混合瘤胃微生物体外利用蛋氨酸、赖氨酸的研究

为研究瘤胃微生物对瘤胃液中添加的蛋氨酸、赖氨酸的利用情况，并进一步探讨添加氨基酸对瘤胃液中尿素氮、部分酶活的影响，将采集到的来自3头装有永久性瘤胃瘘管的成年萨能山羊的瘤胃液混合后，平均分装成18个瓶，随机分成3个处理组，每组6个重复。采用6重复随机因子试验设计。第1组中每瓶添加蛋氨酸2mM（0．25mol／L．Met溶液8mL），第2组中每瓶添加赖氨酸2mM（0．25mol／L Lys溶液8mL），第3组中每瓶加等体积蒸馏水作为空白对照组，体外培养16h。结果表明：添加氨基酸后，混合瘤胃液中的尿素氮（UN）含量显著高于对照组（P〈0．05）；谷氨酸脱氢酶（GLDH）活性随着体外培养时间的延长而迅速增加，并且添加蛋氨酸组的GLDH的活性明显高于其他两组（P〈0．01），而添加赖氨酸组的GLDH活性则极显著低于蛋氨酸组和对照组（P〈0．01）；添加的氨基酸和体外培养时间对于谷丙转氨酶（GPT）活性没有显著的影响（P〉0．05）；添加氨基酸组的谷草转氨酶（GOT）的活性显著低于对照组（P〈0．05），但体外培养时间对各组中GOT的活性没有显著的影响。说明体外培养过程中瘤胃微生物分解赖氨酸的速率要比蛋氨酸快；添加的氨基酸使UN增加，但对GOT、GPT的活性起抑制作用；添加蛋氨酸可增加GLDH活性，但添加赖氨酸却有抑制作用。     

水稻L-半乳糖内酯脱氢酶基因的克隆和原核表达及抗体的制备

应用RT-PCR技术从水稻叶片中克隆了水稻L-半乳糖内酯脱氢酶(GLDH)的全长基因.序列分析表明,水稻GLDH基因全长1752bp,亚克隆其部分成熟蛋白编码基因(789bp),利用基因重组技术构建了E.coli/pET30a-GLDHe,经酶切鉴定,DNA测序,证实重组质粒构建正确;将重组质粒转化大肠杆菌Rosseta,IPTG诱导表达,SDS-PAGE分析证实表达出相对分子质量约为36000的蛋白.用原核表达蛋白作为抗原免疫家兔制备抗体,用Westernblot检测抗体的特异性及效价,结果表明,抗体血清效价为1∶1000,在加IPTG诱导后的菌液中可以检测到相应的蛋白条带((相对分子质量为36000)),在水稻叶片样品中能检测到1条相对分子质量为36000的蛋白条带.     

Serum haptoglobin concentration and liver enzyme activity as indicators of systemic inflammatory response syndrome and survival of sick calves

BackgroundIncreased concentration of haptoglobin (Hp) in serum is associated with survival of critically ill humans and horses. High serum activity of liver‐derived enzyme is associated with sepsis in children and foals.Hypothesis/ObjectivesInvestigate whether admission serum Hp and glutamic dehydrogenase (GLDH) are associated with systemic inflammatory response syndrome (SIRS) and survival of sick calves.AnimalsOne hundred two calves.MethodsRetrospective cross‐sectional study. Electronic medical records from all calves <30 days of age admitted to a teaching hospital for 8 years were reviewed. The signalment, clinicopathological findings, the presence of SIRS, final diagnosis, hospitalization time and outcome were recorded. A Cox proportional hazard ratio (HzR) were calculated to assess the association between clinicopathological variables and survival to discharge.ResultsSerum Hp concentrations were similar between SIRS (0.29 g/L; range, 0.05‐3.6) and non‐SIRS calves (0.22 g/L; range, 0‐4.2; P = .62). GLDH activity was similar between SIRS (12 U/L; range, 1‐1025) and non‐SIRS calves (9 U/L; range, 2‐137; P = .2). Absent suckle reflex (HzR: 6.44, 95% CI: 1.44‐28.86), heart rate (HR) < 100 beats per minute (bpm; HzR: 12.2; 95% CI: 2.54‐58.62), HR > 140 bpm (HzR: 3.59, 95% CI: 1.05‐12.33), neutrophil count <1.7 × 10⁹/L (HzR: 7.36; 95% CI: 2.03‐26.66) and increased gamma‐glutamyl transferase activity (every 50‐unit, HzR: 1.12; 95% CI: 1.03‐1.21) were predictive of nonsurvival.Conclusions and Clinical ImportanceThe use of Hp and GLDH for prediction of survival in sick calves cannot be recommended at this time.     

不结球白菜L-半乳糖酸-1,4-内酯脱氢酶基因(GLDH)cDNA片段克隆与RNAi表达载体构建

根据植物中hpRNA(hairpin RNA)原理,选择GLDH基因cDNA序列同源性较低区域设计1对携带双酶切位点的特异引物,以不结球白菜‘苏州青’叶片为材料提取总RNA,利用RT-PCR技术克隆了GLDH基因片段。构建中间克隆载体,经过3次亚克隆将测序正确的GLDH基因片段分别以正向和反向两种形式插入到间隔基因YYT的两端,经鉴定已插入到植物表达载体中,成功地构建了干扰GLDH基因表达的RNAi植物双元表达载体pRNAi-GLDH,并将表达载体转化到农杆菌菌株LBA4404中,为深入研究GLDH基因功能提供有效的工具。     

Circadian and seasonal variability and influence of sex and race on eight clinical chemistry parameters in budgerigars (Melopsittacus undulatus, Shaw 1805)

The aim of the study was to investigate if there is a clinically relevant influence of daytime or season and if there are sex-dependant differences in some frequently determined clinical chemistry parameters. The study was performed with 99 1.5- to 2-year old healthy budgerigars of three races. A harmonic regression analysis was performed taking into account linear and cyclic trends over the year. Correlations were investigated by the calculation of Spearman correlation coefficients. Summarizing, it can be said that some circadian, seasonal, as well as sex- and race-dependant influences have been shown. However, although the differences are statistically significant, they were too small to have an impact on clinical decision making. As clinical chemistry results are in practice independent from the influence factors investigated, their meaning and use as a diagnostic tool is further substantiated.     

马铃薯GLDH基因的克隆及序列分析

 以马铃薯品种‘Favorita’叶片中的cDNA为模板,采用RT-PCR、巢式PCR、3′RACE和5′RACE技术,获得了L–半乳糖酸–1,4–内酯脱氢酶（EC 1131213,GLDH）基因cDNA 2 563 bp的全长序列,命名为StGLDH（GenBank：FJ755844）。序列分析表明,该基因编码区长1 773 bp,编码590个氨基酸,与其他植物GLDH氨基酸序列具有很高的同源性,尤其与番茄、辣椒、烟草GLDH 氨基酸序列具有90.6% ~ 95.9%的同源性。荧光定量分析表明,该基因在马铃薯不同器官中均有表达,在幼叶和功能叶中表达量最高,在茎中表达量最低;除匍匐茎外,其它器官中抗坏血酸（ascorbic acid,AsA）含量与StGLDH的表达高度一致。