| 14-3-3proteins encoded by different genes in most organisms are ubiquitous in all eukaryotic cells, which are highly conserved in most biological species. Since the discovery of14-3-3proteins, they extensively involved in various physiological processes of resistance due to their functional diversity, such as cellular signal transduction, cell cycle regulation, transcription factors, environmental response, apoptosisand protein transembrane transport. The main function of the14-3-3proteins can be used as adapter/scaffold protein which involved in protein-protein interactions, involving in a set of mechanisms to regulate their biological functions.14-3-3proteins act as the "bridge" to connect the two proteins which are at different position in the cells originally, and then to trigger a series of physiological responses. Therefore,14-3-3protein can be used as a kind of protein interacting with a variety of proteins under stress enviroments, screening out their interaction protein of unknown function. This will further improve the14-3-3b protein interacting protein networks.Aluminum toxicity in acid soils is a major limiting factor for crop production, having influenced the growth and yield of crops for long time. The study on the mechanisms of crops tolerance to aluminium will help to improve the growth and yield of crops affected by aluminum toxicity. Yunnan is the important origin of the broad bean and the soil of Yunnan region is red soil acidified strongly. Therefore, it is necessary to study mechanisms of the broad bean aluminum tolerance deeply. There are much researches related to the organic acids with aluminum tolerance mechanisms, but research related to14-3-3proteins with aluminum tolerance mechanisms has not been reported. In this thesis, the broad beans in Yunnan as experimental material,14-3-3b protein gene selected from the broad bean aluminum stress gene library as the object, we studied14-3-3b protein interacting protein under Al stress. This will provide a new theoretical data for further exploring the mutual relationship of the protein network nodes. At same time it will provide new research directions for further studying the mechanisms of broad bean aluminum tolerance. The main results are as follows: 1. After extracting total RNA from the broad bean root, we amplified gene fragment GF14-b14-3-3b by RT-PCR. Recombinant plasmid pGEX-4T-1-14-3-3b was constructed through TA cloning, restriction enzyme digestion and sequencing as well as connecting14-3-3b gene into the prokaryotic expression vector pGEX-4T-1. The pGEX-4T-1-14-3-3b was transformed into genetically engineered protein E.coli BL21for inducing expression at different temperature and time under1mM of IPTG. The optimal induction conditions for recombinant protein expression of pGEX-4T-1-14-3-3b were1mM IPTG,16°C and inducing10h. SDS-PAGE detection indicated the main form of experesion protein was soluble protein. The target recombinant protein was purified by GST affinity chromatography and total4mg protein with relative pure recombinant protein was obtained.2. In order to study the14-3-3b interacting proteins under Al stress, we extracted total protein from Broad bean root treated with50μM AlCl3. Then the concentration of the total plant protein was calculated by measuring the value of OD595. Thus, the reaction system was established by adding lmg of total plant protein,200μg purified protein of pGEX-4T-1-14-3-3b,3μL GST antibody and3μL14-3-3b antibody, respectively. The GF14-b interacting proteins were screened out by using GST fusion protein pull-down technology and co-immunoprecipitation techniques in vitro.18proteins were identificated from protein interaction by the method of protein spectrum analysis, including two unknown protein,10putative putative protein, an unnamed protein, and five known protein (3-phosphate dehydrogenase, NADH dehydrogenase, calcium-dependent phospholipid binding protein, delta-amino-acetylacid dehydratase dehydratase and DNA methyltransferase).3. The obtained protein was identified by mass spectrometry. Interaction relationships between3-phosphate dehydrogenase or NADH dehydrogenase and14-3-3b protein were verified by far-western blot. There was a weak band at the37kd and81kd location. The levels of interaction between14-3-3b and3-phosphate dehydrogenase or NADH dehydrogenase in the root treated with50μM AlCl3in different time were detected by the GST-pull-down method. There were more close relationships between them with time. At the same time, mRNA expression levels in the root treated with50μM in different time were analysized by RT-PCR. Two genes encoding GAPDH were upregulated in different degrees and nad4gene, one of two genes encoding NADH dehydrogenase, was also upregulated expression, indicating that those proteins indeed interact with14-3-3b protein under aluminum stress. |
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