小麦丙酮酸脱氢酶激酶(PDK)的互作蛋白增殖细胞核抗原(TaPCNA)对小孢子发育周期的调节

目前对于植物体中丙酮酸脱氢酶激酶(pyruvate dehydrogenase kinase,PDK)的研究,除了解其参与调节线粒体丙酮酸脱氢酶复合体的活性外,对其他调控机制尚不清楚.本实验室的前期研究结果表明,小麦丙酮酸脱氢酶激酶(TaPDK)在小麦(Triticum aestivum)遗传型不育系和相应的生理型不育系中的表达是不一致的.为进一步研究SQ-1诱导的小麦花药败育过程中调控TaPDK的作用因子,本研究采用酵母双杂交方法筛选小麦TaPDK的互作蛋白.将含有pGBKT7-PDK质粒的酵母(Saccharomyces cerevisiae)菌株Y2HGold与文库酵母菌株Y187共同融合培养,在SD/-Ade/-His/-Leu/-Trp平板上划线培养,挑选直径大于2mm的克隆,在SD/-Ade/-His/-Leu/-Trp/AbA/X-α-Gal平板上划线,筛选蓝色克隆.其中,筛选得到一个新的结合蛋白,该蛋白被命名为增殖细胞核抗原(proliferating cell nuclear antigen,TaPCNA).将花粉细胞的核复制分裂与TaPCNA的定量表达结合分析发现,TaPCNA在不育系与可育系的叶片中几乎不表达,但在花粉发育的各个时期中呈现高表达,尤其在生理型不育系花药中的表达量显著高于遗传型不育系和可育系,这表明在生理型不育系中,TaPCNA与小孢子的细胞周期发育更加密切相关.这为进一步研究生理型不育小孢子异常发育的细胞周期提供了基础资料.     

Changes of hepatic biochemical parameters and proteomics in broilers with cold-induced ascites

Ascites syndrome is still a problem for chicken industry in various parts of the world. Despite the intensive investigations of this syndrome for many years, its pathogenesis remains unclear. The objective of this study was to analyze the difference in hepatic proteomics between ascites and healthy broilers by two-dimensional electrophoresis （2-DE） and matrix-assisted laser desorption/ionization-time of flight mass spectrometry （MALDI-TOF-MS）. Changes of biochemical parameters of liver and blood were also determined. The results indicated that red blood cell counts （RBC）, hematocrit （HCT） and haemoglobin （HGB） of ascites broilers were significantly greater than healthy broilers. Hepatic malondialdehyde （MDA） level of ascites broilers was significantly increased, and the activity of total superoxide dismutase （T-SOD） was significantly decreased. Hepatic lactic acid （LD） level of ascitic broilers were significantly lower than healthy ones, Serum glucose and cholesterol level of ascites broilers were significantly increased, and serum globulin level was significantly decreased in ascites broilers. There was no significant difference in triglyceride （TG） and blood urea nitrogen （BUN） level. The activity of liver hexokinase （HK） and succinodehydrogenase （SDH） in ascites broilers was significantly decreased, and there was no significant difference in the activity of liver pyruvate kinase （PK） and Na＋-K＋-ATPase. The hepatic proteomics analysis showed that 18 proteins expression difference were identified between ascites and healthy broilers. These proteins were mainly involved in： 1） cytoskeleton; 2） glucose, lipids and amino acid metabolism; 3） cell secretion; 4） cell apoptosis; 5） signal transduction; 6） immune and inflammatory response; and 7） cellular redox homeostasis. Mitochondrial isoform phosphoenolpyruvate carboxykinase （M-PEPCK） mainly participates in gluconeogenesis of chicken liver. In conclusion, liver oxidative damage was significantly aggravated, but antioxidant capacity was decreased in cold-induced ascites broilers. Serum glucose level was significantly increased, with liver M-PEPCK expression higher in ascites broilers, which implied that some potential regulatory reagents may reduce ascites susceptibility and mortality under cold temperature by increasing liver gluconeogenesis level.     

Deletions in the pyruvate pathway of Salmonella Typhimurium alter SPIl-mediated gene expression and infectivity

Background： Salmonella enter/ca serovar Typhimurium is a major foodborne pathogen worldwide. S. Typhimurium encodes type III secretion systems via Salmonella pathogenicity islands （SPI）, producing the major effector proteins of virulence. Previously, we identified two genes of Salmonella pyruvate metabolism that were up-regulated during chicken cell infection： pyruvate formate lyase I （pf/B） and b/functional acetaldehyde-CoA/alcohol dehydrogenase （adhE）. We were therefore interested in examining the role these genes may play in the transmission of Salmonella to humans. Methods： Mutant strains of Salmonella with single gene deletions for pflB and adhE were created. Invasion and growth in human HCT-8 intestinal epithelial cells and THP-1 macrophages was examined. Quantitative PCR was performed on 19 SPI-1 genes. Results： In HCT-8 cells, both mutant strains had significantly higher intracellular counts than the wild-type from 4 to 48 h post-infection. Various SPI-1 genes in the mutants were up-regulated over the wild-type as early as 1 h and lasting until 24 h post-infection. In THP-1 cells, no significant difference in internal Salmonella counts was observed; however, SPI-1 genes were largely down-regulated in the mutants during the time-course of infection. We also found five SPI-1 genes - hilA, hiIC hill）, sicP and rtsA - which were up-regulated in at least one of the mutant strains in log-phase broth cultures alone. We have therefore identified a set of SPI-1 virulence genes whose regulation is effected by the central metabolism of Salmonella.     

3种石斛PEPC羧化酶活力比较

 石斛属植物具有兼性景天酸代谢植物特征,磷酸烯醇式丙酮酸羧化酶是该类植物碳代谢的关键酶。本研究选用3种石斛为材料,测定了其磷酸烯醇式丙酮酸羧化酶活性。结果表明,报春石斛晚间黑暗条件下酶活性较强,凌晨高达25μmolCO2/(m2·s),而在中午光照最充足时,酶的活性相对较弱;金钗石斛酶活性日变化特点与报春石斛相似,但是活性比较弱;鼓槌石斛的酶活性很弱,通常在8μmolCO2/(m2·s)以下,昼夜变化幅度很小,表现为黑暗状态下比光下更强。     

植物细菌型磷酸烯醇式丙酮酸羧化酶基因的生物信息学分析

采用生物信息学方法对GenBank中的拟南芥、油菜、大豆、花生和水稻等11种植物细菌型磷酸烯醇式丙酮酸羧化酶(PEPC)基因的核苷酸和氨基酸序列进行了比对分析,进而对其基因结构、系统进化、理化性质、亚细胞定位、疏水性、功能结构域及蛋白质三级结构等重要参数进行了预测和分析.     

不同配比关系的丙酮酸乙酯复方混悬剂在细菌感染模型中的比较研究

利用青岛农业大学动物医学实验室研制的4种不同配比关系的丙酮酸乙酯（EP）复方混悬剂,对大肠杆菌感染小鼠进行治疗试验,以得到EP与头孢噻呋（CE）的最佳配比量,选出一个最佳组方,并比较不同的EP剂量、给药时间对治疗结果的影响。试验将340只昆明小鼠随机分17组,每组20只,注射大肠杆菌ATCC 25922菌液后,分别于1、4、8 h注射4种EP复方混悬剂（EP分别为40、80、120、160 mg/kg）和1种CE单方混悬剂（CE为5 mg/kg）。观察各组小鼠7 d内的死亡情况并做统计学分析。结果显示,攻毒后1和4 h进行治疗,EP为40、80 mg/kg的复方混悬剂治疗效果显著优于CE单方混悬剂（P<0.05）,但2种EP复方混悬剂间无显著差异（P>0.05）。攻毒后8 h进行治疗,注射4种EP复方混悬剂和1种CE单方混悬剂均无显著治疗效果（P>0.05）,且与阳性对照组相比差异不显著（P>0.05）。结果表明,在小鼠大肠杆菌攻毒试验中,EP 40和80 mg/kg的复方混悬剂治疗效果明显优于CE单方混悬剂,能显著提高小鼠治愈率,2种复方混悬剂间无明显差别,但从更安全、更经济的角度考虑选择低剂量的EP 40 mg/kg复方混悬剂更为合适。     

外源葡萄糖增强高表达转玉米C4型pepc水稻耐旱性的生理机制

为揭示葡萄糖参与植物耐旱性的内在机制,以高表达转玉米C4型磷酸烯醇式丙酮酸羧化酶 (phosphoenolpyruvate carboxylase,PEPC) 基因(C4-pepc)水稻(PC)和受体“Kitaake”(WT)为材料,通过盆栽和水培试验,研究外施葡萄糖联合干旱处理下,功能叶片的光合参数、总可溶性糖及其组分、Ca²⁺、NO含量、己糖激酶活性、B类钙调磷酸酶(calcineurin B-like, CBL)与蔗糖非发酵1 (sucrose nonfermenting-1, SNF1)相关蛋白激酶(SNF1-related protein kinase 3s, SnRK3s)基因表达的变化。结果表明,在盆栽试验中,分蘖期外施3%葡萄糖联合干旱处理对水稻的产量及其构成因子影响不显著,而在孕穗期处理,PC的株高、穗数、每穗实粒重和单株产量均显著高于WT。在水培试验中,外施1%葡萄糖和12% (m/v)聚乙二醇6000 (polyethylene glycol-6000, PEG-6000)模拟干旱处理,均显著提高了PC功能叶片的净光合速率(P_n)、气孔导度(Gs)和羧化效率(CE),叶片内蔗糖和果糖的含量也均显著高于WT。值得关注的是,PC叶片己糖激酶(hexokinase, HXK)活性、CBL1/SnRK3.1/SnRK3.4/ SnRK3.21基因的相对表达量在外施1%葡萄糖联合12% PEG模拟干旱处理下均显著低于12%PEG处理,而NO含量则显著上升。相关性分析也表明,PC中Pn、胞间CO2浓度(Ci)和Gs分别与葡萄糖含量、HXK活性和SnRK3.16基因表达显著相关。外施葡萄糖处理可上调PC糖水平,下调其CBL和SnRK3s基因表达,诱导NO参与叶片气孔调节,从而增强保水能力,保持光合能力稳定,最终表现为耐旱。     

瘦蛋白对犊牛肝细胞PC mRNA表达及其活性的影响

取单层培养72 h生长良好的犊牛肝细胞,采用单因素重复试验,分别添加0,2.5,5.0,10.0,50.0,100.0 ng/mL的牛重组牛瘦蛋白(Leptin),每个处理3个重复(每重复2孔)。再培养12 h后分别提取RNA并制备细胞上清液。应用荧光定量PCR方法检测外源leptin对肝细胞糖异生关键酶丙酮酸羧化酶(Pyruvate car-boxylase,PC)基因表达的影响,同时用比色法检测其对肝细胞PC活性的影响。结果表明:一定浓度的Leptin显著抑制了肝细胞PC mRNA表达,降低了PC活性。     

地毯草黄单胞菌分泌黄原胶的电镜观察

为了研究地毯草黄单胞菌(Xanthomonas axonopodis)FJAT-10151产黄原胶的分泌行为,采用比浊法测定地毯草黄单胞菌的生长曲线,干重法测定黄原胶产量,2,4-二硝基苯肼显色法测定黄原胶中丙酮酸含量变化,通过扫描电子显微镜镜(SEM)和透射电子显微镜镜(TEM)观察地毯草黄单胞菌产黄原胶的分泌行为。FJAT-10151在发酵时间0～26 h为对数期,26～72 h为稳定期。菌株FJAT-10151为短杆状(约2μm),端生鞭毛(约7.9μm),菌体外面形成了一层较厚的荚膜,且许多菌体通过黄原胶连在一起,分泌的黄原胶呈棒状和网状2种形态,聚集或缠绕在细胞表面,其含量随发酵时间的延长而增加。菌株FJAT-10151发酵72 h可获得高品质黄原胶,其产量及丙酮酸含量最高。     

人工接种CGMMV后与西瓜倒瓤相关部分生理指标的研究

 本研究以辽宁省田间主栽西瓜品种“京欣”为试材,通过测定多聚半乳糖醛酸酶(PG)的活性,以及乙烯、丙酮酸及丙酮酸代谢产物—乳酸、乙醛、乙醇含量的变化,明确了西瓜感染黄瓜绿斑驳花叶病毒后上述生理指标的改变与西瓜倒瓤的关系。研究结果表明:接种处理的西瓜果实授粉后7 d和28 d PG出现2次活性高峰,PG活性过高将导致果肉软化;乙烯含量亦在授粉后21~28 d明显高于健康对照,这一变化将导致果肉水渍化败坏;无论接种早晚,西瓜果实内丙酮酸及丙酮酸代谢产物均有不同程度的积累。定植期接种处理的西瓜,授粉后21 d时西瓜果实内丙酮酸含量为健康对照的2.51倍,35 d时达健康对照4.09倍;授粉后7 d乳酸含量已显著高于对照,35 d时接种处理的西瓜果实内的乳酸含量迅速增加至43.5 μmol·g^-1FW,达健康对照的4.7倍;乙醛含量随着西瓜成熟逐渐增加,尤其是授粉后21~35 d乙醛含量增加速度极为迅速,35 d时达健康对照的26.4倍;乙醇含量授粉后7 d最高,达健康对照的10.1倍,随后有所下降。于压蔓期接种处理的西瓜果实,授粉后21 d,丙酮酸、乳酸、乙醛含量明显高于健康对照,但均低于定植期接种处理的,乙醇含量与健康对照无明显差异,也就是说,感染该病毒越早,对西瓜的生长影响越明显。