Differential Proteomic Analysis Of Nostoc Flagelliforme In Different Growth State

Nostoc flagelliforme is a kind of terrestrial nitrogen-fixing cyanobacterium whose colonies are dark brown when it is dry and it turns dark green or brown after absorbing water. It is distributed in arid or semiarid steppes in the west or west-northern parts of China. The study established proteomics method of N. flagelliforme, differently expressed proteins were analysed in daily growth cycle in growth season, ultrastructure, physiological and proteomic differences of N. flagelliforme in response to dehydration and rehydration were studied, differentially expressed proteins (peroxiredoxin, Mn-CAT, ferritin and hypothetical protein NXL-01) gene were cloned, bioinformatics of these proteins were analysed and prokaryotic expression were disscused, RT-PCR and Western blot were also analysed. This study is the first to offer information on a novel, global insight into physiological and proteomic differences of N. flagelliforme in response to daily growth cycle, especially in dehydration and rehydration. The results laid a foundation of growth, development and drought-resistant molecular mechanism and protective mechanism of N. flagelliforme in extreme arid environment further more.1 The establishment of proteomic method in N. flagelliformeIn order to establish a two-dimensional gel electrophoresis (2D-E) protocol for proteomic study of N. flagelliforme, extracting method of total protein, lysis method, loading quantity and the key process of IEF and SDS-PAGE were optimized. The results showed that the protein spots were distributed mainly within the range of pH4～7. Improved TCA method was obviously improve the protein content and resolution patterns, lysis buffer contained 60 mmol/L DTT and loading quantity of sample protein were 1.3 mg /(24 cm IPG trip)could obtain clear map and better separation effect. More than 800 protein spots with pH4～7 were detected by the way of staining with Colloidal Coomassie Blue R-250. The optimized protocol of N. flagelliforme markedly minimized the interference from non-protein substances.The proteomic for N. flagelliforme was primarily established. 2D-E was carried out to isolate proteins, image was analyzed by PDQuest, peptide mass figerprinting (PMF) were obtained by MALDI-TOF-TOF/MS, protein and gene information was identified by protein database searching. Of 89 protein spots from the 2-DE gel, 68 spots were identified successfully. 57 spots could be matched with the proteins of cyanobacterium in NCBI database, and 43 of these could matched with the proteins of Nostoc punctiforme PCC 73102, 7 of these could matched with Nostoc sp. PCC 7120, 2 of these could matched with Anabaena variabilis, 2 of these could matched with Nostoc commune. 4 spots could not matched with species, 3 of these could matched with other species.2 Differential proteomic analysis of N. flagelliforme in daily growth cycleIn daily growth cycle in growth season of N. flagelliforme, physiological analysis showed that a significant increase in net photosynthesis, dark respiration, nitrogenase and glutamine synthetase activities were observed in the morning (7:00). While net photosynthesis, dark respiration, nitrogenase and glutamine synthetase activities registered significant decrease in the afternoon (13:00), and net photosynthesis, dark respiration, nitrogenase and glutamine synthetase activities were slowly increased when the temparature decreased, light intensity decreased and colonies rewetted slowly in the afternoon (19:00). Furthermore, 2-DE was taken to compare the proteome patterns of N. flagelliforme colonies in daily growth cycle. After CBB R-250 stained, more than 1,000 protein spots were detected on each gel (1247 protein spots were detected on gel at 7:00 AM, 1164 protein spots were detected on gel at 13:00 PM, 1188 protein spots were detected on gel at 19:00 PM). Comparative analysis of the 2-DE images of N. flagelliforme proteins was performed by using PDQuest software. In general, the proteome patterns were very similar to all 2-DE images, which indicated that most proteins were accumulated at comparable levels in daily growth cycle. However, quantitative image analysis revealed that a total of 38 protein spots changed in abundance more than two-fold during different growth stages, that 18 protein spots were down-regulated, 7 protein spots were up-regulated, 13 protein spots were down-regulated firstly, and than up-regulated. The differentially expressed proteins were excised from the 2-DE gels, in-gel digested by trypsin, and analyzed by MALDI-TOF-TOF/MS. As a whole, 31 proteins sports were identified (identification rate: 81.58%). Based on their physiological functions, all differential proteins were categorized into the following eleven groups: secretion and regulation (15.79%), antioxidative processes (20.05%), nitrogen metabolism (10.53%), carbohydrate and energy metabolism (10.53%), cell division(2.63%), unclassified (unknown) (21.05%) and unidentified proteins (18.42%). These differentially expressed proteins maybe play some important roles in growth, development and stress defense.3. Ultrastructure, physiological and proteomic analysis of N. flagelliforme in response to dehydration and rehydrationDrought is one of the most severe limiting factors to terrestrial blue-alga growth and distribuation. N. flagelliforme has evolved a unique capability to tolerate desiccation and can survive for several decades under extremely dry conditions, and rapidly recover physiological metabolic activity after reabsorbing water. This study is the first to offer information on a novel, global insight into Ultrastructure, physiological and proteomic differences of N. flagelliforme in response to dehydration and rehydration. Morphological and ultrasturcture changes exhibited that colonies were dry and shrunk with black, and the structure of colonies, filaments, sheath and cell were intact, thylakoid membrane and granules were not obvious change when colonies were subjected to 48 hours of water deprivation. While colonies were swollen with blue-green or brown , the structure of colonies, filaments, sheath and cell were intact when colonies followed by rewatering for 4 hours, but quantity and volume of vacuoles were more under the condition of rehydration for 4 hours than that of dehydration in N. flagelliforme. Physiological analysis showed that a significant increase in net photosynthesis, dark respiration, O-2, SOD, CAT, POD, nitrogenase and glutamine synthetase activities were observed after N. flagelliforme colonies had been subjected to rehydration. While H2O2, ammonium, proline and glutamate contents registered significant decrease in rewetted colonies compared with dried ones. Furthermore, 2-DE reproducibly detected more than 1,000 protein spots in N. flagelliforme, with 45 protein spots being significantly altered in their intensity between dried and rewetted colonies. 32 protein spots were identified by MALDI-TOF-TOF/MS analysis and protein database searching. Most of identified proteins were associated with a variety of functions, including secretion (2.38%), signaling (2.38%), transcription and translation (4.76%), antoxidative processes (11.90%), nitrogen metabolism (9.52%), carbohydrate and energy metabolism (11.90%), lipid metabolism (2.38%), chaperonin (4.76%), and other functional classes. And their biological functions were also discussed. The expression patterns of Mn-CAT and peroxiredoxin were validated by immunoblotting analysis. We anticipate that morphological and ultrasturcture changes, physiological changes and identification of the differentially expressed proteins may lead to a better understanding of the growth and desiccation tolerance mechanisms in N. flagelliforme which is usually subjected to extreme environmental changes in nature.4 Gene cloning and prokaryotic expression of differential expressed proteins related to growth and drought-resistant from N. flagelliformeDegeneracy primer of peroxiredoxin, Mn-CAT, ferritin and hypothetical protein NXL-01 were designed based on identified amino acid sequences in order to colone these gene, the gene and amino acid sequences were analysed, Bioinformatics of peroxiredoxin, Mn-CAT, ferritin and hypothetical protein NXL-01 were analysed, and prokaryotic expression of these gene was carried out. The results indicated that peroxiredoxin, Mn-CAT, ferritin and hypothetical protein NXL-01 gene were cloned and a full length of 639 bp, 693 bp, 540 bp and 327 bp DNA were obtained, respectively (GenBank access number were BankIt 1373957(HM854286), BankIt 1311043 ( GU549477 ) , BankIt 1373981(HM854287)and BankIt 1374705(HM854288), respectively). Homology analysis showed that each of the peroxiredoxin, Mn-CAT, ferritin and hypothetical protein NXL-01 of N. flagelliforme had high consensus regions. The hydrophobicity of peroxiredoxin, Mn-CAT, ferritin and hypothetical protein NXL-01 were analysed. The secondary structure of peroxiredoxin and Mn-CAT were made up ofαhelix,βstrands and random coil, respectively, while the secondary structure of ferritin and hypothetical protein NXL-01 were made up ofαhelix and random coil, respectively. The TMHMM posterior probabilities for seguence of peroxiredoxin, Mn-CAT, ferritin and hypothetical protein NXL-01 were outside proteins. Predicted phosphyorylation sites in the sequence of peroxiredoxin (Ser: 5; Thr: 6; Tyr: 2), Mn-CAT (Ser: 3; Thr: 1; Tyr: 2), ferritin (Ser: 1; Thr: 2; Tyr: 1) and hypothetical protein NXL-01(Ser: 5; Thr: 1) were identified. Peroxiredoxin, Mn-CAT, ferritin and hypothetical protein NXL-01 gene were expressed in E. coli, and a 26.5 kD, 26 kD, 22.4 kD and 12.4 kD heterologous protein were observed, respectively. And western blotting confirmed peroxiredoxin and Mn-CAT. Semi-Quantitative RT-PCR results showed that each of Mn-CAT, peroxiredoxin and ferritin had identical regulation on transcription and translation level, while hypothetical protein NXL-01 had minor expression differential on transcription level, which the aboved-mentioned situations were the response of N. flagelliforme to daily growth cycle; that each of peroxiredoxin, Mn-CAT, ferritin and hypothetical protein NXL-01 had identical regulation on transcription and translation level, which the aboved-mentioned situations were the response of N. flagelliforme to dehydration and rehydration.