【作者】 梁文裕；

【导师】 陈伟；

【作者基本信息】 福建农林大学 ， 植物学， 2011， 博士

【摘要】 发菜是一种陆生固氮蓝藻,主要分布于干旱―半干旱荒漠草原地区,是当地主要的生物固氮资源和拓荒植物。本研究建立了发菜蛋白质组学研究技术,研究了日生长周期中不同生长状态下的蛋白质表达差异、干燥失水和恢复吸水状态下的超微结构、生理学和蛋白质表达差异,克隆了与生长发育和抗逆相关的差异蛋白（Peroxiredoxin、Mn-CAT、Ferritin和Hypothetical protein NXL-01）基因,进行了生物信息学分析,研究其原核表达,并进行了RT-PCR和Western blot验证。1发菜蛋白质组学研究方法的建立为建立适用于发菜蛋白质组研究的双向电泳技术,对发菜蛋白质的提取、裂解、上样量、IEF及SDS-PAGE电泳等关键步骤进行了优化,结果显示:发菜蛋白质主要分布在pH4～7范围内,采用改良TCA法可提高提取液中蛋白质的含量和双向电泳图谱的分辨率,裂解液含60 mmol/L DTT,上样量1.3 mg /（24 cm IPG胶条）时不仅提高了蛋白质的溶解性,而且改善了双向电泳的分离效果,蛋白点清晰,图谱分辨率较好。采用优化后的双向电泳体系提高了发菜蛋白质双向电泳的分辨率和重复性,建立起一套适用于发菜蛋白质组分析的双向电泳方法。利用双向凝胶电泳（2-DE）技术,PDQuest软件分析图像,基质辅助激光解析电离飞行时间串联质谱（MALDI-TOF-TOF/MS）分析得到肽质量指纹图谱（PMF）,通过MASCOT和NCBI进行数据库检索,初步建立了发菜蛋白质组学研究方法。从2-DE图谱中选择89个蛋白点进行蛋白质谱鉴定,共成功鉴定到68个蛋白点,代表44种蛋白,鉴定率为76.40%。57个蛋白点确定为数据库中收录的蓝藻的蛋白,其中43个来源于已知的点形念珠藻的蛋白,7个来源于念珠藻PCC 7120,2个来源于鱼腥藻,3个来源于普通念珠藻,4个未鉴定出物种来源。另有3个蛋白点来源于其他物种。对发菜SOD的PMF和氨基酸序列进行了分析,表明与普通念珠藻SOD的序列覆盖率为41%。2发菜日生长周期差异蛋白质组学研究在生长季节的日生长周期中,早晨发菜凈光合速率与暗呼吸速率旺盛，固氮酶和谷氨?泛?成酶活性高,氮代谢活跃;中午随着气温升高,发菜失水干燥,光照强度加大,凈光合速率与暗呼吸速率均急剧下降,固氮酶和谷氨酰胺合成酶活性降低;下午,气温逐渐下降,光照强度逐渐减弱,发菜再次逐渐吸收少量空气中的水分,凈光合速率与暗呼吸速率均小幅回升，固氮酶和谷氨酰胺合成酶活性回升。运用2-DE对生长季节的日生长周期中不同生长状态发菜差异蛋白分离进行分离,在上午7:00的2-DE图谱上共检测到蛋白点1247个,下午13:00的2-DE图谱上共检测到蛋白点1164个,下午19:00的2-DE图谱上共检测到蛋白点1188个。3个时期2-DE图谱的平均匹配率为71.32 %。对3个时期2-DE图谱Master胶进行统计,匹配的蛋白点较多集中在66.2-26.6kD,占总蛋白点数的68%,pH 4.5～6之间,占总蛋白点数的83%。PDQuest图像软件分析表明,有38个表达量差异在2倍以上蛋白点,其中18个差异蛋白呈下调表达,7个蛋白呈上调表达,13个蛋白先下调然后上调表达。MALDI-TOF-TOF/MS鉴定结果表明,有31个蛋白点被成功鉴定（鉴定率为81.58%）,可以分为6个功能类群,包括分泌和调节蛋白（15.79%）,抗氧化作用相关的蛋白（20.05%）,氮代谢相关的蛋白（10.53%）,能量和糖代谢相关的酶（10.53%）,细胞分裂相关蛋白（2.63%）,未知蛋白（21.05%）等,另有7个蛋白点未鉴定成功（18.42%）。这些差异蛋白在发菜的生长发育和抗逆作用有中具有重要功能。3干燥和恢复吸水发菜超微结构、生理和蛋白质组学分析在发菜持续失水48 h,然后恢复吸水4 h条件下,形态和结构分析表明,持续失水48h（48HAD）的发菜外表干燥、邹缩,呈黑色,但藻体、藻丝体、胶鞘和细胞结构完整,类囊体膜及细胞内颗粒物质无明显变化,恢复吸水4 h后,藻体膨胀、呈蓝绿色或棕褐色,具有光泽,藻体、藻丝体、胶鞘和细胞结构完整,但细胞内液泡的数量和体积比干燥发菜细胞的要多;生理学分析表明,恢复吸水4 h后的发菜自由水含量、自由水/束缚水比值、净光合活性、暗呼吸速率、RuBisCO活性、可溶性糖、ō₂˙、SOD、CAT、POD、固氮酶和谷氨酰胺合成酶活性显著增加,而MDA、H2O2、氨、脯氨酸、谷氨酸盐含量明显下降;比较蛋白质组学研究表明,与失水—复吸水密切相关的32个蛋白被鉴定,包括分泌蛋白（2.38%）、信号转导（2.38%）、转录和翻译（4.76%）、抗氧化过程（11.90%）、氮代谢（9.52%）、碳和能量代谢（11.90%）、脂代谢（2.38%）、分子伴侣（4.76%）、未知蛋白（28.57%）和没有鉴定成功蛋白（21.43%）。基于形态结构、生理和蛋白质组学分析,表明随着干燥失水和恢复吸水,发菜的生长发育呈现静止休眠与复苏生长交替进行,在干燥条件下,生理代谢减弱,生长缓慢甚至停止,而当恢复吸水,代谢水平上调,生理活性增强,生长启动甚至加速。形态结构、生理和蛋白质组分的变化体现了发菜对特殊生态环境的适应。4发菜生长与耐旱相关差异蛋白基因克隆与原核表达通过分析发菜过氧化物氧还蛋白（Peroxiredoxin）、锰过氧化氢酶（Mn-CAT）、铁氧还蛋白（Ferritin）和Hypothetical protein NXL-01在不同生长状态及持续干燥48 h和复吸水4 h后的差异表达水平,根据鉴定的已知氨基酸序列设计简并性引物克隆基因并进行生物信息学分析,研究克隆基因的原核表达和分子验证。结果表明:（1）根据简并性引物克隆获得长度为639 bp的过氧化物氧还蛋白基因,GenBank登陆号为BankIt1373957（HM854286）;693 bp的锰过氧化氢酶基因,GenBank登陆号为BankIt1311043（GU549477）;540 bp的铁氧还蛋白基因,GenBank登陆号为BankIt1373981（HM854287）;327 bp的Hypothetical protein NXL-01基因,GenBank登陆号为BankIt1374705（HM854288）。（2）生物信息学分析表明:①过氧化物氧还蛋白、锰过氧化氢酶、铁氧还蛋白和Hypothetical protein NXL-01序列比较分析显示该基因具有较高的保守性。②过氧化物氧还蛋白在第194的Pro亲水性最强,第122位的Arg疏水性最强;锰过氧化氢酶在第206的Gly亲水性最强,第96位的Val疏水性最强;铁氧还蛋白在第128的Asp和129位的Glu亲水性最强,第28位的Tyr疏水性最强;Hypothetical protein NXL-01在第101位的Glu亲水性最强,第12位的Gly疏水性最强。③过氧化物氧还蛋白二级结构主要由α螺旋、β折叠和随机卷曲构成;锰过氧化氢酶二级结构主要由α螺旋、β折叠和随机卷曲构成;铁氧还蛋白二级结构主要由α螺旋和随机卷曲构成;Hypothetical protein NXL-01二级结构主要由α螺旋和随机卷曲构成。④蛋白跨膜区分析表明过氧化物氧还蛋白、锰过氧化氢酶、铁氧还蛋白和Hypothetical protein NXL-01为膜外蛋白。⑤过氧化物氧还蛋白的Ser有5个磷酸化位点,Thr有6个磷酸化位点,Tyr有2个磷酸化位点;锰过氧化氢酶的Ser有3个磷酸化位点,Thr有1个磷酸化位点,Tyr有2个磷酸化位点;铁氧还蛋白的Ser有1个磷酸化位点,Thr有2个磷酸化位点,Tyr有1个磷酸化位点;Hypothetical protein NXL-01的Ser有5个磷酸化位点,Thr有1个磷酸化位点。（3）将过氧化物氧还蛋白、锰过氧化氢酶、铁氧还蛋白和Hypothetical protein NXL-01基因在大肠杆菌中表达,分别获得一个约26.5 kD、26 kD、22.4 kD和12.4 kD的外源蛋白。（4）对过氧化物氧还蛋白和锰过氧化氢酶基因在大肠杆菌中表达的蛋白进行Western blotting验证,表明外源蛋白为过氧化物氧还蛋白和锰过氧化氢酶。（5）RT-PCR分析表明,在日生长周期的不同状态下,锰过氧化氢酶基因、过氧化物氧还蛋白基因和铁氧还蛋白基因在转录和翻译水平上具有同样表达差异,而Hypothetical protein NXL-01基因在转录水平上无明显差异;在干燥和复吸水状态下,过氧化物氧还蛋白基因、锰过氧化氢酶基因、铁氧还蛋白基因和Hypothetical protein NXL-01在转录和翻译水平上具有同样表达差异。研究结果为进一步研究发菜生长发育和耐旱的分子机理及探讨发菜对极端干旱环境的适应和保护机制奠定基础。更多还原

【Abstract】 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.更多还原