绵羊BMP15基因B2突变可视化检测技术的建立

骨形态发生蛋白15（bone morphogenetic protein 15,BMP15）基因突变对绵羊排卵率、产仔数以及繁殖力有很大影响,本研究旨在建立快速可视化检测BMP15基因B2突变的技术。将改良的扩增阻滞突变系统（amplification refractory mutation system,ARMS）与核酸染料SYBR Green Ⅰ结合,针对BMP15基因B2突变设计ARMS特异性引物,使引物下游3'端的最后一个碱基为A,并在下游引物3'端的第3位碱基处设计额外的错配,以提高引物的特异性;经ARMS PCR扩增后,向产物中加入SYBR Green Ⅰ,通过肉眼观察PCR管内的颜色变化来检测BMP15基因的B2突变。经测序发现所采集的样本均为野生型,因此,利用重叠延伸PCR构建BMP15基因B2突变型模板,测序结果显示BMP15基因B2突变型模板构建成功。ARMS PCR结果显示,ARMS特异性引物能够只扩增突变型模板,而不会扩增野生型模板,且扩增效果良好。ARMS PCR扩增后加入SYBR Green Ⅰ发现已知野生型模板与阴性对照一致,呈SYBR Green Ⅰ原始颜色橙黄色,而已知突变型模板呈亮绿色,且色差明显,肉眼可辨。用建立的可视化检测BMP15基因B2突变的技术检测50个小尾寒羊基因组DNA （随机向其中20个样本中加入构建的BMP15基因B2突变型模板）,将可视化检测的突变型样本编号与混合样本时记录的编号对比,结果表明该技术能够准确检测绵羊BMP15基因的B2突变,且准确率高达100%。将构建的BMP15基因B2突变型的模板进行梯度稀释,对本试验可视化检测体系的灵敏度进行检测,发现ARMS可视化检测技术的灵敏度达到0.006 ng/μL。因此,本研究建立的技术能够可视化地检测绵羊BMP15基因B2突变,且准确率高,有望为高繁殖力绵羊的选育提供技术支持。     

猪戊型肝炎病毒ORF3蛋白影响HepG2细胞核黄素代谢的lncRNA-mRNA调控网络的鉴定

为初步鉴定并挖掘出猪戊型肝炎病毒(Hepatitis E virus,HEV)ORF3蛋白影响HepG2细胞核黄素代谢信号通路的lncRNA-mRNA调控网络,本试验通过构建腺病毒过表达载体,制备高滴度过表达腺病毒,介导猪 HEV-ORF3在HepG2细胞中实现过表达。Western blotting检测ORF3蛋白过表达成功后,运用lncRNA高通量组学测序,筛选出差异表达的lncRNA并进行靶向差异基因预测,对lncRNA靶向差异基因进行GO功能和KEGG通路富集分析,初步鉴定出与核黄素代谢信号通路相关的lncRNA-mRNA调控网络。Western blotting结果显示,在约为12 ku处出现目的条带,说明成功实现腺病毒介导猪HEV-ORF3在HepG2细胞中过表达。lncRNA高通量组学测序结果显示,共发现102个显著差异表达lncRNAs表达量上调,80个显著差异表达lncRNAs表达量下调。GO功能和KEGG通路富集分析显示,初步挖掘出lncRNA(MSTRG.13995.2)和lncRNA(MSTRG.1960.1)可能是与核黄素代谢信号通路相关的显著差异表达lncRNA,分别通过顺式调控其靶向基因APC-5和FLAD1来影响核黄素代谢信号通路。本试验初步鉴定出lncRNA(MSTRG.13995.2)-APC5和lncRNA(MSTRG.1960.1)-FLAD1可能是影响HepG2细胞核黄素代谢信号通路的lncRNA-mRNA调控网络。     

Preparation and Identification of Canine Parvovirus NY Strain VP2 Protein Polyclonal Antibody

This study was aimed to prepare canine parvovirus (CPV) VP2 protein polyclonal antibody.The recombinant expression vector pET28a-CPV-VP2 was constructed and transfromed into E.coli BL21 (DE3),the expression of recombinant proteins was induced by IPTG from which the fusion protein was identified by SDS-PAGE.The target protein was purified and emulsify with adjuvant,the prepared immunogen was inoculated into rabbit by subcutaneous injections to prepare of VP2 protein specific polyclonal antibody.The immuno-activity,titers,neutralization titers of the prepared polyclonal antibody were determined by immunoperoxidase monolayer assay (IPMA).The results showed that the expressed recombinant protein VP2 (rVP2) existed in the form of inclusion body with a molecular weight of 72 ku.The prepared polyclonal antibody titer was 1 600 dilution,the virus titer was 10⁷ TCID₅₀/mL,the neutralizing titer was 1∶2 884.The antibodies showed specific reaction with CPV.In conclusion,rVP2 specific polyclonal antibody showed wonderful immunocompetence,specificity and neutralizing activity,providing foundation for the development of genetic vaccine and clinical therapeutic method.     

Effects of lncRNA-GTL2 Transgenosis on Gut Microbiota Composition in Mice

This research was aimed to study whether the lncRNA would have effects on the structure and constitution of the intestinal microbial colonies in mice.High throughout sequencing was used to sequence and analyze the intestinal microbial colonies in both transgenic and non-transgenic mice of three months old.We conducted comparison and t-test at the level of phylum and genus.The result showed that transferred into long non-coding RNA genes GTL2 (lncRNA-GTL2),the mouse intestinal microbial colony structure and composition had no significant differences in the overall,within the same gender,but there were individual differences.Therefore,this study didn't find that long non-coding RNA transfered had a significant impact on the intestinal microflorain the phylum and genus level.     

维生素B_2对中华蜜蜂工蜂寿命及学习记忆能力的影响

本试验旨在研究维生素B_2对中华蜜蜂工蜂寿命及学习记忆能力的影响。试验分为工蜂饲养试验和学习记忆试验2部分。工蜂饲养试验:将1日龄中华蜜蜂工蜂分成3组,每组5个重复,每个重复约200只。对照组(Ⅰ组)饲喂1∶1的糖水,2个试验组在1∶1糖水的基础上分别添加100(Ⅱ组)、400 mg/kg(Ⅲ组)的维生素B_2,每天记录各组工蜂的死亡情况,直到全部死亡。工蜂学习记忆试验:分组方法同工蜂饲养试验,并按照工蜂饲养方法对1日龄工蜂喂养7 d后,利用吻伸反应方法测定工蜂短时学习记忆及长时学习记忆能力,并通过荧光定量PCR检测学习成功后工蜂学习记忆相关基因的相对表达量。结果表明:工蜂的平均寿命随维生素B_2添加量的升高而延长,而且试验组(Ⅱ组、Ⅲ组)的平均寿命显著高于对照组(P0.05),但Ⅱ组与Ⅲ组间差异不显著(P0.05)。Ⅲ组工蜂的长时学习记忆及短时学习记忆能力均显著高于对照组和Ⅱ组(P0.05),而且Ⅱ组工蜂的长时学习记忆能力也显著高于对照组(P0.05)。Ⅲ组工蜂的多巴胺受体基因2(Acdop3)、c AMP反应元件结合蛋白(Ac CREB)相对表达量均显著高于对照组和Ⅱ组(P0.05),而且Ⅱ组工蜂的Acdop3相对表达量也显著高于对照组(P0.05)。由此得出,维生素B_2影响中华蜜蜂工蜂的寿命及学习记忆能力,在人工饲喂蜂群时需要提供适量的维生素B_2。     

Comparison of emission estimates for non‐CO2 greenhouse gases from livestock and poultry in Korea from 1990 to 2010

It has often been claimed that non‐carbon dioxide greenhouse gases (NCGGs), such as methane, nitrous oxide and fluorinated greenhouse gases, are significant contributors to climate change. Here we nvestigate emission estimates of methane and nitrous oxide from livestock and poultry production, which is recognized as a major source of those NCGGs, in Korea over the period of 1990 through 2010. Based on the data on livestock and poultry populations, emission estimates of methane and nitrous oxide are first derived based on the Tier 1 approach. Then, the Tier 2 approach is adopted to obtain emission estimates of methane and nitrous oxide from cattle, which are known to be the largest sources of these NCGGs and account for about 70% of emissions from livestock and poultry in Korea. The result indicates that the Tier 2 estimates of methane and nitrous oxide emissions from enteric fermentation and manure management are significantly different from the Tier 1 estimates over the analysis period.     

Effect of prostaglandin F2alpha on ovarian, adrenal, and pituitary hormones and on luteal blood flow in mares

The effect of a single injection of prostaglandin F2alpha (PGF) during mid-diestrus on systemic concentrations of progesterone, LH, FSH, estradiol, and cortisol and on blood flow to the corpus luteum was studied in 10 controls and 10 PGF-treated mares. Blood flow was assessed by estimating the percentage of corpus luteum with color-Doppler signals of blood flow during real-time scanning of the entire structure and by the diameter of the vascular pedicle near its attachment to the ovary. Treatment was done 8 days after ovulation and 0 h was immediately before the treatment. Examinations and collection of blood samples were done at 0 h, every 5 min until 1h, and then at 1.5, 2, 4, 8, 12, 24, 48, and 72 h. The concentrations of estradiol did not change, but progesterone, LH, FSH, and cortisol increased significantly within 5 min. Concentrations of LH and FSH in the PGF group remained elevated until a temporarily lower concentration at 8 or 4h, respectively, rebounded to 12h, and then slowly decreased. Cortisol remained elevated, until a decrease between 1 and 4h. Progesterone in the PGF group increased significantly until 10 min after 0 h and then decreased by 40 min to below the concentrations in controls. Within the PGF group, progesterone decreased significantly by 45 min to below the concentrations at 0 h. The values for each of the two indicators of blood flow did not differ significantly between the PGF and control groups until a decrease at 24h in the PGF group. Results did not support the hypothesis that the immediate transient post-PGF increase in progesterone was associated with an increase in luteal blood flow. Luteolysis, as indicated by decreasing progesterone, began well before the beginning of a decrease in luteal blood flow.     

Transthoracic echocardiographic measurement of patent ductus arteriosus in dogs

BACKGROUND: Patent ductus arteriosus (PDA) size and morphology influence the selection of the kind and the size of the embolization device used to effect shunt closure. HYPOTHESIS: That echocardiographic measurement of PDA in dogs is accurate. ANIMALS: Forty-five client-owned dogs with PDA. METHODS: Prospective observational study. Echocardiographic and angiographic data were compared. RESULTS: Measurement of the ductus in color Doppler echocardiography (CD-E) and 2-dimensional echocardiography (2D-E) was achieved from left parasternal views in 43 of 45 unsedated dogs (96%). In these 43 dogs, the angiographic minimal PDA diameter was 3.72 +/- 1.59 mm, and the diameter of the PDA ampulla was 8.46 +/- 3.01 mm. The CD-E minimal PDA diameter ranged from 2.3 to 9.5 mm (median, 4.0 mm). There was a significant mean difference to the angiographic measurements (1.15 +/- 0.95 mm; P < .0001). An agreement in a 1-mm range was found in 21 of 43 dogs (48%). The 2D-E minimal PDA diameter was 3.73 +/- 1.78 mm, and the mean difference to the angiographic measurements was not significant (0.00 +/- 0.72 mm; P = .98). An agreement in a 1-mm range was found in 31 of 43 dogs (72%). The 2D-E measurement of the PDA ampulla revealed a significant mean difference to the angiographic data (1.95 +/- 2.43 mm, P < .0001). An agreement in a 2-mm range was found in 21 of 43 dogs (49%). CONCLUSIONS AND CLINICAL IMPORTANCE: The 2D-E from the left cranial parasternal view is an excellent noninvasive method to estimate the PDA minimal diameter before doing catheter intervention.