Comparative Proteomic Analysis Of Transgenic Cotton Leaves At Different Developmental Stages

Abstract:Upland cotton (Gossypium hirsutum L) is the important economic crop in China. More than70percent of the cultivated area of cotton is transgenic cotton in China, and there is an obvious increasing trend. Upland cotton is the model plant in fiber research. However, the studies on proteomics, genomics, physiology, and genetics in cotton is relatively weak when compared with other model plant (Arabidopsis, Oryza Sativa et.al). The databases of cotton two-dimensional electrophoresis profiles and proteome need to be improved because of limited studies on cotton proteomics. Most proteomics research of cotton focuses on fiber development mechanisms and stress reaction. There were few reports about cotton developmental phases.In this study, the transgenic lines (07-19and08-6) and common line (ZHNO7-222, which was the parent of07-19) were used as experimental materials. To reveal the differences of proteome in cotton leaf at different developmental stages, two-dimensional electrophoresis (2-DE), bioinformatics technique and mass spectrometry (MS) were used for comparative proteomic study. The results showed that26proteins changed in abundance at different developmental stages, with9(34.6%) proteins having increased expression and17(65.4%) proteins having decreased expression at flower and boll stage. Seventeen spots were identified by MALDI-TOF-MS/MS after in-gel tryptic digestion, and these spots belonged to15kinds of unique proteins:mitochondrial glycine decarboxylase complex P-protein-like protein, chloroplast chlorophyll A-B binding protein, light-harvesting complex I protein Lhcal, ribulose-1,5-bisphosphate carboxylase/oxygenase large subunit, ribulose-1,5-bisphosphate carboxylase, large subunit, ascorbate peroxidase, transcription factor APFI-like protein, Peptidyl-prolyl cis-trans isomerase, phenylcoumaran benzylic ether reductase-like protein, Clp ATPase, NAD dependent epimerase/dehydratase, putative, ATP synthase CF1epsilon subunit, Isocitrate dehydrogenase, uracil phosphoribosyltransferase and50S ribosomal protein L21. These proteins are involved in a wide range of tricarboxylic acid cycle, protein synthesis/folding, ATP synthesis, defense, photo synthetic carbon metabolism and so on. The reason that caused the changes of proteome should be related to growth and development of cotton. The varied proteomic between leaves of seedling stage, flowering and bolling stage indicated a better understanding of the growth and development mechanism of cotton.In order to reveal the changes of proteome expression in three breeding lines (07-19,08-6and ZHNO7-222) at seedling stage and flowering and boiling stage, the comparative proteomics of07-19, ZHNO7-222and08-6were studied by using2-DE. Fourty seven defferently expressing proteins which were identified by MALDI-TOF-MS/MS were screened among three lines at seedling stage. Twenty five unique functional proteins were distinguished for47identified proteins. These proteins were involved in a wide range of tricarboxylic acid cycle, glycolytic pathway, cell development, cytoskeleton, signal transduction, growing development, defense and photosynthetic carbon metabolism. Thirty four defferently expressing proteins which were identified by MALDI-TOF-MS/MS were screened between the three lines at flowering and bolling stage, and they were identified out22unique proteins. These proteins were involved in ATP synthesis, tricarboxylic acid cycle, cell division/differentiation, defense, nucleotide metabolism, growing development, photosynthetic carbon and metabolism. Genetic recombination in genetically modified process and hybrid process were the possible causes of varied proteome.We cloned three gene fragment (APX, ICDH and rbcL) in this study. The length of the three amplified fragments were581bp,1055bp and1121bp, respectively. The the matching rates of the three genes were97.9%,99.1%and99.3%, respectively when aligned with genes of upland cotton in NCBI database. The expressions level of the three genes were detected by qPCR. The qPCR results showed that the APX and ICDH were down-regulated and rbcL was up-regulated at flowering and bolling stage, which were similar to the expression results of proteomic.