Cloning and bioinformatics analysis of cDNA encoding cattle Smad4 gene

The cDNA of cattle Smad4 gene was cloned by RT-PCR, 3′ RACE and 5’t RACE and got a 3503-bp full-long cDNA sequence. The cloned cattle Smad4 cDNA sequence had been send to GenBank and got an accession number: DQ494856. Cattle Smad4 gene consists of 12 exons and codes 553 amino acids. Cattle Smad4 cDNA shares 99%, 96%, 95%, 91% and 91% similarity in nucleic acid sequences, and 99%, 98%, 98%, 99% and 98% similarity in amino acid sequences with sheep, pig, human, rat and mouse, respectively. Smad4 cDNA was found in the testes, pancreas, liver, small intestine, ovary, lymph, cardiac muscle, skeleton muscle and thymus gland, which indicated that Smad4 was broadly expressed in cattle. __________ Translated from Acta Veterinaria et Zootechnica Sinica, 2007, 38(4): 321–325 [译自: 畜牧兽医学报]     

CHANGES IN GROWTH AND GENE EXPRESSION INDUCED BY SULFUR DEFICIENCY IN GARLIC

ABSTRACT

Sulfur deficiency in garlic Allium sativum L. caused a reduction in growth together with chlorosis and necrosis of leaves. Large differences in shoot sulfur and sulphate concentrations between deficient and high sulfur treatments were only observed after 54 days growth. Using the mRNA differential display technique, a novel cDNA was isolated from shoots grown in sulfur depleted nutrient solution for 24 days. This novel cDNA was constitutively expressed in the shoots during further growth in sulfur depleted solution, but it was undetectable following 30 days recovery with sulfur supplementation. The cDNA sequence demonstrated a high degree of identity with a coat protein gene of a garlic latent carlavirus. The results suggest a possible relationship between low plant sulfur status and the induction of a latent carlavirus in garlic.     

鸡传染性支气管炎病毒的分离与鉴定

鸡传染性支气管炎对全球养鸡业带来巨大损失，虽已有商品化疫苗用于临床预防该疾病，免疫鸡群高发病率、高死亡率现象仍然常见。本试验旨在为重庆地区的传支流行毒株进行调查。通过病毒的分离培养和高变区部分序列测序，发现流行于重庆地区的鸡传染性支气管炎毒株与现有的疫苗毒株存在较大差异。某些位点的基因突变与病毒特性的改变之间是否存在联系还需要进一步的研究。     

猪水泡病病毒RT-PCR检测方法的建立

根据基因库中的猪水泡病病毒(SVDV)高度保守的VP1基因的序列,设计了与SVDV互补的2对特异性引物,通过对影响PCR扩增因素的筛选,成功地扩增出251bp和366bp特异性条带,将其克隆入pMD18-T载体中,并进行序列测定,与已发表的SVDV基因比较发现,核苷酸的同源性为100%,证实为SVDV的VP1段基因。通过检测FMDV、VSV、BHK21细胞等,对照的扩增结果均为阴性,验证其特异。对SVDVRNA进行10倍系列稀释后检测,结果SVDVRNA在10-4稀释度时仍能检测到阳性,说明具有较好的敏感性。此项试验能稳定、高效、快速地检测出猪水泡病病毒。     

半定量RT-PCR法分析猪肝脏中铜锌超氧化物歧化酶mRNA表达水平

细胞内铜锌超氧化物歧化酶（CuZnSOD）是动物体内一种重要的抗氧化酶。本实验室构建了优化的半定量RT-PCR法，以18S rRNA为内标，研究猪肝脏组织中CuZnSOD基因mRNA表达水平。提取猪肝脏组织总RNA，经反转录后进行扩增，先寻求PCR线性扩增范围，确定RT．PCR的最佳循环数和Mg^2＋浓度。扩增后用VDS摄像系统扫描PCR扩增产物的电泳条带灰度。通过CuZnSOD PCR产物的灰度与18S rRNA PCR产物的灰度之比，即可计算出CuZnSOD mRNA的相对含量。     

应用竞争PCR检测丙酸盐对体外培养牛肝细胞丙酮酸羧化酶mRNA水平的影响

根据牛肝细胞内PC(丙酮酸羧化酶)基因组与PCcDNA相比多含有一个内含子序列．在其中再插入一外源DNA序列．从而使最终构建的突变体片段的长度大于PCcDNA的长度，成功构建了PCcDNA的竞争DNA模板，然后应用竞争PCR方法研究了丙酸盐对体外培养新生牛单层肝细胞PCmRNA水平的影响。使单层肝细胞培养液中丙酸钠浓度分别为0、1．5、2．5、3．5、4．5、8．5、11．5mmol／L，处理24h，提取总RNA、逆转录，在同一体系中用相同引物扩增目的带和竞争模板带。结果表明．随着丙酸钠浓度的升高．PCmRNA水平呈上升趋势，提示肝细胞内PCmRNA的表达水平受培养液中丙酸钠浓度的影响。     

RT-PCR法快速鉴别新城疫强弱毒株的试验

通过对大量不同毒力NDV F基因核苷酸序列的分析，根据强弱毒株F0裂解位点的序列差异设计合成了三对引物，建立了快速诊断新城疫并能鉴别其强弱毒株的反转录-聚合酶链式反应(RT-PCR)方法，整个试验过程可在5h内完成。试验表明，该方法具有快速特异和操作简便的特点，不仅适用于对鸡胚毒的检测，而且适用于对病鸡组织匀浆液的检测，是新城疫鉴别诊断和流行病学调查的颇具潜力的分子诊断方法。     

一株基因Ⅶ型新城疫病毒的分离及分子鉴定

从一起新城疫暴发的病例中分离出1株新城疫病毒（NDV），命名为铜山分离株（TS）。运用RT-PCR技术，克隆了该分离株F基因的重要功能片段（约0.5kb)，并进行了序列测定。序列分析表明，TS分离株在F蛋白裂解位点的氨基酸顺序为^112-R-R-Q-K-R-F^117，与NDV强毒株特征相符，且具有101外的K（赖氨酸）和121处V（缬氨酸）2个特征性氨基酸，与基因Ⅶ型NDV的特性完全相符。