嗜水气单胞菌刺激下鲤血清质子转运载体15a2(Slc15a2)的表达分析

为解析鲤质子转运载体15a2 (Slc15a2)在嗜水气单胞菌侵染过程中对机体免疫应答机制的影响,实验通过制备鼠源Slc15a2-1和Slc15a2-2多克隆抗体,采用酶联免疫吸附测定(ELISA)技术检测嗜水气单胞菌感染后鲤血清中Slc15a2-1和Slc15a2-2蛋白表达变化。结果显示,(1)感染后2个拷贝的最高蛋白表达水平与空白对照组相比,分别增加了4.81和2.48倍;与Slc15a2-1相比Slc15a2-2的表达量从0～48 h一直处于较高表达水平。(2)从整体来看,Slc15a2的2个拷贝具有相似的表达趋势,均为先升高后降低、再升高再降低;但Slc15a2-1在3 h达到第1个表达高峰,而Slc15a2-2则在6 h达到第1个表达高峰。(3) 6～24 h的4个时期中,Slc15a2-1和Slc15a2-2蛋白表达呈现互补的表达趋势,即在同一时期一个拷贝的表达量显著下降,另一个拷贝的表达量显著升高,但总体维持较高表达水平。结果表明,本实验制备的鼠源Slc15a2-1和Slc15a2-2多克隆抗体具有较高的亲和力和特异性,当鲤受到嗜水气单胞菌侵染后,2个拷贝整体具有相似的...     

Differences in stable isotope ratios of Dall’s porpoises (Phocoenoides dalli) between coastal and oceanic areas of the North Pacific

Carbon and nitrogen stable isotope ratios in the muscle of Dall’s porpoises were measured. Samples were collected from the catches of the hand harpoon fishery, incidental catches of drift net, and scientific research on the use of drift nets. Samples were from the North Pacific, Sea of Japan, Sea of Okhotsk, and Bering Sea. Although no variation in δ¹⁵N was observed, δ¹³C was significantly different between population groups near Japan and the oceanic North Pacific and Bering Sea. The difference may be due not only to local variation in prey species, but also to an overall difference in carbon stable isotope ratios that originate from coastal benthic or oceanic pelagic‐based food webs. We differentiated Dall’s porpoise population groups from both areas using carbon stable isotope ratios with an error rate of <5%. Although further study is needed, our results suggest that carbon stable isotope ratios could possibly be an indicator of whether a Dall’s porpoise belongs to a coastal benthic or oceanic pelagic food web.     

山羊源伪狂犬病病毒的分离与鉴定

为确诊一例发病山羊是否感染伪狂犬病病毒,采集发病羊体的肺脏和脑组织进行伪狂犬病病毒gE基因PCR检测,并将病料接种至PK-15细胞分离病毒,以及进行小鼠感染试验和gD基因分析。结果表明,PCR检测结果 PRV阳性,病料接种PK-15细胞24h后,细胞开始出现细胞病变;将病毒感染小鼠,36h后小鼠出现局部奇痒、死亡;gD基因序列分析发现,分离毒株与GenBank中的PRV gD基因序列同源性均在98%以上,氨基酸同源性在99%以上;在分离毒株gD基因的808bp~837bp位置上存在缺失与变异、高变重复区。本研究成功分离获得一株羊源伪狂犬病毒,为云南省羊伪狂犬病防控和基础研究提供资料。     

绵羊BMP15基因外显子单核苷酸多态性研究

以控制Belclare和 Cambridge绵羊高繁殖力的骨形态发生蛋白15( Bone morphogenetic protein 15,BMP15)基因为候选基因,采用PCR-SSCP技术检测BMP15基因在中国部分绵羊品种 (蒙古羊、内蒙古细毛羊、呼伦贝尔羊、中国美利奴羊)中的单核苷酸多态性,同时研究突变位点对绵羊繁殖力的影响.结果表明:在4个绵羊品种中均检测到BMP15的突变,检测到的BMP15突变在4个绵羊品种中均呈现3种基因型.这表明BMP15基因外显子Ⅰ中虽然存在突变位点,但这一突变位点对绵羊繁殖力高低没有显著影响.     

鞍带石斑鱼isg15基因cDNA克隆及其在虹彩病毒感染下的表达分析

本研究利用PCR和RACE技术首次获得了鞍带石斑鱼(Epinephelus lanceolatus)干扰素刺激基因15(interferon stimulated gene 15,isg15)全长序列.isg15基因序列长为910 bp,包含一个468 bp的开放阅读框,可编码155个氨基酸,预测分子量为17.09 k...     

基于PK15细胞的猪圆环病毒2型全悬浮培养工艺

【目的】筛选1株适合PCV2病毒生产的悬浮细胞株,摸索PCV2悬浮生产工艺(病毒种毒来源、MOI及收获时间),为大规模悬浮培养技术制备疫苗提供试验依据。【方法】使用有限稀释法对PK15原细胞稀释后接种于96孔板,每2 d观察细胞生长和形态,待细胞90%长满后,将细胞从96孔板陆续扩至24孔板、12孔板、6孔板,最后到方瓶,筛选3株可贴壁生长的、形态较好的PK15克隆。3株克隆(PK15-1C8、2F11、1E5)按照1×10⁶细胞/m L的密度,直接接种在PK15的无血清培养基中,并置于37℃,5%二氧化碳,120 r/min的摇床培养箱中继续培养,每天监测细胞密度和活率,每3 d传代,使细胞逐渐适应悬浮环境,驯化为可全悬浮、无血清培养的悬浮细胞株;细胞传代稳定并建库后,接种PCV2病毒,通过对比3株悬浮克隆细胞培养PCV2病毒含量的差异,筛选1株克隆细胞,用于生产PCV2;针对不同种毒(来源于贴壁细胞或悬浮细胞),摸索感染MOI(0.1、0.2、0.5)及收获时间(48、72、96、120h),确定PCV2最佳生产工艺。【结果】(1)贴壁细胞置于无血清培养基中,...     

茶树氮吸收效率的早期鉴定技术研究

氮是植物生长的重要营养元素,在茶树栽培过程中常需施用大量氮肥,不仅消耗大量的资源,施用不当还会造成一系列环境问题。培育氮肥高效利用的茶树品种是解决这一问题的重要途径,而建立快速筛选高效株系的早期鉴定方法对于缩短育种茶树育种年限具有重要意义。本研究分析龙井43（LJ43）和中茶108（ZC108）两个茶树品种在不同氮素水平下对氨态氮和硝态氮的吸收与利用数据,通过与¹⁵N同位素标记技术的比对,验证非损伤微测技术（NMT）和实时荧光定量（qRT-PCR）技术在早期鉴定茶树株系氮素吸收利用能力方面的可行性与实用性,以期建立茶树氮吸收效率的室内早期鉴定技术。试验结果表明,¹⁵N同位素标记技术的稳定性和可重复性分别为89.51%、99.26%,而NMT的稳定性、可重复性分别为95.22%、96.76%;两种方法测定结果均显示茶树具有明显的喜铵特性;硝酸根转运蛋白基因CsNRT3.2和CsNRT2.4在两个品种中均表现出诱导上调表达效应,相比中茶108,龙井43中CsNRT2.4和CsNRT3.2具有更高的表达量,表明LJ43对外界氮源的响应高于ZC108。综上所述,认为NMT技术可在短时间内处理并测得茶树的瞬时吸收速率,且试验材料损耗少,可以用于茶树氮瞬时吸收速率的早期鉴定;CsNRT2.4和CsNRT3.2的表达量一定程度上反映了茶树对硝态氮吸收的能力。本研究可为氮高效茶树品种的早期鉴定技术建立提供依据。     

烤烟烟碱合成及其氮素来源与移栽期和氮肥的关系研究

采用田间试验与15N同位素示踪微区试验,在湖北襄樊植烟生态区老湾村(N 31°27,′E 111°14′,海拔1130 m)研究了3个不同移栽期和施用氮肥对烤烟烟碱含量和烟碱氮素来源的影响。研究结果表明,与5月5日移栽相比,推迟移栽期至5月15日2~5日,烟碱含量、烟碱氮占总氮比例以及烟碱肥料氮比例分别平均增加10%8~5%、5%1~10%和21%5~6%;与不施氮相比,施用氮肥提高烟碱含量12%5~9%,提高烟碱氮占总氮比例5%~127%。烤烟烟碱氮占总氮的比例随生育进程逐渐增加,而各部位烟叶烟碱肥料氮比例随生育进程和叶位上升逐渐下降。土壤氮是烟碱氮的主要来源,对烟叶烟碱含量有决定性影响。结果说明在当地条件下,控施氮肥和适当提前移栽期更有利于降低烟叶(尤其是中、上部叶)中的烟碱含量。     

可变电荷与恒电荷稻田土壤硝态氮和铵态氮淋失规律

A variable-charge (VC) and a permanent-charge paddy soil (PC) were selected to study nitrate (NO3--N) and ammonium (NH4+-N) leaching with N isotopes for one consecutive year. An irrigation and intermittent drainage pattern was adopted to mimic natural occurrence of rainfall during the upland crop season and drainage management during the flooded rice season. Treatments to each soil type were no-N controls (CK), 15N-labeled (NH4)2SO4 (NS) and milk vetch (NV) applied at a rate equivalent to 238 kg N ha–1 to unplanted lysimeters, totaling six treatments replicated in triplicates. Results indicated that the soil type dominated N leaching characteristics. In the case of PC, NO3--N accounted for 78% of the total leached inorganic N; NS was prone to leach three times more than the NV, being 8.2% and 2.4% of added 15N respectively; and > 85% of leached NO3--N came from native N in the soil. In the case of VC, NH4+-N made up to 92% of the total inorganic N in leachate. Moreover, NH4+-N leaching was detected throughout the whole incubation, and was particularly high during the flooded season. NO3--N leaching in VC occurred later at a lower rate compared to that in PC. The findings of this study indicate that NO3--N leaching during the drained season in permanent-charge paddy soils and NH4+-N leaching in variable-charge soils deserve more attention for regional environmental control.