Genetic Engineering Approaches to Pig Production*

Modern biotechnology promises a number of new applications in animal breeding and production. Although conventional pig breeding has achieved a high level of efficiency and productivity numerous problems have been encountered with animal health and the loss of meat quality. Selection based on phenotypic performance data of individual animals does not take into account the importance of specific genes and their relevance within a complex regulatory system. In most cases it is therefore difficult to trace back the genetic origins of clinically important disorders. The application of genetic engineering techniques in pig production will facilitate diagnosis, improvement of productivity, and animal health by allowing direct genetic manipulation. Attention must be focussed on the physical and genetic analysis of the procine genome. The isolation and characterisation of genes, DNA-markers, polymorphic DNA-fragments, and their chromosomal assignment will be important prerequisites and tools for the elucidation of genetic disorders. Especially the detection of heterozygous carriers of recessive disorders and their elimination from the breeding stock will increase selection accuracy and decrease the generation intervals. But also the rapid and simple detection of infectious diseases, which is sometimes difficult if not impossible at present, will improve animal health and welfare. Although the production of transgenic animals either by DNA-microinjection into zygotes or the use of embryonal stem cells manipulated in vitro is less straightforward than DNA-based diagnosis it will play an important role in the direct manipulation of the porcine genome and genes. Breeding programmes including the use of transgenic livestock have already been developed. There is no doubt that genetic engineering has reached a degree of practical feasibility, allowing it to play an important role in pig breeding in particular and animal production in general.     

鸡Rab6基因的克隆和特征分析

Rab是真核生物细胞器进行物质交换和信息传递重要的小分子结合蛋白。研究采用RT-PCR方法从22周龄母鸡脂肪组织中克隆了Rab6基因的cDNA序列。序列长度为774bp,包含1个627bp完整的CDS,编码208个氨基酸。该序列(Genbank No:DQ470138)与鸭、人、小鼠、狗的Rab6有85.8%～93.1%的同源性,而相应蛋白的同源性达97.6%以上。蛋白预测的分子量和等电点分别为23490Da、5.42,与鸭、人、小鼠、狗的Rab6蛋白高度相似,在78～102氨基酸处有1个跨膜结构。分析结果表明Rab6基因及其蛋白质在动物进化中是高度保守的。     

猪殃殃对AHAS抑制剂靶标抗性的快速分子检测

为建立猪殃殃靶标抗性快速检测方法,并明确小麦田猪殃殃Galium aparine var.tenerum对AHAS抑制剂靶标的突变类型及分布,从河南、陕西、安徽、江苏和山东5省不同田块采集疑似对AHAS抑制剂产生抗性的猪殃殃植株,采用特异性引物PCR扩增靶标酶AHAS基因保守区片段,并以直接测序法检测采集样品,通过与拟南芥AHAS基因序列比对分析后明确其突变位点。结果显示,在5省25个农田的样品中共有19个农田检测到AHAS突变,分布在河南、安徽和江苏3省;在检测样品中发现突变发生在2个位点,共有3种突变类型,分别是197位脯氨酸(CCC)突变为丙氨酸(GCC)或丝氨酸(TCC),或者是574位色氨酸(TGG)突变为亮氨酸(TTG),检测结果与田间药效反应基本一致。这种用特异性引物扩增目的片段测序的方法,由于其可以在生长当季进行检测,适用于田间靶标突变抗性猪殃殃的快速检测与监测。     

Identification of divergent variants of Grapevine virus A

Eight isolates of Grapevine virus A (GVA), which induced different symptoms in leaves of Nicotiana benthamiana, were recovered from various grapevines. The dsRNA patterns of two isolates, which consistently induced mild vein clearing (referred here as mild isolates of GVA) were similar, but different from those of other isolates of GVA. Analysis based on overall nucleotide (nt) sequence identity in the 3 terminal part of the GVA genome, comprising part of ORF3 (putative movement protein, MP), entire ORF4 (capsid protein, CP), entire ORF5 and part of 3 UTR, revealed that GVA isolates separate into three groups (I, II, III), sharing 91.0–99.8% nt sequence identity within groups and 78.0–89.3% nt sequence identity between groups. Mild isolates of the virus were group III and shared only 78.0–79.6% nt sequence identity with the other isolates. The comparison of predicted amino acid sequences for MP and CP revealed many amino acid alterations, revealing distinct local net charges of these proteins for mild isolates of the virus. Based on both conserved and divergent nt regions in the CP and ORF5, oligonucleotide primers were designed for the simultaneous RT-PCR detection of all GVA isolates and for the specific detection of the most divergent virus variants represented here by mild isolates of the virus.     

Deep‐sequencing revealed Citrus bark cracking viroid (CBCVd) as a highly aggressive pathogen on hop

Newly emerging or re‐emerging diseases are a constant and significant threat to agricultural production, so prompt and accurate identification of the causative agents is required for rapid and appropriate disease management. Classical methods of pathogen detection can be successfully supplemented by next‐generation sequencing (NGS), whereby sequence analysis can help in the discovery of new or emerging diseases. In 2007, hop growers in Slovenia reported the appearance of severely stunted hop plants, a phenomenon that spread rapidly within hop gardens and among farms. Classical diagnostic methods were unable to detect a new pathogen; therefore, single step high‐throughput parallel sequencing of total RNA and small RNAs from plants with and without symptoms was employed to identify a novel pathogen. The sequences were assembled de novo and also mapped to reference genomes, resulting in identification of a novel sequence of Citrus bark cracking viroid (CBCVd) in the stunted hop plants. Furthermore, the presence of this novel pathogen on hop was confirmed by RT‐PCR analysis of 59 plants with symptoms from 15 hop gardens, representing the main outbreak locations identified by systematic disease monitoring, and small RNA Illumina sequencing of the bulked RNA sample. The high infectivity of the newly identified CBCVd was also confirmed by biolistic inoculation of two hop cultivars, which developed aggressive symptoms in controlled conditions. This study shows the feasibility of deep sequencing for the identification of causative agents of new diseases in hop and other plants.     

猪繁殖与呼吸综合征病毒感染的诊断及其分子特点

采用流行病学调查、临床症状观察、病理学检查、病毒分离、分离毒株测序以及遗传演化分析的方法,对北京及其周边地区4个猪场剖检的4头猪进行分析。结果:流行病学和临床症状表现为急性高热性传染病;病理学观察为非化脓性脑炎和间质性肺炎等多器官严重的病理变化;RT-PCR和病毒分离确定该疫情的主要病原是猪繁殖与呼吸综合征病毒(PRRSV)。与典型PRRSV感染不同的是:成年猪感染率达50%以上,病死率达80%以上;PRRSV分离株全基因序列分析表明属于PRRSV北美洲型,特别是PRRSV NSP2不连续缺失30个氨基酸,说明此次流行的PRRSV毒株可能为高致病性毒株。     

祁连山中段退化高寒草地土壤细菌群落分布特征

为探究祁连山中段不同退化高寒草地土壤细菌群落分布特征,采用Illumina HiSeq PE250高通量测序平台对轻度、中度和重度退化草地土壤细菌群落变化特征进行研究,并对土壤细菌群落与土壤酶活性、土壤理化因子间关系进行分析。结果表明:随着退化程度加剧,植被盖度、高度、地上生物量和多样性指数均明显降低(P<0.05);土壤理化性质和酶活性变化各异且差异显著(P<0.05)。高通量测序共得到257971条有效序列,219017条优质序列和2004个OTUs。细菌群落丰富度指数依次为轻度>中度>重度,多样性指数为轻度>重度>中度,Beta多样性分析表明各样地间差异为轻度>重度>中度。其中,放线菌门(Actinobacteria)、厚壁菌门(Firmicutes)、酸杆菌门(Acidobacteria)和变形菌门(Proteobacteria)为3种退化草地土壤的优势菌门,在轻度、中度和重度退化草地土壤中分别占77.25%、84.27%和78.66%;乳球菌属为3种退化草地土壤的优势菌属,在轻度、中度和重度退化草地土壤中分别占14.29%、38.84%和7.39%。冗余分析表明:土壤酶活性和土壤理化性质均对细菌群落的组成具有影响,其中土壤pH是影响土壤细菌群落分布的主要驱动因子。祁连山中段不同退化高寒草地土壤细菌群落的变化主要受土壤理化性质和酶活性的影响。     

猪的全基因组测序研究进展

猪具有独特的生物学特征,在畜牧业生产和医学研究中占有重要地位。全基因组测序即de novo测序和全基因组重测序为解释猪的生物学特性和促进猪的分子育种发挥了重要作用。本文重点阐述全基因组测序在猪基因组学研究中的应用,分析全基因组测序技术及其在猪的全基因组测序工作中的优势和不足,并对未来猪的分子遗传育种研究工作进行展望。     

高通量测序技术结合传统方法筛选及鉴定奶酒中曲霉菌

奶酒含有丰富的营养,且具有驱寒活血、开胃消食等保健功效。但由于传统的可培养方法对其微生物全貌的认识具有一定的局限性。因此,本文采用高通量测序技术,对奶酒的真菌生物多样性进行检测。结果表明,酵母菌属(Saccharomyces)和曲霉属(Aspergillus)是奶酒最主要的真菌群落,其中,酵母菌属为第一丰度的真菌,丰度约为62%。第二丰度的真菌为曲霉属,丰度为33%。通过进一步分离并采用16S鉴定出了一株曲霉菌,这可为奶酒发酵剂的开发提供依据。