Multi‐level mixed models for evaluating factors affecting the mortality and weaning weight of piglets in large‐scale commercial farms in central China

This study investigated the factors affecting piglet mortality (square root of mortality, SQRM) and average weaning weight (AWW) in commercial farms in central China. Information on sow diets, management and climate from 2478 weaning batches completed in 16 pig farms was collected from 2009 to 2013. Multi‐level mixed models, which included batch level (level 1) and farm level (level 2), were used to analyze the factors associated with SQRM and AWW. The mean values of SQRM and AWW were 2.52% (SD = 0.96) and 7.31 (SD = 0.77), respectively. Lactation sow diets supplemented with oregano essential oils (OEOs) decreased the SQRM (P < 0.05) and increased the AWW of piglets (P < 0.01). The SQRM was lower in period 2 (June to September, hot) than in period 1 (February to May, warm) and period 3 (October to January, cold; P < 0.05 and 0.001, respectively). The AWW was lower in periods 2 and 3 than in period 1 (P < 0.01). In conclusion, supplying OEOs to lactation diets can increase the weaning weight and reduce the mortality of piglets. The sources of variations in SQRM and AWW are of greater concern in the warm season than in the hot season.     

猪瘟病毒E2基因的克隆及分子特性分析

为研究四川地区猪瘟病毒（classical swine fever virus,CSFV）及E2基因的遗传变异情况,试验对CSFV阳性病料抽提RNA,通过RT-PCR扩增CSFV E2基因并将其克隆到pMD18-T载体,经菌落PCR、双酶切、序列测定后利用生物信息学软件分析了E2基因的分子特性。结果显示,扩增的E2基因大小为1 125 bp,编码375个氨基酸,与GenBank中CSFV GXWZ02、CSFV/2.1、HEBZ、YC11WB、SXYL2006、0406/CH/01/TWN亲缘关系较近,核苷酸同源性分别为98.3%、96.5%、94.7%、92.4%、92.1%和90.9%,而与疫苗株HCLV E2的同源性较低,为87.6%。密码子偏向性分析显示,E2基因的ENc值为53.161,密码子使用频率与大肠杆菌、酵母菌和人差异较大的分别有33、25和25个;此外,E2基因共有34个稀有密码子,且有多个稀有密码子多次串联成簇的情况出现。结果表明,E2基因在密码子使用中整体上不存在较明显的偏向性,且密码子使用模式更接近真核生物,本试验克隆的E2基因与疫苗株相比,部分核苷酸出现了变异,但主要抗原域氨基酸未发生改变,该结果为进一步开展CSFV E2功能研究及后期基因工程苗的研制提供了基础数据。     

2016-2017年江西地区猪繁殖与呼吸综合征病毒分子流行病学调查及ORF5基因变异分析

为了解近年来江西地区猪繁殖与呼吸综合征病毒（porcine reproductive and respiratory syndrome virus,PRRSV）分子流行病学和其遗传变异情况,本次调查于2016-2017年从江西省各地区规模化猪场采集453份疑似猪繁殖与呼吸综合征（PRRS）的病料,采用RT-PCR方法对所有病料进行检测。结果发现,其中321份病料为PRRSV阳性,阳性率为70.86%,各地区的阳性率在19.15%~84.85%之间。挑选14份阳性样品测序后,经ORF5基因序列分析,江西地区各PRRSV毒株ORF5基因的核苷酸同源性为83%~100%,PRRSV流行毒株与参考毒株的同源性在59.9%~98.5%之间。基于ORF5基因的进化树分析表明,14个测序毒株均为美洲型毒株,其中有4株为基因亚型Ⅰ,即高致病性毒株（HP-PRRSV）;3株为基因亚型Ⅱ,即经典毒株;3株为基因亚型Ⅲ,即NADC30-like毒株;4株为新出现的基因亚型Ⅳ。氨基酸序列比对分析表明,基因亚型Ⅰ、Ⅱ、Ⅲ和Ⅳ毒株ORF5基因编码的GP5蛋白氨基酸在3个表位及2个重要的抗原相关区域存在较大变异,其中以NADC30毒株为代表的基因亚型Ⅲ毒株和以GD1404毒株为代表的基因亚型Ⅳ毒株均表现出独有的氨基酸变异,这些变异可能会影响GP5蛋白的免疫原性。本次调查结果表明,2016-2017年江西地区PRRSV流行出现了新形势,美洲型毒株出现了多基因亚型共同存在的局面,以高致病性毒株（HP-PRRSV）为主,NADC30-like毒株和新基因亚型等新毒株的比例较高,同时还存在经典毒株;持续实时监测PRRSV的毒株流行和变异情况,可为临床诊断、药物和疫苗开发及PRRS的科学防控提供依据。     

酪氨酸磷酸化在精子获能过程中的作用

在精子细胞发生顶体反应并使卵母细胞受精之前,获能是一个重要的生理先决条件。获能是精子在雌性生殖道中进一步成熟的复杂现象,其赋予精子以增强活性的能力,使精子能够与卵母细胞透明带（ZP）相互作用,进行顶体反应并与卵母细胞质膜融合,进而完成受精过程。然而精子获能的分子机制十分复杂,目前还未完全明确,但获能后的精子会有诸多结构及生化方面的变化,如蛋白酪氨酸磷酸化、精子膜胆固醇外流、活性氧的产生及精子膜超极化。蛋白质通过磷酸化或去磷酸化调节精子获能和顶体反应等一些重要的现象,这是精子到达、结合、穿透和融合卵母细胞所必需的过程。因此蛋白磷酸化是获能的一个非常重要的过程,尤其是在酪氨酸残基处的磷酸化是获能过程中发生的最重要的事件之一,且酪氨酸磷酸化可能是细胞中信号转导途径的主要甚至是唯一的指标。作者主要针对蛋白磷酸化、精子中酪氨酸磷酸化的发现、作用和定位,以及影响获能过程中酪氨酸磷酸化的几个因素进行阐述。     

Metabolite profiling of the response to high-nitrogen fertilizer during grain development of bread wheat (Triticum aestivum L.)

Wheat yield and quality are dependent largely on nitrogen (N) availability. In this study, we performed the first metabolomic analysis of the response to high-N fertilizer during wheat grain development using non-targeted gas chromatography-mass spectrometry (GC–MS). Quality parameter analyses demonstrated that high-N fertilizer application led to a significant increase in grain protein content and improvement in starch and bread-making quality. Comparative metabolomic profiling of six grain developmental stages resulted in identification of 74 metabolites, including amino acids, carbohydrates, organic acids and lipids/alcohol, which are primarily involved in carbon and N metabolism. Under high-N fertilizer treatment, numerous metabolites accumulated significantly during grain development. Principal component analysis revealed two principal components as being responsible for the variances resulting from N-fertilizer treatments. Metabolite–metabolite correlation analysis demonstrated that the high-N treatment group had a greater number of positive correlations among metabolites, suggesting that high-N fertilizer treatment induced a concerted metabolic change that resulted in improved grain development. Particularly, the high-N treatment-mediated significant accumulation of metabolites involved in the TCA cycle, starch and storage protein synthesis could be responsible for the improvement of grain yield and quality. Our results provide new insight into the molecular mechanisms of wheat grain development and yield and quality.     

Combined Campylobacter jejuni and Campylobacter coli Rapid Testing and Molecular Epidemiology in Conventional Broiler Flocks

Campylobacter spp. are important causes of bacterial zoonosis, most often transmitted by contaminated poultry meat. From an epidemiological and risk assessment perspective, further knowledge should be obtained on Campylobacter prevalence and genotype distribution in primary production. Consequently, 15 Austrian broiler flocks were surveyed in summer for their thermophilic Campylobacter spp. contamination status. Chicken droppings, dust and drinking water samples were collected from each flock at three separate sampling periods. Isolates were confirmed by PCR and subtyped. We also compared three alternative methods (culture‐based enrichment in Bolton broth, culture‐independent real‐time PCR and a lateral‐flow test) for their applicability in chicken droppings. Twelve flocks were found to be positive for thermophilic Campylobacter spp. during the entire sampling period. Seven flocks (46.6%) were contaminated with both, C. jejuni and C. coli, five flocks harboured solely one species. We observed to a majority flock‐specific C. jejuni and C. coli genotypes, which dominated the respective flock. Flocks within a distance <2 km shared the same C. jejuni genotypes indicating a cross‐contamination event via the environment or personnel vectors. Multilocus sequence typing (MLST) of C. jejuni revealed that the majority of isolates were assigned to globally distributed clonal complexes or had a strong link to the human interface (CC ST‐446 and ST4373). The combination of techniques poses an advantage over risk assessment studies based on cultures alone, as, in the case of Campylobacter, occurrence of a high variety of genotypes might be present among a broiler flock. We suggest applying the lateral‐flow test under field conditions to identify ‘high‐shedding’ broiler flocks at the farm level. Consequently, poultry farmers and veterinarians could improve hygiene measurements and direct sanitation activities, especially during the thinning period. Ultimately, real‐time PCR could be applied to quantify Campylobacter spp. directly from chicken droppings and avoid non‐interpretable results achieved by culture‐dependent methods.     

不同钾水平下花魔芋差异表达基因分析

为了探索魔芋对不同水平钾素营养的反应机制,提取施用低、中、高水平硫酸钾5d后的富源花魔芋叶片总RNA,合成了c DNA,采用优化的c DNA-SRAP技术体系分析了在低钾、中钾、高钾水平下富源花魔芋叶片的差异表达基因,并对其进行了生物信息学分析。结果显示:29对引物组合共扩增出507条基因片段,其中差异片段126条。相对于中钾处理,低钾、高钾处理的差异片段包括抑制型表达片段19条,诱导型表达片段14条,上调表达型片段29条,下调表达型片段64条。选取表达量高的13条差异片段进行回收测序,并在NCBI上进行BLAST比对,共比对出9条差异片段的同源序列,其中4条与已知功能基因(谷氨酰胺酰-t RNA合成酶、β-ATP合成酶、GTP结合蛋白、核苷酸还原酶)有较高同源性,5条与假定基因或预测基因同源性较高,剩余序列未找到同源序列,可能是一些新的魔芋与钾营养吸收利用相关的基因,具体功能还有待于进一步研究。     

Molecular characterisation of coconut (Cocos nucifera L.) varieties using ISSR and SSR markers

The present study was carried out to standardise a DNA isolation protocol for coconut and to characterize five coconut varieties using 18 inter-simple sequence repeat (ISSR) and 14 simple sequence repeat (SSR) markers. DNA was extracted from tender young leaf samples collected from the fronds of five different trees of each coconut variety. A protocol using 0.095 g ml^?1 glucose, 0.025 g ml^?1 polyvinylpyrrolidone, 0.0045 g ml^?1 sodium bisulphite, 0.0055 g ml^?1 sodium dodecyl sulphate, and 50 µl ml^?1 sarcosine produced good quality DNA. The average polymorphism percentages revealed using ISSR or SSR markers between the five varieties were 31.9% or 92.9%, respectively. Using ISSR markers, the overall similarity between all five varieties ranged from 0.657 to 0.775, whereas it was 0.037–0.304 using SSR markers. The levels of polymorphism detected using ISSR markers among the five samples each of ‘Banawali’, ‘Gangabondum Green Dwarf’, ‘Pratap’, ‘Konkan Bhatye Coconut Hybrid-I’, and ‘East Coast Tall’ were 23.2%, 24.2%, 25.6%, 27.1%, and 21.2%, respectively. The levels of polymorphism detected using SSR markers among the five samples of the same five varieties were 85.7%, 86.9%, 85.7%, 100%, and 92.9%, respectively. This study indicated that genetic variation existed both between and within samples of each of the five varieties of coconut. SSR markers were superior to ISSR markers. The extent of genetic variation obtained within a variety was not expected, so it is essential to maintain seed purity via artificial pollination.     

瘤胃微生物生态研究方法评述

瘤胃微生物生态研究方法主要经历了微生物纯培养、混合培养、微生物分子生物学技术[(从瘤胃中提取DNA,进行PCR-16S rDNA(rRNA)技术]、变性梯度凝胶电泳(DGGE)技术),在不同程度上揭示了瘤胃微生物群落丰富的多样性和生态功能.但由于各种方法本身的局限性,限制了人类对瘤胃微生物的全面了解,现代生物技术和传统微生物研究方法的配合将为瘤胃微生物生态研究提供较好的前景.     

多枝赖草高分子量核DNA的有效制备

以多枝赖草Leymus multicaulis黄化苗幼嫩叶片为材料,在液氮中研磨成粉末,加入冰冷的细胞核抽提缓冲液,经一系列钢制细胞筛过滤,离心分离细胞核,相差显微镜镜检后,用低熔点琼脂糖包埋,蛋白酶K原位裂解,经脉冲场电泳分离,用1%低熔点琼脂糖回收获得了较纯净的分子量大于2 Mb的核DNA,Southern杂交显示没有叶绿体和线粒体DNA的污染.利用该方法制备的高分子量核DNA,可用于后续可转化人工染色体(TAC)文库的构建和基因组分析.