wall(SCW) thickening,and maturation.It is theoretically and practically significant to elucidate the molecular mechanisms underlining cotton fiber cell development.Previously,two genes encoding cell wall proline-rich proteins,which were designated as GhPRP3 and GhPRP5,were isolated form cotton fiber and ovule libraties. Northern blotting and Real-time RT-PCR results showed that the two GhPRP genes were preferentially expressed in fibers and also developmentally regulated.In this study,their expression,subcellular localization and the functions were investigated.The main results were as follows:1.The expression patterns of GhPRP3 and GhPRP5 in response to different biotic and abiotic treatments such as phytohormones,salt and drought stress were investigated using in vitro ovule culture techniques.The results indicated that GA3 and drought treatment can up regulate GhPRP3 while KT,ABA and dought stress can up regulate GhPRP5 implying that the two fiber-preferential GhPRP genes were regulated in response to phytohormones and environmental stress.2.To further investigate the tissue-preferential and developmental regulated expression of GhPRP5,an 2bp fragment upstream of the GhPRP5 translation start codon (ATG) was isolated by Genome walking.Analysis of the isolated promoter sequence revealed many putative cis-acting regulatory elements were present.The expression of the GhPRP5∷GUS was examined in transgenic Arabidopsis plants.Histochemical assays demonstrated GUS expression driven by the promoter was most intense in trichomes. Because trichomes in Arabidopsis and tobacco show a lot of structural and genetic similarities to cotton fibers,it can be concluded that this promoter is fiber specific.3.To test the localization of GhPRPs protein in cotton cells,GhPRP3/5-eGFP fusion protein was expressed in transgenic cotton cells under the control of 35S CaMV promoter.The eGFP fluorescence was monitored under a Leica Confocal laser scanning microscopy.WT cotton callus cells showed no background fluorescence,while green fluorescence was observed on the surfaces(cell wall or plasma membrane) as well as in cytoplasm of the transgenic cotton cells.However,when the cells were plasmolyzed with 4%NaCl for 10 min,green fluorescence was localized in the cytoplasm and the plasma membrane,indicating that these two GhPRPs protein may be localized in the plasma membrane and in the cytoplasm.4.In order to figure out the functions of these two genes,an overexpression vector was constructed and transformed into Arabidopsis thaliana.7 and 9 lines of transgenic plants were obtained respectively,Preliminary RT-PCR results showed that GhPRP3/5 highly expressed in the transgenic plants,now the phenotypic analyses are underway.In addition,an RNAi vector was constructed and was introduced into cotton.After one-year of transformation,several lines of transgenic cotton plants were obtained.Preliminary PCR results showed most of the transgenic plants were positive,and also the phenotypic analyses are going on.