植物中依赖于RNA的RNA聚合酶研究进展

依赖于RNA的RNA聚合酶(RDR)能够以单链RNA为模版合成互补RNA链,产生双链RNA。双链RNA在细胞内被类似RNaseⅢ的酶DCL加工成20～24 nt的小分子干扰RNA(siRNA)。siRNA可以在转录水平或转录后水平抑制靶基因的表达。RDR通过基因沉默途径参与植物的生长发育调节、逆境应答以及表观遗传修饰等许多生物学过程。加深对植物RDR表达模式、生化活性及生物学功能等方面的理解将有利于植物基因沉默的发展和运用。     

RNA干涉技术与棉花高油育种

 棉子脂肪酸既是重要的食用油,又是重要的工业原料。但是关于棉子遗传改良的研究报道甚少。研究表明,PEPcase和ACCase的相对活性决定种子的蛋白质与油脂的含量,应用RNAi技术抑制PEPcase催化活性,可以提高脂肪酸总量。本文概述了棉花油分与RNAi技术的研究现状,并阐述了应用RNAi技术改良棉花高油分育种的技术路线。     

棉花FAD 2-1基因的克隆及其ihpRNA和amiRNA干扰载体的构建

 采用RT-PCR方法克隆了棉花△-12脂肪酸去饱和酶基因(GhFAD2-1)的cDNA全长序列,该基因含有1158 bp的完整开放阅读框。棉花FAD2-1是种子中编码催化油酸形成不饱和脂肪酸的关键酶的基因,选取GhFAD2-1基因中的一段特异的515 bp片段,构建了种子特异性启动子NAPIN调控的ihpRNA干扰表达载体pFGC1008-NAPIN-FAD2-1,同时构建了针对GhFAD2-1基因的人工miRNA表达载体pCAMBIA1302- amiRNA-FAD2-1。     

Application of time-frequency distribution correlating and matching method in signal extraction

The time-frequency coupling relation between useful non-stationary components and noises bring great difficulties to the realization of de-noising for non-stationary signals,which can not be solved by classic de-noising method in time or frequency domain. The principles of short time Fourier transform(STFT),Wigner-Ville transform,Chirplet adaptive decomposition are analyzed,and then a novel de-noising method for non-stationary based on joint time-frequency distribution is proposed. In this method,the analyzed signal WVD is seen as the combination of auto-WVD and cross-term WVD. Firstly,STFT energy spectrum of the analyzed signal is used as template to cross-correlate with its corresponding WVD in order to obtain the satisfactory time-frequency distribution with high time-frequency resolution and without cross-term interferences. Secondly,the useful components are decomposed as Chirplet function using the two-dimension least square fitting method,and then are extracted out to reconstruct for noise suppression. Finally,the computer simulation results verify the effectiveness of this proposed method. Its application in gearbox fault diagnosis indicates that with the method the extracted cycle of the gearbox vibration impulses has a good consistency with the corresponding fault frequency.     

Interference elimination of PCB and its ramification in determining trace PBBs/PBDEs by GC/MS

The normal method to determine the trace brominated flame retardants such as polybrominated biphenyls (PBBs) and polybrominated diphenylethers (PBDEs) contained in the electronic and electrical products is gas chromatography/mass spectrometry (GC/MS). However, the method is based on the determinations of the persistent organic pollutants (POPs) such as polychlorinated biphenyls (PCBs), and polychlorinated naphthalene (PCNs) etc. The interferences of those compounds are inevitable. Therefore, in order to overcome this problem, two methods e.g. 1) The combination use of gas chromatography negative ion chemical ionization mass spectrometry (GC-NICI-MS) and gas chromatography-electron impact-mass spectrometry (GC-EI-MS) is employed, 2) a matrix solid-phase dispersion (MSPD) method with YMC ODS-C18 as carrier is developed. The result show that, by the former method, the brominated isomers or ramifications are distinguished remarkably from other halogen compounds because that anion fragment retention peaks of ［Br］-, ［HBr2］- and molecule chain fragment retention peaks of ［M+2］+,［M+4］+,［M+6］+,［M+8］+ are observed simultaneously, and thus the selectivity to determine bromine-containing retardant flames is greatly improved. Using the latter method, the gas chromatographic peaks of multiple polychlorinated biphenyls and polybrominated diphenylethers can be efficiently separated. Thus provides a project to solve interferences of POPs in brominated flame retardants’ determinations. The standard addition experimental results of 10 kinds of BDEs/PCBs belonging to 8 sorts of electronic and electrical equipment show this method has a high precision and reliability due to 60%～98% recovery and <9.5% relative error, which meet the needs regulated by the IEC Commission. It provides a technical support for electronic and eletrical industries in China to further comply with RoHS directive.     

基于线粒体RNAs序列分析雁形目家禽的分歧时间

雁形目鸟类具有重要的社会经济意义和科学研究价值,部分种类已被驯化成为重要的家禽,然而雁形目内部物种之间的关系仍存在许多争议。本研究利用线粒体RNAs序列标定雁形目部分鸟类的分歧时间,旨在进一步查明该类群系统进化关系。首先, 采用酚仿法分别从太湖鹅和朗徳鹅血液中提取基因组DNA,PCR 扩增获得线粒体RNAs基因序列。 然后,从GenBank 中提取10 种鸟类线粒体DNA的同源序列,以鸵鸟为外群,采用贝叶斯法结合RNA结构信息重建系统发生关系;最后,以黑雁属与雁属的分歧时间为锚定点,利用r8s软件分析了雁形目物种间的分歧时间。 结果表明,中国鹅与欧洲鹅的分歧时间为0.61百万年,番鸭与家鸭的分歧时间为15.5百万年。依据家鹅祖先分歧时间,联系当时发生的地学事件,作者认为家鹅祖先物种分化与更新世冰川运动有关。研究结果有助于我国水禽遗传资源的保护与利用。     

葡萄病毒AMP基因RNA干扰载体的构建

RNA干扰（RNAi）介导的植物抗病毒研究是近年来引起广泛关注的一项植物抗病毒基因工程策略。本文以GVA运动蛋白（MP）基因为模板扩增出了418 bp的基因片段,根据RNA干扰的基本原理,将目的片段正反向分别插入到载体pFGC5941内含子的两侧以便其转录过程中高效产生RNA发夹结构。经PCR扩增证明已成功构建以GVA MP基因为靶标的干扰载体pFGC5941-GVAFR,并成功转入农杆菌EHA105中,为后续探讨干扰载体的干涉效果奠定了基础。     

蚜虫基因RNAi载体的构建及其在转基因烟草中dsRNA的表达分析

RNAi机制可以通过dsRNA诱导同源mRNA的降解,从而导致该基因转录后沉默。RNA干扰作用从发现以来就具有了利用转录后基因沉默（PTGS）来进行抗虫的一种潜在可能性。针对dsRNA的这种作用,研究中提取蚜虫RNA,克隆出蚜虫Cathepsin B基因的干扰片段并构建出RNAi载体,转入本氏烟草中,在转基因烟草体内表达dsRNA。成功构建RNAi载体后,通过农杆菌转化和叶盘法将RNAi载体导入烟草中并采用组织培养方法培养出转基因烟草,通过PCR鉴定显示15株生根的转基因烟草中14株显示阳性。提取转基因烟草的RNA进行RT-PCR和Northern表达分析显示转基因烟草植株中dsRNA表达的存在。实验结果说明了在转基因植物体内表达RNAi载体可以产生dsRNA,从而在蚜虫进食植物时达到利用dsRNA抗蚜虫的作用。     

Inhibition of hirame rhabdovirus growth by RNA aptamers

RNA aptamers are artificial nucleic acids that specifically bind to a wide variety of targets. They are an effective tool for pharmaceutical research and development of antiviral agents. Here, we describe four Hirame rhabdovirus (HIRRV)‐RNA aptamers (H1, H2, H3 and H4) that we obtained from an in vitro process called the systematic evolution of ligands by exponential enrichment (SELEX). The HIRRV‐RNA aptamers specifically bind to HIRRV. Hirame natural embryo (HINAE) cells treated with virus and the RNA aptamer showed a decrease in appearance of cytopathic effect when compared with control (treated only with virus). Rhodovulum sulfidophilum was transformed with genes for the RNA aptamers, and the aptamers were detected in the culture medium, indicating that they were secreted from the cells. Thus, the recombinant R. sulfidophilum might be a powerful tool for the prevention of HIRRV in aquaculture.     

RNA干扰技术及其在蜜蜂基因功能研究中的应用前景

RNA干扰作用(RNA interference,RNAi)是生物界一种古老且进化上高度保守的现象,自发现以来便成为研究基因功能的有效手段。作者重点介绍了RNA干扰技术发现的历史过程和作用机制,阐述了其在昆虫功能基因组学方面的应用进展,展望了其在蜜蜂基因功能研究中的应用前景。