应用DPO-PCR方法特异性检测志贺氏菌

In this study, a dual-priming oligonucleotide (DPO)-based PCR method for specific detection of Shigella was established using ipaH gene of Shigella as the target gene. The results showed that detection sensitivity of the DPO-PCR method was 1.65×10² CFU/mL. Compared with conventional PCR primers, the DPO primers were easily designed, which simplified the design procedure of PCR primers. DPO primers were not sensitive to annealing temperature, which could efficiently amplify the target gene in the annealing temperature range from 50 to 70 ℃. Moreover, due to the special structure of DPO primers, it had a higher specificity than conventional PCR primers, and none nonspecific amplifications were produced in reaction. In the practical application, tests on 133 samples including frozen/fresh meat, fruits and vegetables, fresh milk, eggs and cooked food by the DPO-PCR method showed that 15 samples were Shigella positive, which were in accordance with the testing results using GB method (GB/T 4789.5-2012), showing good practicality and reliability. The DPO-PCR method provided a new tool for fast and accurate detection of Shigella.     

Association Analysis between Differences of GnRHR-Ⅱ Gene Expression with Reproduction Traits Heterosis in Duck Populations

The objective of this study was to clone the sequence of duck type Ⅱ gonadotropin releasing hormone receptor (GnRHR-Ⅱ) gene, and to analyze the association of its expression levels with reproduction trait heterosis. RT-PCR method was used to clone GnRHR-Ⅱ gene fragments, and the expression levels during early laying days and laying fastigium period in Gaoyou duck, Jinding duck and crossed populations were tested by Real-time PCR method. Sequencing and homology analysis showed that the cloned duck GnRHR cDNA was about 500 bp in length, and it belonged to the avian type Ⅱ GnRHR isoforms. It was 96% identical to the cGnRHR-Ⅱ sequence reported in domestic chicken. From the early laying days to laying fastigium period, expression levels of GnRHR-Ⅱ gene in pituitary increased for Jinding duck, Gaoyou duck and crossed populations, with the highest expression levels in Jinding×Gaoyou hybrid group (P<0.01) . The expression levels of GnRHR-Ⅱ gene in hypothalamus also increased for Jinding duck, Gaoyou duck, however, it decreased in two crossed populations. The expression levels of GnRHR-Ⅱ gene in ovary were significantly higher for Jinding duck and two crossed populations than Gaoyou duck (P<0.01), but it decreased during laying fastigium period. Compared to Jinding duck and Gaoyou duck breeds, the two crossed populations had positive heterosis in 42-week production and 42-week egg fertilization percent, and negative heterosis in the first laying age and 42-week fertilized egg hatching rate traits. It suggested that the short-lived higher expression of ovary GnRHR-Ⅱ gene before early laying days be correlated with heterosis of the first laying age and 42-week production traits, and its higher expression levels in pituitary could aid to keep laying fastigium.     

饲粮半胱氨酸水平对獭兔生产性能和SLC1A4基因表达的影响

试验旨在研究饲粮中半胱氨酸含量对獭兔生长性能、毛皮品质和SLC1A4基因表达的影响。选取90日龄健康獭兔144只,随机分为4组(每组36个重复,每个重复1只)。对照组饲喂基础饲粮(半胱氨酸含量为0.25%),试验组饲喂半胱氨酸含量分别为0.60%、0.70%和0.80%的试验饲粮(在基础饲粮中分别添加0.35%、0.45%和0.55%半胱氨酸)。预饲期7d,正饲期40d。试验期间每隔10d,每组随机屠宰4只獭兔,取样测定皮毛品质和SLC1A4基因表达。结果表明:(1)饲粮半胱氨酸水平为0.70%的试验组末重和平均日增重显著提高(P0.05);料重比显著降低(P0.05);四个组间平均日采食量差异不显著(P0.05)。(2)饲粮半胱氨酸水平为0.60%和0.70%的试验组饲喂到130日龄时皮张面积显著增大(P0.05);三个试验组獭兔在100日龄时被毛密度均显著大于对照组(P0.05);半胱氨酸水平为0.70%的试验组被毛密度在130日龄时显著大于其他三个组(P0.05);半胱氨酸对獭兔皮张厚度无显著性影响(P0.05)。(3)饲粮半胱氨酸含量对獭兔皮肤、肝脏和十二指肠组织中SLC1A4基因的表达量影响极显著(P0.01)。SLC1A4基因的表达量均呈现先增高后降低的趋势,饲粮中半胱氨酸水平为0.70%的试验组达到最高值。这一趋势在皮肤组织中、肝脏和十二指肠组织中分别于110日龄和100日龄时表现得较为显著。由此可见,獭兔饲粮中半胱氨酸的最优水平为0.70%,且在110日龄前使用效果最佳。     

H1N1亚型猪流感病毒HA和NA基因双重RT-PCR定型检测方法的建立与应用

为了建立适用于临床诊断的H1N1亚型猪流感病毒快速检测方法,本研究根据GenBank已登录的H1N1亚型猪流感病毒HA和NA基因序列设计RT-PCR扩增引物,以H1N1亚型猪流感病毒、H3N2亚型猪流感病毒、猪瘟病毒和猪繁殖与呼吸综合征病毒为试验对照,通过优化RT-PCR反应条件和反应体系,建立了H1N1亚型猪流感病毒HA和NA基因双重RT-PCR定型检测方法。同时,运用H1N1亚型猪流感病毒血凝和血凝抑制试验方法和本研究建立的方法对165份猪病料样品进行了对比验证。结果表明,本研究建立的H1N1亚型猪流感病毒双重RT-PCR具有良好的特异性、敏感性、重复性,所扩增的目的基因片段大小分别为428 bp和678 bp左右,可检出最小基因组RNA浓度为2.9×10-5μg/μL。本研究建立的方法和H1N1亚型猪流感病毒血凝和血凝抑制试验方法均从同一份猪肺脏样品中检测出H1N1亚型猪流感病毒,其余样品中均未检出H1N1亚型猪流感病毒,两种方法符合率为100%。本研究建立的方法适用于H1N1亚型猪流感病毒双基因定型检测,可在H1N1亚型猪流感病毒流行病学调查和临床诊断中应用。     

p53调控Nfkbiz基因表达的研究

核因子-κB（NF-κB）在炎症反应中起着关键的促进作用,并且受其他多种因素的调节。Nfkbiz基因编码的IκB？蛋白是IκB家族成员之一,在炎症反应中起着调节NF-κB的作用。本研究采用荧光实时定量PCR分析Nfkbiz基因的表达情况,发现在p53特定刺激源作用下Nfkbiz基因mRNA丰度显著降低。分析Nfkbiz基因序列,发现其基因中存在可能的p53反应元件,通过构建荧光素酶报告质粒及双荧光素酶报告试验和染色质免疫共沉淀试验,证明Nfkbiz基因中含有p53反应元件并且受p53蛋白调节。以上结果初步证明Nfkbiz是受p53蛋白负向调控的靶基因。     

Lipid-based transfection as a method for gene delivery in zebrafish (Danio rerio) embryos

A major challenge to the widespread production of transgenic, knockout and knockdown zebrafish has been the absence of a simple and effective procedure for introducing macromolecules into the fertilized egg. None of the existing techniques for gene transfer in fish embryos has proven to be a major advance over cytoplasm microinjection, which is a technically demanding and time‐consuming procedure. This report addresses this need, considering that the development of protocols for lipid‐based transfection with fish embryos would considerably simplify gene transfer in this complex biological model. In this study, lipid‐based transfection with two different reporter vectors was carried out in zebrafish embryos at different developmental stages. The parameters tested included different plasmid/transfection reagent ratios as well as the influence of an added transfection enhancer reagent. When embryos were transfected in the blastula stage with a pEGFP‐N1 vector, more than 35% successfully incorporated the plasmid and expressed the fluorescent protein 24 h after transfection. The transfection enhancer did not show any significant effect in our experiments. This work presents an approach to implement this technique as a faster, cheaper and more practical alternative than microinjection.     

棘头梅童鱼肝型脂肪酸结合蛋白基因克隆及组织表达分析

为进一步了解肝型脂肪酸结合蛋白基因(L-FABP基因)在棘头梅童鱼(Collichthys lucidus)营养调控、脂肪酸代谢和免疫应答等方面的功能作用,以棘头梅童鱼全基因组组装数据为基础,获得L-FABP基因的含开放阅读框的cDNA序列,利用生物信息学方法分析编码的蛋白质结构及进化关系,采用荧光定量(qRT-PCR)技术研究了不同组织中L-FABP基因的表达情况。结果表明,L-FABP基因cDNA序列为475 bp,其中5’非翻译区为51 bp,3’非翻译区为40 bp,开放阅读框为384 bp,编码127个氨基酸。其结构由10个β-折叠链和2个α-螺旋链组成,在氨基酸3~126位点间具有保守的脂质/胞质性脂肪酸结合结构域。棘头梅童鱼L-FABP基因与大黄鱼、条纹狼鲈同源性较近,与绿河鲀、尼罗罗非鱼和斑马鱼同源性较远。L-FABP基因在棘头梅童鱼肝脏、肠道、眼、大脑、心脏、肌肉、胃、鳃、脾脏、头肾和中肾中均有表达,其中肝脏中的L-FABP相对表达量最高,显著高于其他组织(P<0.05)。     

RGA法克隆NBS-LRR类抗病基因同源序列及其在葫芦科作物上应用的研究进展

RGA克隆法是利用抗病基因产物的保守结构域人工设计简并引物,以植物gDNA或cDNA为模板进行PCR扩增而克隆植物抗病基因同源序列的方法。随着各种植物基因组测序计划的完成及计算机和生物信息学的发展,植物抗病基因的克隆取得了很大进展,目前人们已经从植物中克隆得到100多个抗病基因。研究表明,大多数抗病基因都存在NBS-LRR、STK、LZ、TIR等功能保守的结构域,其中大部分都为NBS-LRR类型,利用NBS-LRR保守结构域设计PCR引物已经从植物中扩增出大量的RGA。简要综述了已克隆NBS-LRR类抗病基因的结构特点和功能、RGA法克隆NBS-LRR类抗病基因同源序列及其在葫芦科作物上应用的研究进展,并探讨其未来的发展与应用。