嗜水气单胞菌蛋白质组分子解剖图谱的初步建立

采用蛋白质组学技术,建立了嗜水气单胞菌PPD(134/91)在稳定期的蛋白质组分子解剖图谱.将嗜水气单胞菌接种于LB培养基28℃培养18h,取菌总蛋白进行2 DPAGE.从2 DPAGE图谱中随机选取124个蛋白质点进行肽质量指纹图谱鉴定,检索发现其中10个确定为数据库中收录的嗜水气单胞菌的基因或蛋白,其比例约为1/12.实验结果对嗜水气单胞菌菌株之间的比较研究和功能蛋白质组研究具有重要参考价值.     

小麦灌浆期籽粒粒重差异的代谢蛋白质组学初步研究

高粒重（48号）和低粒重（154号）小麦株系是01-35（大粒）和6044（小粒、多穗）的稳定杂交后代,具有相似的遗传背景.取灌浆期（开花后17天）的小麦籽粒,提取代谢蛋白,双向电泳后,进行图谱分析.结果显示：两品系中分别鉴定了380和367个蛋白点,其中52个蛋白点为两品系差异显著性表达.将其中13个差异点采用基质辅助激光解析电离飞行时间质谱（matrix assisted laser desorption/ionizaton time of flight mass spectrometry,MALDI-TOF-MS）进行了肽质指纹图分析,通过采用Mascot软件对MSDB数据库查询,其中9个蛋白质点得到了鉴定,包括葡聚糖合成酶、葡萄糖-1-磷酸-腺苷转移酶小亚基前体、一种胚胎晚期富集蛋白等.本研究初步建立了小麦籽粒灌浆其代谢蛋白蛋白质组分析的双向电泳（2-DE）方法,通过质谱分析、数据库搜索的结果对粒重形成的机制进行了探讨,并对以后的研究工作提出了改进方案.     

南方水牛奶酪蛋白及大豆蛋白肽指纹图谱的差异性

通过LTQ Orbitrap XL组合型傅里叶变换高分辨质谱与反相高效液相色谱技术联用建立了南方水牛奶酪蛋白和大豆蛋白主要组分肽质指纹图谱,并对其氨基酸组成与序列进行了比较.结果表明:酪蛋白经胰蛋白酶酶解后得到的28个肽段中没有检测到与酶解大豆分离蛋白181个肽段相匹配的肽段;酪蛋白(CN)的主要组分是αs1-CN、β-CN、κ-CN,大豆蛋白的主要组分为大豆球蛋白(包括大豆球蛋白G1~G5亚基)和β伴大豆球蛋白(α,α',β链);水牛奶酪蛋白和大豆蛋白在亚基组分的氨基酸数量、组成比例和排列方式均呈现出明显的差异,大豆蛋白的各组分亚基的氨基酸全序列均大于酪蛋白各组分的氨基酸全序列,且大豆蛋白各组分的分子质量均显著高于酪蛋白各组分.LTQ Orbitrap XL质谱技术对乳源蛋白肽质指纹的分析,为今后鉴定分析牛奶蛋白中是否添加廉价蛋白提供了高精度和高准确度的检测方法,也为复杂混合物中的痕量组分检测提供了理论依据.     

基于支持向量机的多类凋亡蛋白亚细胞位置预测

基于支持向量机,以全部和局部氨基酸序列的n肽组分、序列的亲疏水性分布等五种特征提取方法构成特征向量表示蛋白质序列,对六类细胞凋亡蛋白的亚细胞位置进行预测.结果表明,基于氨基酸二肽组成成分构成的特征向量集(以符号DIPE表示)的预测结果高于其它四种特征向量集的预测结果,在Jackknife检验下,总预测成功率达到了89.3%;与现有的方法比较,发现对于Mitochondrial类凋亡蛋白,支持向量机方法有更好的预测效果.     

肝癌细胞株HepG-2的G1期细胞蛋白质组双向电泳图谱的建立

采用双向电泳分离肝癌细胞株 HepG2 的 G1 期细胞的蛋白质,经考马斯亮蓝染色及图像扫描与分析显示:在 pH3-10,13 cm 的 IPG 胶条上,可分离到 227 个重复性及分辨率均较好的蛋白斑点,蛋白斑点的匹配率为 84%.建立了 HepG2 的G1 期细胞蛋白质组双向电泳图谱,为其蛋白质组学的进一步研究奠定了基础.     

低温胁迫下棉花子叶蛋白质差异表达的双向电泳分析

采用TCA-丙酮沉淀法,改良优化了棉化蛋白质组研究中的双向电泳技术.通过对根和子叶全细胞蛋白的提取、蛋白的溶解、胶条的选择及电泳等环节的优化,得到了重复性很高、分辨率很好的蛋白质图谱.进一步对5 d苗龄的棉花进行4℃低温处理12 h后,提取子叶的蛋白质,利用双向电泳技术分离全细胞蛋白.并用PDQuest软件分析比较棉花子叶在低温处理下的蛋白表达谱,得到了49个有显著差异的蛋白点.其中,低温处理上调表达的蛋白点有27个,减弱表达的蛋白点有22个,推测这些差异蛋白点可能与低温胁迫相关.     

DMSO处理后中国仓鼠卵巢细胞的蛋白质组变化分析

在培养基中添加二甲基亚砜(DMSO)可使基因工程中国仓鼠卵巢细胞(CHO)外源蛋白乙肝表面抗原(HBsAg)的比生产速率提高4倍以上.为了进一步研究DMSO的作用机理,采用双相凝胶电泳技术分离在培养基中添加1.5%的DMSO后CHO细胞的总蛋白,胶态考马斯亮蓝G-250染色,GS-800凝胶扫描仪获取凝胶图像,并用PDQUEST软件分析,测得每张图谱平均斑点数量为1852±123,以其中一块胶为参考胶进行4块胶间的斑点匹配,其平均匹配的点数达1326±100,平均匹配率为71.6%以上.通过分析得到18个蛋白质点在添加了DMSO后表达量有明显变化(其中12个蛋白表达量明显降低,6个蛋白表达量明显增加.用基质辅助激光解析电离飞行时间质谱测定其胶内酶解后的肽质指纹谱图和串级质谱图,用GPS软件查询NCBI数据库进行了鉴定.鉴定结果表明,这些差异蛋白中一些是与细胞周期有关的蛋白,一些是与细胞能量代谢相关的蛋白,还有一些是与蛋白翻译后修饰有关的酶,这些结果进一步深化了对DMSO提高CHO细胞表达HBsAg的机理的理解.     

藻蓝色素蛋白通过调控miR-642a-5p抑制LTEP-a2细胞的增殖和迁移

研究藻蓝色素蛋白抑制非小细胞肺癌LTEP-a2体外增殖迁移的机制.以LTEP-a2细胞为模型,采用高通量miRNA转录组学对藻蓝蛋白处理后细胞中差异表达的miRNA进行了筛选;对筛选出的差异miRNA,利用体外转染mimics的方法对细胞的增殖、迁移能力进行验证.研究结果显示,藻蓝蛋白处理LTEP-a2细胞后能够显著增加细胞内miR-642a-5p的表达水平;过表达miR-642a-5p能够显著抑制细胞的体外增殖、迁移能力;此外,藻蓝蛋白可以通过上调miR-642a-5p表达抑制核受体NF-κB信号通路,降低蛋白磷酸化水平,进而抑制LTEP-a2细胞的增殖迁移能力.研究能够为藻蓝色素蛋白的应用以及非小细胞肺癌的治疗提供一定的理论基础.      

嗜酸氧化亚铁硫杆菌ATCC23270周质蛋白的选择性提取及差异表达

通过考察萃取时间、高渗液与低渗液的摩尔比以及总的渗透液体积对细胞质苹果酸脱氢酶的比活性(相对于细胞蛋白质总量)的影响,建立选择性提取Acidithiobacillus ferrooxidans ATCC23270周质蛋白的渗透休克实验方法;结合透射电镜和双向电泳实验方法,分析渗透处理前后细胞的形态和不同生长期的细胞周质空间蛋白表达差异.研究结果表明,合适的渗透休克实验条件为:细胞质量为0.1 g(湿重),渗透液的总体积为10 mL,渗透液的作用时间为15 min,高渗液与低渗液的摩尔比为1:1;在高渗环境下,细胞收缩,周质空间变小;在低渗液环境下,细胞周质空间膨胀,外膜被胀破,周质空间内蛋白释放出来;双向电泳显示周质蛋白总体上以小相对分子质量蛋白为主,在不同的生长时期内的周质蛋白表达存在差异.     

锯缘青蟹对病原菌感染的急性反应功能蛋白质组

为研究锯缘青蟹对常见重要病原菌的急性反应蛋白质组．将锯缘青蟹随机分为四组．分别注射生理盐水、副溶血弧菌、鳗弧菌和嗜水气单胞菌．继而提取肌肉蛋白进行双向电泳．通过比较各组肌肉蛋白的双向电泳图谱获得三个差异表达的蛋白．对这三个差异蛋白进行肽质量指纹图谱及其生物信息分析．鉴定为Calexeitin、无翅蛋白片断、速激肽相关肽．这些结果对研究锯缘青蟹的抗病蛋白及其机理具有重要意义．     

Proteomic analysis of blast-resistant near-isogenic lines derived from japonica rice,var. Yunyin,infected with Magnaporthe oryzae

Rice blast is one of the most devastating dis- eases in the world and outbreaks occur frequently. The differences in protein expression between blast resistant and susceptible near-isogenic lines （NILs） of japonica rice var. Yunyin infected with Magnaporthe oryzae were ana- lyzed using proteomics, indicated that 67 different proteins were identified from 75 obtained proteins using the matrix- assisted laser desorption/ionization time-of-flight mass spectrometry technique. Seven specific expression proteins, 43 up-regulated proteins and 17 down-regulated proteins were identified among the 67 proteins. The bioinformatical analysis demonstrated that these 67 different proteins were involved in many biological physiological processes including five proteins related to photosynthesis, 25 pro- teins related to metabolism, six proteins related to anti- oxidants, 10 proteins related to protein synthesis and modification, five proteins related to signal transduction,four proteins related to adversity stress and 12 non-func- tional proteins. These identified proteins were directly or indirectly related to stress. Five proteins related to different physiological processes were selected. Their cDNA sequences were predicted and their expression patterns were analyzed using real-time PCR, demonstrated that the genes would response to M. oryzae and the response blindingly different between blast resistant and blast sus- ceptible NILs.     

Proteomic comparison of four maize inbred lines with different levels of resistance to Curvularia lunata （Wakker） Boed infection

Protein profiles of leaves in four maize inbred lines with different disease resistance to pathogen Curvularia lunata(Wakker)Boed were studied by two-dimensional electrophoresis(2-DE)and mass spectrometry.Proteins were extracted from the forth leaf of maize seedlings 24 h after fungal inoculation,and fractionated by polyethylene glycol to precipitate the most abundant leaf protein,Rubisco,before gel separation.Protein profiles from 2-DE showed that total numbers of protein spots were increased in all four inbred lines inoculated with C.lunata CX-3 strain compared with the control.The numbers of changed protein spots in abundance were higher in resistant inbred lines than in susceptible ones,which implied that resistant inbred lines were more sensitive than susceptible ones to pathogen infection.Among proteins identified by MALDI-TOF MS,germin-like protein GLP and translation initiation factor eIF-5A were supposed to play important roles in maize resistance against C.lunata infection.     

Proteomic comparison of four maize inbred lines with different levels of resistance to Curvularia lunata(Wakker)Boed infection

Protein profiles of leaves in four maize inbred lines with different disease resistance to pathogen Curvularia lunata(Wakker)Boed were studied by two-dimensional electrophoresis(2-DE)and mass spectrometry.Proteins were extracted from the forth leaf of maize seedlings 24 h after fungal inoculation,and fractionated by polyethylene glycol to precipitate the most abundant leaf protein,Rubisco,before gel separation.Protein profiles from 2-DE showed that total numbers of protein spots were increased in all four inbred lines inoculated with C.lunata CX-3 strain compared with the control.The numbers of changed protein spots in abundance were higher in resistant inbred lines than in susceptible ones,which implied that resistant inbred lines were more sensitive than susceptible ones to pathogen infection.Among proteins identified by MALDI-TOF MS,germin-like protein GLP and translation initiation factor eIF-5A were supposed to play important roles in maize resistance against C.lunata infection.     

对白粉病敏感、抗性栽培小麦及杂交后代的蛋白质组分析

感病小麦京411、抗病小麦Brock及杂交后代近等基因系感染白粉菌后,发现京411感病,Brock和近等基因系抗病,因此白粉病抗性呈显性．对上述3种小麦的叶片进行蛋白质组分析,在感小麦的京411中有一个8kD／pI 5．3的特异蛋白,该蛋白在抗病品种Brock和杂交后代近等基因系中不存在,后者的蛋白质谱型与Brock相同．上述结果表明,幼苗抗白粉病特性的遗传与蛋白质谱型的遗传相类似,此结果也许可以为生产上筛选抗白粉病小麦品种提供生化依据．     

菜豆锈病菌侵染对寄主生理代谢的影响

从细胞膜透性、呼吸强度、光合作用及游离氨基酸和蛋白质代谢等几方面,对菜豆锈病菌(Uromyces appendiculatus)侵染与菜豆生理代谢变化的关系进行了研究.结果表明U.appendiculatus侵染可导致被侵染菜豆叶片细胞膜透性的增加,但这种增加在抗感程度不同的品种中出现的早晚和程度不同; 抗感品种在接种后呼吸强度都增加,但不同品种呼吸强度升高的时间和程度有异;U.appendiculatus对寄主光合作用的影响体现在叶绿体含量的降低及光合产物(糖和淀粉)的异常积累;接种前后菜豆叶片中蛋白质含量无明显变化,但个别游离氨基酸的含量在抗病品种中有变化.     

Comparative proteomic analysis of the response in resistant and susceptible maize inbred lines to infection by Curvularia lunata

Proteins differentially expressed from maize leaves in response to the infection by Curvularia lunata strain CX-3 were identified through a high-resolution two-dimensional gel electrophoresis (2-DE) method. Two inbred lines, 78599-1 and E28, were used, respectively, as resistant and susceptible lines to CX-3 infection. Proteins were extracted from the fourth leaves of six- or seven-leaf stage plants sampled at 24, 36, 48, 60, and 72 h after inoculation with CX-3. Twenty-seven differentially expressed protein spots resolved on the 2-DE gels were identified by MALDI-TOF MS/MS. The results showed that these proteins are associated with photosynthesis, respiration,oxidative and drought stress tolerance as well as signal transduction in maize. Among stress-related proteins, the 22 kDa drought-inducible protein, putative glutathione peroxidase (GPX), and translation initiation factor (eIF-5A) were up-regulated in the resistant inbred line and were implicated in host defense response to C. lunata infection. It suggests that drought-inducible and oxidation stress-related proteins might directly contribute to maize resistance to C. lunata.     

不同烟草品种罹黑胫病后几种酶活性的变化

文章就黑胫病表现不同抗性的两烟草品种接种黑胫病菌后,对其叶片内苯丙氨酸解氨酶(PAL)、多酚氧化酶(PPO)、过氧化物酶(POD)的活性进行了测定。结果表明,接种后抗病品种的PAL、PPO及POD活性水平皆明显高于对照,感病品种的PAL、PPO及POD活性水平也高于对照,但接种后抗病品种的PAL、PPO及POD活性水平前期增加速度明显高于感病品种。另外,接种后抗病品种和感病品种与各自对照相比皆未出现新的酶带,只是几个酶带的强弱发生了变化。     

松材线虫在抗性黑松体内移动及寄主水势变化

以接种松材线虫的1年生抗性黑松苗为实验材料,用感病黑松苗作对照来研究松材线虫在抗性黑松体内的移动及寄主水势变化.结果表明:接种初期,松材线虫在树体内的数量少,移动速度慢;在抗病黑松体内,线虫主要向下移动,且虫体数量明显少于在感病黑松体内的数量;在感病黑松体内线虫自接种点向上、下移动,且向下移动的速度和虫体数量明显大于向上的.随着时间的推移,线虫开始繁殖,扩散能力增强.水势测定结果显示,感、抗病黑松水势变化总趋势是先上升后下降再上升.     

4个小麦品种的抗白粉病遗传分析

抗病品种郑315、CP87-26-12和91138-16-2-13-12,与感病品种豫麦49号杂交的F1代表现抗病,F2代抗、感单株的分离比例为31,表明这3个品种均携带1对显性抗病基因.N97189-2和豫麦18号的杂交F1代对白粉病表现高感,F2代抗、感单株的分离比例为13,由此推断N97189-2对白粉病的抗性由1对隐性基因控制.     

抗松针褐斑病湿地松体内氧化酶的变化

松针袍斑病是湿地松上的重要病害，分析湿地松的９个抗病无性系和４个易感株２年生针叶中过氧化物酶和多酚氧化酶的活性，结果表明湿地松针叶中过氧化物酶活性很高，多酚氧化酶活性相对较弱，而且抗病植株的过氧化物酶和多酚氧化酶活性都明显高于感病植株，过氧化物酶活性与多酚氧化物酶活性之间呈明显正相关，相关系数０．９，０湿地松的１年生针叶多酚氧化酶和过氧化物酶生都非常低，而且在抗与感病针叶间酶活性无明显差别，用病原