猪繁殖与呼吸障碍综合征病毒基因芯片检测技术的建立与优化

根据猪繁殖与呼吸综合征病毒（PRRSV）的核衣壳蛋白编码基因序列设计了一对特异性引物P1／P2。扩增出大小为294bp的目的片段；再针对这个基因片段。设计合成4条寡核苷酸探针。其中反向引物的5’端用荧光素Cy3标记。以荧光标记不对称PCR技术为基础。通过将单链PCR产物与芯片杂交实现对PRRSV的检测。建立PRRSV的基因芯片检测方法。利用该方法对39份猪组织样品进行检测。与RT—PCR检测方法相比。本方法具有良好的特异性和敏感性。试验结果表明用该方法快速检测病料组织中PRRSV是可行的，对该病的进行快速诊断和分子流行病学调查具有重要意义。     

富含ω-3多不饱和脂肪酸克隆猪制备的关键技术研究

作者综述了制备富含ω-3多不饱和脂肪酸克隆猪的研究意义和国内外的研究现状，并介绍了自己的研究进展。用化学合成的方法获得了C briggsae的ω-3脂肪酸去饱和酶基因sFat-1，并构建了哺乳动物细胞表达载体。通过显微注射的方法制备了转基因小鼠，sFat-1基因能够在转基因小鼠中产生更多更长链的而且更有价值的DHA以及DPA。在确认sFat-1基因功能的基础上，将载体转染到猪胎儿成纤维细胞，利用阳性的细胞克隆通过核移植方式制备富含ω-3多不饱和脂肪酸克隆猪。     

生长因子EGF、bFGF对猪孤雌胚体外发育的影响

在胚胎发育的不同阶段，分别在培养基中添加EGF和bFGF，研究EGF和bFGF对猪孤雌胚体外发育的作用。结果表明：在1细胞阶段添加EGF或bFGF，添加EGF能够显著提高孤雌胚的卵裂率（P〈0．05）；2～4细胞阶段添加EGF和bFGF，添加EGF组和添加bFGF组的囊胚率都显著高于对照组（P〈0．05），而添加bFGF组的囊胚率和囊胚细胞数都略高于对照组和添加EGF组。说明EGF和bFGF有利于猪孤雌胚的体外发育，而且，bF-GF能够通过提高囊胚细胞数而提高猪孤雌胚的质量。     

绿头野鸭与我国主要地方鸭品种遗传多样性研究

利用17个微卫星标记,采用PCR扩增,8%非变性聚丙烯酰胺凝胶电泳、银染,对绿头野鸭种群和我国9个主要地方鸭品种的遗传多样性进行了检测,统计了各群体的平均有效等位基因(Ne)、平均遗传杂合度(H)、多态信息含量(PIC)、群体间的遗传距离(Ds)和相似系数(I)。采用UPGMA和NJ法构建系统发生树。结果表明:17个微卫星座位在绿头野鸭种群和我国9个主要地方鸭品种中的多态信息含量均为高度多态,所有鸭种的平均PIC、H、Ne分别为0.6506、0.6896、4.1±1.5,基因多态性和遗传多样性相对较高;家鸭各品种间的遗传距离在0.1631~0.6844范围内,绿头野鸭与家鸭品种间的遗传距离分别在0.3096~0.8219之间。根据系统发育树推断中国主要地方鸭品种不是单一起源于绿头野鸭。中国主要地方鸭品种大致可以分为5支。一支为北京鸭、荆江鸭,一支为中山鸭、建昌鸭及高邮鸭,一支为靖西大麻鸭和金定鸭,连城鸭为一支,绍兴鸭为另一支。     

禽传染性支气管炎DNA疫苗在鸡体内的分布和安全性

采用本实验室构建的3种禽传染性支气管炎病毒基因的真核表达质粒pIBVS1、pIBVM、pIBVN,按各50 mg/L配制成质量浓度为150 mg/L的DNA疫苗,经腿部肌肉分点注射免疫1周龄SPF雏鸡。分别在免疫后24 h,73、0及60d,采集试验鸡的血液、心、肝、脾、肺、肾、胸腺、性腺（卵巢/睾丸）及注射部位肌肉进行组织总DNA抽提,以组织总DNA为模板进行PCR扩增。另外,在对照组DNA模板中加入不同拷贝数的质粒,确定PCR反应的灵敏性。以纯化后的组织总DNA为模板,PCR法检测质粒DNA整合到鸡细胞染色体基因组上的可能性,评价疫苗的安全性。结果表明,该疫苗在24 h内迅速分布于全身,并能在血液及所有检测的组织器官内持续分布60 d;纯化后的组织基因组DNA经PCR扩增均呈阴性,未发现整合现象,证实该DNA疫苗的安全性好。     

提高益生素作用效果的研究进展

益生素具有无污染、无毒副、无药残等特点,是抗生素的良好替代品。但益生素作用效果还存在许多争议,加强其作用效果的研究迫在眉睫。目前,人们主要通过高效菌株的选育、复合益生素的开发与益生素有协同作用的饲料添加剂研究来提高益生素的功效。寻求与益生素有协同作用的饲料添加剂被认为是最有效的方法,而广泛的应用于生产实践中。     

不同光增方式和周期对蛋鸡生产性能和血液生化指标的影响

研究光增方式和光照周期对蛋鸡蛋品质和血液生化指标的影响.将20周龄海兰褐商品蛋鸡320只,随机分为8组,每组4个重复,每个重复10只鸡.试验光照周期采用渐增和骤增2种,光周期设11L13D、13L11D、15L9D、17L7D 4个水平.结果表明①不同光增方式对蛋形指数和哈氏单位有显著影响(P＜0.05),以渐增条件下较好.不同光增方式对血液生化指标没有显著影响(P＞0.05).②不同光照周期对蛋黄颜色有显著影响(P＜0.05),以15L9D处理组蛋黄颜色最好.不同光照周期对血液生化指标有一定影响,以13L11D处理组较好.     

Classification of Morchella species from Yunnan and Zhejiang Provinces Based on rDNA ITS Sequences

Fourteen Morchella samples (eleven from Yunnan and three from Zhejiang Provinces) were selected on the basis of differences in fruit body morphology. Ribosomal DNA internal transcribed spacers (ITS) were amplified in each case using the universal primer pair, ITS-1 and ITS-4, and the amplification products were purified and sequenced. Comparisons with sequence data in GenBank revealed that the 11 Morchella isolates collected from Yunnan belonged to four species: Morchella elata, Morchella conica, Morchella crassipes and Morchella costata. The three isolates collected from Zhejiang Province (M12, M13 and M14) were designated as unknown Morchella species. When Verpa conica (AJ544206) (from the genus Verpa belonging to the same family as Morchella) was taken as the outgroup, the 14 isolates formed three groups, M. elata, M. costata (Group 1); Morchella esculenta, M. conica (Group 2); and M. crassipes, M12, M13 and M14 (Group 3).     

Genetic Analysis of Fruit Body Color in a Hybrid Strain of Flammulina velutipes

A series of hybrid Flammulina velutipes dikaryons were obtained by crossing yellow and white monokaryons.The color of fruit bodies generated from these dikaryons,and the ratio between dark-colored stipe regions and the entire stipe of individual fruit bodies,were determined.Our data suggest that the expression of fruit body color in the hybrid dikaryons was under the control of factors present in both the white and yellow parent monokaryons,and that multiple alleles exist in both white and yellow color-controlling factors.     

Construction and identification of eukaryotic expression vector of bcl-2 siRNA

AIM: To construct eukaryotic expression vector of small interfering RNA(siRNA) specific to bcl-2 and investigate the effect of recombinant plasmid on suppressing bladder cancer cell growth.METHODS: siRNA of bcl-2 gene was designed according to the principle of RNAi-based medicine, and was converted into cDNA coding expression of small hairpin RNAs(shRNA) of siRNA. The cDNA was synthesized and inserted into plasmid pGenesil-1. The recombinant eukaryotic expression vectors of pGenesil-1545 and pGenesil-1555 were controlled by the U6 promoter of RNA polymerase Ⅲ, identified by the restriction map and the sequence analysis, and transfected into T24 cells. After T24 cells were transfected for 72 h, expression of bcl-2 mRNA was assayed by RT-PCR; and MTT was used to observe the proliferation of T24 cells.RESULTS: The recombinant plasmids of pGenesil-1545 and pGenesil-1555 were identified by the restriction map and the sequence analysis. The sequences completely coincided with the designs. The expression of the bcl-2 mRNA in T24 cells transfected with recombinant plasmid decreased nearly 80%, and the growth of T24 cells was suppressed significantly.CONCLUSION: The siRNA eukaryotic expression vector against bcl-2 gene is successfully constructed. It effectively downregulates the expression of bcl-2 in T24 cells and suppresses the cell growth.