| The functions of life are executed by proteins, proteomics is the study of cells, tissues or organisms in the protein composition and changes in the laws of science. Currently proteomics research are widely used in plants, but in cotton has not been widely used. As one of the three core technologies about proteome research, two-dimensional gel electrophoresis is the only one commonly-used method at present that can continuously separates thousands of proteins in a piece of gel, widely used in all aspects of biological research.Using two-dimensional gel electrophoresis to analyze the gene expression on the plant development process can reveal the intrinsic mechanisms of plants development in protein level. In research of the molecular mechanism of plant male sterility, the whole protein two-dimensional gel electrophoresis is being used more and more widely. But so far, the study of male-sterile proteomics in cotton has not been reported at home and abroad. Using proteomics to analyze the total proteins of cotton buds, aimed to finding the abortion-related proteins in the process of pollen development, provided the basis for the further studies on the mechanism of abortion occurrence of cotton and the other plants.This study has established two-dimensional electrophoresis for anther proteomics of upland cotton. We used upland cotton anther as the material, compared and optimized the key steps, such as sample preparation,the volume of the sample, and so on, hoped that improve the resolution and repeatability. We tried to establish a 2-DE method that can be applicable in the cotton proteome analysis.The materials were frozen in liquid nitrogen immediately and grinded in a mortar, then the proteins were extracted by TCA/acetone precipitation and phenol extraction-methanol/ammonium acetate precipitation. Then proteins were separated by two-dimensional gel electrophoresis. It is expected that phenol extraction-methanol/ammonium acetate precipitation method could be one of the options for protein extraction from cotton anthers, and the ISO-DALT-IEF is better than IPG-DALT-IEF. The high-resolution and well-reproducible patterns of proteins were observed when the silver staining is taken and 500ug protein sample were added in electrophoresis. The 2-DE maps are analyzed using ImageMaster2D platinum 5.5 software, and distinguished approximate 1300-1400 spots.The proteins of genic male sterility (GMS) upland cotton anther of the sterile line and fertile line were separated by two-dimensional electrophoresis. The silver-stained proteins spots were analyzed using ImageMaster2D platinum 5.5, there were about 1300 detectable spots on each 2DE gel, and about 79 spots were differential expressed. With direct MALDI-TOF mass spectrometry analysis and protein database searching,58 protein spots out of 79 were identified.Among those proteins, there were putative retinoblastoma binding protein, ibulose-1,5-bisphosphate carboxylase/oxygenase activase 2, ATP-dependent RNA helicase eIF4A-13, glyceraldehyde 3-phosphate dehydrogenase, NADH dehydrogenase subunit 1, gibberellin 20-oxidase, gibberellin 3-hydroxylase 1, cytosolic ascorbate peroxidase 1, polygalacturonase, ketoacyl-CoA synthase, calmodulin-binding heat shock protein, and so on. They were closely associated with signal transduction, Gene regulation, carbohydrate and energy metabolism, Photosynthesis, and so on, all of which are cell activities that are essential to pollen development. Some of the identified proteins were deeply discussed on the relationship to GMS. This study gave new insights into the mechanism of GMS in cotton and demonstrated the power of the proteomic approach in plant biology studies. |
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