广西德保猪MC1R基因的克隆与分析

近年来含有天然黑色素的动植物黑色食品得到青睐,为探讨广西地方品种德保猪全黑毛色形成的分子机制,本文克隆了广西德保猪MC1R基因并进行了系统生物信息学分析。根据NCBI已公布的猪MC1R基因序列（GI：347618788）设计特异性引物,以从德保猪血液中提取的基因组DNA为模板,通过PCR扩增克隆获得长1 519 bp的MC1R基因序列片段。多重序列比较结果显示克隆片段与已公布的猪、牛、人、狗、绵羊、小鼠和鸡的相似性依次分别为99%、87%、86%、85%、84%、81%和77%,表明MC1R基因在不同哺乳动物间具有较高的保守型。德保猪MC1R蛋白结构预测结果显示,其理论分子质量为34.65 ku,等电点为8.70,为弱碱性蛋白,N-末端不存在信号肽,定位于细胞质膜,具有1个低复杂度结构域和7个跨膜结构域,具有典型的细胞膜受体蛋白结构。多项研究报道表明MC1R基因是动物毛皮颜色重要的决定基因,德保猪MC1R基因克隆为揭示其全黑色形成的分子机制奠定了较好的实验基础。     

罗氏沼虾白肉病病原研究初报

为了探明罗氏沼虾白肉病发病原因,特别采集白肉病的典型病虾苗进行细菌分离、除菌组织悬液制备及人工感染试验,结果表明:从罗氏沼虾白肉病病虾的肌肉、肝胰腺中分离到的细菌浸泡感染正常罗氏沼虾苗不能复制疾病。病虾除菌组织过滤液浸泡感染正常虾苗,7d后可发病,并出现典型的白肉病症状,氯仿处理不能破坏病毒的感染力,表明罗氏沼虾白肉病是一种由病毒引起的疾病。     

微生物原生质体融合技术在净化水质上的应用

原生质体融合技术是20世纪70年代发展起来的一项新技术,在微生物育种方面显示出了巨大的潜力.了解微生物原生质体融合技术的原理、分析其优点和所涉及的技术参数,对微生物原生质体融合技术在水质净化中的应用有重要意义.     

3种养殖模式罗非鱼品质比较分析与评价

【目的】探讨池塘工程化循环流水养殖（水槽组）、稻田养殖（稻田组）和池塘养殖（池塘组）3种不同养殖模式对罗非鱼肌肉营养品质的影响,为罗非鱼养殖业的提质增效提供参考依据。【方法】以吉富品系尼罗罗非鱼（GIFT,Oreochromis niloticus）为研究对象,选取平均体质量为595±88、625±75和644±21 g的罗非鱼分别饲养于水槽组、稻田组和池塘组养殖模式下,通过每克氮氨基酸评分模式、完全蛋白质评分模式和质构特性参数评价对肌肉营养成分进行分析比较,判断罗非鱼肌肉的营养价值。【结果】在常规营养成分方面,3组罗非鱼肌肉水分和粗灰分含量无显著差异（P> 0.05）,水槽组罗非鱼肌肉粗脂肪含量显著高于池塘组和稻田组（P< 0.05,下同）,稻田组罗非鱼肌肉粗蛋白含量显著高于水槽组和池塘组。在氨基酸组成方面,天门冬氨酸（Asp）、谷氨酸（Glu）、亮氨酸（Leu）和赖氨酸（Lys）含量最高,稻田组罗非鱼肌肉必需氨基酸总量（ΣEAA）显著高于池塘组和水槽组,3组罗非鱼肌肉ΣEAA/氨基酸总量（ΣTAA）范围为0.39～0.41,ΣEAA/非必需氨基酸总量（ΣNEAA）为0.65～0.68。在必需氨基酸评价方面,水槽组罗非鱼肌肉必需氨基酸总量（2689 mg/g N）和必需氨基酸指数（EAAI,87.96）最高,池塘组次之,稻田组最低。在肌肉质构特性方面,水槽组罗非鱼肌肉硬度显著低于稻田组和池塘组,胶黏性显著高于稻田组和池塘组。【结论】池塘工程化循环流水养殖模式下生产的罗非鱼肉质综合评价最佳,养殖效果好,且符合当前节能减排、提质增效的要求,可在适宜区域内大力推广应用。     

Expression and significance of TLR4 in Sombatis cell-based model of epilepsy

AIM：To study the change of Toll-like reporter 4 (TLR4) expression in Sombatis cell-based model of epilepsy and to explore the role of TLR4 in this model. METHODS：After cultured in vitro for 9 d, the neurons of newborn SD rats were randomly divided into control group and model group. The neurons in model group were cultured in low-magnesium medium for 3 h and then returned to the normal medium culture. The expression of TLR4 at mRNA and protein levels was detected by fluorescence quantitative PCR and the method of immunohistochemistry, respectively. RESULTS：The immunohistochemical results showed that the protein expression of TLR4 was enhanced in the Sombati’s cells. The results of fluorescence quantitative PCR showed that the mRNA expression of TLR4 time-dependently increased in the Sombatis cells (P<0.05). CONCLUSION：The expression of TLR4 in the neurons is up-regulated in Sombatis cell-based model of epilepsy in rats, suggesting that TLR4 is associated with the pathogenesis of epilepsy.     

Effects of GOLPH3 gene silencing mediated by lentivirus on proliferation,invasion and metastasis of human gastric cancer cell line SGC-7901

AIM：To investigate the effect of Golgi phosphoprotein 3 (GOLPH3) gene silencing on the proliferation, invasion and metastasis of human gastric cancer SGC-7901 cell in vitro. METHODS：SGC-7901 cells were transfected with lentiviral vector carrying GOLPH3 shRNA to construct a stable GOLPH3-silencing cell line LV-GOLPH3-RNAi. The expression of GOLPH3 at mRNA and protein levelss were detected by real-time PCR and Western blotting, respectively. The cell proliferation was analyzed by MTT assay. Transwell migration and invasion experiments were performed to measure the migration and invasion abilities, respectively. RESULTS：The stable GOLPH3-silencing cell line was successfully established. The expression of GOLPH3 at mRNA and protein levels was reduced significantly (P<0.05), leading to the inhibition of cell proliferation in LV-GOLPH3-RNAi group compared with scrambled group and blank control group, as well as the capacities of migration (56.7±1.5 vs 186.0±3.4 and 183.3±4.2, P<0.05) and invasion (33.5±3.0 vs 85.0±3.9 and 83.1±4.4, P<0.05). CONCLUSION：GOLPH3 silencing suppresses the capacities of proliferation, migration and invasion of human gastric cancer SGC-7901 cells, suggesting that GOLPH3 may be a potential tumor marker and independent prognostic factor.     

Role of miRNA-24 in regulation of endothelial nitric oxide synthase expression and vascular endothelial cell proliferation

AIM：To investigate whether miRNA-24 is involved in the regulation of endothelial nitric oxide synthase (eNOS) expression and vascular endothelial cell proliferation. METHODS：A plasmid that highly expressed miRNA-24 was constructed, and was transfected into the human umbilical vein endothelial cells (HUVECs) by liposome. The cell proliferation was detected by MTT assay. The expression of eNOS and Sp1 at mRNA and protein levels was exa-mined by real-time PCR, immunohistochemistry and Western blotting.RESULTS：Compared with control group, the proliferation of endothelial cells in miRNA-24 group was significantly decreased by 41.97 % (0.47±0.04 vs 0.81±0.03, P<0.01), and the expression of eNOS at mRNA and protein levels was decreased by 44.8% (0.48±0.01 vs 0.87±0.03, P<0.05) and 71.92% (0.16±0.06 vs 0.57±0.08, P<0.05), respectively. Meanwhile, the mRNA and protein levels of Sp1 were significantly decreased by 53.00% (0.45±0.02 vs 0.93±0.01, P<0.05) and by 62.31% (0.13±0.07 vs 0.31±0.09, P<0.05), respectively. In miRNA-24 inhibitor group, the above indexes were decreased compared with control group, but significantly increased compared with miRNA-24 group. CONCLUSION：miRNA-24 significantly inhibits the proliferation of HUVECs and the eNOS expression. Sp1 possibly acts as one of the important factors in the regulation of eNOS expression by miRNA-24.     

凡纳滨对虾HSP1噬因序列与低温表达分析

【目的】克隆分析凡纳滨对虾HSPIO~因,为研究其在寒冷耐受过程中发挥调控功能提供参考依据。【方法】搜索EST库获得凡纳滨对虾HSPIO~因序列,再通过RACE—PCR扩增获得凡纳滨对虾HSP10基因cDNA全长序列,对其进行生物信息学分析,并通过荧光定量PCR分析HSPIO基因的组织表达及低温胁迫下表达量的变化。【结果】凡纳滨对虾HSP10基因cDNA全长720bp,包含306bp的开放阅读框,编码102个氨基酸。以各物种的HSP10蛋白序列构建系统进化树,能较好反映各物种间的进化关系。荧光定量PCR分析结果显示,凡纳滨对虾HSPIOmRNA在各组织中呈遍在表达,其中以肌肉中的表达量最高。低温表达谱分析结果显示,凡纳滨对虾HSPIOmRNA在低温处理凡纳滨对虾的各组织中均呈下调表达,当处理温度降至13℃,其在肝胰腺中表达量降至最低。【结论】凡纳滨对虾HSP10在结构和进化上较保守,其mRNA表达量在低温胁迫下呈下降趋势,可能在寒冷耐受过程中发挥负调控作用。     

153种中草药对罗非鱼无乳链球菌和海豚链球菌的抑制活性研究

【目的】研究中草药对罗非鱼无乳链球菌和海豚链球菌的抑菌活性,为生产中罗非鱼链球菌病害的防治提供参考。【方法】采用琼脂扩散法,测定153种中草药乙醇提取物对罗非鱼(Tilapiasp.)无乳链球菌(Streptococcus agalactiae)和海豚链球菌(S.iniae)的体外抑菌作用,并采用二倍稀释法测定中草药乙醇提取物的最小抑菌浓度(MIC)和最小杀菌浓度(MBC)。【结果】153种中草药中,博落回(Macleaya cordata)、十大功劳(Mahonia fortunei)、三颗针(Berberis spp.)、紫草(Lithospermum erythrorhizon)、田七须(Panax notoginseng)、补骨脂(Psoralea corylifolia)、田基黄(Hypericum japonicum)和五倍子(Galla chinensis)提取物对无乳链球菌表现出了明显的抑制活性,其中博落回的抑菌效果最显著,抑菌圈直径为(25.1±0.6)mm;博落回、千斤拨(Flemingia philippinensis)、田七须、甘草(Bidens bipinnata)、田基黄、败酱草(Patrinina villosa)、紫草、苦参(Sophora flavescens.)、补骨脂、北五味子(Schisandra chinensis)、五倍子、鬼针草(Bidens bipinnata)、虎刺(Damnacanthus indicus)、三颗针、翠云草(B.bipinnata)、羌活(Notopterygium incisum Ting ex)、香薷(Elsholtzia patrini Garcke)对海豚链球菌具有明显的抑菌活性,其中博落回的抑菌作用最强,抑菌圈直径为(24.3±1.1)mm。对抑菌活性较强的中草药的MIC和MBC进行了测定,结果表明,三颗针和博落回提取物的抑菌和杀菌效果均较好。三颗针、博落回对无乳链球菌和海豚链球菌的MIC和MBC分别为0.31,0.63;0.63,1.25和1.25,2.50;0.94,1.88mg/mL。【结论】博落回、紫草、田基黄、补骨脂、三颗针、田七须和五倍子对无乳链球菌和海豚链球菌均具有较好的抑菌效果。     

Cross-species comparison of Smad3 expression in hepatocellular carcinoma tissues

AIM： To study the expression of Smad3 and its significance in hepatocellular carcinoma (HCC) in different species including human, rat and tree shrew, and to verify the feasibility of cross-species oncogenomic approach. METHODS：Real-time fluorescent quantitative polymerase chain reaction and Western blotting were applied to detect the expression of Smad3 at mRNA and protein levels in HCC tissues, corresponding HCC adjacent liver tissues and normal liver tissues collected from different species including human, rat and tree shrew. RESULTS：The mRNA expression of Smad3 in HCC tissues of human, rat and tree shrew was lower than those in the corresponding HCC tissues adjacent liver tissues. The mRNA level of Smad3 in HCC tissues of rat was lower than those in its normal liver tissues, while that in HCC adjacent tissues of tree shrew appeared higher than those in the normal liver tissues. No significant difference of Smad3 expression in other tissues was observed. The protein levels of Smad3 in HCC of human and rat were lower than those in the corresponding HCC adjacent liver tissues and the normal liver tissues. However, the protein expression of Smad3 was at a low level in HCC tissues of tree shrew and was lower than that in the HCC adjacent liver tissues and the normal liver tissues, although without statistical difference. The differences of Smad3 expression between HCC adjacent liver tissues and normal liver tissues in all the 3 species were not statistically significant. CONCLUSION：Smad3 is lowly expressed in HCC tissues of different species, suggesting that it might play a pivotal role in hepatocarcinogenesis and be applied as a key molecular target in HCC prevention and treatment.