Association Analysis between Differences of GnRHR-Ⅱ Gene Expression with Reproduction Traits Heterosis in Duck Populations

The objective of this study was to clone the sequence of duck type Ⅱ gonadotropin releasing hormone receptor (GnRHR-Ⅱ) gene, and to analyze the association of its expression levels with reproduction trait heterosis. RT-PCR method was used to clone GnRHR-Ⅱ gene fragments, and the expression levels during early laying days and laying fastigium period in Gaoyou duck, Jinding duck and crossed populations were tested by Real-time PCR method. Sequencing and homology analysis showed that the cloned duck GnRHR cDNA was about 500 bp in length, and it belonged to the avian type Ⅱ GnRHR isoforms. It was 96% identical to the cGnRHR-Ⅱ sequence reported in domestic chicken. From the early laying days to laying fastigium period, expression levels of GnRHR-Ⅱ gene in pituitary increased for Jinding duck, Gaoyou duck and crossed populations, with the highest expression levels in Jinding×Gaoyou hybrid group (P<0.01) . The expression levels of GnRHR-Ⅱ gene in hypothalamus also increased for Jinding duck, Gaoyou duck, however, it decreased in two crossed populations. The expression levels of GnRHR-Ⅱ gene in ovary were significantly higher for Jinding duck and two crossed populations than Gaoyou duck (P<0.01), but it decreased during laying fastigium period. Compared to Jinding duck and Gaoyou duck breeds, the two crossed populations had positive heterosis in 42-week production and 42-week egg fertilization percent, and negative heterosis in the first laying age and 42-week fertilized egg hatching rate traits. It suggested that the short-lived higher expression of ovary GnRHR-Ⅱ gene before early laying days be correlated with heterosis of the first laying age and 42-week production traits, and its higher expression levels in pituitary could aid to keep laying fastigium.     

使用粪球形态指标快速判别天山马鹿的性别

通过观察来自于新疆乌鲁木齐市南山山区的未知性别的天山马鹿种群粪便样本,依粪球形态可分为2类：子弹状、枣核状。子弹状呈短粗型,长宽比较小;枣核状呈细长型,长宽比较大。140份样品中子弹状86份、枣核状54份。通过扩增SRY基因进行分子鉴定知雄性94份、雌性46份。形态分类与实际性别吻合率88.34%,即子弹状为雄性,枣核状为雌性。并以样品长、宽平均值的比值（R）为指标快速聚类,建立了判别方程,统计指出判别结果与实际性别吻合率85.48%。结果提示今后的野外研究可直接利用粪球形态判定天山马鹿性别,长宽比判别方程可作为辅助。     

High level of treatment failure with commonly used anthelmintics on Irish sheep farms

Background

In 2013 a Technology Adoption Program for sheep farmers was established to encourage the implementation of best management practices on sheep farms in Ireland. There were 4,500 participants in this programme in 2013. As part of this programme, farmers had the option to carry out a drench test to establish the efficacy of their anthelmintic treatment.

Results

Flock faecal samples were collected before and after treatment administration and gastrointestinal nematode eggs enumerated. In total there were 1,893 participants in the task, however only 1,585 included both a pre- and post-treatment faecal sample. Of those, 1,308 provided information on the anthelmintic product that they used with 46%, 23% and 28% using a benzimidazole (BZ), levamisole (LEV) and macrocyclic lactone (ML) product respectively. The remaining farmers used a product inapplicable for inclusion in the task such as a flukicide or BZ/LEV combination product. Samples were included for analysis of drench efficacy if the pre-treatment flock egg count was ≥200 eggs per gram and the interval post-sampling was 10–14 days for BZ products, 4–7 days for LEV products and 14–18 days for ML products. These criteria reduced the number of valid tests to 369, 19.5% of all tests conducted. If the reduction post-treatment was ≥95% the treatment was considered effective. Only 51% of treatments were considered effective using this criterion. There was a significant difference in efficacy between the anthelmintic drug classes with BZ effective in only 30% of treatments, LEV effective in 52% of cases and ML effective in 76% of cases.

Conclusions

Gastrointestinal nematode anthelmintic treatments, as practiced on Irish farms, have a high failure rate. There was a significant difference between the efficacies of the anthelmintic classes with BZ the least effective and ML the most effective.     

鸡CD4蛋白基因的克隆及其mRNA在淋巴器官中的表达

从鸡胸腺、脾脏、哈德氏腺、外周血液和法氏囊等免疫器官中分离淋巴细胞,应用RT-PCR对这些免疫器官中鸡CD4分子mRNA的表达进行了研究。同时,用RT-PCR对鸡脾淋巴细胞CD4分子cDNA进行了克隆和序列测定。结果表明,在上述器官中,法氏囊淋巴细胞不表达鸡CD4分子mRNA,其他器官中均有表达。通过序列分析表明,本试验扩增得到的基因为编码鸡CD4分子的基因。     

牛抵抗素基因的克隆及原核表达

从健康新生牛大网膜脂肪组织中提取总RNA,根据已发表的牛Resistin mRNA序列设计、合成引物,通过RT-PCR进行cDNA扩增,获得了343 bp的片段。将该片段克隆于pMD18-T载体后进行序列分析,确认PCR产物为牛Resistin cDNA。从阳性克隆中提取质粒,经BamHⅠ和XhoⅠ双酶切,回收343 bp的目的片段,定向克隆到pGEX-6P-1表达载体中,提取质粒并再次转化到BL21(DE3)中,成功地筛选出阳性克隆。经IPTG诱导阳性菌,通过SDS-PAGE检测出牛的Resistin基因的表达。     

高邮鸭PRL基因内含子1的SNP检测及其与产蛋性状的相关分析

利用PCR—RFLP技术对高邮鸭催乳素（Prolactin，PRL）基因内含子1进行了SNP检测和测序分析，并对该基因与高邮鸭产蛋性状的相关性进行了研究。结果表明：PRL基因内含子1存在两个Dra Ⅰ酶切位点，其中1个位点具有DraⅠ多态性。该位点由于在1326位点发生了T→C的碱基突变，产生了AA、AB和BB3种基因型。基因型BB和等位基因B的频率最高；BB基因型个体30周龄蛋重极显著高于AB基因型个体（P〈0．01）；AB基因型个体的双黄率显著高于BB基因型个体（P〈0．05）；3种基因型间产蛋数、最长连产天数和开产体重无显著差异。     

H9N2亚型禽流感病毒NS1基因真核表达载体的构建及其在293细胞瞬时表达

在293细胞中瞬时表达A/Chicken/Henan/1001/2010（H9N2）NS1基因蛋白,并对表达蛋白活性进行测定。试验利用PCR技术从NS1-T载体质粒上扩增NS1基因,将其克隆至pCAGGS载体,构建重组质粒NS1-pCAGGS;经酶切和测序鉴定正确后,重组质粒NS1-pCAGGS与脂质体按照一定比例混合后转染293细胞,用间接免疫荧光方法对瞬时表达细胞进行荧光信号反应。结果显示,禽流感NS1基因可以很好地在293细胞中瞬时表达,具有良好的反应原性。     

霍乱弧菌DPO-PCR特异性检测方法的建立与应用

采用一种新型的引物设计方法——双启动寡核苷酸引物（dual-priming oligonucleotide,DPO）,以霍乱弧菌mdh基因为靶序列,建立了霍乱弧菌DPO-PCR特异性检测方法,分析了DPO引物退火温度不敏感性、检测灵敏度及特异性,并对检测方法进行了初步应用。灵敏度结果显示,DPO-PCR方法对霍乱弧菌的最低检出限为1.07×10^2 CFU/mL。退火温度不敏感性试验中,与常规PCR引物相比,DPO引物在45～65℃退火温度均能够高效扩增出靶基因片段。特异性结果显示,DPO-PCR方法的特异性比常规PCR方法强,不产生任何非特异性扩增。利用建立的霍乱弧菌DPO-PCR检测方法对采集的550份样本进行检测,检出43份霍乱弧菌阳性样本,经行业标准法（SN/T2425-2010）复检,检测结果相同,表明所建立的DPO-PCR检测方法具有良好的实用性,为霍乱弧菌的快速准确检测提供了新途径。     

金黄色葡萄球菌临床分离耐氟喹诺酮类药物株的gyrA和grlA基因突变

对 5株临床分离的耐氟喹诺酮类药物的金黄色葡萄球菌 (MIC值范围 :4～≥ 12 8mg/ L) ,提取各细菌染色体DNA,对 gyr A和 grl A基因进行 PCR扩增 ,产物与 p MD18- T载体连接 ,转化至大肠杆菌 JM10 9感受态细胞 ,筛选阳性克隆 ,测序。序列分析结果表明 ,5株菌均发生了基因突变。突变位点 grl A:Ser80 (TCC)→ Phe(TGC) ,Ser80 (TCC)→ Tyr(TAC) ;gyr A:Ser84 (TCA)→ L eu(TTA )、Ser84 (TCA)→ Ala(GCA)、Glu88(GAA)→ L ys(AAA) ,其中 2株对氟喹诺酮类药物的 MIC值低 (4～ 6 4 m g/ L) ,均发生 grl A单一点突变 ,其余 3株在 gyr A和 grl A上产生多突变位点 ,且对氟喹诺酮类药物的 MIC值较高 (8～≥ 12 8mg/ L )。说明单一的点突变只能引起低水平或中等水平的耐药 ,高水平的耐药需要多位点的突变。 5株耐药菌株均发生 grl A Ser80→ Tyr(Phe)点突变 ,提示环丙沙星、诺氟沙星等氟喹诺酮类药物作用于金黄色葡萄球菌的首要靶酶是拓扑异构酶 Iv