利用TRV过表达和沉默系统验证植物油脂合成相关基因的功能

为探究烟草脆裂病毒过表达和沉默系统进行油脂相关基因研究的可能性,该研究克隆了拟南芥的WRI1和FAD2基因,分别构建了TRV过表达和沉默载体. 利用瞬时侵染技术侵染本氏烟草,取材料进行RT-PCR和脂肪酸含量检测. 结果显示在过表达WRI1基因的本氏烟草中,侵染叶和非侵染叶的WRI1基因及其相关参与脂肪酸合成基因ACP1、KAS1和BCCP2等都有明显上调,且脂肪酸含量检测结果显示分别增加16%和28%. 在沉默FDA2基因的本氏烟草植株中,发现多不饱和脂肪酸含量分别减少约25%和24%. 因此,利用TRV系统对油脂相关基因进行研究是可能的.     

烟草玉米黄质环氧化酶基因的转录表达模式及其功能研究

通过Real-time PCR检测了普通烟草(Nicotiana tabacum)红花大金元的玉米黄质环氧化酶(Zeaxanthin epoxidase,ZE)ZE基因在盛花期不同器官中的表达量.利用烟草脆裂病毒诱导的基因沉默(VIGS)技术抑制本氏烟草(Nicotiana benthamiana)ZE基因的表达,在该基因沉默后,Real-time PCR检测其上游基因的表达变化;同时检测烟草中质体色素(β-胡萝卜素、紫黄质、新黄质、叶黄质、叶绿素a和叶绿素b)含量的变化.结果显示:ZE基因在盛花期的第10位叶片、第15位叶片和花萼中表达量较高.与对照组相比,在ZE基因沉默后,其上游的基因八氢番茄红素合成酶(PSY)、八氢番茄红素脱氢酶(PDS)、ζ-胡萝卜素脱氢酶(ZDS)、类胡萝卜素异构酶(CRTISO)、番茄红素β-环化酶(β-LCY)、胡萝卜素β-环羟化酶(β-OHase)和紫黄质脱环氧化酶(VDE)的表达量降低;烟草中质体色素的含量降低.以上结果说明:ZE基因作为类胡萝卜素合成通路下游的基因在该通路中发挥着重要的调控作用,该基因表达量的变化可以影响烟草中质体色素的含量,与烟草的光合生理过程也存在着密切关系.     

JcERF1基因RNA干扰载体的构建、转化及沉默效果研究

构建了麻疯树(Jatropha curcas)一个新的ERF类转录因子基因RNA干扰(RNAi)载体,并将之转入过表达JcERF1基因烟草体内, 得到了RNA干扰型烟草. 利用实时荧光定量PCR(QPCR)检测了JcERF1基因的表达量, 发现该基因在干扰型烟草中表达量明显降低. 通过野生型、过表达型和干扰型烟草的种子萌发实验和高盐胁迫实验发现: 与过表达型烟草相比, 干扰型烟草的抗盐能力明显降低, 且干扰型烟草的游离脯氨酸和可溶性糖含量在高盐处理后上升的幅度明显低于过表达型烟草. 以上研究结果说明JcERF1基因在干扰型烟草体内已被有效沉默, 同时说明该基因具有提高转基因烟草抗盐性的功能.     

西瓜花叶病毒HC-Pro基因的克隆与原核表达

利用RT-PCR方法获得了西瓜花叶病毒(WMV)陕西分离物HC-Pro基因,大小为1 371 bp.将HC-Pro基因克隆到pMD18-T Simple Vector,测序分析发现与其它国家HC-Pro核苷酸同源性为90.7%~94.8%.将HC-Pro基因定向插入EcoR I/Sal I切开的pET30a中,构建了原核表达载体pET30-WHC,转化大肠杆菌BL21.经IPTG诱导2~8 h后,成功表达了分子量约为57 kD的HC-Pro蛋白.通过不同时间诱导发现,加入IPTG 2 h后蛋白开始表达,继续诱导到8h后表达量变化不大.以诱导的蛋白为抗原免疫家兔,制备了HC-Pro蛋白的抗血清,ELISA法测其效价为1/6 400,Western blot分析能与HC-Pro发生血清学反应.     

药食两用模式植物苦蘵基因功能研究方法的建立

苦蘵中的酸浆苦素R可能有抗癌功效,基因组测序结果预测其生源代谢合成途径缺失环节可能分布于3个基因家族的89个相关同源基因里,这给其药效成分的生源合成阐明带来困难.本实验构建了由植物病毒介导的苦蘵基因功能研究体系,应用狗尾草花叶病毒(Foxtail mosaic virus,FoMV)来实现相关外源基因(eGFP)的稳定表达;同时,基于烟草脆裂病毒(Tobacco rattle virus, TRV)建立苦蘵的功能基因表达沉默技术,实验中有效地抑制苦蘵内源的八氢番茄红素脱氢酶基因(PDS)的表达水平.该方法可对候选基因实现差异化表达,对上下游的相关性基因进行分析,可以用于苦蘵的功能基因筛选和鉴定.     

RNA干涉研究进展

RNA干涉(RNAi)是生物体内的一种通过双链RNA(dsRNA)来抵抗病毒入侵和抑制转座 子活动的自然机制.快速发展的RNAi技术为抑制特异性基因的表达提供了有利的工具.到目前为 止,在真菌、拟南芥、线虫、锥虫、水螅、涡虫、果蝇、斑马鱼、小鼠等真核生物中都发现存在基因沉默 机制.RNAi作为基因沉默的工具,为基因功能研究、基因治疗、药物研究与开发等许多领域开辟了 一条新路.     

组成型表达CsCOR1基因烟草的制备

将已克隆的一个被冷显著上调的基因CsCOR1整合到穿梭载体pBI121的CaMV35S启动子的下游,然后采用农杆菌介导法转化进烟草细胞,从而制得了转CsCOR1基因烟草植株,并筛选到组成型表达CsCOR1基因的烟草转化子,为进一步研究该基因的功能奠定了基础.     

慢病毒介导的RNA干扰抑制白血病细胞增殖的应用

通过慢病毒载体介导的RNA干扰(RNAi)技术,沉默成人T细胞白血病(ATL)中关键的病毒基因HBZ,并研究RNA干扰后对白血病细胞增殖的影响.首先根据HBZ基因序列设计RNA干扰片段,构建其慢病毒载体,并将其包装成病毒.然后用HBZ siRNA慢病毒感染白血病细胞株TL-Om1,通过RT-PCR和Western blot检测HBZ基因和蛋白表达情况,MTT技术检测沉默HBZ后TL-Om1细胞的增殖情况,利用RT-PCR检测病毒感染后下游生长相关基因的表达.实验结果表明:利用慢病毒介导的RNA干扰技术能有效降低HBZ的表达,进而抑制了白血病细胞的增殖;沉默HBZ后,细胞内E2F1,PCNA和Myc等下游生长相关基因的表达受到了明显抑制.这将为慢病毒介导的RNA干扰技术应用于临床治疗成人T细胞白血病提供重要的实验依据.     

抑制NtVTC1基因的表达延缓烟草生长发育

目的研究VTC1通过调控细胞内抗坏血酸(Ascorbic acid, AsA)的合成参与植物生长发育和响应逆境的重要功能。方法构建烟草VTC1基因的RNAi干扰表达载体RNAi-NtVTC1并转化烟草,通过抑制烟草VTC1基因的表达分析其影响植物发育的重要功能。结果将构建的干扰载体RNAi-NtVTC1转化烟草,成功获得了转基因株系RI。半定量PCR(RT-PCR)和实时荧光定量PCR(qRT-PCR)检测结果表明转基因烟草株系RI中NtVTC1基因的表达显著被抑制。AsA含量测定结果表明:与野生型烟草相比,转基因烟草株系RI的AsA含量均显著降低,为野生型的30%～40%。对烟草的表型观察发现,转基因烟草株系RI的发育显著受到抑制,尤其是在植株整体发育、叶片延展发育、开花等方面表现出显著延迟的表型。烟草抗氧化系统酶活性测定结果表明:转基因烟草株系RI中抗氧化系统酶活性如抗坏血酸过氧化物酶(APX)、脱氢抗坏血酸还原酶(DHAR)、过氧化物酶(CAT)和谷胱甘肽还原酶的酶活性(GR)都显著降低。结论 VTC1基因在植物的生长发育过程中发挥着重要作用,为进一步深入研究VTC1基因的功能提供了良好参考。     

Recognition of small interfering RNA by a viral suppressor of RNA silencing

RNA silencing (also known as RNA interference) is a conserved biological response to double-stranded RNA that regulates gene expression, and has evolved in plants as a defence against viruses. The response is mediated by small interfering RNAs (siRNAs), which guide the sequence-specific degradation of cognate messenger RNAs. As a counter-defence, many viruses encode proteins that specifically inhibit the silencing machinery. The p19 protein from the tombusvirus is such a viral suppressor of RNA silencing and has been shown to bind specifically to siRNA. Here, we report the 1.85-A crystal structure of p19 bound to a 21-nucleotide siRNA, where the 19-base-pair RNA duplex is cradled within the concave face of a continuous eight-stranded beta-sheet, formed across the p19 homodimer interface. Direct and water-mediated intermolecular contacts are restricted to the backbone phosphates and sugar 2'-OH groups, consistent with sequence-independent p19-siRNA recognition. Two alpha-helical 'reading heads' project from opposite ends of the p19 homodimer and position pairs of tryptophans for stacking over the terminal base pairs, thereby measuring and bracketing both ends of the siRNA duplex. Our structure provides an illustration of siRNA sequestering by a viral protein.     

DNMT1 and DNMT3b cooperate to silence genes in human cancer cells

Inactivation of tumour suppressor genes is central to the development of all common forms of human cancer. This inactivation often results from epigenetic silencing associated with hypermethylation rather than intragenic mutations. In human cells, the mechanisms underlying locus-specific or global methylation patterns remain unclear. The prototypic DNA methyltransferase, Dnmt1, accounts for most methylation in mouse cells, but human cancer cells lacking DNMT1 retain significant genomic methylation and associated gene silencing. We disrupted the human DNMT3b gene in a colorectal cancer cell line. This deletion reduced global DNA methylation by less than 3%. Surprisingly, however, genetic disruption of both DNMT1 and DNMT3b nearly eliminated methyltransferase activity, and reduced genomic DNA methylation by greater than 95%. These marked changes resulted in demethylation of repeated sequences, loss of insulin-like growth factor II (IGF2) imprinting, abrogation of silencing of the tumour suppressor gene p16INK4a, and growth suppression. Here we demonstrate that two enzymes cooperatively maintain DNA methylation and gene silencing in human cancer cells, and provide compelling evidence that such methylation is essential for optimal neoplastic proliferation.     

马铃薯Y病毒辅助成分蛋白酶与加工番茄OPA3-like蛋白结合及其定位研究

为了研究加工番茄OPA3-like蛋白与马铃薯Y病毒(Potato virus Y,PVY)的辅助成分蛋白酶(helper component proteinase,HC-Pro)的相互作用,进行了2种蛋白的结合及其定位研究。通过PCR的方法从加工番茄中克隆OPA3-like基因,同时利用Blast方法对其分析,进行了OPA3-like蛋白基因氨基酸序列同源性分析。通过构建双分子荧光互补载体pSPYCE-35S-OPA3-like和pSPYNE-35S-HC-Pro,利用农杆菌介导法共侵染洋葱表皮细胞60 h后,在共聚焦显微镜下观察洋葱表皮细胞中的黄色荧光。结果显示:加工番茄OPA3-like基因ORF长510 bp,编码169个氨基酸,编码蛋白属OPA3蛋白家族。激光共聚焦显微镜下可观察到沿细胞膜呈小泡状分布黄色荧光,说明PVY的HC-Pro蛋白与加工番茄OPA3-like蛋白结合并且共定位在细胞膜附近。这为进一步研究PVY的HC-Pro蛋白与加工番茄OPA3-like蛋白的相互作用机制奠定了一定的基础。     

Suppressor of RNA silencing encoded by Rice gall dwarf virus genome segment 11

Rice gall dwarf virus （RGDV） is an important rice pathogen in China and Southeast Asia. However, little is known about the molecular mechanisms of RGDV interactions with plant cells. Here, we have identified an RGDV protein, Pns11, which acts as a suppressor of RNA silencing in coinfiltration assays with the reporter, green fluorescent protein （GFP）in transgenic Nicotiana benthamiana line 16c carrying GFP. Pns11 suppressed local and systemic silencing induced by sense RNA. The spread of mobile RNA silencing signals was blocked or inactivated by Pns11. Expression of Pns11 also enhanced Potato virus X pathogenicity in IV. benthamiana. This suppressor could reduce, but not eliminate, siRNA in the local and systemic RNA silencing suppression assays, suggesting that Pns11 functions by interfering with initial stages of RNA silencing.     

Identification of RPS14 as a 5q- syndrome gene by RNA interference screen

Somatic chromosomal deletions in cancer are thought to indicate the location of tumour suppressor genes, by which a complete loss of gene function occurs through biallelic deletion, point mutation or epigenetic silencing, thus fulfilling Knudson's two-hit hypothesis. In many recurrent deletions, however, such biallelic inactivation has not been found. One prominent example is the 5q- syndrome, a subtype of myelodysplastic syndrome characterized by a defect in erythroid differentiation. Here we describe an RNA-mediated interference (RNAi)-based approach to discovery of the 5q- disease gene. We found that partial loss of function of the ribosomal subunit protein RPS14 phenocopies the disease in normal haematopoietic progenitor cells, and also that forced expression of RPS14 rescues the disease phenotype in patient-derived bone marrow cells. In addition, we identified a block in the processing of pre-ribosomal RNA in RPS14-deficient cells that is functionally equivalent to the defect in Diamond-Blackfan anaemia, linking the molecular pathophysiology of the 5q- syndrome to a congenital syndrome causing bone marrow failure. These results indicate that the 5q- syndrome is caused by a defect in ribosomal protein function and suggest that RNAi screening is an effective strategy for identifying causal haploinsufficiency disease genes.     

Chromosomal landscape of nucleosome-dependent gene expression and silencing in yeast

Eukaryotic genomes are packaged into nucleosomes, which are thought to repress gene expression generally. Repression is particularly evident at yeast telomeres, where genes within the telomeric heterochromatin appear to be silenced by the histone-binding silent information regulator (SIR) complex (Sir2, Sir3, Sir4) and Rap1 (refs 4-10). Here, to investigate how nucleosomes and silencing factors influence global gene expression, we use high-density arrays to study the effects of depleting nucleosomal histones and silencing factors in yeast. Reducing nucleosome content by depleting histone H4 caused increased expression of 15% of genes and reduced expression of 10% of genes, but it had little effect on expression of the majority (75%) of yeast genes. Telomere-proximal genes were found to be de-repressed over regions extending 20 kilobases from the telomeres, well beyond the extent of Sir protein binding and the effects of loss of Sir function. These results indicate that histones make Sir-independent contributions to telomeric silencing, and that the role of histones located elsewhere in chromosomes is gene specific rather than generally repressive.     

Two virus-encoded RNA silencing suppressors, P14 of Beet necrotic yellow vein virus and S6 of Rice black streak dwarf virus

Functional analysis for gene silencing suppressor of P14 gene of Beet necrotic yellow vein virus and S6 gene of Rice black streak dwarf virus was carried out by agro- infiltration with recombinant vectors of Potato virus X. The phenotype observation of green fluorescent protein (GFP)expression and Northern blot showed that the gene silencing of gfp transgenic Nicotiana benthamiana induced by homologous sequence was strongly suppressed by the immixture infiltration of either the P14 or the $6. In the suppressed plants, the gfp mRNA accumulation was higher than that in the non-suppressed controls and the symptoms caused by PVX infection became more severe, especially the gfp DNA methylation of plant genome was significantly inhabited when co-infiltrated with RBSDV S6 gene. These results suggested that these two virus genes were potentially to encode for proteins as RNA silencing suppressors.