RNA干扰与基因敲除

RNAi是指通过双链RNA介导特异性降解靶mRNA,导致转录后水平基因沉默的现象。其作用途径有RdRP依赖的RNAi的途径与非RdRP依赖的RNAi途径2种。利用RNAi的基因敲除技术在dsRNA序列选择、质粒或病毒为载体的dsRNA体内合成、发夹样siRNA的转录、dsRNA的导入方法等方面取得了很大进展,在研究人类或其他生物基因组中未知基因及蛋白质的功能等领域具有诱人的应用前景。     

Split-marker 重组技术构建泛素C 末端水解酶基因缺失菌株

目的:对烟曲霉(Aspergillus fumigatus)泛素末端水解酶(creB)基因进行敲除。方法:通过氨基酸序列分析软件初步分析烟曲霉CreB蛋白结构.利用split-marker重组技术构建重组片段,并通过PEG-原生质体方法对烟曲霉野生菌株进行转化,采用PCR方法对转化子进行筛选,最后选取初步筛选的转化子进行测序鉴定。结果:结构分析显示烟曲霉CreB蛋白具有泛素特异蛋白酶(ubiquitin-processing protease)UBP亚家族六个结构域。本实验构建了转化片段并转化,在抗性平板中获得了25个Hyg抗性转化子,进一步采用PCR方法筛选到20个转化子,最终通过测序分析获得一株creB基因缺失菌株。结论:Split-marker重组技术是对烟曲霉creB基因进行敲除的快速有效的方法。获得的creB缺失菌株可用于基因功能研究。     

伤寒沙门菌bcfD基因敲除突变株的构建

目的:构建伤寒沙门菌Ty2菌株菌毛亚单位bcfD基因敲除突变株.方法:利用交错PCR得到bcfD基因缺失且含其两侧翼序列的片段,将该片段与pMD 18-T连接,亚克隆到pYG4,电转入大肠埃希菌S17-1/λpir菌株,阳性菌株与受体菌伤寒沙门氏菌Ty2进行固相杂交后筛选.结果:成功获得敲除bcfD基因序列954bp的敲除突变株.结论:交错PCR有利于细菌基因精确敲除突变株的构建,bcfD基因敲除株的构建将为进一步研究该基因在伤寒沙门菌中的功能奠定了基础.     

黄曲霉菌aflR基因启动子序列变异与黄曲霉毒素产生相关联

为探讨黄曲霉菌aflR基因启动子序列变异与黄曲霉毒素产生的关系,收集黄曲霉菌、米曲霉菌和寄生曲霉菌若干株。在有利于黄曲霉毒素产生的条件下培养后,提取各菌株的总RNA,RT-PCR法检测aflR基因的mRNA表达水平;并应用ELISA法检测各菌株产生黄曲霉毒素B1的情况。提取各菌株的基因组DNA,PCR扩增aflR基因启动子序列并测序。应用基因分析软件将不产毒素的黄曲霉菌与产毒黄曲霉菌的aflR基因启动子序列进行比较,找出不产毒菌株aflR基因启动子序列的变异位点。ELISA法和RT-PCR法结果表明,产毒的黄曲霉菌菌株均有明显的aflR基因转录,而在2株不产毒的黄曲霉菌菌株中,一株aflR基因无转录,另一株仅有较低水平的转录。序列比较结果表明,不产毒黄曲霉菌菌株的aflR基因启动子序列存在如下共同变异位点:-90、-236、-253、-262、-282位。米曲霉菌产生黄曲霉毒素B1和aflR基因转录的检测均为阴性,并且其aflR基因启动子序列中存在与上述不产毒黄曲霉菌菌株相同的变异位点。寄生曲霉菌产生黄曲霉毒素B1和aflR基因转录的检测均呈阳性,并且其aflR基因启动子序列的上述5个位点与产毒黄曲霉菌完全一致。在不产毒素的黄曲霉菌aflR基因启动子序列中发现了5个共同变异位点,实验结果提示这些变异位点可能与黄曲霉毒素的产生有关。     

RNA干扰:脑学习和记忆的可能机制

dsRNA介导同源靶基因沉默的RNA干扰 (RNAi)是转录后基因水平沉默的主要作用方式 ,具有普遍的生物学意义。RNAi是dsRNA介导的核酸酶作用于dsRNA(>2 6nt)同源不成熟mR NA的酶解过程 ,mRNA降解为 2 1 2 3nt的dsRNA而使基因表达沉默。RNAi所具有的特性和脑学习和记忆的特征 ,提示RNAi可能是RNA介导的脑记忆移转的潜在机制     

H1启动子siRNA载体的构建及应用

利用双链RNA(dsRNA)调控基因表达已经成为研究基因功能的有力工具。用人H1启动子构建了pBS／H1PS小干扰RNA(siRNA)表达载体,用于在哺乳动物细胞中产生特异性dsRNA转录产物。通过对293细胞中的PSMA7分子进行表达抑制,证明该siRNA载体能够有效产生针对靶基因的RNA干扰(RNAi)效应。     

非完全互补shRNA对RNA聚合酶II启动子调控的RNAi的影响

RNA干扰（RNAi）是一种转录后基因沉默技术,可有效诱导序列特异性基因沉默．由RNA聚合酶Ⅱ启动子调控表达的小发卡RNA可有效介导RNAi效应,为组织特异性基因沉默提供了一条新的途径．但是,由RNA聚合酶Ⅱ启动子调控表达的小发卡RNA（shRNA）在序列上与靶基因非完全互补对RNAi效应的影响鲜有报道．本文初步探索RNA聚合酶Ⅱ启动子调控表达的shRNA碱基发生突变或缺失对RNAi效应的影响．研究表明,靶向hTERT mRNA的碱基突变shRNA显著降低RNAi效应,而靶向GFP mRNA的碱基缺失shRNA对RNAi效应没有显著影响．本研究为非完全互补shRNA对RNAi效应的进一步深入研究提供了理论与实验依据．     

绿僵菌微循环产孢基因Pyk的克隆及RNA干扰分析

微循环产孢是真菌遭遇逆境时发生的一种产孢机制,具有繁殖快速及抗逆性强等诸多优点。研究绿僵菌的微循环产孢机制并加以利用,对增强该菌在生物防治中的应用效果具有重要的意义。采用绿僵菌基因组数据库与NCBI数据库中同源蛋白比对获得Pyk基因DNA序列;分析Pyk基因结构并设计引物,通过RT-PCR扩增克隆Pyk全长cDNA序列。序列分析显示该基因cDNA全长1 934 bp,开放阅读框长为1 752 bp(GenBank登录号:HQ153828),编码产物为583个氨基酸的丙酮酸激酶,该酶与子囊菌门中其他真菌具有较高的相似性(57%-77%)。构建Pyk基因的RNAi载体,基因枪转化野生型绿僵菌获得3个突变菌株,RT-PCR证实3个干扰突变菌株中Pyk基因的干扰效率分别为:51%、56%、33%。对突变菌株的微循环产孢模式做了进一步分析,结果显示:与野生菌株相比,突变菌株产生更多的孢子形态类型,菌落周边的白色菌丝也相对较少。     

利用RNAi技术沉默小菜蛾类钙粘蛋白基因

RNA干扰（RNA interference, RNAi）是一种调控基因表达的方法, 其通过体外合成一段与内源靶基因同源的双链RNA（dsRNA）或siRNA, 导入生物体内, 使内源靶基因中同源mRNA降解, 从而达到阻抑基因表达的目的。类钙粘蛋白（cadherin-like protein）是位于昆虫中肠刷状缘膜囊(brush border membrane vesicles, BBMV)上与钙粘蛋白(cadherin)结构相似的物质, 是多种昆虫体内Bt杀虫蛋白的受体。本研究利用基因特异引物通过RT-PCR扩增了小菜蛾类钙粘蛋白基因的2个片段（CAD1和CAD2）, 合成相对应的双链RNA（double-stranded RNA, dsRNA）; 并将dsRNA通过显微注射导入小菜蛾3龄幼虫体内, 测定了不同靶位点、不同剂量、不同检测时间对目的基因mRNA表达量的影响。结果表明: 将70 nL CAD1对应的dsRNA注射到幼虫体内48 h后, 基因表达量显著下降, 72 h后恢复。免疫印迹检测结果表明, 类钙粘蛋白在注射dsRNA 48 h后幼虫BBMV中的含量明显下降。本实验成功实现了小菜蛾类钙粘蛋白基因的沉默, 该体系的成功建立为利用RNAi技术分析小菜蛾及其他鳞翅目昆虫基因的功能提供了参考。     

RNA干涉及其应用前景

RNA干涉是指由特定双链RNA(dsRNA)引起的转录后基因沉默现象。研究表明,Dicer断裂dsRNA产生的小干涉RNA可以抑制哺乳动物体细胞和胚胎中的基因的表达。RdRP在扩增RNAi中起着关键性的作用,RdRP活性复制较长的触发性dsRNA或以一种非引物的方式复制短的siRNA,即以siRNA为引物的RdRP反应使靶mRNA转变为dsRNA,同时复制触发性dsRNA。所有的产物又可作为Dicer的底物,起始RdRP级联反应。本文综述了RNAi可能的作用机制,并对RNAi在分析功能基因组、药物治疗等方面的应用前景进行了展望。     

A Developmentally Regulated Gene Cluster Involved in Conidial Pigment Biosynthesis in Aspergillus fumigatus

Aspergillus fumigatus, a filamentous fungus producing bluish-green conidia, is an important opportunistic pathogen that primarily affects immunocompromised patients. Conidial pigmentation of A. fumigatus significantly influences its virulence in a murine model. In the present study, six genes, forming a gene cluster spanning 19 kb, were identified as involved in conidial pigment biosynthesis in A. fumigatus. Northern blot analyses showed the six genes to be developmentally regulated and expressed during conidiation. The gene products of alb1 (for "albino 1"), arp1 (for "aspergillus reddish-pink 1"), and arp2 have high similarity to polyketide synthases, scytalone dehydratases, and hydroxynaphthalene reductases, respectively, found in the dihydroxynaphthalene (DHN)-melanin pathway of brown and black fungi. The abr1 gene (for "aspergillus brown 1") encodes a putative protein possessing two signatures of multicopper oxidases. The abr2 gene product has homology to the laccase encoded by the yA gene of Aspergillus nidulans. The function of ayg1 (for "aspergillus yellowish-green 1") remains unknown. Involvement of the six genes in conidial pigmentation was confirmed by the altered conidial color phenotypes that resulted from disruption of each gene in A. fumigatus. The presence of a DHN-melanin pathway in A. fumigatus was supported by the accumulation of scytalone and flaviolin in the arp1 deletant, whereas only flaviolin was accumulated in the arp2 deletants. Scytalone and flaviolin are well-known signature metabolites of the DHN-melanin pathway. Based on DNA sequence similarity, gene disruption results, and biochemical analyses, we conclude that the 19-kb DNA fragment contains a six-gene cluster which is required for conidial pigment biosynthesis in A. fumigatus. However, the presence of abr1, abr2, and ayg1 in addition to alb1, arp1, and arp2 suggests that conidial pigment biosynthesis in A. fumigatus is more complex than the known DHN-melanin pathway.     

Silencing of essential genes by RNA interference in Haemonchus contortus

In this study we assessed three technologies for silencing gene expression by RNA interference (RNAi) in the sheep parasitic nematode Haemonchus contortus. We chose as targets five genes that are essential in Caenorhabditis elegans (mitr-1, pat-12, vha-19, glf-1 and noah-1), orthologues of which are present and expressed in H. contortus, plus four genes previously tested by RNAi in H. contortus (ubiquitin, tubulin, paramyosin, tropomyosin). To introduce double-stranded RNA (dsRNA) into the nematodes we tested (1) feeding free-living stages of H. contortus with Escherichia coli that express dsRNA targetting the test genes; (2) electroporation of dsRNA into H. contortus eggs or larvae; and (3) soaking adult H. contortus in dsRNA. For each gene tested we observed reduced levels of mRNA in the treated nematodes, except for some electroporation conditions. We did not observe any phenotypic changes in the worms in the electroporation or dsRNA soaking experiments. The feeding method, however, elicited observable changes in the development and viability of larvae for five of the eight genes tested, including the 'essential' genes, Hc-pat-12, Hc-vha-19 and Hc-glf-1. We recommend the E. coli feeding method for RNAi in H. contortus and provide recommendations for future research directions for RNAi in this species.     

The effectiveness of double-stranded short inhibitory RNAs (siRNAs) may depend on the method of transfection

RNA interference (RNAi) is a recently described powerful experimental tool that can cause sequence-specific gene silencing, thereby facilitating functional analysis of gene function. Consequently, we became interested in using RNAi to determine the function of aberrantly expressed ErbB3 in the KAS-6/1 human myeloma cell line. Despite the wealth of information available on the use of RNAi, dsRNA target design, and the transfection of dsRNA in vitro, little information is available for transfecting dsRNA into nonadherent cells from any species. In the present study, we report that gene silencing of ErbB3 was not observed in myeloma cells when dsRNA targeting ErbB3 was introduced using conventional transfection agents and protocols that have proved successful for several adherent cell lines. Silencing of ErbB3, however, was observed in T47D cells, an adherent breast carcinoma cell line, using the same transfection methods, indicating that our target sequence was functional for gene silencing of ErbB3. Interestingly, ErbB3 was silenced in myeloma cells when the dsRNA target was introduced by electroporation. Thus, our studies illustrate the striking dependence of dsRNA-mediated gene silencing in some cells on the methods of dsRNA transfection.     

Development of methods for RNA interference in the sheep gastrointestinal parasite, Trichostrongylus colubriformis

The efficiency of RNA interference (RNAi) delivery to L1 through L3 stage worms of the sheep parasitic nematode Trichostrongylus colubriformis was investigated using several techniques. These were: (i) feeding of Escherichia coli expressing double stranded RNA (dsRNA); (ii) soaking of short interfering (synthetic) RNA oligonucleotides (siRNA) or in vitro transcribed dsRNA molecules; and (iii) electroporation of siRNA or in vitro transcribed dsRNA molecules. Ubiquitin and tropomyosin were used as a target gene because they are well conserved genes whose DNA sequences are available for several nematode parasite species. Ubiquitin siRNA or dsRNA delivered by soaking or electroporation inhibited development in T. colubriformis but with feeding as a delivery method, RNAi of ubiquitin was not successful. Feeding was, however, successful with tropomyosin as a target, suggesting that mode of delivery is an important parameter of RNAi. Electroporation is a particularly efficient means of inducing RNA in nematodes with either short dsRNA oligonucleotides or with long in vitro transcribed dsRNA molecules. These methods permit routine delivery of dsRNA for RNAi in T. colubriformis larval stage parasites and should be applicable to moderate to high-throughput screening.     

Gene silencing with RNA interference in the human pathogenic fungus Aspergillus fumigatus

Aspergillus fumigatus is an opportunistic pathogenic fungus which causes fatal invasive aspergillosis among immunocompromised patients. To obtain a better understanding of the key elements involved in A. fumigatus virulence and to identify possible drug targets, it is necessary to be able to generate gene-deletion strains. Unfortunately, the molecular techniques available do not include a rapid method to disrupt and identify essential genes. RNA interference, a process in which the presence of double-stranded RNA homologous to a gene of interest results in specific degradation of the corresponding message, has been successfully tested on A. fumigatus. We have shown that expression of double stranded RNA corresponding to portions of the ALB1/PKSP and FKS1 genes results in reduced mRNA levels for those genes, with phenotypic consequences similar to that of gene disruption. The two genes could also be subjected to simultaneous interference through expression of chimeric double-stranded RNA. Use of RNA interference in Aspergillus will allow easier examination of the phenotypic consequences of reducing expression of a gene of interest, especially for essential genes.     

Aspergillus fumigatus arp1 modulates conidial pigmentation and complement deposition

Aspergillus fumigatus is an important pathogen causing invasive pulmonary aspergillosis in immunocompromised patients. The fungus propagates by conidia, which are the infectious structures inhaled by the human host. Opsonophagocytosis is thought to contribute to clearance of the inhaled conidia, a process that is facilitated by complement deposition on conidial surfaces. We now show that conidial colour mutants exhibit significant increases in C3 binding capacity compared with wild type. A reddish-pink mutation that led to enhanced C3 binding was complemented by a cosmid clone. A 3.3 kb DNA fragment from the subsequently rescued cosmid was sufficient to restore the bluish-green conidial pigment. The bluish-green transformant exhibited a level of C3 binding similar to that of the parental strain. A gene, designated arp1 , was responsible for the complementation. Comparison of the genomic and cDNA sequences of arp1 revealed that it has two introns and encodes a putative protein of 168 amino acids. Arp1 is very similar to scytalone dehydratase, an enzyme involved in 1,8-dihydroxynaphthalene-melanin synthesis in Colletotrichum lagenarium and Magnaporthe grisea . Northern hybridization analysis revealed that arp1 is developmentally regulated, being expressed during conidiation. Disruption of arp1 resulted in reddish-pink conidia and increased C3 binding. Our studies suggest that arp1 modulates the bluish-green pigmentation of conidia as well as complement deposition.     

Characterization of essential genes by parasexual genetics in the human fungal pathogen Aspergillus fumigatus: impact of genomic rearrangements associated with electroporation of DNA

We have evaluated the usefulness of parasexual genetics in the identification of genes essential for the growth of the human fungal pathogen Aspergillus fumigatus. First, essentiality of the A. fumigatus AfFKS1 gene, encoding the catalytic subunit of the beta-(1,3)-glucan synthase complex, was assessed by inactivating one allele of AfFKS1 in a diploid strain of A. fumigatus obtained using adequate selectable markers in spore color and nitrate utilization pathways and by performing haploidization under conditions that select for the occurrence of the disrupted allele. Haploid progeny could not be obtained, demonstrating that AfFKS1 and, hence, beta-(1,3)-glucan synthesis are essential in A. fumigatus. Second, random heterozygous insertional mutants were generated by electroporation of diploid conidia with a heterologous plasmid. A total of 4.5% of the transformants failed to produce haploid progeny on selective medium. Genomic analysis of these heterozygous diploids led in particular to the identification of an essential A. fumigatus gene encoding an SMC-like protein resembling one in Schizosacccharomyces pombe involved in chromosome condensation and cohesion. However, significant plasmid and genomic DNA rearrangements were observed at many of the identified genomic loci where plasmid integration had occurred, thus suggesting that the use of electroporation to build libraries of A. fumigatus insertional mutants has relatively limited value and cannot be used in an exhaustive search of essential genes.     

Genetic interference in Trypanosoma brucei by heritable and inducible double-stranded RNA

The use of double-stranded RNA (dsRNA) to disrupt gene expression has become a powerful method of achieving RNA interference (RNAi) in a wide variety of organisms. However, in Trypanosoma brucei this tool is restricted to transient interference, because the dsRNA is not stably maintained and its effects are diminished and eventually lost during cellular division. Here, we show that genetic interference by dsRNA can be achieved in a heritable and inducible fashion. To show this, we established stable cell lines expressing dsRNA in the form of stem-loop structures under the control of a tetracycline-inducible promoter. Targeting a-tubulin and actin mRNA resulted in potent and specific mRNA degradation as previously observed in transient interference. Surprisingly, 10-fold down regulation of actin mRNA was not fatal to trypanosomes. This type of approach could be applied to study RNAi in other organisms that are difficult to microinject or electroporate. Furthermore, to quickly probe the consequences of RNAi for a given gene we established a highly efficient in vivo T7 RNA polymerase system for expression of dsRNA. Using the alpha-tubulin test system we obtained greater than 98% transfection efficiency and the RNAi response lasted at least two to three cell generations. These new developments make it possible to initiate the molecular dissection of RNAi both biochemically and genetically.     

Gene silencing in Caenorhabditis elegans by transitive RNA interference

When a cell is exposed to double-stranded RNA (dsRNA), mRNA from the homologous gene is selectively degraded by a process called RNA interference (RNAi). Here, we provide evidence that dsRNA is amplified in Caenorhabditis elegans to ensure a robust RNAi response. Our data suggest a model in which mRNA targeted by RNAi functions as a template for 5' to 3' synthesis of new dsRNA (termed transitive RNAi). Strikingly, the effect is nonautonomous: dsRNA targeted to a gene expressed in one cell type can lead to transitive RNAi-mediated silencing of a second gene expressed in a distinct cell type. These data suggest dsRNA synthesized in vivo can mediate systemic RNAi.