珍稀濒危鸟类微量取样及DNA提取的研究

为解决珍稀濒危鸟类分子生物学研究中存在的取样局限性,采用非伤害性取样和非损伤性取样,对大鸨10~70μL的血液和羽毛的不同部位(羽根、羽髓、羽鞘、实心羽茎和羽毛上脐周围绒羽)的DNA提取进行了研究。结果表明,鸟类血液样本与哺乳动物样本极为不同,两者相同体积的血液中,鸟类样本DNA含量是哺乳动物样本的几百倍,研究发现,大鸨可以从微量的血液中(10μL)提取出较多的DNA(45.75μg)。对于羽毛的不同部位,羽髓、羽鞘和羽根可以提出一定纯度的核DNA和mtDNA;实心羽茎可以提出一定纯度的mtD-NA。因此该研究将拓宽珍稀濒危鸟类采样的范围,为珍稀濒危鸟类的分子生物学研究提供了更广阔的样品来源。     

不同部位取样对鸡基因组DNA提取的效果的影响

采用伤害性取样和非伤害性取样,采集鸡的肝脏、血液、羽髓、羽鞘、羽根、实心羽茎和羽毛上脐周围绒羽等不同部位样品,并采取对样品不同处理程序提取基因组DNA。结果表明,鸡的微量血液(50μl)和羽髓样品提取基因组DNA与肝脏(50 mg)样品提取的基因组DNA的量相当;羽鞘、羽根、实心羽茎和羽毛上脐周围绒羽可以提出一定量的基因组DNA,其提取量少于肝脏和羽髓组织,但可以满足各种涉及PCR反应的分子生物学试验,肝脏羽髓消化最适时间为2.5 h,其他样品需消化过夜。该研究结果为禽类分子生物学研究拓宽了样本来源。     

非损伤性取样提取动物DNA方法探讨

近年来,随着分子生物学技术的发展,非损伤性取样逐渐成为野生动物分子生物学研究中获取试验材料的主要方法.文章主要通过介绍当前非损伤取样提取动物DNA的方法,比较各种方法的优缺点和局限性,并分析了各种方法的研究特点和应用前景,以期对下一步研究提供有价值的参考.     

一种禽类血液基因组DNA高效提取通用方法的建立

为了建立一种适合禽类血液的廉价、高效、通用的基因组DNA提取方法,试验采用鸡、鸭、鹅、鸽、火鸡、麻雀6种禽类血液为材料,提取的禽类血液DNA经微量分光光度计分析、基因组DNA凝胶成像分析以及PCR扩增效果检测。结果表明:提取的6种禽类血液DNA质量较好(OD260/OD280值范围为1.80~1.84,OD260/OD230值范围为2.21~2.30),基因组DNA电泳主条带和PCR扩增产物条带清晰、整齐、无拖带。说明本方法能够完全适用于大批量禽类血液基因组DNA的提取,且DNA纯度较好,满足后续相关分子生物学试验要求。     

苯酚/氯仿抽提法提取牛血与鸡血DNA部分条件的比较研究

为了探讨苯酚/氯仿抽提法中加入血液和红细胞裂解液(PBS)的量对DNA提取效果的影响以及找到针对牛血和鸡血最合适的DNA提取条件,试验以安格斯牛和静原鸡为研究对象,采用苯酚/氯仿抽提法提取血液基因组DNA。结果表明:在试验第1步加入200μL鸡血1次,用1 500μL PBS重复裂解2次,可以得到高质量鸡的基因组DNA。当加入鸡血的量过大时,提取的DNA浓度过高且不纯,蛋白质等外源核酸的污染较严重。在试验第1步加入500μL牛血1次,用1 500μL PBS裂解,重复2次,可以得到高质量牛的基因组DNA。苯酚/氯仿抽提法可以节省时间,还可以避免血液的浪费。     

从白羽王鸽的血液中提取基因组DNA方法的比较研究

禽类血液中的蛋白质含量较高,用传统方法提取其血液中的基因组DNA比较繁琐、纯度不高,而且提取的产物中有大量蛋白质残留。本研究采用改良的CTAB法从白羽王鸽全血中提取全基因组DNA,并以传统的酚-氯仿抽提的方法和TAKARA公司基因组提取试剂盒法作对照。结果表明：qCTAB法可以得到大量的、较完整的基因组DNA,并且纯度达到2．00,浓度达到了144．67ng/μL,完全可以满足PCR扩增限制酶切等实验的要求。     

适于滞育期蚕卵的基因组DNA提取方法

家蚕以卵滞育,蚕卵保存时间长,取样方便,但是DNA含量较低,抽提较为困难,利用不多。从处于滞育期(丙2胚子前)的蚕卵中快速高效地获取基因组DNA,对于提高家蚕研究效率具有实用价值。本试验对两种SDS法和一种CTAB法的抽提效果进行比较,结果表明SDS-II提取效果较好,单粒蚕卵提取基因组DNA即可用作PCR模板,5粒蚕卵获得DNA可以基本满足分子生物学研究需要。     

鹅血液基因组DNA的简单快速提取方法研究

本研究针对家禽血液红细胞有细胞核的特点,研究适合鹅血液基因组DNA的廉价、简便、快速的DNA提取方法,并为其它禽(鸟)类相关操作提供参考。通过裂解细胞,利用简便快速的操作方法将蛋白质和核酸分离,去除杂质,沉淀核酸,获得大量基因组DNA,并通过基因扩增检测其质量和效果。该方法提取的基因组DNA电泳检测条带明量,无拖尾,含量及纯度均较高,能够用于分子生物学实验研究。与传统的酚-氯仿DNA抽提方法相比,本实验建立的血液基因组DNA提取方法具有成本低、操作步骤简便和快速的特点,特别是对大量样品的提取更为实用。     

利用SSR标记分析一年生黑麦草遗传多样性的取样策略

一年生黑麦草(Lolium multiflorum)为异花授粉植物,其DNA多态性研究的取样策略与遗传多样性分析的可靠性和效率直接相关。从一年生黑麦草国家审定品种“长江2号”和“特高”中,分别提取50个单株DNA,同时提取5、10、15、20、25和30个单株叶片混合样本的DNA,利用SSR标记进行遗传多态性分析。研究结果表明,取样梯度间多态性信息含量指数(PIC)差异不显著(P>0.05),但当混合单株样本为20株时,PIC达到最高值;SSR平均等位基因数和单个SSR座位上等位基因种类随样品内单株混合数目增加而增加,当取样量在20株及以上时,等位基因数和等位基因种类趋于稳定,电泳图谱表现基本一致;不考虑稀有等位基因（基因频率≤10%）的漏检,采用20株混合样本能够最大限度保持检出较高频率（＞10%）等位基因,且重演性较好。鉴于此,利用SSR标记分析一年生黑麦草遗传多样性时采用20个单株混合样提取的DNA样本能够有效反映群体间差异。     

基于SCoT标记分析紫花苜蓿遗传多样性的取样数量

本研究采用目标起始密码子多态性(Start codon targeted,SCoT)标记,对600份不同紫花苜蓿(Medicago sativa L.)材料(共6个种质)的基因组DNA进行扩增,分析了不同取样数量下(10,15,20,30,40,50,60,70,80,90和100)紫花苜蓿的遗传多样性,旨在筛选出代表紫花苜蓿种群遗传多样性的最佳取样单株数。结果发现,6条SCoT引物共扩增出80个条带,平均每条引物的扩增条带数为13.3个,多态性条带百分率为100%。在评价不同取样梯度下的紫花苜蓿遗传多样性覆盖度时发现,当取样数量增加至40时,6个供试紫花苜蓿种质的遗传多样性覆盖率均达到95%以上。此外,本试验采用UPGMA与STRUCTURE聚类分析方法,分析了不同取样梯度下紫花苜蓿的遗传结构。结果表明,样本量为40时能够有效反映紫花苜蓿群体间差异。     

Routes and prevalence of shedding of psittacine beak and feather disease virus.

Psittacine beak and feather disease (PBFD) virus was recovered from the feces and crop washings from various species of psittacine birds diagnosed with PBFD. High concentrations of the virus also could be demonstrated in feather dust collection from a room where 22 birds with active cases of PBFD were being housed. The virions recovered from the feces, crop, and feather dust were confirmed to be PBFD virus by ultrastructural, physical, or antigenic characteristics. Virus recovered from the feather dust and feces hemagglutinated cockatoo erythrocytes. The specificity of the agglutination was confirmed by hemagglutination inhibition, using rabbit antibodies against PBFD virus. During the test period, 26% (8 of 31) of the birds screened were found to be excreting PBFD virus in their feces, and 21% (3 of 14) of crop washings were positive for PBFD virus. Some birds in the sample group had active cases of diarrhea, whereas others had normal-appearing feces. Diarrhea was found to be the only significant indicator of whether a bird was likely to be excreting virus from the digestive tract. These findings suggest that exposure of susceptible birds to PBFD virus may occur from contact with contaminated feather dust, feces, or crop secretions. Viral particles that were morphologically similar to parvovirus (20- to 24 nm-icosahedral nonenveloped virions) also were recovered from feces of some of the birds.     

Isolation of genomic DNA from feathers.

The use of feathers in veterinary clinical practice simplifies the sampling of avian genomic DNA, especially when blood extraction is difficult because of the age or the size of the bird. A rapid and accurate protocol was used to isolate high-quality genomic DNA from feathers. The technique includes a lysis step of the feather quill, which differs in temperature and time of incubation depending on the feather size. Purification of genomic DNA is performed with phenol: chloroform: isoamyl alcohol extraction and ethanol precipitation. This protocol consistently provided significant amounts of high-quality genomic DNA from more than 800 birds belonging to 120 different species. Genomic DNA isolated with this method was used for Southern blotting and also in several polymerase chain reaction systems devoted to sex determination and paternity testing.     

Detection of DNA of the finch polyomavirus in diseases of various types of birds in the order Passeriformes

Between 2000 and 2004 a disease occurred in an aviary in Germany affecting various bird species belonging to the order Passeriformes including Collared Grosbeaks (Mycerobas affinis), Eurasian Bullfinches (Pyrrhula pyrrhula griseiventris), Brown Bullfinches (Pyrrhula nipalensis), Grey-headed bullfinches (Pyrrhula erythaca) and Yellow-bellied Tits (Periparus venustulus).The major clinical signs included increased mortality of fledglings and young birds, as well as feather disorders and feather loss in adult birds. In addition, adult Eurasian Bullfinches showed in one year a disease course, in which the major symptom was inflammation of the skin beginning on the basis of the beak and spreading over the head occurring a few days before death. Bacteriological and parasitological investigations did not reveal any consistent findings. Using a newly developed polymerase chain reaction protocol, DNA of the recently discovered finch polyomavirus (FPyV) was demonstrated in several affected birds. Because of the consistent detection of FPyV-DNA and the similarity of the symptoms with those observed during infection with the closely related avian polyomavirus in other bird species, an etiological role of FPyV in the observed disease is assumed.     

Preen gland function in layer fowls: factors affecting preen oil fatty acid composition

1. Preen oil derived from the preen gland has previously been shown to differ in its composition between birds of different ages, sex and diet. As a part of a larger study on preening behaviour and its components, the relative percentages of fatty acids in preen oil were examined in laying hens that differed in age, beak trimmed status, feather pecking status (pecker and pecked) and method of sampling (either direct from the preen gland or from lipid extracted from feathers). 2. Five experiments are described. Fatty acids extracted from lipid were analysed by gas chromatography (GC). Fatty acid composition was affected by age, by whether or not a bird was feather pecked (but not if it was a feather pecker) and by lipid source. 3. Changes in preen oil composition with age (shown here) may be due to changes in circulating concentrations of hormones. Differences in preen oil composition between feather pecked and non-pecked birds may influence plumage odour and therefore taste, making the plumage of some birds more attractive to pecking than others. 4. The proportions of some fatty acids derived from feathers differed to those extracted from the preen gland. Lipid found on feathers is most likely a combination of that from the preen gland and from sebaceous secretions from the skin.     

目标取样法和扫描取样法在圈养黑熊行为研究中的比较

应用目标取样法和扫描取样法同时对5只圈养黑熊(Ursus thibetanus)的15种行为进行了观察和记录,并对两种方法所得结果进行比较和讨论,评估两种方法在动物行为学研究中的可靠性。结果表明:两种方法所得的行为实际频次间存在显著差异(χ2=4.388,P=0.036,df=1),相对频次间无显著差异(χ2=0.468,P=0.494,df=1);行为持续时间的长短是影响两种方法所得结果间差异的主要因素,其与相对偏差值(Rela-tive Deviation,RD)之间存在显著的负相关关系(实际频次:r=-0.900,P=0.000,df=13;相对频次:r=-0.898,P=0.000,df=13);两种方法所得行为发生频次间存在显著正相关关系(r=0.965,P=0.000,df=13);扫描取样法所得行为间的差异性分析结果中70.5%与目标取样法的分析结果一致,29.5%存在分歧,且在分歧组中,参与差异性分析的两种行为的持续时间之差极显著大于一致组(χ2=10.082,P=0.001,df=1)。     

内蒙古敖汉旗地区基础母牛存栏量调查方案的研究

对畜禽存栏量的调查是农业统计调查的重要内容之一,其统计调查方法主要是抽样调查。本研究旨在通过实例抽样调查,拟出合适的抽样方法对全国的基础母牛存栏量进行调查估计。采用二阶PPS(sampling with probability proportional to size)抽样、分层等比例抽样、分层二阶典型抽样对内蒙古敖汉旗肉牛存栏量进行调查,比较3种方法的准确度。结果表明, 在此3种方法10%的样本量下进行比较时,3种抽样方法按相对误差大小顺序排列为:分层二阶典型抽样＜分层等比例抽样＜二阶PPS抽样,其相对误差分层二阶典型抽样比分层等比例抽样、二阶PPS抽样分别小60.38%、92.83%;置信区间按大小顺序排列为:分层二阶典型抽样＜分层等比例抽样＜二阶PPS抽样。总体而言,分层二阶典型抽样的准确度相对较高,相对误差最小,仅为0.0412。故在实际应用当中,分层二阶典型抽样目前较为适用。     

Feather folliculoma in a captive-bred barn owl (Tyto alba).

Proliferative cutaneous lesions are frequently encountered in examination of avian species. Tumors of the skin have been reported in many bird species, although comparatively less is known about the incidence of integumentary neoplasia in nondomesticated species than in production or companion species. An adult male, 9-yr-old, captive-bred barn owl (Tyto alba) was presented for examination of a proliferative skin mass of several months' duration on the left wing. An excisional biopsy was performed, and the owl recovered uneventfully. Light microscopic examination of tissue sections of the mass revealed a focal, unencapsulated, well-demarcated, multiloculated mass that consisted of variably sized cystic spaces lined by stratified squamous epithelium and containing concentric laminations of keratin and foci of feather differentiation. A histopathologic diagnosis of feather folliculoma was made. This neoplasm has not previously been described in owls. Its incidence and documentation are significant in that this neoplasm should be considered as a differential diagnosis of proliferative skin lesions in this raptorial species.     

朝鲜鹌鹑黑素皮质素受体1基因多态性及其在不同羽色个体中的表达

通过混合样品DNA测序方法寻找黑素皮质素受体l（MC1R）基因的突变位点,采用Alu1-RFLP对突变位点在4种羽色（栗羽、黄羽、白羽、黑羽）鹌鹑群体中的基因分布进行了研究;利用qRT-PCR技术测定了MC1R基因在12日龄时4种羽色鹌鹑胚胎皮肤组织中的表达情况。结果表明,在鹌鹑MC1R基因上发现1个T/C突变位点,该位点没有导致编码蛋白氨基酸序列改变,A1u1-RFLP分析发现,该突变位点的不同基因型在4种羽色鹌鹑群体间的分布有显著差异（P〈0.05）。4种羽色鹌鹑皮肤组织中MC1R基因的表达量存在明显差异,栗羽鹌鹑皮肤组织中该基因的表达量明显高于黑羽鹌鹑皮肤中的表达量（栗羽〉黄羽〉白羽〉黑羽）。本试验没有发现导致日本鹌鹑黑羽突变的Glu92Lys突变位点,表明朝鲜鹌鹑的黑羽突变与报道的日本鹌鹑黑羽突变的机制不同,朝鲜鹌鹑的黑羽可能与其他基因的突变有关。