真核表达NDV F基因重组质粒的构建及其在CEF细胞中的转染

通过PCR方法自含NDVZF基因的克隆质粒中扩增NDVF基因 ,将其与真核表达载体pcDNA3..1/V5_His_TOPO重组。经核酸内切酶酶切 ,阳性克隆鉴定准确无误 ,核酸序列测定结果与原始基因比较 ,同源性大于 99% ,启始密码和终止密码未出现变异 ,将该重组质粒命名为pcDNANDVZF。将pcDNANDVZF在脂质体作用下转染CEF细胞 ,用间接免疫荧光试验检测 ,结果证明pcDNANDVZF可在CEF细胞中大量表达F蛋白。     

Reproductive roles predict sexual dimorphism in internal and external morphology of lake whitefish,Coregonus clupeaformis

Abstract – The different reproductive roles of the sexes can predict the direction and magnitude of sexual dimorphism of external and internal morphology. Males should have enlarged structures that enhance the acquisition of mating opportunities, whereas females are predicted to have enlarged organs that are associated with the production of eggs. We tested these predictions in male and female lake whitefish, a species in which both sexes have similar overall body size and shape. After controlling for body size, male lake whitefish had significantly longer jaws and pectoral and pelvic fins, larger hearts, and more muscle than females. Sexual dimorphism in relative muscle mass may be one of the most fundamental morphological differences between males and females. Females had relatively heavier livers than males. Because the liver is important for the breakdown of fats and vitellogenesis, selection should favour an enlarged liver in females for the processing of energy and the production of large numbers of eggs.     

瓯江鲫鱼胸鳍的波动性不对称、两性异形和生育

研究浙江瓯江鲫鱼胸鳍的波动性不对称、两性异形和生育力。雌雄两性的鳍长、鳍条数的不对称均属波动性不对称。雌雄两性的胸鳍长和鳍条数的不对称指数之间相关关系不显著。雌性个体显著大于雄性个体,特定体长的雄性胸鳍长显著大于雌性,雄性的鳍条数显著大于雌性。鳍长和鳍条数的不对称指数雌雄两性差异不显著,雌雄两性鳍条数对称个体的比例差异不显著。以体长和时间为控制变量,怀卵数量与鳍长和鳍条数的不对称指数具有显著的负相关,与鳍长和鳍条数的相关不显著。以体长和发育时间为控制变量,平均卵重与鳍条数的不对称指数具有显著的正相关,与其他变量的相关不显著。分析表明,雄性较大的胸鳍可能与雄性的性选择有关,胸鳍的波动性不对称可能提供了一种发育历程的指示,间接影响雌性生育力。     

玉蜀黍赤霉有性重组体的诱导及其生物学特性

 本研究以硝酸盐营养缺陷型突变体(nit)和多菌灵抗性为遗传标记,在5个所选菌株中设计了3个玉蜀黍赤霉病菌Gibberella zeae的杂交组合,使各菌株之间进行杂交,从而诱导有性重组体。从各杂交组合的后代中任意挑选出3个有性重组体,比较了这些有性重组体与其亲本在无性和有性阶段的主要生物学性状。结果表明,玉蜀黍赤霉中的nit基因及对杀菌剂多菌灵的抗药性基因可以通过有性杂交的方式重组,即发生了有性重组。有性重组体与其亲本在菌落生长、培养性状和致病性方面没有显著差异;但某些有性重组体中,产孢量和产子囊壳能力方面存在一定差异。总体看来,有性重组体仍然保持了较高的适合度。因此,可以认为有性重组在玉蜀黍赤霉群体对多菌灵抗药性发生发展以及群体遗传进化中可能起着重要的作用。     

虎皮鹦鹉发声行为学比较研究

为了解虎皮鹦鹉发声行为的性别差异,采用计算机鸣声分析技术对虎皮鹦鹉的鸣叫行为做了研究,结果表明,雄性虎皮鹦鹉的叫声明显比雌鸟的复杂多变,但是鸣声幅度比雌鸟的小;随着虎皮鹦鹉的成长,雌雄鸟的叫声越来越复杂,幅度越来越高。说明虎皮鹦鹉的叫声具有明显的性双态性,并且叫声随着年龄的增大越来越成熟,30日龄虎皮鹦鹉的鸣声复杂程度已接近成鸟水平。     

基于Maize6H-60K芯片精准分型的玉米DH群体遗传规律研究

为解析玉米双单倍体（doubled haploid,DH）群体的遗传规律,使用自主研发的精准分型策略对‘先玉335’DH群体的Maize6H-60K芯片结果进行筛选校正和解析。结果表明：1）通过剔除单态、杂合、缺失标记,使用精准分型策略对标记准确聚类,获得的18 947个SNPs标记覆盖玉米全基因组。2）该群体在10条染色体上平均交换次数为1.84～1.03次,在6号染色体随体区域的交换次数显著减少。3）该群体在1、2、3、8号染色体上存在明显的偏分离现象;通过高频次、高密度的交换,染色体两臂端粒区域理论分离系数表现出回归到50%的趋势。利用获得的重组交换及分离规律可提高从DH群体中筛选到预期育种材料的准确性。     

检测鸭呼肠孤病毒抗体间接ELISA方法的建立

RT—PCR扩增鸭呼肠孤病毒（Duckreovirus,DRV）的dB基因,克隆到pET-32a（＋）表达载体,再转化大肠杆菌Transetta（DE3）;含有重组质粒pET—σB的大肠杆菌经IPTG诱导,获得大小为55000的以包涵体形式表达的σB重组蛋白。Westernblotting显示,σB重组蛋白能够与兔抗DRV多抗血清特异性结合。以高亲和NI—NTA树脂在变性条件下纯化、梯度尿素复性的σB重组蛋白为包被抗原,建立了检测DRV抗体的间接ELSIA方法。分别以间接ELSIA方法和血清中和试验对DRV感染鸭血清和SPF鸭血清进行检测,两者的符合率为100％。该间接ELSIA方法对鸭瘟病毒、鸭病毒性肝炎病毒和禽流感病毒阳性血清均无交叉反应。     

禽网状内皮组织增生症流行现状及检测技术研究进展

禽网状内皮组织增生症(RE)是禽类一种重要的致瘤性和免疫抑制性疾病。近年来,国内外鸡群中网状内皮组织增生症病毒(REV)流行比较严重;REV与马立克病病毒(MDV)、鸡贫血病病毒(CAV)和J亚群禽白血病病毒(Avianleukosis virus subgroup J,ALV-J)几种免疫抑制性病毒的混合感染在我国部分养鸡场中均普遍存在,使得鸡群处于免疫力低下的状态,导致禽流感、新城疫等重要疫苗免疫失败,并容易造成继发感染,使疫病的诊断和防控难度加大。另外,REV可以整合到MDV和鸡痘病毒(Fowlpox virus,FPV)基因组中,从而导致商品化疫苗受到污染,并可用做将外源基因插入到鸡和哺乳动物细胞内的表达载体;基于REV传统检测方法的基础上,新型的分子生物学检测技术如PCR、实时荧光PCR、LAMP提高了诊断REV的敏感性和特异性。     

表达H5亚型禽流感病毒HA基因的重组马立克氏病病毒的构建

采用聚合酶链反应或反转录聚合酶链反应扩增出H5亚型禽流感病毒(AIV)的HA基因、网状内皮增生症病毒的长末端重复序列(LTR)、马立克氏病病毒(MDV)Rispens CVI988毒株基因组的sorf 1和sorf 2序列、两端带loxp位点的lac/smGFP标志基因,构建含这些基因的转移载体质粒pMHA;以MDV Rispens CVI988毒株的基因组DNA和PMHA质粒DNA共转染鸡胚成纤维细胞(CEF),采用同源重组方法将LTR、lac/smGFP和HA基因插入到MDV基因组,获得重组病毒rMDV-HA/GFP;以cre介导的同源重组去除lac/smGFP标志基因,再转染CEF,获得仅带LTR启动子和HA基因的重组MDV疫苗毒株rMDV-HA.rMDV-HA仍保留了MDV RispensCVI988疫苗毒株的复制特点,并能稳定表达AIV的HA.     

Recombinant pseudorabies virus expressing P12A and 3C of FMDV can partially protect piglets against FMDV challenge

One of the crucial factors for evaluation of an effective genetically engineered vaccine is whether susceptible animals are protected from virus challenge after vaccination. In this study, a recombinant pseudorabies virus (PRV-P12A3C) that expressed capsid precursor polypeptide P12A and nonstructural protein 3C of foot-and-mouth disease virus (FMDV) was used as a vaccine. The expression of P12A3C and its immunogenicity and protective efficacy against FMDV challenge were measured. Humoral and cellular immune responses were evaluated after each immunization. Subsequently, each piglet was challenged with 1000 ID₅₀ (50% infection dose) FMDV serotype O, named OR/80, which is used to produce vaccine in China. PRV-P12A3C induced a high level of neutralizing antibody and FMDV-specific lymphocytes. Inactivated vaccine provided 100% protection, and the vector strain (TK⁻/gE⁻/gI⁻) showed no protection. PRV-P12A3C induced 60% protection, compared with piglets that were vaccinated with TK⁻/gE⁻/gI⁻. The severity of clinical signs for the remaining two piglets was lighter and the appearance of vesicles was delayed.