GLGI技术鉴定奶山羊乳腺中的к-酪蛋白基因

采用GLGI技术扩增本实验室构建的Long-SAGE标签库中的к-酪蛋白基因.使用SMARTTM RACE cDNA Ampliication Kit通过RT-PCR把奶山羊乳腺组织中的rnRNA逆转录成cDNA.分别用3′RACE和5′ACE扩增к-酪蛋白基因的3′端和5′端.扩增产物经TA克隆后转化大肠杆菌,随机挑取阳性克隆后进行测序分析.通过序列比对、拼接,获得全长的к-酪蛋白基因.应用GLGI技术成功获得了完整的奶山羊乳腺CSN3序列,为CSN3结构分析和功能研究奠定了重要基础.     

奶山羊乳腺组织中基因CCDC85B的克隆

克隆奶山羊乳腺中基因CCDC85B。从本实验室前期建立的基因文库中挑选出CCDC85B的17 bp标签,采用GLGI和RACE技术进行扩增,获得基因的全长。结果表明,扩增片段长度为1 282 bp序列,其中编码区为606 bp,编码202个氨基酸残基,BLAST比对结果显示,该序列与已知牛、人CCDC85B的同源性分别为100%和83%,证明此基因确为奶山羊乳腺组织中的CCDC85B,为该基因的进一步结构分析和功能研究奠定基础。     

奶山羊和荷斯坦奶牛乳腺组织mRNA差异显示体系建立

利用mRNA差异显示技术对关中奶山羊和荷斯坦奶牛泌乳末期乳腺组织进行差异表达基因分析,为进一步进行奶山羊泌乳分子生物学研究及筛选克隆差异表达基因、揭示膻味脂肪酸调控的分子机理奠定基础.研究通过对各影响因素进行单因素分析,建立了适合奶山羊和荷斯坦奶牛的乳腺差异显示分析体系,得到的最佳体系.在20 μL DDRT-PCRR反应总体系中各组分的添加量分别为:13.0 μL DEPC-H2O;1.0 μL 10×PCK Buffer(加Mg2+);1.5 μL dNTPs(各2.5 mmol/L);1.5 μL锚定引物H-T11M(20 μmol/L);1.5 μL随机引物(10 μmol/L);1.0 μL cDNA;0.5 μL Taq DNA Polymerase(2 U/μL).     

阪崎肠杆菌α-葡萄糖苷酶的原核表达及其多克隆抗体制备

以阪崎肠杆菌(ATCC29544)的基因组DNA为模板通过PCR扩增出α-葡萄糖苷酶基因malA,将其克隆入表达载体PET22b(+),构建重组表达质粒pET22b-malA将重组表达质粒转化大肠杆菌BL21 (DE3),进行优化表达.采用尿素洗涤包涵体并切胶回收纯化融合蛋白,然后再免疫新西兰大白兔制备多克隆抗体.通过ELISA测定效价,并通过免疫荧光法鉴定与菌体表面结合能力结果表明克隆的目的基因全长1677bp,与Genebank的malA比较具有100％的同源性.带His标签的融合蛋白以包涵体形式表达,其最优化的表达条件是37℃下用0.8mmmol/L IPTG诱导5h.ELISA测定效价为5×105;免疫荧光法鉴定表明多克隆抗体能与阪崎肠杆菌表面结合.本实验为阪崎肠杆菌的免疫磁珠检测奠定了基础.     

关中奶山羊乳主要成分的质量分数及理化特性研究

用Milko-Scan乳质综合测定仪对220份关中奶山羊生鲜乳的主要成分的质量分数进行了测定。结果表明,关中奶山羊生鲜乳中的脂肪、蛋白质、非脂乳固体、乳糖和干物质的平均质量分数分别为4.88%,2.74%,7.80%,4.36%和12.91%。乳的化学成分质量分数与饲养管理条件有密切关系。关中奶山羊乳中的脂肪质量分数和干物质质量分数呈正相关。同时对100份关中奶山羊生鲜乳的部分理化指标进行了测定,结果表明,关中奶山羊乳的比重为1.0285～1.0333,乳的滴定酸度为13.455±0.236°T。     

西农萨能奶山羊乳和荷斯坦牛乳挥发性游离脂肪酸组成比较及分子机理分析

以西农萨能奶山羊和荷斯坦牛为对象,研究山羊乳和牛乳挥发性游离脂肪酸组成的差异,并分析其分子机理.采用固相微萃取/GC-MS分析二者挥发性游离脂肪酸组成,对西农萨能奶山羊脂肪酸合成酶乙酰-CoA/丙二酰-CoA转移酶区域(AT-MT) cDNA克隆,对其AA序列及蛋白二级结构与荷斯坦牛进行比较.结果表明西农萨能奶山羊奶中癸酸(40.89 g/100 g)是影响其风味的主效成分.西农萨能奶山羊AT/MT AA序列、蛋白二级结构与荷斯坦牛存在明显差异.西农萨能奶山羊脂肪合成与代谢相关酶基因应是羊奶膻味形成的关键因素,传统育种和分子育种方法将是提高山羊乳品质的有效途径.     

烤烟ζ-胡萝卜素脱氢酶基因的克隆与生物信息学分析

利用同源克隆和电子克隆方法,从烤烟栽培品种韭菜坪2号(Nicotiana tobacum cul.Jiucaiping2)中克隆获得烤烟ζ-胡萝卜素脱氢酶(zeta-carotene desaturase,ZDS)基因的全长cDNA序列(命名为T-zds1),并对其编码蛋白的一级、二级进行了预测。该基因在GenBank中的登录号为JF975566,全长为2312 bp,编码的蛋白含588个氨基酸,蛋白登录号AEG73891。BLASTp搜索结果及进化分析结果表明,烤烟T-zds1基因编码氨基酸序列与番茄、向日葵、胡萝卜ZDS基因编码氨基酸序列的相似性分别为97%,92%,90%。     

奶山羊品种和养殖区对羊乳支链脂肪酸组成的影响

支链脂肪酸作为新兴生物活性物质备受关注。羊乳营养价值丰富,其支链脂肪酸组成和含量与母乳最为接近,分析影响羊乳中支链脂肪酸组成的因素对羊乳资源高值化利用具有重要的研究意义。本研究采用气相色谱-质谱法对萨能奶山羊、关中奶山羊2个品种和山东、陕西地区4个养殖区奶山羊的羊乳支链脂肪酸组成进行比较分析。结果表明:萨能奶山羊的羊乳中支链脂肪酸成分组成较关中奶山羊的羊乳中支链脂肪酸成分组成丰富,山东萨能奶山羊anteiso-C17∶0显著高于陕西省3个养殖区,陕西省3个养殖区羊乳的异构支链脂肪酸占比存在显著差异性(P<0.05)。本文从支链脂肪酸组成角度揭示了萨能奶山羊的乳营养更丰富,陕西地区萨能山羊乳和山东地区关中山羊乳中支链脂肪酸组成更加贴近母乳。     

嗜热链球菌胸苷酸合成酶基因的克隆与序列分析

本研究以嗜热链球菌(S．thermophilus)染色体DNA为模板,利用PCR技术扩增出胸苷酸合成酶(thymidylate synthase,thyA)基因,将其克隆入T载体中,转化DH5α,筛选阳性克隆,提取质粒,进行酶切鉴定,PCR扩增鉴定,进行测序,与已知序列进行同源性比较,结果表明成功克隆了thyA基因,全长900bp,与国外报道的S．thermophilus ATCC19258 thyA基因同源性达99．9%。这为构建以thyA基因为选择压力的非抗生素抗性载体提供了理论依据。     

球等鞭金藻Cry1-like基因克隆及生物信息学分析

隐花色素(cryptochrome)基因家族作为蓝光受体的一大类,广泛存在于动植物体内,介导了植物和动物的各种光响应。文章通过实验克隆出球等鞭金藻(Isochrysis galbana)一条隐花色素基因,该基因DNA全长2916 bp,cDNA全长2316 bp,由771个氨基酸组成。对其进行相关生信分析,预测该基因是球等鞭金藻Cry1基因(文中称为Cry1-like基因),克隆得到的Cry1-like基因能够为后续球等鞭金藻隐花色素基因结构与功能研究提供依据。     

Caprine and ovine Greek dairy products: The official German method generates false-positive results due to κ-casein gene polymorphism

Caseins are widely used for species identification of dairy products. Isoelectric focusing (IEF) of para-κ-casein peptide is used as the official German method for the differentiation between caprine (isoform A) and ovine (isoform B) dairy products, based on their different isoelectric points. The discrimination between Greek goat and ewe dairy products using IEF has, however, been shown to be problematic because of the existence of the ewe isoform in milk from Greek indigenous dairy goats. This could be due to nucleotide polymorphisms within the goat κ-casein gene of Greek indigenous breeds, which alter the isoelectric point of the para-κ-casein peptide and lead to false positive results. Previous DNA analysis of the goat κ-casein gene has shown high levels of polymorphism; however, no such information is available for Greek indigenous dairy goats. Therefore, 87 indigenous dairy goats were sequenced at exon IV of κ-casein gene. In total, 9 polymorphic sites were detected. Three nonsynonymous point mutations were identified, which change the isoelectric point of the goat para-κ-casein peptide so that it appears identical to that of the ewe peptide. Ten composite genotypes were reconstructed and 6 of them included the problematic point mutations. For the verification of genetic results, IEF was carried out. Both goat and ewe patterns appeared in the problematic genotypes. The frequency of these genotypes could be characterized as moderate (0.23) to high (0.60) within Greek indigenous breeds. However, this is not an issue restricted to Greece, as such genotypes have been detected in various non-Greek goat breeds. In conclusion, IEF based on the official German method is certainly inappropriate for ovine and caprine discrimination concerning Greek dairy goat products, and consequently a new method should be established.     

中国奶羊业发展战略

分析了中国奶羊业的现状及问题，并有针对性地提出了中国奶羊业发展的８项战略，分别为：实验跨世纪的提高全民乳营养战略；北羊南进战略；养羊扶贫战略；奶山羊肉改战略，民族乳制品开发战略；羊奶进城战略；基地养羊战略；奶山羊产品系列开发战略。通过实施这些战略，将促使中国奶羊业更快更好地发展。     

Analysis of the MUC1 gene and its polymorphism in Capra hircus

The objective of our study was to demonstrate the existence of a repetitive region in the goat MUC1 gene and to develop a polymerase chain reaction (PCR) protocol to analyze its polymorphism in different breeds. Using 2 primers derived from the bovine MUC1 sequence, a PCR fragment was obtained and cloned. The sequence analysis shows that the repetitive region of goat MUC1 is an array of 60 bp repeats in accordance with the data reported for other species. The polypeptide sequences encoded by the consensus repeats of goat and bovine were exactly alike. A PCR protocol to improve the detection of goat MUC1 polymorphism was developed, and a total of 178 animals from 6 Italian breeds were analyzed. Fifteen different alleles, ranging in size from 1500 to 3000 bp, were found. The high number of alleles observed shows that the goat MUC1 is a hypervariable gene. These results are the basis for further investigations on the possible role of MUC1 polymorphism in the genetic control of disease susceptibility and production traits in the goat species.     

Past, present, and future perspectives of small ruminant dairy research

The objectives of this paper are to review small ruminant dairy research in relation to the dimensions of the dairy goat and dairy sheep industries in the United States and the world. At least 10 countries depend on goats and sheep for between 30 to 76% of total milk supply. Leading among developed countries is Greece producing 178 kg milk per person per year with 61% from sheep and goats. Most developing countries need research, extension service, and public support to improve apparent productivity of goats and sheep. Domestic supply from all milk sources is <100 kg/person per year, and annual apparent yields average <100 kg of milk/goat, <50 kg of milk/sheep, which makes supplies of animal protein and calcium from domestic sources very low. Statistical data on goat and sheep production for United States are not available. The small population of DHIA tested US dairy goats averaged in recent years >700 kg of milk/goat per year, and some dairy sheep breeds may produce as much as 650 kg/yr. The need for more milk availability appears to be reflected in the dramatic increases of dairy goat populations during the last 20 yr: 52% for the world, 56% for developing, 17% for developed countries, while sheep populations decreased by 3% for the world, by 6% in developed, but increased 14% in developing countries. Research has been sparse on the unique qualities of goat and sheep milk compared with cow milk. Much development work by various agencies has been devoted to reducing mortality and improving feed supplies in harmony with the environment; this work is mostly published in proceedings of scientific meetings, often not in English. Results have shown in many cases that dairy goats and dairy sheep can be very profitable, even in developing countries with difficult climate and topographical conditions.     

Sensory analysis and species-specific PCR detect bovine milk adulteration of frescal (fresh) goat cheese

The Brazilian market for dairy products made from goat milk is increasing despite the seasonality of production and naturally small milk production per animal, factors that result in high-priced products and encourage fraud. In Brazil, no official analytical method exists for detecting adulteration of goat dairy products with cow milk. The aim of this study was to design a strategy to investigate the adulteration of frescal (fresh) goat cheeses available in the Rio de Janeiro retail market, combining analysis of cheese composition and the perception of adulteration by consumers. Commercial goat cheeses were tested by using a duplex PCR assay previously designed to authenticate cheeses, by targeting the mitochondrial 12S ribosomal RNA genes of both species simultaneously. The PCR test was able to detect 0.5% (vol/vol) cow milk added during goat cheese formulation. The analysis of 20 locally produced goat cheeses (20 lots of 4 brands) showed that all were adulterated with cow milk, even though the labels did not indicate the addition of cow milk. To estimate the ability of consumers to perceive the fraudulent addition of cow milk, a triangle test was performed, in which cheeses formulated with several different proportions of goat and cow milk were offered to 102 regular consumers of cheese. Detection threshold analysis indicated that almost half of the consumers were able to perceive adulteration at 10% (vol/vol) cow milk. Effective actions must be implemented to regulate the market for goat dairy products in Brazil, considering the rights and choices of consumers with respect to their particular requirements for diet and health, preference, and cost.     

河南奶山羊乳酶活力和乳蛋白组分的研究

测定河南奶山羊乳的4种酶酶活力,并采用SDS-PAGE方法对乳中主要蛋白组分进行分析。结果表明：在初乳和常乳中4种酶酶活力分别为：γ-谷胺酰转肽酶(γ-GT)为322.46U/100mL和247.71U/100mL,碱性磷酸酶(AKP)为248.62U/100mL和200.14U/100mL,过氧化物酶(LP)为281.76U/mL和205.07U/mL,淀粉酶(AMY)为71.20U/100mL和22.15U/100mL。初乳中AKP活力显著高于常乳(0.01＜P＜0.05),γ-GT、LP、AMY的活力极显著高于常乳(P＜0.01)。主要乳蛋白按分子质量由小到大均依次显示出乳清蛋白(α-La)、β-乳球蛋白(β-LG)、IgG-L(轻链)、酪蛋白(CN)、IgG-H(重链)、血清白蛋白(SA)等区带。在初乳和常乳中的含量分别为：α-La为4.52%和4.51%,β-LG为18.78%和9.59%,IgG-L为7.52%和3.95%,CN为11.29%和11.49%,IgG-H为6.95%和4.07%,SA为5.48%和5.39%。乳中β-LG相对含量较高,达到20.96%,α-La含量较低。初乳中的免疫球蛋白IgG(IgG-L、IgG-H)相对含量显著高于常乳(P＜0.01或0.01＜P＜0.05),β-LG极显著高于常乳(P＜0.01)。乳球蛋白和免疫球蛋白随着泌乳天数的增加呈现明显的下降趋势,而其他蛋白变化不显著。乳蛋白组分的相对含量在山羊个体间存在差异。     

Consumer attitudes toward dairy products from sheep and goats: A cross-continental perspective

This study aimed to assess consumer knowledge, attitudes, and perceptions toward dairy products from sheep and goats. A web-based survey was conducted in Latin America (Mexico and Chile), Europe (Italy, Spain, Greece, and Denmark), and Asia (Bangladesh). From March to June 2021, adult participants answered an online survey available in 5 languages. In total, 1,879 surveys were completed. Categorical and ordinal data were analyzed as frequencies and percentages. To determine the relationship between the variables for purchasing and consumption behaviors of respondents who declared that they consume dairy products, a multiple correspondence analysis was carried out. Most completed surveys were from Mexico and Italy (30% and 33.7%, respectively). Most respondents were between 18 and 29 yr old, female, highly educated, and employed. The majority of respondents (70.8%) declared that they consume dairy products from small ruminants. Consumers preferred products from both sheep and goats (49.4%); however, it was observed that in Mexico, Denmark, and Bangladesh, more than 50% preferred goat dairy products. The most-consumed products were mature and fresh cheeses. Mature cheese was the most-preferred product in Chile; in Mexico, Italy, Greece, and Denmark, it was fresh cheese. Unlike the rest of the countries, in Bangladesh, dairy product consumption from small ruminants was observed by more than 30% of respondents. In Mexico, a higher percentage of people do not consume sheep or goat dairy products because they are unfamiliar with them. In Mexico, Chile, and Bangladesh, limited market availability was also a variable responsible for nonconsumption. In European and Asian countries, sheep and goat dairy products are not consumed because consumers dislike them, in addition to a greater awareness of sustainability and climate change issues. The multiple correspondence analysis defined 5 dimensions. Dimension 1 was associated with the geographic location of the respondent (country and continent), the type of milk (sheep or goat), and the consideration of well-being and health as characteristics associated with the consumption of dairy products from small ruminants. Dimension 2 was associated with the respondent's country of origin and the frequency of consumption. Dimension 3 was associated with gender, education, and employment status. Dimension 4 was associated with the respondent's age, the association of the “healthy” concept of sheep and goat dairy products, and the consideration of the nutritional benefits of dairy as responsible for considering them healthy. Dimension 5 was associated with a “strong smell and taste” of sheep and goat dairy products. This study showed that consumer attitudes toward dairy products from sheep and goats vary between continents. In conclusion, results showed consumer interest in animal welfare and environmental impact issues related to small ruminant farming as well as a general attraction to local products. It seems that these factors contribute to consumers' perception of the quality of dairy products, so the industry and select farmers should carefully consider incorporating them into their supply chain.     

Application of a polymerase chain reaction to detect adulteration of ovine cheeses with caprine milk

A polymerase chain reaction (PCR) procedure has been applied for the detection of caprine milk in ovine cheeses by using primers targeting the mitochondrial 12S ribosomal RNA gene. The use of a primer pair specific for goat in PCR analysis of cheese samples, enabled the amplification of a goat 122 bp fragment with a sensitivity threshold of approximately 1%, whereas no amplification signal was achieved with sheep's, cow's and water buffalo's milk DNA. This study demonstrates the usefulness of the proposed PCR assay for the qualitative detection of goats’ milk in ewes’ cheeses, and may therefore provide a simple and accurate approach applicable to the authentication of cheese or other dairy products in routine monitoring programs.