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.     

RNAi技术在转基因西瓜抗病毒病上的研究与应用

对RNAi技术的概念、研究进展及西瓜病毒病的危害及其发生特点、RNAi技术在西瓜抗病毒病上的研究进展进行了阐述,并对存在的一些问题作了简要探讨与展望。     

靶向S基因外源性microRNA抑制TGEV增殖效果的研究

应用生物信息学软件对猪传染性胃肠炎病毒(TGEV)S基因进行分析,筛选出可能与S基因有相互作用的外源miRNA:amiRNA-S-28035,amiRNA-S-28038,amiRNA-S-28165。之后,利用脂质体将构建好的相应的表达载体瞬时转染至PK-15细胞;通过Q-PCR和间接免疫荧光方法检测其对S基因的抑制作用;CPE分析和TCID50测定检测其对TGEV增殖的抑制效果。结果发现,3个外源性miRNA均降低了S基因mRNA的转录和蛋白的表达,其中amiRNA-S-28038对TGEV mRNA的平均抑制率可达64.6%,最高可达69.9%,表明外源性microRNA可以通过靶向TGEV基因组来抑制TGEV的复制。研究结果为猪传染性胃肠炎的预防和治疗提供了新的思路。     

Competition for food between Eurasian perch (Perca fluviatilis L.) and ruffe (Gymnocephalus cernuus [L.]) over different substrate types

Abstract –  Food consumption by Eurasian perch ( Perca fluviatilis L.) and ruffe ( Gymnocephalus cernuus [L.]) was studied in single and mixed-species treatments in the laboratory, where alternative food resources, chironomids and zooplankton, were offered simultaneously. The effects of structural complexity, which was represented by substrate grain size, and of feeding level on food consumption were analysed. Across all experiments, the outcome of competition between perch and ruffe depended on food abundance and on the structural complexity of the environment. Perch and ruffe both changed their food consumption in the presence of a heterospecific competitor. With high food supply, perch consumed more benthic food than ruffe. With low food supply, the consumption of perch decreased strongly, while that of ruffe remained high on fine sediment. Under all conditions tested, the mechanism of competition appeared to be of interference rather than of exploitative nature. It is suggested that with decreasing lake productivity caused by re-oligotrophication, habitat shifts of both species will occur, which will alleviate interspecific competition. Ruffe will forage over fine sediment and perch over coarse sediment, whereby both species will achieve the highest foraging efficiency under conditions of low food supply.     

Effects of HIF-1α silencing on proliferation of rat hepatoma cells

AIM：To study the effect of hypoxia-inducible factor 1α (HIF-1α) silencing on the proliferation of hepatoma cells under hypoxia. METHODS：Rat hepatoma cell line CBRH-7919 was used in this study. Hypoxia model was established by treating the cells with cobalt chloride (CoCl2). The expression of HIF-1α was silenced by small interfe-rence RNA. Real-time RT-PCR and Western blotting were used to detect the mRNA and/or protein expression of HIF-1α, vascular endothelial growth factor (VEGF), p21 and cyclin D1 in CBRH-7919 cells under hypoxia. The proliferation of CBRH-7919 cells was measured by the technique of 5-bromo-2’-deoxyuridine (BrdU) incorporation. RESULTS：The expression of HIF-1α and VEGF at mRNA and protein levels was significantly increased under hypoxia (P<0.05). Silencing of HIF-1α significantly inhibited the expression of HIF-1α, VEGF and cyclin D1 at mRNA and/or protein levels, while increased the protein expression of p21 (P<0.05). The BrdU-positive cells in HIF-1α siRNA transfection group were significantly less than those in control group. CONCLUSION：HIF-1α silencing significantly inhibits the proliferation of hepatoma cells under hypoxia.     

Potential allelopathic influence of certain pasture weeds

Laboratory and growth chamber research was conducted to determine the potential allelopathy of tissue harvested from certain weeds commonly found in pastures. Results of bioassays indicated that the weeds dogfennel and mayweed chamomile are potentially allelopathic to alfalfa and Italian ryegrass seedlings. Leaf-tissue extracts from the two weed species reduced seedling growth more than stem and root extracts. In most experiments alfalfa seedlings were influenced more than Italian ryegrass seedlings and seedling growth was influenced more than seed germination. Leaf tissue from the weed species mixed into potting soil at a concentration as low as 0.25% (w/w) significantly reduced alfalfa and Italian ryegrass plant development and foliage production. Extract and tissue concentration used in this research were estimated to be similar to concentrations expected to occur within the pasture ecosystem and below concentrations that would result in hypertonicity in the bioassays.     

散生类竹子地下鞭系生长影响因子研究综述

在搜集、分析大量相关文献的基础上,就影响散生类竹地下鞭系生长的主要因素,如林分气象、林地土壤、地形条件及林分经营干扰等因子,论述了目前我国在这一领域的研究现状,指出了研究中的薄弱环节,并提出今后的重点研究方向。     

改进邻体干扰模型在北京侧柏水源涵养林中的应用

以北京市密云半城子侧柏水源涵养林为研究对象,提出了改进的邻体干扰模型,着重讨论了干扰指数与侧柏的胸径、树高、生境利用率等因子的关系。结果表明,侧柏林内的邻体干扰指数与林木的胸径、树高、生境利用率呈负相关关系。本研究为该地区水源涵养林的经营提供了具有参考价值的量化依据。     

LncFAM200B对牦牛肌内前体脂肪细胞脂质沉积的影响

【目的】拟通过腺病毒介导的超表达及siRNA干扰试验,分析lncFAM200B对牦牛肌内脂肪细胞脂质沉积的影响,为解析lncFAM200B对牦牛肌内脂肪细胞脂质沉积的调控机制奠定基础。【方法】采集牦牛背最长肌组织,采用酶消化法和差速贴壁法分离牦牛肌内前体脂肪细胞;构建包装lncFAM200B超表达腺病毒,设计合成其siRNA干扰序列;通过实时荧光定量PCR（RT-qPCR）分析lncFAM200B超表达与干扰后脂肪分化标志基因PPARγ、C/EBPα、AP2,lncFAM200B潜在靶基因SIRT1、PTEN的表达水平;采用油红O染色、甘油三酯（TAG）测定、CCK-8检测等方法检测细胞内脂滴沉积情况、甘油三酯含量变化及细胞增殖情况。【结果】从牦牛背最长肌成功分离获得肌内前体脂肪细胞;lncFAM200B超表达后,脂肪分化标志基因C/EBPα、AP2表达量显著升高（P<0.05）,随着诱导分化时间的增加,lncFAM200B潜在靶基因SIRT1表达量呈先降低后升高的趋势,PTEN表达量呈现先增高后降低趋势;细胞脂滴沉积显著增加（P<0.05）,且胞内形成较大脂滴;超表达4 d后,细胞内甘油三酯含量显著增加（P<0.05）。干扰lncFAM200B可显著降低脂肪分化标志基因PPARγ、C/EBPα、AP2的表达水平（P<0.05）,降低细胞脂肪沉积,且诱导分化第6天细胞内甘油三酯含量显著降低（P<0.05）;干扰lncFAM200B后其潜在靶基因SIRT1呈现先升高后降低的表达趋势,PTEN则相反。CCK-8增殖实验表明,lncFAM200B超表达72 h后,细胞增殖效率显著降低（P<0.05）,而干扰lncFAM200B后72 h后细胞增殖效率显著增强（P<0.05）。【结论】lncFAM200B可能通过影响脂肪分化标志基因C/EBPα和AP2,脂肪合成相关基因SIRT1和PTEN的表达从而影响牦牛肌内脂肪沉积,但其具体机制还需要进一步研究。     

布鲁菌感染小鼠RAW264．7细胞对IRGl基因的表达调控作用

针对小鼠RAW264．7细胞IRGl基N设计4个RNA干扰靶位,筛选出最佳干扰序列构建shRNA慢病毒载体质粒并包装获得慢病毒颗粒,进而经嘌呤霉素筛选获得稳转细胞系,实现IRGl基因在RAW264．7细胞基因表达的沉默。并通过布鲁菌l6M株及M5株感染基因沉默细胞对IRGl基因在布鲁菌感染中的作用进行研究。结果表明,慢病毒介导的shRNA高效、稳定地沉默了IRGl基因的表达,布鲁菌侵染RAW264．7细胞后IRGl基因表达上调。未试验为1RG1基因及相美调控基因抗布鲁菌病作用研究奠定了基础。