Screening And Identification Of Differentially Expressed Proteins In Insect-resistant Transgenic Cotton(Gossypium Hirsutum L.) Of Different Regions

Gossypium hirsutum L is the important economic crops in china.it is susceptible to pests and diseases in its growth. The insect-resistant transgenic cotton is effective in improving the yield and reduce pesticide. It can also remarkable speed up the breeding process. The GM product has being widely applied. But the safety of GM product has becoming a worldwide concern, the biosafety GM product are often used as barriers to trade in some countries, thus causing international trade disputes. Many countries have formulated relevant laws and regulations to moitor the GM product, However, the detection method and security management of teconology of GMO and its products is lags far behind the demand of GMO safty supervition. Therefore, the establishment of standards of measurement and traceability system for GMO has become an urgent task.Proteomic approaches including two-dimensional electrophoresis(2-DE), matrix assisted laser desorption/ionization time of flight mass spectrometry(MALDI-TOF/MS) and bioinformatic techique were used in this paper. A two-dimensional gel electrophoresis has been developed to analyze the proteomics of cotton, Gossypium hirsutum L. leaf. The protein expression profile of insect-resistant transgenic cotton (Gossypium hirsutum L.) and it’s parent in different regions using2-DE and MALDI-TOF/MS is analysised in our study, in order to screen and identify the important proteins associated with the regions.The study provided a new clue to investigate the analytical techniques used for determining geographical origin of GMO.In this study, A two-dimensional gel electrophoresis has been developed to analyze the proteomics of cotton, Gossypium hirsutum L. leaf. The results showed that a high purity of cotton protein was extracted by TCA-acetone precipitation, and by cleaning the protein sample with solvent methanol or acetone. The parameters for obtaining a high resolution of cotton2-D electrophoresis maps were optimized as follows:24cm strip with linear (IPG) in pH5～8,300μg sample loading, TBP substituting for DTT in the IPG buffer, and combination of two carrier ampholytes in the pH range of5～8and3～10with the volume ratio of1:1. This optimal2-DE system gave a much better protein profile with a high resolution, protein yield and spot intensity.By using two-dimensional electrophoresis and mass spectrometry techniques, the proteomes in cotton Seedling Leaves of three varieties from four regions in2009including Linqing City, Shandong Province, Shihezi City, Xinjiang Province, Yuanyang City, Henan Province and Hangzhou City, Zhejiang Province were comparatively analyzed. Results showed that the proteomes had a significant difference in four regions.113distincted protein spots were analyzed via MALDI-TOF/MS and database retrieval, among them84proteins were successively indentified, including39different proteins. A model were established to distinguish region by detecting the abundance value of protein spots in cotton leaf from above regions, the accuracy was94%.Proteome variations maps associated with three varieties from three regions in2010including Linqing City, Shandong Province, Shihezi City, Xinjiang Province and Hangzhou City, Zhejiang Province were comparatively analyzed. Compared2009, there was a same tendency in the expression level of63protein spots in the protein profiles.among of them49proteins were successively indentified including29different proteins. Compared Linqing City, Shandong Province, the abundance value of44protein spots variated in Shihezi City, Xinjiang Province, including19different proteins.the abundance value of49protein spots variated in Yuanyang City, Henan Province, including25different proteins.the abundance value of37protein spots variated in Hangzhou City, Zhejiang, including19different proteins. Most of these indentified proteins involved in development processes, energy metabolism, resistance response, photosynthesis.the change of the correlations between the abundance value of84protein spots and different regions under different environmental conditions were also investigated.