Conversion of Goat Fibroblasts into Lineage‐Specific Cells Using a Direct Reprogramming Strategy

Direct reprogramming is an efficient strategy to convert one cell type to another. In this study, due to the failure of maintaining the undifferentiated state of goat embryotic stem‐ and induced pluripotent stem‐like cells in vitro, we explored an alternative way to directly convert goat fibroblasts to lineage‐specific cells. The ‘Yamanaka factors’ was ectopically expressed in fibroblasts for a short term to situate cells in a metastable state. By culturing with lineage‐specific media for 1–2 weeks, the cardiomyocyte‐like cells and neurocyte‐like cells were generated and confirmed by the quantitative RT‐PCR and immunocytochemical staining. The metastable‐state cells could also be converted into oocyte‐like cells (OLCs) after culturing in media with retinoic acid (RA) and bovine follicular fluid (bFF) for 2–3 weeks. The generated OLCs were surrounded by cumulus granulosa cell‐like cells and formed a structure resembling goat cumulus‐oocyte complex from ovaries. This primary follicular structure could be developed further in oocyte mature medium and expressed germ cell‐specific markers. In addition, we found that the induction efficiency was higher and OLC cell size was bigger in bFF than in RA treatment. Altogether, the direct reprogramming of goat fibroblasts into lineage‐specific cells can facilitate stem cell research in domestic animals.     

猪繁殖与呼吸综合征病毒双基因多组合DNA疫苗的构建

本实验根据PRRSV结构蛋白的特性和IFNγ及IL-2这两种细胞因子的特性,构建表达PRRSVGP4-GP5、GP5-M、GP5-N、GP5-IFNγ、GP5-IL-2、N-IFNγ、N-IL-2的重组质粒。这些重组质粒在真核细胞中得到了表达。用这些真核表达质粒可以免疫小猪,通过检测其对猪体免疫应答的诱导能力和接种后动物对PRRSV的抵抗力来对基因疫苗的效能进行研究,以期筛选到有效的基因疫苗并为今后进一步的研究提供思路和理论基础。     

猪日本脑炎病毒RT-PCR检测方法的建立

设计了1对引物,利用RT-PCR技术检测乙型脑炎病毒(JEV)。从GenBank中查出收录的31株猪乙型脑炎病毒E基因的已知序列,用DNA star软件对这31株猪乙型脑炎病毒E基因进行同源性分析,以确定扩增的靶序列。以这段靶区域为模板,利用Primer 5软件设计了1对引物,用减毒株SA14-14-2建立了检测乙脑病毒的RT-PCR方法,经敏感性,特异性试验测定,证明该方法敏感,特异;该法可检出样品稀释至256倍的鼠脑毒,相当于0.06个TCID50,对4株河北地区JEV分离株进行检测,结果所设计引物对4株病毒均能扩增出预期的片段。     

青海省草地鼠害现状及其治理

概述了青海省草地生态系统在当地经济发展、生态环境以及长江、黄河流域生态环境稳定和居民生活安全中的重要地位。结合当前鼠害防治工作的现状，提出了一些合理的鼠害防治策略和举措。     

垄作模式下甜高粱对农田渍害胁迫的响应

我国长江中下游地区常年降水较多,易形成农田渍害。为探究渍害胁迫对甜高粱(Sorghum dochna)产量性状和抗氧化酶系统的影响,设计了平作和垄作两个处理,在甜高粱三叶期对其进行渍水处理,测定并比较各处理的抗氧化酶系统活性和产量性状。结果表明,苗期遭受渍害胁迫,垄作处理下甜高粱的茎粗、单株干重、叶数、叶面积指数均显著高于平作(P0.05)。株高和茎叶比在两个处理间无显著差异(P0.05);与平作处理相比,渍害胁迫下甜高粱叶片的抗氧化酶[超氧化物歧化酶(SOD)、过氧化物酶(POD)、过氧化氢酶(CAT)]活性更强。研究表明,垄作耕作方式能够提高甜高粱耐受渍害胁迫的能力,获得更高的产量。     

兔病毒性出血症母源抗体动态与抗病毒感染的关系和对策

兔病毒性出血症疫苗免疫母兔所产６、１５、３０、４５、６０、日龄共１２６只兔的母源抗体随日龄增长而下降，至３０￣４５日龄，４８￣８５％的兔为阴性，其平均值为２＾３。强毒攻击耐过母兔所产的未吃初乳、已吃初乳、１５、３０、４５、６０、９０及１１４日龄以上兔１８６只的母源抗体也随日龄增长而消失，但消失时间长，其平均值至１１４ｄ后才降至２＾３。已吃初乳仔兔抗体平均值比未吃初乳高，未吃初乳兔的抗体与母兔基本持     

中国荷斯坦奶牛EEF1D基因的SNPs检测及其与产奶性状的关联分析

试验旨在研究中国荷斯坦奶牛真核生物翻译延伸因子1D（eukaryotic translation elongation factor 1 delta,EEF1D）基因的多态性及其与产奶性状的相关性。利用Sequenom MassARRAY SNP分型技术对宁夏地区1 252头中国荷斯坦奶牛EEF1D基因的多态性进行了检测,并对其多态位点不同基因型和组合基因型与产奶性状进行了关联分析。结果显示,EEF1D基因的5'侧翼区存在2个SNPs位点,即EEF1D-1和EEF1D-3;经检测发现,EEF1D-1存在2种基因型,EEF1D-3存在3种基因型。χ²检验表明,中国荷斯坦奶牛在EEF1D-1位点偏离Hardy-Weinberg平衡状态（P < 0.05）,在EEF1D-3位点未偏离Hardy-Weinberg平衡状态（P > 0.05）;EEF1D-1和EEF1D-3位点多态信息含量（PIC）分别为0.10和0.28,分别呈现低度多态和中度多态。在试验群体中,EEF1D-1位点对乳脂率和乳蛋白率性状的效应均达到极显著水平（P<0.01）,对305 d产奶量性状的效应达到显著水平（P<0.05）;EEF1D-3位点对305 d产奶量、乳脂率和乳蛋白率性状的效应均达到极显著水平（P<0.01）;EEF1D基因的优势基因型组合GG-AG和GG-GG个体乳脂率均显著高于GG-AA组合个体（P<0.05）。说明EEF1D基因可以作为影响中国荷斯坦奶牛产奶性状的候选基因用于标记辅助选择。     

陇东肉牛PON1基因结构与功能的生物信息学分析

为阐明陇东肉牛对氧磷酶1（paraoxonase 1,PON1）基因的结构与功能,本研究运用PCR扩增陇东肉牛PON1基因所有外显子序列并测序,通过Seqman软件完成序列拼接,结合生物信息学方法预测和分析其碱基组成、开放阅读框及编码蛋白的理化性质、原子组成、氨基酸残基数、亲水性和疏水性、分子质量、等电点、二级结构、高级结构等。结果显示,陇东肉牛PON1基因CDS区序列全长1 068 bp,编码355个氨基酸残基,其中数目最多的为亮氨酸（Leu）,数目最少的为半胱氨酸和色氨酸（Cys和Trp）。碱基组成中A+T含量（56.55%）高于G+C含量（43.26%）。与GenBank中牛PON1基因序列（登录号：EU289337）比对分析发现,在PON1基因外显子4、6、8、9中存在突变,但均未导致氨基酸发生变化,为同义突变。PON1蛋白的原子组成是C₁₈₁₀H₂₇₉₆N₄₅₄O₅₃₃S₁₂,分子质量为39.83 ku,理论等电点为5.24,为稳定的水溶性蛋白质。PON1蛋白二级结构中无规则卷曲、延展链、α-螺旋和β-转角分别由159、116、55和25个氨基酸构成,最终形成了以无规则卷曲和延展链为主的混合型。PON1蛋白的疏水性结果表明,第8位苏氨酸（Thr）疏水性最强（最高分值2.878）,第299位脯氨酸（Pro）亲水性最强（最低分值-2.222）。本试验结果为深入研究陇东肉牛PON1蛋白功能及探讨PON1基因突变对甘肃地方肉牛胴体、肉质性状的影响奠定基础。     

甘肃省临泽县部分饲草饲料的营养成分分析

从甘肃临泽县８个乡采集饲草饲料样品,用常规分析方法测定其中的干物质、粗蛋白、粗纤维、钙和磷含量,测出了常用的８种饲料共６４个样品的上述营养成分含量。结果表明,不同乡的同种饲草饲料间,多数营养成分相近,且与甘肃省及全国平均水平相近,个别值间存在着一定差异。所得结果可以作为评价当地畜禽饲料营养价值的参考值。     

Transmissible gastroenteritis virus infection induces apoptosis through FasL- and mitochondria-mediated pathways

Transmissible gastroenteritis virus (TGEV) has been reported to induce apoptosis in swine testis (ST) cells. However, the mechanisms underlying TGEV-induced apoptosis are still unclear. In this study we observed that TGEV infection induced apoptosis in porcine kidney (PK-15) cells in a time- and dose-dependent manner. TGEV infection up-regulated FasL, activated FasL-mediated apoptotic pathway, leading to activation of caspase-8 and cleavage of Bid. In addition, TGEV infection down-regulated Bcl-2, up-regulated Bax expression, promoted translocation of Bax to mitochondria, activated mitochondria-mediated apoptotic pathway, which in turn caused the release of cytochrome c and the activation of caspase-9. Both extrinsic and intrinsic pathways activated downstream effector caspase-3, followed by the cleavage of PARP, resulting in cell apoptosis. Moreover, TGEV infection did not induce significant DNA fragmentation in ammonium chloride (NH(4)Cl) pretreated PK-15 cells or cells infected with UV-inactivated TGEV. In turn, block of caspases activation also did not affect TGEV replication. Taken together, this study demonstrates that TGEV-induced apoptosis is dependent on viral replication in PK-15 cells and occurs through activation of FasL- and mitochondria-mediated apoptotic pathways.