紫花苜蓿MsLEA4启动子的克隆及表达分析

为了深入研究紫花苜蓿（Medicago sativa）MsLEA4基因的功能,本试验采用染色体步移技术从紫花苜蓿基因组中扩增出MsLEA4启动子序列,构建GUS植物超表达载体,并根据序列分析结果对紫花苜蓿进行相应逆境胁迫。结果表明：紫花苜蓿MsLEA4启动子含有响应脱落酸（abscisic acid,ABA）调控的顺式作用元件ABRE,响应赤霉素（gibberellin,GA）调控的顺式作用元件P-box,参与胚乳合成的顺式作用元件GCN4和Skn-1,以及涉及光调控和光周期的顺式作用元件;外源ABA、GA、持续光照以及黑暗均能诱导MsLEA4基因的表达;GUS基因只在拟南芥花和荚果中表达。因此,MsLEA4基因能参与紫花苜蓿的逆境调控,与该基因启动子序列中所含的一系列顺式作用元件相关。本研究为进一步探索MsLEA4基因在紫花苜蓿逆境胁迫、光调控以及组织特异性表达中的作用奠定了基础。     

草地早熟禾PpGA2ox基因启动子的克隆及功能分析

为了研究草地早熟禾(Poa pratensis L.)GA2-氧化酶基因(PpGA2ox)的启动子,利用染色体步移的方法从草地早熟禾中克隆得到1109bp的PpGA2ox基因启动子序列。Plant CARE在线预测结果表明该启动子序列中存在CAAT-box、TATA-box等启动子基本顺式作用元件以及响应干旱胁迫和茉莉酸甲酯诱导的调控元件。构建PpGA2ox基因启动子与GUS报告基因融合的植物表达载体转化拟南芥,阳性植株GUS组织化学染色结果表明PpGA2ox基因启动子可以驱动GUS基因在拟南芥中表达并且表达具有组织特异性。与对照相比,激素处理及非生物胁迫下GUS基因表达量发生变化,推测PpGA2ox基因的表达可受到激素及非生物胁迫调控。     

苦豆子赖氨酸脱羧酶基因启动子在拟南芥中的表达分析

赖氨酸脱羧酶(LDC)基因是苦豆子中氧化苦参碱(OMA)生物合成的第一个关键酶基因。在已有苦豆子赖氨酸脱羧酶基因(SaLDC)基础上克隆得到该基因上游1260 bp的启动子序列,GenBank登录号为KY038928,前期在苦豆子愈伤组织中的瞬时表达研究显示该启动子具有启动活性。生物信息学分析发现该启动子区域除了拥有启动子区的基本顺式作用元件TATA-box和CAAT-box外,还具有多个与光信号、逆境应答等相关的顺式作用元件。为进一步研究SaLDC启动子的功能,构建了该启动子与β-葡萄糖苷酸酶(GUS)报告基因融合的植物表达载体并通过农杆菌介导遗传转化拟南芥,同时对光诱导和聚乙二醇(PEG)胁迫的转基因拟南芥进行GUS活性染色和定量分析。结果显示,在T₂代转基因拟南芥幼苗的不同生长阶段和成株的各组织器官中均可检测到GUS酶活性,且随幼苗生长时间的延长,叶片中的表达活性下降;在成株叶片和花萼中的表达活性强于根、茎、花瓣和角果。光和PEG胁迫均能诱导转基因拟南芥中GUS的表达;GUS酶活性定量测定显示,短时间的PEG胁迫(1~2 h)GUS酶活性显著上调(P<0.05),而连续胁迫8 h时GUS酶活性下调至最低(P<0.01),比胁迫前下降了28.2%。以上结果表明SaLDC启动子既有时空表达特异性又有组织表达特异性,光诱导和干旱胁迫对结构基因的表达有重要的调控作用。     

家蚕脂肪酶-1基因(Bmlipase-1)的启动子活性分析

家蚕脂肪酶-1(Bmlipase-1)在蚕体中肠组织中特异性表达,具有抵抗家蚕核型多角体病毒(Bm NPV)侵染的活性。用PCR方法扩增了Bmlipase-1启动子的8个不同区域片段,以绿色荧光蛋白基因(EGFP)为报告基因,分别构建重组家蚕杆状病毒并注射感染家蚕幼虫后,通过荧光观察、荧光定量PCR和ELISA方法,检测报告基因在家蚕中肠中的表达量,以明确对Bmlipase-1具有转录调控活性的启动子片段。结果表明:Bmlipase-1启动子-1 453~192 bp区域的活性最强;在-1 453~-679 bp区域有与Bmlipase-1在中肠组织特异性表达相关的启动子保守序列及1个CAAT box和大量的GATA序列等正调控元件,推测该区域可能与Bmlipase-1的组织特异性表达有关;-81~493 bp区域涵盖核心启动子区、第1外显子区和第1内含子区,含有大量Pbx-1转录因子结合位点、OTC序列和GATA序列,推测该区域发挥对Bmlipase-1转录的基础调控作用;-1 980~-1 452 bp区域存在负调控元件。研究结果有助于进一步揭示Bmlipase-1的转录调控机制。     

几个杨树木质部特异启动子片段表达载体的构建与功能分析

木质部纤维素合酶基因(CesA)在植物正常生长期的木质部特异表达,受到外界压力时可诱导在韧皮部表达,因而被用作杨树抗桑天牛转基因研究中的特异性表达启动子。将前期分离的CesA上游DNA片段JCesAP、YCesAP和MDCesAP分别替换植物表达载体pCAMBIA1302中的组成型启动子CaMV35S,构建成新的植物组织特异性表达载体pJCesAP-GFP、pYCesAP-GFP和pMDCesAP-GFP,然后采用农杆菌介导法转化烟草,均得到完整的再生烟草植株。经PCR初步检测证明目的基因已整合到烟草基因组中。荧光检测JCesAP、YCesAP和MDCesAP片段均能启动GFP蛋白的表达,在395 nm激发光下出现黄绿色荧光,显示3个CesA片段均具有在木质部特异表达的启动子活性。     

马铃薯SGT3基因表达及其启动子功能分析

鼠李糖转移酶(rhamnosyltransferase SGT3)是植物糖苷生物碱合成的关键酶,它主要调控α-茄碱和α-查茄碱从其β形式的转化。本文研究马铃薯栽培种SGT3基因的表达特点及其启动子的功能。实时定量PCR结果发现在红光照射24 h后,SGT3的表达量是黑暗处理的26.8倍,说明红光显著诱导了SGT3的表达;为进一步分析该基因的光调控机理,本项研究克隆到SGT3上游长度为2449 bp的启动子序列,通过分析发现该启动子的转录起始位点位于翻译起始位点上游-152 bp,同时确定了该启动子核心序列及上游增强子、抑制子序列,受病原菌、损伤、干旱、ABA 激素及一系列光调控的顺式元件。构建不同长度(349,572,979,1312和1870 bp)的该启动子驱动报告基因GUS的植物表达载体并转化烟草。结果证明,不同长度的SGT3启动子都可以启动GUS表达,但没有CMV 35S启动的GUS表达量高;其中在P572和P979的表达强度较高,这可能与该片段含有启动子的正调控元件(GATA BOX,5′UTRPY-RICH STRETCH)有关,GUS表达强度在P1312和P1870中明显减弱,预测到该区段存在抑制基因表达的负调控元件(WRKY710S);SGT3启动子的组织特异性实验表明GUS染色主要集中在烟草叶片的叶脉部分,茎中的表皮、韧皮部及木质部,但髓部几乎不表达,根中主要分布在根冠、分生区以及维管束组织中。上述结果为将来研究SGT3在糖苷生物碱合成过程中的调节功能提供了依据。     

家蚕丝素蛋白H链基因启动子区域的克隆及活性分析

家蚕(Bombyxmori)丝素蛋白H链基因(fib-H)具有在后部丝腺组织专一性、高效性表达的特点。为利用其时空调控机制,通过PCR扩增方法克隆fib-H启动子片段,并进行序列分析,进而构建由fib-H启动子控制报告基因DsRed的重组载体pSK-FH-DsRed-PolyA,通过家蚕BmN细胞和丝腺进行瞬时表达。结果表明:克隆的fib-H启动子序列具有典型的丝腺特异性表达启动子的特征,并可以驱动DsRed报告基因在BmN细胞和家蚕后部丝腺组织中瞬时表达。     

小尾寒羊MYL1基因启动子区的克隆、分析及蛋白表达

为研究绵羊肌球蛋白轻链1(Myosin Light Chain 1,MYL1)基因在骨骼肌中的表达调控和功能,本实验选择3只11月龄小尾寒羊,对其启动子区域进行了克隆和序列特征分析;利用Western blotting法对其编码蛋白在小尾寒羊不同组织(肝、胃、心、肺、背最长肌、脾、卵巢)中的表达进行比较分析。结果表明:MYL1基因含有多个启动子,其最佳启动子可能位于2102~2152 bp处,并且可结合有AP-2、TFIID等多种转录因子;其编码蛋白的分子量约21 ku;该蛋白只在小尾寒羊骨骼肌中表达,在其他组织中均不表达。本研究认为MYL1基因具有多个启动子,虽然其转录后存在多个可变剪接体,但翻译蛋白只有1种,属于绵羊骨骼肌组织特异性表达的基因。     

TLR5基因启动子区甲基化修饰与苏太断奶仔猪E. coli F18抗性的关系

旨在分析探讨TLR5基因启动子区甲基化修饰对断奶仔猪F18大肠杆菌抗性的调控作用。本研究首先利用qPCR和Western blot检测分析了TLR5基因在F18大肠杆菌抗性型和敏感型苏太断奶仔猪小肠组织(十二指肠和空肠)中的差异表达,然后利用生物信息学分析和双荧光素酶报告系统检测确定TLR5基因核心启动子区、CpG岛及其作用元件,进而检测并分析TLR5基因启动子区甲基化修饰与TLR5基因在F18大肠杆菌抗型和敏感型断奶仔猪小肠组织中表达水平的相关性。结果表明,TLR5基因在敏感型断奶仔猪十二指肠和空肠组织中的mRNA表达水平分别显著(P0.05)和极显著(P0.01)高于在抗性型个体中的表达,且十二指肠和空肠组织中敏感组蛋白表达水平均显著高于抗性组(P0.05)。TLR5基因启动子区包含2个CpG岛和16个作用元件,启动子区第2个CpG岛第6个CG位点甲基化水平对TLR5基因的表达具有一定的调控作用,该位点位于转录因子Sp1结合的核心启动子区域。本研究结果表明,猪TLR5基因的表达水平和F18大肠杆菌的抗性有关,其低表达可能有利于F18大肠杆菌抗性;TLR5基因启动子区第2个CpG岛第6个CG位点甲基化能够显著抑制TLR5基因的表达,并最终影响断奶仔猪对大肠杆菌的抗性。     

Phase I evaluation of CCNU (lomustine) in tumor-bearing cats

1-(2-Chloroethyl)3-cyclohexyl-1-nitrosourea (CCNU) is an alkylating agent in the nitrosourea subclass. A prospective evaluation of CCNU was done to determine the maximally tolerated dosage of CCNU in tumor-bearing cats. Response data were obtained when available. Twenty-five cats were treated with CCNU at a dosage of 50-60 mg/m3 body surface area. Complete hematologic data were available for 13 cats. Neutropenia was the acute dose-limiting toxicity. The median neutrophil count at the nadir was 1,000 cells/microL (mean, 2,433 cells/microL; range, 0-9,694 cells/microL). The time of neutrophil nadir was variable, occurring 7-28 days after treatment, and counts sometimes did not return to normal for up to 14 days after the nadir. Based on these findings, a 6-week dosing interval and weekly hematologic monitoring after the 1st treatment with CCNU are recommended. The nadir of the platelet count may occur 14-21 days after treatment. The median platelet count at the nadir was 43,500 cells/microL. No gastrointestinal, renal, or hepatic toxicities were observed after a single CCNU treatment, and additional studies to evaluate the potential for cumulative toxicity should be performed. Five cats with lymphoma and 1 cat with mast cell tumor had measurable responses to CCNU. Phase II studies to evaluate antitumor activity should be completed with a dosing regimen of 50-60 mg/m3 every 6 weeks.     

Furosemide continuous rate infusion in the horse: evaluation of enhanced efficacy and reduced side effects

Continuous rate infusion (CRI) of furosemide in humans is considered superior to intermittent administration (IA). This study examined whether furosemide CRI, compared with IA, would increase diuretic efficacy with decreased fluid and electrolyte fluctuations and activation of the renin-angiotensin-aldosterone system (RAAS) in the horse. Five mares were used in a crossover-design study. During a 24-hour period, each horse received a total of 3 mg/kg furosemide by either CRI (0.12 mg/kg/h preceded by a loading dose of 0.12 mg/kg IV) or IA (1 mg/kg IV q8h). There was not a statistically significant difference in urine volume over 24 hours between methods; however, urine volume was significantly greater after CRI compared with IA during the first 8 hours ([median 25th percentile, 75th percentile]: 9.6 L [8.9, 14.4] for CRI versus 5.9 L [5.3, 6.0] for IA). CRI produced a more uniform urine flow, decreased fluctuations in plasma volume, and suppressed renal concentrating ability throughout the infusion period. Potassium, Ca, and Cl excretion was greater during CRI than IA (1,133 mmol [1.110, 1,229] versus 764 mmol [709, 904], 102.7 mmol [96.0, 117.2] versus 73.3 mmol [65.0, 73.5], and 1,776 mmol [1,657, 2.378] versus 1,596 mmol [1,457, 1,767], respectively). Elimination half-lives of furosemide were 1.35 and 0.47 hours for CRI and IA, respectively. The area under the excretion rate curve was 1,285.7 and 184.2 mL x mg/mL for CRI and IA, respectively. Furosemide CRI (0.12 mg/kg/h) for 8 hours, preceded by a loading dose (0.12 mg/kg), is recommended when profound diuresis is needed acutely in horses.     

Prevalence of gastric lesions in racing Alaskan sled dogs

Human and equine athletes are reported to have a high prevalence of gastric disease, and anecdotal evidence suggests a similar phenomenon applies to racing sled dogs. To investigate the prevalence of gastric disease in racing sled dogs, we conducted 2 gastroscopy studies on dogs competing in the annual Iditarod Sled Dog Race. A pilot study of dogs that were either dropped from the 2000 Iditarod Sled Dog Race because of illness or that finished the race indicated that, approximately 5 days after competing, 10 of 28 dogs (35%) had endoscopic evidence of gastric ulceration, erosion, or hemorrhage. The next year, an endoscopic study of 73 dogs participating in the 2001 Iditarod race was performed in order to evaluate a larger population of dogs. Data from 70 of these dogs could be used; 34 (48.5%) had ulceration, erosion, gastric hemorrhage, or some combination of these findings. When this group of 70 dogs was compared retrospectively to a control group of 87 dogs presented to the Texas A&M University (TAMU) Veterinary Medical Teaching Hospital, the Iditarod sled dogs had a significantly higher prevalence (P = .049) of gastric lesions. These findings suggest that, similar to athletes of other species, elite canine athletes have an increased prevalence of gastric disease compared to the canine population at large.     

The Epidemiology of Gastrointestinal Nematodes of Dairy Cattle in Central Kenya

The epidemiology of H. placei and of other gastrointestinal nematodes in yearling dairy cattle was examined on two farms in Kiambu District, central Kenya during each of 13 one-month periods from April 1993 to April 1994. On each farm, 32 newly weaned dairy calves were given a single dose of albendazole and then placed on experimental pastures. Twelve of the animals were designated for bi-monthly slaughter (n = 2) and analysis of worm population characteristics and 20 were designated for blood and faecal collection and for weighing. Two parasite-free tracer calves were grazed alongside the weaner calves each month throughout the study period and were also slaughtered for analysis of worm populations. Faecal egg counts, haematological and serum pepsinogen determinations, herbage larval counts, and animal live weight changes were recorded monthly. The study revealed that Haemonchus placei, Trichostrongylus axei, Cooperia spp. and Oesophagostomum radiatum were responsible for parasitic gastroenteritis and that H. placei was the predominant nematode present in the young cattle on both farms. Faecal egg counts from resident cattle and necropsy worm counts revealed that pasture larval levels were directly related to the amount of rainfall. The total worm burdens in the animals were highest during the rainy season (March–June and October–December) and lowest during the dry seasons (July–September and January–February). The very low recovery of immature larvae of H. placei from the tracer calves indicated that arrested development is not a feature of the life cycle of this parasite in central Kenya. The maintenance of the parasite population depended on continuous cycling of infection between the host and the pasture. The agroclimatic conditions of the study area were such that, in general, favourable weather conditions for the development and survival of the free-living stages of gastrointestinal nematodes existed all year round.     

Diversity of Ectoparasites in Sheep Flocks in Sa~o Paulo,Brazil

The occurrence of ectoparasites in sheep flocks is frequently reported but seldom quantified. Sheep production used to be a predominantly family activity in the state of Sa~o Paulo (Brazil), but it began to become a commercial activity in the past decade. Thus, information about the ectoparasites existing in sheep flocks has become necessary. The present data were obtained by means of questionnaires sent to all sheep breeders belonging to the `Associaça~o Paulista de Criadores de Ovinos' (ASPACO; Sa~o Paulo State Association of Sheep Breeders). Response reliability was tested by means of random visits paid to 10.6% of the respondents. Most of the properties (89.5%) reported the presence of one or more ectoparasites. Screw-worm (Cochliomyia hominivorax) was the most frequent ectoparasite (72.5%), followed by bot fly larvae (Dermatobia hominis, 45.0%), ticks (Amblyomma cajennense) and Boophilus microplus, 31.3%) and finally lice (Damalinia ovis, 13.8%). Combined infestations also occurred, the most common one being screw-worm with bot fly larvae (36.0%) followed by bot fly larvae with ticks (13.9%), screw-worm with ticks (9.3%), bot fly larvae with lice (6.9%), and ticks with lice (5.0%). The most common triple combination was screw-worm, bot fly larvae and ticks (12.8%). Breeds raised for meat or wool were attacked by bot fly larvae and ticks more often than other breeds. Lice were only absent from animals of indigenous breeds. The relationships among these ectoparasites are discussed in terms of sheep breeds, flock size, seasonality and the ectoparasitic combinations on the host.     

Evaluation of biohydrogenation rate of canola vs. soya bean seeds as unsaturated fatty acids sources for ruminants in situ

An experiment was conducted to study disappearance of C14 to C18 fatty acids, lag times and biohydrogenation (BH) rates of C18 fatty acids of ground soya bean and canola seeds in situ. Three ruminally fistulated Dallagh sheep were used to determine ruminal BH of unsaturated fatty acids (UFAs). Differences in the disappearance of fatty acids through the bags and lag times were observed between the oilseeds. We saw that the longer the incubation time of the oilseeds in the rumen, the lower the content of C18:2 and C18:3. Significantly higher lag times for both C18:2 and C18:3 were observed in ground canola compared to ground soya bean. BH rates of C18:2 and C18:3 fatty acids in soya bean were three times higher than those of canola. These results suggest that the fatty acid profile of fat source can affect the BH of UFAs by rumen micro‐organisms. So that UFAs of canola had higher ability to escape from ruminal BH. It seems that fatty acid profile of ruminant products is more affected by canola seed compared to soya bean seed.     

Serum biochemical values and mineral element contents of tissues in yaks

Serum biochemical values and the wet weight to dry weight ratios of tissues were determined in yaks in Shandan county of Gansu province. The liver, kidney, heart and muscle contents of seven elements in yaks were also analysed. Most serum biochemical values were similar to those of cattle, camels and sheep, but the calcium concentration was considerably above the normal range for other ruminants. The liver contained the highest concentrations of copper, manganese and cobalt and the kidney of selenium, iron and calcium.Abbreviations Alb albumin - ALT alanine aminotransferase - AST aspartate aminotransferase - BUN blood urea nitrogen - Chol cholesterol - Glob globulin - -GT -glutamyl transpeptidase - IP inorganic phosphorus - TP total protein     

Avian brood parasitism — a growing research area in behavioral ecology (part Ⅱ)

正We are pleased to publish the second special issue on avian brood parasitism and to be responsible guest editors for the two special issues of Chinese Birds (Vol. 3, No. 4, 2012 and Vol. 4, No. 1, 2013), entitled "Avian Brood Parasitism - A Growing Research Area in Behavioral Ecology". The first issue was published in December 2012. The goal of the two special issues is to publish accumulated knowledge and some of the recent developments in     

Internal Parasites and Haematological Values in Cattle Slaughtered in Buea Subdivision of Cameroon

Tropical Animal Health and Production -