Hairpin RNAs derived from RNA polymerase II and polymerase III promoter-directed transgenes are processed differently in plants

RNA polymerase III (Pol III) as well as Pol II (35S) promoters are able to drive hairpin RNA (hpRNA) expression and induce target gene silencing in plants. siRNAs of 21 nt are the predominant species in a 35S Pol II line, whereas 24- and/or 22-nucleotide (nt) siRNAs are produced by a Pol III line. The 35S line accumulated the loop of the hpRNA, in contrast to full-length hpRNA in the Pol III line. These suggest that Pol II and Pol III-transcribed hpRNAs are processed by different pathways. One Pol III transgene produced only 24-nt siRNAs but silenced the target gene efficiently, indicating that the 24-nt siRNAs can direct mRNA degradation; specific cleavage was confirmed by 5' rapid amplification of cDNA ends (RACE). Both Pol II- and Pol III-directed hpRNA transgenes induced cytosine methylation in the target DNA. The extent of methylation is not correlated with the level of 21-nt siRNAs, suggesting that they are not effective inducers of DNA methylation. The promoter of a U6 transgene was significantly methylated, whereas the promoter of the endogenous U6 gene was almost free of cytosine methylation, suggesting that endogenous sequences are more resistant to de novo DNA methylation than are transgene constructs.     

香蕉枯萎病菌Argonaute基因功能分析及其调控小RNA发生

尖孢镰刀菌古巴专化型Fusarium oxysporum f.sp.cubense是威胁香蕉生产的重要土传病原真菌,其中4号生理小种(Foc4)能感染几乎所有的栽培品系.Argonaute蛋白(AGO)介导的RISC复合体在RNAi干扰中起到重要作用.Foc4含有两个进化上高度保守的AGO蛋白,本研究利用同源重组技术获...     

玉米直链淀粉生物合成多基因转化载体构建

淀粉分支酶基因sbe是影响玉米直链淀粉含量的主要因素,淀粉分支酶分为3种即sbeI、sbeIIa和sbeIIb,其中sbeIIb对直链淀粉含量影响效应最大,抑制玉米淀粉分支酶sbeIIb基因的表达可减少支链淀粉的含量,从而达到提高直链淀粉的目的;ADP-葡萄糖焦磷酸化酶(AGPase)是直链淀粉合成的关键酶,通过提高AGP表达量同样可提高玉米直链淀粉含量。以此为目的分别克隆了sbeIIb一段375bp高度保守区,玉米SBE基因一段175bp内含子,AGP完整开放阅读框,大麦胚乳特异启动子和ADPG基因终止子。构建了sbeIIb基因正、反义的hpRNA发夹结构,将该发夹结构与上述基因分别连接到pCAM-BIA3301上;构建得到包含sbeIIb基因干扰结构与AGP基因过表达的pCAMB-RSA多基因胚乳特异表达载体。为此,pCAMB-RSA载体的成功构建将为高直链淀粉玉米的培育奠定基础。     

多种外源因素对转基因植物抗性影响的研究进展

病毒病对植物的危害在近些年日益严重,培育抗病毒转基因植物是众多防治方法中较为有效的一种。转基因植物在不同机制介导下会产生抗性,尤其是病毒基因介导的抗性试验最为普遍。众多研究证明,抗性受多种因素作用且表现为不同水平。对插入序列来源、转录后hpRNA长度、茎环比和拷贝数等多个因素在近些年的研究进展进行了综述,重点对各自在转基因植物抗性产生中的特点及影响程度进行了分析,并对其他因素进行了展望。     

RNA silencing platforms in plants

Since the discovery of RNAi, its mechanism in plants and animals has been intensively studied, widely exploited as a research tool, and used for a number of potential commercial applications. In this article, we discuss the platforms for delivering RNAi in plants. We provide a brief background to these platforms and concentrate on discussing the more recent advances, comparing the RNAi technologies used in plants with those used in animals, and trying to predict the ways in which RNAi technologies may further develop.     

转录后基因沉默(PTGS)及其在作物遗传改良中的应用

转录后基因沉默(PTGS)或RNA干扰(RNAi)技术的发展为创造植物遗传变异体提供了新途径。PTGS于1998年被明确为双链RNA(dsRNA)诱导的序列特异性基因沉默,短短几年内有关研究取得了突破性进展。结合利用PTGS技术进行的淀粉合成关键酶基因沉默研究,概述了PTGS的作用机理和特点、dsRNA表达载体设计、沉默效应的遗传稳定性及在作物改良应用方面的研究进展。研究表明,基因沉默效应可在子代间稳定遗传并可通过杂交进行重组,显示了其在农作物改良方面的应用潜力。     

应用套叠PCR技术构建番木瓜eIF4E和eIFiso4E基因的hpRNA植物表达载体

植物真核翻译起始因子4E(eIF4E)在蛋白质合成的起始中发挥重要作用,参与植物-病毒互作,影响病毒的侵染过程.为了研究eIF4E在植物病毒侵染中的功能,建立了一种快速的套叠PCR新方法,成功构建了番木瓜eIF4E和eIFiso4E基因的hpRNA结构,并将其连接到改造的植物表达载体pBI121上,为利用RNA干扰技术研究番木瓜eIF4E和eIFiso4E基因在病毒侵染中的作用奠定了基础.     

Rapid one-step construction of hairpin RNA

Hairpin RNA (hpRNA) is commonly used for gene-function exploration and gene engineering. In this study, a novel method was developed to construct intron-containing hairpin RNA (ihpRNA) rapidly and efficiently based on Overlap Extension PCR (OE-PCR). This method, Mixed One-step OE-PCR (MOOE-PCR), can amplify two inverted repeats of DNA fragments and a spliceable intron in parallel, and then assemble them to generate ihpRNA constructs in the same tube without the purification of intermediate products. This method required a PCR process of 38-40 cycles and ordinary PCR reagents. A total of 10 ihpRNA constructs were amplified successfully using this method, with the stems ranging from 50 bp to 484 bp in length. Our results suggest that this novel method is a useful strategy for constructing ihpRNA.     

反向重复结构在抗植物病毒上的研究进展

双链RNA(dsRNA)的形成是启动RNA沉默的关键因子,而插入了反向重复结构的表达载体能有效地转录产生hpRNA,从而能高效启动RNA沉默。分析了反向重复结构构建的特点,综述了近些年利用该结构启动RNA沉默后在抗植物病毒上的研究进展。