冷藏对郁金香生物学性状及F1种子萌发的影响

以‘AAFKE’‘Eskimo Chief’等5个郁金香品种为试材,研究了冷藏对其生物学性状及F_1种子萌发的影响。结果表明:增加冷藏时间可以有效推迟郁金香花期,冷藏时间每增加2周,盛花期就会相应的推迟10d,生育周期也缩短。但冷藏时间对植株的生物学性状、杂交结实率、F_1代种子萌发率影响不大。杂交结实率与品种倍性有关,郁金香二倍体与二倍体的杂交结实率高于二倍体与三倍体的杂交结实率。     

Substitution between Virulent and Avirulent PPRSV Vaccine Strains with Reverse Genetics System

The Nsp9 gene of porcine reproductive and respiratory syndrome virus（PRRSV）coded the RNA polymerase,which played an important role in the virus replication.In order to determine the interaction between the Nsp9 gene and virulence,replication capacity and cell tropism,the Nsp9 gene sequence of PPRSV vaccine strain CH-1R was cloned,and ligated to a virulent strain XH-GD infectious clone after restriction enzyme digestion,and transformed the virus under the basis of the reverse genetics.The results showed that the virus was successfully saved.And the results of biological characteristics test showed that titers and growth rate of saved virus were obviously higher than that of parental strain rXH-GD,but were lower than vaccine strain CH-1R.While plaques of two strains were similar in size.The polymerase of vaccine strain CH-1R could help to improve the drop degree of the virus,and this test could lay the foundation for further study of the effect of Nsp9 gene on virus virulence.     

甘蓝夜蛾核型多角体病毒京农8217株的分离及其特性研究

1982年在京郊从甘蓝夜蛾虫体上分离到一株核型多角体病毒一“京农8217”株。多角体直径0.87～2.25微米,病毒粒子平均长250纳米,为多粒包埋型。凝集试验表明:与黑龙江分离的MbNPV之间凝集滴度有4倍以上差别,补结试验表明:与单粒包埋的HaNPV无抗原交叉。交叉感染试验结果:对甘蓝夜蛾、棉铃虫、烟青虫的LC_(50)无显著差异。防治菜田及棉田棉铃虫均有70％左右效果。     

牦牛源干燥棒状杆菌的分离与鉴定

一只牦牛屠宰后肝有干酪样结节,为了探究其形成原因,本研究对该牦牛肝进行细菌的分离培养,对所分离可疑致病菌的16SrRNA以及rpoB基因序列进行扩增和序列分析,并用Vitek 2全自动微生物鉴定仪进行生化表型的鉴定,用K-B法进行药敏试验。结果显示,经细菌分离培养得到一株革兰阳性、棒状、可疑致病菌;其16S rRNA扩增片段大小1 483bp,GenBank序列编号为MH000696,该菌的16SrRNA与干燥棒状杆菌GD34株(KF928790.1)等的相似性最高,序列一致性为99%,系统进化树显示,本菌与干燥棒状杆菌在同一进化分支上;rpoB基因扩增片段大小434bp,GenBank序列编号为MH006608;Vitek 2全自动微生物鉴定仪鉴定为变异库克菌,可信度为93%。该菌对妥布霉素、氨曲南、头孢哌酮、米诺环素等多种药物高度敏感。综上所述,分离的细菌为干燥棒状杆菌,但基因序列,如rpoB等以及生化表型均存在一定程度的变异,本研究为兽医临床上该菌的分离和鉴定提供了依据,且为该菌的致病性研究奠定了基础。     

基于GBS技术对梅花鹿、马鹿及其杂交后代基因组SNP特征的分析

旨在利用基因分型测序（genotyping by sequencing,GBS）技术对梅花鹿、马鹿及其杂交后代（F1、F2）基因组的SNP特征进行分析。本试验采用GBS技术对梅花鹿（63个）、马鹿（12个）及其杂交后代（F1代112个,F2代38个,未知类型个体1个）共226个个体的血液基因组DNA进行测序,并利用本实验室前期110只梅花鹿、197只马鹿和1只F1代杂交鹿的测序数据,以梅花鹿全基因组为参考序列进行比对分析。结果,226个个体共产生Clean data 322.683 Gb,平均每个样品1 427.802 Mb;将所有样本作为一个群体检测SNP变异,共检测出SNP位点23 943 582个,质控过滤后得到SNP位点31 630个。对31 630个SNPs使用最大似然（maximum likelihood,ML）法构建的分子进化树显示,梅花鹿、马鹿、F1及F2代区分明显。对梅花鹿和马鹿的SNPs进行比对分析,筛选出可用于鉴别马鹿、梅花鹿、F1、F2的物种特异SNP位点1 032个（马鹿特异SNP位点474个,梅花鹿特异SNP位点558个）,计算结果显示,F1代个体包含马鹿特异SNPs的比例主要在40%~60%之间,F2代个体含马鹿特异SNPs的比例主要在10%~30%之间,马鹿个体中不含梅花鹿的特异SNPs,梅花鹿中55.49%的个体不含马鹿特异SNPs,17.34%的个体含马鹿特异SNPs的比例低于1%,13.29%的个体含马鹿特异SNPs的比例在1%~10%之间,其余个体含马鹿特异SNPs的比例为10%~20%（其中有一个个体含马鹿特异SNPs的比例为33.3%）。该研究为花马杂交鹿后代的鉴定提供了可靠标记,并定量估计了F1和F2代个体含马鹿特异SNPs的比例,马鹿个体中不含梅花鹿的特异SNPs,这对梅花鹿、马鹿及其杂交后代（F1、F2）的鉴别具有重要意义。     

利用微卫星分析中国家兔的遗传多样性

利用10个微卫星DNA标记对七个家兔品种进行了遗传检测.利用变性聚丙烯酰胺凝胶电泳检测微卫星的PCR扩增产物,计算了七个家兔品种(9个家兔群体)样本在10个微卫星基因座上的等位基因频率、微卫星的多态信息含量(PIC)、各群体遗传杂合度、群体间的遗传距离、并根据遗传距离对这几个群体进行了聚类分析.结果表明:所选择的10个微卫星位点在所检测群体中均表现了较好的多态性,平均多态信息含量为0.569905.平均杂合度范围在0.5885～0.6826.表明所研究的家兔品种的遗传多样性较丰富,还可以做进一步的选择利用.根据遗传距离所做的聚类分析图表明吉戎-Ⅰ系和吉戎-Ⅱ系、哈尔滨大白兔、塞北兔的亲缘关系依次较近,与它们的培育历史相吻合.     

Apoptosis in rat hepatocytes with ischemia/reperfusion injury

AIM: To investigate the distributive rules of apoptosis index (AI) in liver with ischemia/reperfusion (I/R) injury and evaluate the factors related to the hepatocyte apoptosis. METHODS: Sixty SD rats in specific pathogen free grade were randomly divided into three groups: control group (n=18), sham operation group (n=18) and I/R group (n=24). In I/R group, liver injury was induced by blocking blood inflow in rat liver for 20 min, then reperfusion for 22 h. Rats in the control group didnt receive any management. Rats in the sham operation group only subjected sham operation. All rat blood samples and livers were obtained for determination. Blood serum ALT, AST, TBIL, TNF-α, IFN-γ, IL-4, plasma endotoxin concentration, MDA level and SOD activity in liver were detected. Hepatic histological analysis was conduced through HE staining. Apoptosis was detected by TUNEL methods. RESULTS: Focal necrosis occurred in six rats livers in I/R group, in control group and sham group no necrosis cell was found in livers. The hepatic AI of I/R group was significantly increased compared with other groups. The AI in region under hepatic amicula was higher than that in central veins region and portal area. The necrotic regions contained apoptotic cells and AI was higher than that of other regions. Hepatic AI was significantly associated with ALT, AST, TNF-α, IFN-γ and SOD/MDA. CONCLUSION: In liver with I/R injury, the apoptotic cells in the region under hepatic amicula and the focus of necrosis are significantly higher than those in other regions, apoptotic cells and necrosis cells co-exist in the same zone. Hepatic AI may be significantly associated with ALT, AST, TNF-α and SOD/MDA.     

黑貉TYRP1基因的克隆及生物信息学分析

为探明黑貉酪氨酸酶相关蛋白1（tyrosinase-related protein 1,TYRP1）基因CDS序列并对其进行生物信息学分析,试验采集黑貉背部皮肤组织提取RNA后反转录为cDNA,根据GenBank中犬科TYRP1基因mRNA序列（登录号：NM_001194966.1）设计1对特异性引物,通过RT-PCR扩增TYRP1基因CDS区,将其插入到克隆载体中,对阳性菌落经PCR验证和序列测定后进行生物信息学分析。结果显示,黑貉TYRP1基因CDS序列全长为1 614 bp,编码537个氨基酸,所编码蛋白属于不稳定可溶性的亲水蛋白质。TYRP1基因与狐的遗传距离较近,说明该基因在不同物种及进化过程中具有高度保守性。TYRP1蛋白二级结构中,无规则卷曲所占比例最高,为54.0%;α-螺旋次之,为29.8%。TYRP1蛋白三级结构主要由大量无规则卷曲和α-螺旋构成,与二级结构相一致。TYRP1蛋白具有氧化还原酶活性,参与DNA修复和复制、蛋白质合成和降解,可与TYR、DCT、TP53、TRPC1等多种蛋白相互作用。本试验结果为进一步探索TYRP1基因在哺乳动物毛色遗传机制中的功能提供了理论依据。     

牛支原体临洮分离株NOX-1的克隆表达、酶学活性及其亚细胞定位研究

为研究牛支原体(M. bovis)临洮分离株(LT strain)NOX-1基因特征表达产物的酶活性,及其在细胞中的具体存在部位,参照GenBank中M. bovis NOX-1HuBei株NOX-1基因序列(GenBank登录号:NC015725.1)设计引物,应用PCR扩增M.bovis临洮株的NOX-1基因,在完成测序后构建NOX-1基因原核表达载体pET-NOX-1,并转化大肠杆菌BL21(DE3)感受态细胞,经异丙基硫代半乳糖苷(IPTG)诱导后表达。表达产物纯化后进行酶活测定并免疫新西兰兔,制备多克隆抗体,继而应用Western blot及间接ELISA对NOX-1在M. bovis内的分布进行初步定位。结果显示,M. bovis临洮株NOX-1基因全长1365 bp,重组质粒经IPTG诱导在大肠杆菌BL21(DE3)中成功表达,重组蛋白His-NOX-1的分子量约为50 kDa;重组蛋白酶促反应最适酶促温度为30℃、pH为7.5,双倒数法求得重组蛋白Km和Vmax分别为256.41μmol/L、34.25μmol/(L·min);Western blot及间接ELISA结果表明His-NOX-1在M. bovis细胞膜上和细胞浆中均有分布且胞浆含量较高。本研究结果为进一步研究M. bovisNOX-1的生物学功能奠定了一定的基础。     

宁夏枸杞多酚Q-TOF/MS~E分析及对细胞抗氧化能力的影响

为探究宁夏枸杞潜在的营养价值,从多酚组成的角度研究宁夏枸杞的抗氧化机理,采用四级杆串联飞行时间质谱MS~E方法(Q-TOF/MS~E)分析宁夏枸杞果实中的多酚成分,测定其提取物(LBE)中总多酚、总黄酮含量,并从细胞水平评价其抗氧化活性。结果表明,宁夏枸杞果实中共鉴定出28种多酚类化合物,其中8种酚酰胺衍生物为首次发现及鉴定。体外试验结果显示,LEB中总多酚、总黄酮含量分别为3 320 mg GAE·100g-1DW和570 mg RE·100g-1DW。进一步研究发现,LBE能有效降低氧化应激状态下人克隆结肠癌细胞(Caco-2)中活性氧自由基(ROS)水平和还原型谷胱甘肽(GSH)水平,且效果呈剂量依赖性,这从细胞水平证明LBE可以显著增强Caco-2细胞的抗氧化能力。综上,宁夏枸杞中含有大量多酚类化合物,并具有较强的抗氧化活性,可以广泛应用于营养食品、制药、化妆品以及各类天然产品中。