日粮营养对动物脂肪组织基因表达的影响

脂肪组织是一个强大的分泌器官,它能分泌许多与能量、脂肪代谢相关的酶、激素等.调控能量代谢和脂肪组织生长.本文综述了日粮对动物脂肪组织中基因表达作用的研究状况.并阐述了营养成分对这些基因上下游表达的作用机制.     

BADH/pepB双价基因无选择标记表达载体转化苜蓿的初步研究

选用公农1号紫花苜蓿,以5～7d莆龄的无菌子叶为外植体,通过农杆菌介导法将含有甜菜碱醛脱氢酶(BADH)和酸性蛋白酶(pepB)双价基因的无选择标记表达载体导入苜蓿子叶中,并用含有Na2CO3和NaHCO3碱性盐溶液的培养基进行筛选,得到抗盐碱转化植株.碱性盐浓度48mmol/L宜于子叶愈伤诱导,有利于转化植株的获得.对转化植株进行PCR检测,初步证明目的基囚序列已经整合剑苜楷基因组中.移栽成活的13棵碱性盐抗性植株经PCR检测有3棵植株呈阳性.     

Characterization of the cathelicidin cluster in the Japanese quail (Coturnix japonica)

The Japanese quail has several advantages as a low‐fat meat bird with high immunity against diseases. Cathelicidins (CATHs) are antimicrobial peptides that play an important role in innate immunity. The aim of this study was to characterize the CATH cluster in the Japanese quail (Coturnix japonica). The Japanese quail CATH (CjCATH) cluster, contains four CATH genes, as in the chicken. The coding sequences of CjCATHs exhibited >85.3% identity to chicken CATHs. The predicted amino acid sequences of the four CjCATH genes contained the cathelin‐like domain characteristic of CATH proteins. Polymorphisms were detected in the open reading frames (ORFs) of all CjCATH sequences. Two amino acid substitutions were observed in the antimicrobial region of the mature peptide of CjCATH2, and predicted to influence peptide function. CjCATH1 is expressed in lung, heart, bone marrow and bursa of Fabricius (BF). CjCATH2 is expressed in bone marrow. CjCATH3 is expressed in lung, heart, bone marrow, BF, tongue and duodenum. CjCATHB1 is expressed in bone marrow and BF. This study is the first to characterize CATH genes in the Japanese quail, and identifies novel antimicrobial peptide sequences belonging to the cathelicidin family, which may play a role in immunity in this species.     

猪ASC及Ub基因的克隆及真核表达重组质粒的构建

炎症小体不仅参与机体的天然免疫反应,同时还参与机体抗病毒等多种宿主防御反应。在多种疾病相关信号通路反应中都伴随着蛋白质的泛素化修饰。NOD样受体(NLRs)介导的炎症小体信号通路中,凋亡相关点状样蛋白(ASC)是关键的接头蛋白。为了解猪瘟病毒(CSFV)感染对ASC泛素化水平的影响,通过RT-PCR从猪血管内皮细胞(ECs)中扩增获得ASC和泛素(Ub)的全长cDNA双链片段,连接至克隆载体pMD-18T,获得重组克隆质粒pMD-ASC和pMD-Ub。双酶切pMD-ASC获得ASC目的序列,连接至pcDNA3.1-Flag,双酶切pMD-Ub获得Ub目的序列,连接至pcDNA3.0-HA,获得重组表达质粒pcDNA3.1-Flag-ASC和pcDNA3.0-HA-Ub。将pcDNA3.1-Flag-ASC和pcDNA3.0-HA-Ub转染ECs,Western blot在蛋白水平检测ASC和Ub的表达。结果表明,成功构建了带有Flag标签的ASC真核表达质粒和带有HA标签的Ub真核表达质粒,且在ECs中获得高效表达,为进一步体外研究CSFV调控猪血管内皮细胞ASC泛素化的机制提供了实验基础。     

Constitutive expression of interferons in swine leukocytes

Interferon (IFN)-α and IFN-γ positive cells were revealed by flow cytometry in peripheral blood mononuclear cells (PBMC) of Specific Pathogen Free (SPF) pigs. A low prevalence of IFN-γ positive cells was also detected in PBMC of some Porcine Reproductive and Respiratory Syndrome virus-infected pigs and uninfected, control pigs. IFN-α positive cells showed phenotypes of both monocytes and plasmacytoid dendritic cells. The presence of IFN-α in PBMC was also confirmed by Western blotting. By immunoprecipitation, IFN-α was detected as 32 and 55-57 kDa bands in PBMC of healthy SPF piglets. These samples were also IFN-γ positive; the cytokine was revealed as 24, 37 and 54 kDa bands. The unusual molecular mass values of intracellular interferons were probably due to oligomerization, as previously described for human IFN-α. Swine intracellular IFN-α displayed the expected antiviral activity on bovine MDBK cells. The results indicate that interferons are constitutively expressed in swine leukocytes with peculiar molecular features.     

高羊茅腺苷甲硫氨酸脱羧酶基因FaSAMDC的克隆与差异表达分析

在进行高羊茅光敏色素C基因5′端克隆时,筛选到腺苷甲硫氨酸脱羧酶基因的5′端序列,根据5′端序列设计引物,扩增出该基因的3′端,拼接得到高羊茅腺苷甲硫氨酸脱羧酶基因全序列,命名为FaSAMDC(GenBank登录号:HQ606139),利用实时荧光定量PCR技术研究FaSAMDC基因在长日照与短日照条件下昼夜24 h的表达差异,为进一步揭示不同光周期调控FaSAMDC基因的表达奠定理论基础。FaSAMDC的cDNA全长1 964 bp,具有微型上游阅读框、小型上游阅读框和主阅读框3个开放阅读框,分别位于226～234,234～380和555～1 742 bp,微型上游阅读框和小型上游阅读框存在1个碱基重叠,主阅读框编码395个氨基酸,含有保守功能域:酶原剪切位点功能域和SAMDC蛋白快速降解有关的PEST功能域。对编码蛋白氨基酸序列的同源性分析表明,FaSAMDC与其他单子叶植物SAMDC蛋白的亲缘关系较近。在长日照和短日照处理条件下,FaSAMDC都具有3个表达峰。短日照处理条件下的表达峰比长日照处理条件下的表达峰早几个小时,但峰高低于长日照条件。由此说明,FaSAMDC基因的表达受光周期调控,与生物钟控制的昼夜节律有关。     

蒙古马突触融合蛋白17(STX17)基因多态性及在不同毛色皮肤组织中的表达分析

为研究突触融合蛋白17(STX17)基因与蒙古马毛色之间的相关性及其作用机制.本研究利用半巢式PCR技术及实时荧光定量PCR技术对蒙古马STX17基因第6内含子的多态性以及不同毛色蒙古马皮肤组织中STX17基因的相对表达量进行了检测.巢式PCR结果显示:STX17基因在青毛蒙古马第6内含子处存在4.6 kb的重复片段,在其它毛色中不存在该重复片段;荧光定量PCR结果显示:STX17基因在蒙古马青毛皮肤组织中表达量最高,在骝毛皮肤组织中的表达量最低.结果证明蒙古马STX17基因与其青毛表型具有典型的相关性.     

犬IFN-α优化基因的真核表达及抗病毒活性的研究

本研究根据酵母系统密码子的偏嗜性,对成熟蛋白基因核苷酸序列进行密码子偏嗜性改造,利用毕赤酵母分泌型表达载体pPICZαC来表达犬IFN-α成熟蛋白基因。并采用非洲绿猴肾细胞(Vero) -水疱性口炎病毒(VSV)系统以细胞病变抑制法为基础来测定重组犬α干扰素的效价,同样方法测定了表达产物对PRV和CDV的抑制作用。本试验实现了犬α干扰素在酵母中的高表达,表达量高达58 mg/L,SDS-PAGE检测大小约为24 ku,比预测分子质量大,推测发生了糖基化。表达产物具有较强的种属特异性,在Vero细胞上对PRV具有较高的抗病毒活性,达到了3.6×10⁷ U/L,对CDV也有很好的抑制作用。本研究为犬IFN-α以后的临床应用研究奠定基础。     

Divergence of a strain of Pseudomonas aeruginosa during an outbreak of ovine mastitis

Bacterial infections causing mastitis in sheep can result in severe economic losses for farmers. A large survey of milk samples from ewes with mastitis in Sardinia, Italy, indicated an increasing prevalence of Pseudomonas aeruginosa infections. It has been shown previously that during chronic, biofilm-associated infections P. aeruginosa populations diversify. We report the phenotypic and genomic characterisation of two clonal P. aeruginosa isolates (PSE305 and PSE306) from a mastitis infection outbreak, representing distinct colony morphology variants. In addition to pigment production, PSE305 and PSE306 differed in phenotypic characteristics including biofilm formation, utilisation of various carbon and nitrogen sources, twitching motility. We found higher levels of expression of genes associated with biofilm formation (pelB) and twitching motility (flgD) in PSE305, compared to the biofilm and twitching-defective PSE306. Comparative genomics analysis revealed single nucleotide polymorphisms (SNPs) and minor insertion/deletion variations between PSE305 and PSE306, including a SNP mutation in the pilP gene of PSE306. By introducing a wild-type pilP gene we were able to partially complement the defective twitching motility of PSE306. There were also three larger regions of difference between the two genomes, indicating genomic instability. Hence, we have demonstrated that P. aeruginosa population divergence can occur during an outbreak of mastitis, leading to significant variations in phenotype and genotype, and resembling the behaviour of P. aeruginosa during chronic biofilm-associated infections.     

凋亡素基因真核表达载体的构建及在人肿瘤细胞中的表达

为探讨凋亡素对肿瘤的基因治疗效果,构建了凋亡素基因的真核表达载体。将凋亡素基因重组入 ｐｃ Ｄ Ｎ Ａ３ 载体,并通过脂质体介导凋亡素在人喉癌、人肺癌细胞系中表达;通过 Ｒ Ｔ Ｐ Ｃ Ｒ 在转染细胞中检测到了凋亡素的 ｍ Ｒ Ｎ Ａ,这为应用凋亡素进行肿瘤的基因治疗奠定了基础。