Font Size: a A A

Proteome Analysis Of Plant Development Mutants

Posted on:2009-03-11Degree:DoctorType:Dissertation
Country:ChinaCandidate:X J XiaoFull Text:PDF
GTID:1100360272992158Subject:Analytical Chemistry
Abstract/Summary:PDF Full Text Request
Proteomics study is playing an increasingly important role in the functional genomics era. Proteome studies aim at the complete set of proteins encoded by the genome and thus complement the transcriptome studies. At present, the change at the protein expression level caused by gene mutation is one of the most important parts in proteome studies.Rice and Arabidopsis are two important model plants. In the present study, we analyzed the difference at the protein expression level in rice development mutants and Arabidopsis development mutants screened. The results of this study were listed as follows:(1) The screening of Arabidopsis development mutantsWe used Arabidopsis mutant cry1cry2 as material in this paper and constructed a library of arabidopsis mutants by activation tagging. Through screening and observation, some mutants with visible morphological phenotypic variations were obtained, including six mutants related to flowering time and two dwarf mutants. Plasmid rescue, inverse PCR (IPCR) and Thermal asymmetric interlaced-PCR (TAIL-PCR) were used to identify the flanking genomic sequences of mutated target genes. At the same time, the semi-quantitative RT-PCR was used to analyse expression of the T-DNA insertion flanking genes.We conclude that phenotypic variations in mutants were caused by the increased expression of T-DNA insertion flanking genes.The result laid the foundation for further study of regulation of flowering time and inhibition of hypocotyl elongation.(2) Proteome analysis of rice and Arabidopsis sterile mutantsWe used proteomic analysis to investigate the changing patterns of young panicle proteins during different developmental stages [the formation stage of pollen mother cell(â…¤), the stage of meiotic division of pollen mother cell(â…¥), the filling stage of pollen (â…¦)] under sterile and fertile conditions. Proteins were extracted from the spikelets samples and separated by 2-D gel electrophoresis (2-DE). Image acquisition and analysis, under sterile and fertile conditions, 43 protein spots were differently expressed in Zhu-1S during No.â…¤stage; 32 protein spots were differently expressed in Zhu-1S during No.â…¥stage; 20 protein spots were differently expressed in Zhu-1S during No.â…¦stage. Based on spot quantity and quality, 50 protein spots were analyzed by matrix associated laser desorption/ionization time of flight mass spectrometry (MALDI-TOF-MS) and 20 spots were identified. Most of these proteins are closely associated with sugar metabolism, energy metabolism, protein biosynthesis, cell wall formation and stress responses, which are essential cell activities to the pollen development. The expression of genes corresponding to three protein spots was analyzed by semi-quantitative RT-PCR. One of them the mRNA and protein levels was correlated well confirmed, but the other two in some instance, the level of mRNAs and the expression level of the related proteins were inconsistent. This difference could be affected by post-translational modifications. We identified 8 proteins related to fertility, of which UDP-glucuronic acid decarboxylase, putative caffeoyl-coA O-methyl- transferase 1 and OSJNBa0060D06.10 were closed to fertility change. The results indicate that temperature control of rice fertility is co-regulated by many other related genes and elucidate the molecular mechanism of thermo-sensitive genic male-sterile rice fertility change at the protein level.To study the difference at the protein expression level of the cry1cry2 and scc106-D, we used a proteome approach based on 2-DE to compare their protein patterns. The 22 different protein spots were identified by MALDI-TOF-TOF-MS. There are 17 protein spots were decreased in scc106-D and 3 protein spots only appeared in cry1cry2. The identified proteins are involved in several processes, i.e. protein biosynthesis, stress responses, photosynthesis, photorespiration and metabolisms of carbon, sulfur and energy. Most proteins showed enhanced degradation in scc106-D, especially 36% photosynthetic proteins such as Rubisco large subunit and 14% proteins related with energy. The decrease of photosynthesis rate and energy may be the cause of dwarf, sterile, later flower of mutant. The results indicate that light control of phenotypic variations in mutants caused by the increased expression of T-DNA insertion flanking genes is co-regulated by many other related genes.(3) Proteomic analysis of rice and Arabidopsis dwarf mutantsThe stem of thermo-sensitive genic male-sterile rice Zhu-1S and its dwarfing variant SV14 and H628S were separated by 2-DE. 33 differential spots were identified by MALDI-TOF-TOF-MS. There were 27 protein spots were decreased in SV14 and 2 protein spots only appeared in Zhu-1S. There were 25 protein spots were decreased in H628S and 3 protein spots only appeared in Zhu-1S. The identified proteins were divided into two following categories: one is regulatory proteins, for example, caffeic acid 3-O-methyltransferas, HSP70, glyoxalase I , actin, and so on. They are involved in protein biosynthesis and processing, cellular structural organization and stress responses. The other is function proteins, for instance, ascorbate peroxidase, cytoplasmic malate dehydrogenase, cytosolic phosphoglycerate kinase, S-adenosyl methionine synthetase, ATP synthase, and so on. They are involved in photosynthesis, photorespiration, redox homeostasis, and metabolisms. The regulatory proteins promoted the devolopment of stem and their decrease impacted the stem elongation. The decrease of function proteins destroyed the metabolism homeostasis in rice and impacted the stem elongation. The results elucidate the molecular mechanism of rice dwarf at the protein level and lay the foundation for further study of molecular breeding of dwarf rice.To study the difference at the protein expression level of the cry1cry2 and scc8-D, we used a proteome approach based on 2-DE to compare their protein patterns. The 22 different protein spots were identified by MALDI-TOF-TOF-MS. There are 16 protein spots are decreased in scc8-D and 3 protein spots only appeared in cry1cry2. The identified proteins are involved in several processes, i.e. redox homeostasis, stress responses, photosynthesis, photorespiration, and metabolisms of carbon. 32% proteins showed enhanced degradation in scc8-D, especially the photosynthetic proteins such as Rubisco large subunit. The results suggest that the inhibition of hypocotyl elongation was caused by the decreased expression of regulatory proteins and function proteins, which is under light control.
Keywords/Search Tags:mutant, rice, Arabidopsis, proteomics, activation tagging, IPCR, TAIL-PCR, RT-PCR
PDF Full Text Request
Related items