Characterization of MicroRNA* Species in Peking Duck Skin

A substantial fraction of miRNA* species are conserved in animals and can repress activities of target genes. This study aims to investigate the miRNA* species in duck skin by using Solexa sequencing. We obtained a total of 96 miRNA* species in two skin small RNA libraries and identified 56 miRNA/miRNA* (miR/miR*) pairs. Nucleotide bias of miRNA* indicated that the priority was C>A>U>G for the first nucleotide and U>C>A>G for the last nucleotide. Comparison analyses showed that 3′-U accounted for a higher proportion in the 56 miR/miR* pairs. Among the top 20 expressed miRNA* species, 17 were shared by two libraries and most of the miRNA* species were highly conservative, especially in the “seed region”. miR-199a* were expressed highly in our samples, which was also previously shown abundant in mouse hair follicle. Furthermore, four miRNA* species were predicted to target their genes in signal pathways of feather follicle development and feather morphogenesis despite very low levels.     

鸭肠炎病毒UL41基因缺失株的构建及其体外增殖能力分析

旨在构建鸭肠炎病毒（DEV）UL41基因缺失毒株,并对其生物学特性进行分析,本研究以克隆有DEV UL41基因的重组黏粒D1为骨架,利用Red/ET重组技术构建缺失UL41基因的重组黏粒D1 dUL41;将UL41基因缺失重组黏粒D1 dUL41与其他4个克隆有DEV基因组片段的亲本黏粒共转染鸭胚成纤维细胞（DEF）,拯救获得UL41基因缺失毒株rDEV-SD19/dUL41。将该基因缺失病毒感染DEF后,提取病毒基因组进行PCR鉴定及测序,并利用间接免疫荧光试验检测该病毒感染细胞中UL41基因表达情况;绘制拯救的基因缺失病毒的生长曲线,分析其体外复制特性。PCR及测序结果显示,本研究成功构建了缺失UL41基因的重组黏粒D1 dUL41。将该基因缺失黏粒与其他含有DEV基因组的亲本黏粒共转染DEF后能够产生典型的蚀斑病变。PCR及测序结果显示,UL41基因成功从DEV基因组中缺失;间接免疫荧光试验发现,基因缺失病毒rDEV-SD19/dUL41感染DEF后,未见UL41蛋白表达。综上表明,本研究成功构建了DEV UL41基因缺失病毒。体外生长曲线显示,rDEV-SD19/dUL41在DEF中的复制能力明显低于亲本病毒,提示UL41蛋白在DEV复制中发挥重要作用。UL41基因缺失DEV的构建为进一步研究UL41基因在DEV感染和致病中的作用机制奠定了基础。     

靶向cofilin2基因RNAi对鸭病毒性肠炎病毒增殖的影响

旨在探讨cofilin2基因的表达在鸭病毒性肠炎病毒(DEV)增殖过程中的影响。本研究通过已获得的鸭源cofilin2全基因序列(GenBank登录号:MF434775),设计出4对shRNA序列,插入载体中,并转染到鸭胚成纤维细胞中,经FQ-PCR方法和荧光显微镜观察筛选出具有较高干扰效率的shRNA序列,用干扰效率较高的shRNA序列转染到鸭胚成纤维细胞中抑制cofilin2基因的表达,再检测cofilin2在DEV复制过程中表达情况。结果显示:4对shRNA干扰质粒经转染至鸭胚成纤维细胞后,经荧光显微镜观察,构建的4对shRNA干扰质粒均成功转染并得到表达;4对干扰质粒沉默率分别为4.96%±0.78%、41.12%±2.94%、17.55%±2.07%、54.02%±1.73%,以cofilin2-86的沉默效率最高;cofilin2-86转染到鸭胚成纤维细胞,经DEV感染不同时间,其DEV核酸表达量明显下降。综上所述,靶向cofilin2基因RNA干扰在DEV复制和增殖过程发挥重要作用。     

1～14日龄三穗鸭能量需要量的研究

试验旨在研究饲粮表观代谢能水平对1～14日龄三穗鸭生长性能的影响,以估测三穗鸭的表观代谢能需要量。采用单因子完全随机试验设计,选取336只健康1日龄雄性三穗鸭,随机分为6个处理,每个处理7个重复,每个重复8只鸭。采用玉米-豆粕型基础饲粮,表观代谢能水平分别为10.92、11.38、11.80、12.26、12.68、13.14MJ/kg。试验期为1～14日龄。结果表明:随着表观代谢能水平的提高,1～14日龄三穗鸭平均日采食量和耗料增重比呈线性降低(P<0.05),且表观代谢能到达11.80 MJ/kg时,耗料增重比和平均日采食量基本到达平台期;饲粮表观代谢能水平对1～14日龄三穗鸭末重和平均日增重均无显著影响,呈线性和二次曲线升高趋势(P>0.05),饲粮表观代谢能水平对总表观代谢能摄入无显著影响。由此可见,饲喂高表观代谢能饲粮可有效改善1～14日龄三穗鸭的饲料转化效率;以耗料增重比为评价指标,利用直线-折线模型估算的1～14日龄三穗鸭表观代谢能的需要量为11.59 MJ/kg。     

Isolation,Identification, 16S rRNA Gene Sequencing and Genetic Evolution Analysis of Aeromonas aeruginosa from Duck

【Objective】 The experiment was aimed to study the pathogenic bacteria of acute death of ducklings in a duck farm in Henan province and its phylogenetic status.【Method】 The liver and spleen of dead ducks in the diseased duck farm were collected.The bacterial morphology, Gram-staining, biochemical characteristics, sequence analysis of 16S rRNA gene, drug sensitivity test and pathogenicity test were carried out.【Results】 The isolated bacteria grew on blood agar medium with smooth, convex, milky white and α-hemolytic Gram-positive cocci.Biochemical test results showed that the isolated bacteria were positive for maltose, sucrose, raffinose, nitrate reduction reaction, MP-VP test, urea, lysine decarboxylase, ornithine decarboxylase and aescin, and negative for xylose, lactose, glucose, sorbitol, hydrogen sulfide, citrate, peptone, mannitol, phenylalanine and methyl red.The results of 16S rRNA gene sequencing and BLAST comparison results showed that the 16S rRNA gene sequence of the isolated strain was 94.8%-99.9% similar to that of Aerococcus viridans published on NCBI, and the similarity with strains Mnlv1, Mnlv2 and W66 was the highest, up to 99.9%.The similarity with strain GXBl-1 was the lowest, which was 94.8%.Phylogenetic tree analysis showed that the isolated bacteria belonged to the same genus and group on the same branch as strains of Aerococcus viridans FL09, 15MS and Mnlv2 etc., and had the closest genetic relationship with FL09.The results of drug sensitivity showed that the isolated strain was sensitive to fosfomycin, ampicillin and amoxicillin, moderately sensitive to penicillin, neomycin and amikacin, it was resistant to rifampicin, neomycin, bacitracin, erythromycin and enrofloxacin.The results of pathogenicity test showed that the strain was lethal and increased with the increase of inoculation concentration.【Conclusion】 This study determined that the pathogen causing the acute death of ducklings in a duck farm in Henan province was Aerococcus viridans, which provided a reference basis for clinical diagnosis and treatment of the diseases caused by this bacteria.     

北京鸭与樱桃谷鸭的分子遗传关系分析

为了分析北京鸭和樱桃谷鸭的遗传关系,采集包括北京鸭、在英国培育而成的肉鸭品种樱桃谷鸭及其它5个中国地方鸭品种共248个个体,利用11个微卫星标记对这7个鸭品种进行遗传关系分析.FST分析结果表明北京鸭与樱桃谷鸭的遗传分化程度最低,只有0.0795;遗传距离(Ds)分析发现北京鸭与樱桃谷鸭的遗传距离最近,为0.1079;群体分化时间分析表明二者的分化时间为98年,北京鸭与其它地方鸭品种的分化时间在282至694年之间;聚类结果显示北京鸭、樱桃谷鸭和建昌鸭聚为一类;巢湖鸭、高邮鸭、绍兴鸭和金定鸭聚为另一类.这些结果表明了北京鸭与引进品种樱桃谷鸭有非常密切的遗传关系,为樱桃谷鸭培育自北京鸭这一说法提供了分子遗传学的证据,同时本研究还发现北京鸭与樱桃谷鸭在遗传背景上已经发生了一定程度的分化.     

鸭肝炎病毒VP1蛋白B细胞抗原表位预测及变异分析

以鸭肝炎病毒(DHV)SN株VP1基因序列为基础,运用Garnier-Robson、Chou-Fasman和Karplus-Schulz方法预测VP1蛋白的二级结构,并分别运用Kyte-Doolittle方法、Jameson-Wolf方法和Emini方法预测蛋白的亲水性、抗原指数和表面可及性,最后结合吴玉章的方法综合分析预测其B细胞抗原表位.结果显示,VP1蛋白C端第132～137、177～186、209～219区段有较好的亲水性、表面可及性和较高的抗原指数,并且在二级结构上含有易形成抗原表位的无规则卷曲和β-转角,可能是VP1蛋白的B细胞抗原优势表位;DHV SN株与弱毒疫苗株在推测的VP1 B细胞抗原表位177～186和209～219两个区域氨基酸序列变异较大.     

一株鸭新城疫强毒的分离鉴定和分子特性分析

从山东地区发病鸭中分离到一株新城疫病毒(SDLP09),经测定,其鸡胚半数致死量(ELD50)、鸡胚平均死亡时间(MDT)、1日龄雏鸡脑内接种致病指数(ICPI)、6周龄鸡静脉接种致病指数(IVPI)分别为107.84/0.1 mL、48.5 h、1.80、2.36,表明该新城疫病毒为强毒.通过分析其融合蛋白(F)发现,F蛋白多肽裂解位点为112RRQKRF117,符合NDV强毒株裂解位点氨基酸序列.F基因分型及氨基酸同源性比较发现,SDLP09株属于基因Ⅶ型,与野鸭源强毒NDV同源性更高,提示着该毒株可能来源于野鸭.     

不同饲养方式对鸭蛋成分和蛋黄色泽影响的研究

采用单因素试验设计,研究圈养、网上平养和放养3种不同饲养方式对鸭蛋成分和蛋黄色泽的影响。常规方法检测鸭蛋成分。结果表明：饲养方式对蛋清干物质含量有显著的影响（P〈0.05）,放养鸭的蛋清干物质含量显著低于圈养和网上平养（P〈0.05）,圈养和网上平养之间差异不显著;饲养方式对蛋黄干物质含量影响不显著（P〉0.05）,但放养组最低,圈养组稍高,网上平养组最高;饲养方式对蛋清氨基酸的绝对含量有显著的影响（P〈0.05）,放养鸭蛋的蛋清氨基酸总量低于圈养鸭和网上平养鸭（P〈0.05）,圈养鸭和网上平养鸭差异不显著;但饲养方式对蛋清各种氨基酸占氨基酸总量的比值影响较小;饲养方式对蛋黄色泽、蛋黄干物质脂肪含量和鸭蛋风味有显著的影响（P〈0.05）,放养鸭的蛋黄色泽等级和蛋黄干物质中脂肪含量显著高于圈养鸭和网上平养鸭（P〈0.05）,鸭蛋风味也优于圈养鸭和网上平养鸭;圈养和网上平养之间差异不显著;综合评价3种饲养方式的蛋鸭所产的鸭蛋,总的营养物质含量相差不大,但放养鸭蛋口感好,味道美。