Functional analysis of two chitinase genes during the pupation and eclosion stages of the beet armyworm Spodoptera exigua by RNA interference

Insect chitinases are a multigene family that is encoded by a rather large and diverse group of genes. The main function of chitinases is to digest the chitin contained in tissues such as the cuticles and gut lining during molting. In this study, we examined the role of a chitinase (SeChi) and a bacterial type chitinase (SeChi-h) during the pupation and eclosion stages of Spodoptera exigua. First, efficient silencing of the SeChi and SeChi-h genes through specific double-stranded RNA (dsRNA) injection led to a significant reduction in the mRNA levels of SeChi and SeChi-h. Additionally, different phenotypic defects were observed at the pupal and adult stages after injection of the SeChi and SeChi-h dsRNAs. After injecting SeChi dsRNA in the pupal stage, the cuticle of the head split open and the pupal cuticle was visible under the old larval cuticle. However, after injecting the SeChi-h dsRNA, animals died without exhibiting any special phenotypes. At the adult death stage, animals injected with dsSeChi could not shed their pupal shell completely, and their old cuticles remained attached to their head or chest. However, the main lethal phenotype was that insects did not emerge after dsSeChi-h injection. Additionally, the average survival rates of S. exigua were 52.02% and 40.38% at the pupal and adult stages, respectively, after injection with SeChi dsRNA. For the insects injected with SeChi-h dsRNA, the survival rates were 72.38% and 48.52%, respectively. These results suggest that SeChi and SeChi-h may have different biologic functions during the pupal-adult molting.     

Functional analysis of cholesterol biosynthesis by RNA interference

Inborn errors of cholesterol biosynthesis caused by dysfunctionality of single enzymes are known to cause severe malformation syndromes like X-linked chondrodysplasia punctata (CDPX2), CHILD syndrome or Smith–Lemli–Opitz-syndrome (SLOS). In this study we established the method of RNA interference (RNAi) for analyzing the molecular mechanisms underlying disrupted cholesterol biosynthesis. For different genes involved in the cholesterol biosynthesis pathway-NAD(P) dependent steroid dehydrogenase-like (NSDHL), 17-beta hydroxysteroid dehydrogenase type 7 (HSD17B7) and emopamil binding protein (EBP)-shRNA sequences were designed and tested for their effectiveness. For a better comparability of the experiments and to avoid different transfection efficiencies, examined shRNA sequences which reached a knock down of at least 80% were stably transfected in a HeLa cell line with a tetracycline-regulated expression (HeLa T-REx). These stable transfected cell lines represent novel tools for the analysis of cholesterol biosynthesis.     

RNA interference of two acetylcholinesterase genes in Plutella xylostella reveals their different functions

Acetylcholinesterase (AChE, EC 3.1.1.7) is an important enzyme with a typical function of degrading the neurotransmitter acetylcholine. Although two ace genes were reported in Plutella xylostella, their function differences remain largely unknown. The chemically synthesized siRNAs (si‐Pxace1 and si‐Pxace2) were injected into the second instar larvae to knock down Pxace1 and Pxace2, either respectively or simultaneously. The mRNA abundance of Pxace1 and Pxace2 was significantly reduced to 7–33.5% of the control levels at 72 h after siRNA injection. The AChE activities were significantly decreased at 96 h after treatment. Silencing of Pxace1 or Pxace2 resulted in mortality of 33.9 and 22.9%, respectively. The survivors in siRNA‐treated groups had apparent growth inhibition such as reduction in larvae weights and lengths, malformation and motor retardation. Knockdown of Pxace1 apparently affected more on larvae growth than that of Pxace2, suggesting that Pxace1 had more important roles than Pxace2. Both Pxace1 and Pxace2 genes might have atypical functions in regulating larvae growth and motor ability.     

Prnp knockdown in transgenic mice using RNA interference

RNA interference has become a widely used approach to perform gene knockdown experiments in cell cultures and more recently transgenic animals. A designed miRNA targeting the prion protein mRNA was built and expressed using the human PRNP promoter. Its efficiency was confirmed in transfected cells and it was used to generate several transgenic mouse lines. Although expressed at low levels, it was found to downregulate the endogenous mouse Prnp gene expression to an extent that appears to be directly related with the transgene expression level and that could reach up to 80% inhibition. This result highlights the potential and limitations of the RNA interference approach when applied to disease resistance.     

Inheritance and effect on ripening of antisense polygalacturonase genes in transgenic tomatoes

The role of the cell wall hydrolase polygalacturonase (PG) during fruit ripening was investigated using novel mutant tomato lines in which expression of the PG gene has been down regulated by antisense RNA. Tomato plants were transformed with chimaeric genes designed to express anti-PG RNA constitutively. Thirteen transformed lines were obtained of which five were analysed in detail. All contained a single PG antisense gene, the expression of which led to a reduction in PG enzyme activity in ripe fruit to between 5% and 50% that of normal. One line, GR16, showed a reduction to 10% of normal PG activity. The reduction in activity segregated with the PG antisense gene in selfed progeny of GR16. Plants homozygous for the antisense gene showed a reduction of PG enzyme expression of greater than 99%. The PG antisense gene was inherited stably through two generations. In tomato fruit with a residual 1% PG enzyme activity pectin depolymerisation was inhibited, indicating that PG is involved in pectin degradation in vivo. Other ripening parameters, such as ethylene production, lycopene accumulation, polyuronide solubilisation, and invertase activity, together with pectinesterase activity were not affected by the expression of the antisense gene.     

RNA interference-mediated simultaneous silencing of four genes using cross-shaped RNA

The structural flexibility of RNA interference (RNAi)-triggering nucleic acids suggests that the design of unconventional RNAi trigger structures with novel features is possible. Here, we report a cross-shaped RNA duplex structure, termed quadruple interfering RNA (qiRNA), with multiple target gene silencing activity. qiRNA triggers the simultaneous down-regulation of four cellular target genes via an RNAi mechanism. In addition, qiRNA shows enhanced intracellular delivery and target gene silencing over conventional siRNA when complexed with jetPEI, a linear polyethyleneimine (PEI). We also show that the long antisense strand of qiRNA is incorporated intact into an RNA-induced silencing complex (RISC). This novel RNA scaffold further expands the repertoire of RNAi-triggering molecular structures and could be used in the development of therapeutics for various diseases including viral infections and cancer.     

In vitro analysis of RNA interference in Drosophila melanogaster

Double-stranded RNA (dsRNA) triggers the destruction of mRNA sharing sequence with the dsRNA, a phenomenon termed RNA interference (RNAi). The dsRNA is converted by endonucleolytic cleavage into 21- to 23-nt small interfering RNAs (siRNAs), which direct a multiprotein complex, the RNA-induced silencing complex to cleave RNA complementary to the siRNA. RNAi can be recapitulated in vitro in lysates of syncytial blastoderm Drosophila embryos. These lysates reproduce all of the known steps in the RNAi pathway in flies and mammals. Here we explain how to prepare and use Drosophila embryo lysates to dissect the mechanism of RNAi.     

Functional response and mutual interference of Peristenus spretus (Hymenoptera: Braconidae), a parasitoid of Apolygus lucorum (Heteroptera: Miridae)

Peristenus spretus Chen & van Achterberg (Hymenoptera: Braconidae) is a solitary endoparasitoid, which is considered for augmentative biological control of Apolygus lucorum Meyer-Dür (Heteroptera: Miridae) in Chinese cotton fields. Since the association of P. spretus with A. lucorum was only recently discovered, the biology of the parasitoid remains unknown. In order to understand its reproductive biology, the mutual interference and functional response of P. spretus were investigated by altering either the parasitoid or the host density while keeping the other constant. In both experiments, the effects of parasitoid and host densities on parasitism, superparasitism, progeny production and sex ratio were assessed. P. spretus exhibited a Holling type II functional response to changing host densities, indicating that parasitism increases with increasing host density until the parasitoid reaches its maximum reproductive capacity. The model suggested that a single P. spretus female could parasitise a maximum of 88 nymphs per day or four nymphs per hour. Increasing the wasp-nymph ratio from 1:10 to 1:80 significantly increased the offspring production more than fivefold from ±5.8 to ±35.6; further increasing the host densities (above 80 nymphs) did not significantly increase offspring production. Strong mutual interference of foraging P. spretus females occurred only at high parasitoid densities. Parasitoids foraging alone produced an average progeny of 33.4, whereas parasitoids foraging in groups of 16 produced only 2.6. The optimal wasp-nymph ratio for mass-rearing P. spretus is 4:100, given that resources of parasitoids and nymphs are unlimited.     

RNA干扰在植物中的作用机理及其应用研究进展

RNA干扰(RNAi)是广泛存在于生物中的一种现象,它是小干扰RNA诱导的转录后基因沉默,是生物抵抗异常DNA的一种保护机制,同时在生物生长发育过程中调控基因的表达.本文综述了近年来有关RNA干扰的发现、作用过程及其机理,分析了它与反义寡核苷酸、核酶、脱氧核酶的小同,并介绍了RNA干扰在植物基因功能、植物抗病毒、作物品种改良等方面的应用,为siRNA干扰的进一步利用提供参考资料.     

Moment analysis for kinetics of gene silencing by RNA interference

RNA interference (RNAi) was quantitatively evaluated from a kinetic viewpoint. A simple kinetic evaluation based on moment analysis was proposed, assuming suppression and recovery phases of gene expression. We defined the area under the curve of the inhibitory effect (AUC(IE)) as an index of the total intensity of RNAi and the mean response time of the inhibitory effect (MRT(IE)) as an index of its duration. The proposed kinetic analysis helps to understand the RNAi effect in a quantitative and time-dependent manner, which will be beneficial for designing RNAi-based gene silencing for both experimental and therapeutic purposes.     

Suppression of myostatin with vector-based RNA interference causes a double-muscle effect in transgenic zebrafish

Myostatin belongs to the transforming growth factor (TGF)-β superfamily and is a potent negative regulator of skeletal muscle development and growth. We utilized microinjection of an antisense RNA-expressing vector to establish a hereditarily stable myostatin gene knockdown zebrafish strain with a double-muscle phenotype. Real-time PCR and immunostaining revealed that the myostatin messenger (m)RNA and protein levels in homozygous transgenic zebrafish were 33% and 26% those of the non-transgenic controls, respectively. Also, the mRNA levels of myogenic regulatory factor markers such as MyoD, myogenin, Mrf4, and Myf5 were dramatically elevated in myostatin-suppressed transgenic fish compared to the non-transgenic controls. Although there was no significant difference in body length, homozygous transgenic zebrafish were 45% heavier than non-transgenic controls. Histochemical analysis showed that the cross-sectional area of the muscle fiber of homozygous transgenic fish was twice as large as that of non-transgenic controls. This is the first model zebrafish with a hereditarily stable myostatin-suppressed genotype and a double-muscle phenotype.     

Functional analysis of the single calmodulin gene in the nematode Caenorhabditis elegans by RNA interference and 4-D microscopy

Calmodulin (CaM), a small calcium-binding protein, is the key mediator of numerous calcium-induced changes in cellular activity. Its ligands include enzymes, cytoskeletal proteins and ion channels, identified in large part by biochemical and cell biological approaches. Thus far it has been difficult to assess the function of CaM genetically, because of the maternal supply in Drosophila and the presence of at least three nonallelic genes in vertebrates. Here we use the unique possibility offered by the C. elegans model system to inactivate the single CaM gene (cmd-1) through RNA interference (RNAi). We show that the RNAi microinjection approach results in a severe embryonic lethal phenotype. Embryos show disturbed morphogenesis, aberrant cell migration patterns, a striking hyperproliferation of cells and multiple defects in apoptosis. Finally, we show that RNAi delivery by the feeding protocol does not allow the efficient silencing of the CaM gene obtained by microinjection. General differences between the two delivery methods are discussed.     

RNA干扰在疾病治疗上的应用

RNA干扰(RNA interference,RNAi)是一种双链RNA分子在mRNA水平上引发的特异性基因沉默现象。RNAi在基因治疗方面表现出了光明的前景,已成功地应用于多种疾病的临床治疗。本文主要介绍了RNAi在疾病治疗上的应用及研究进展。     

真菌中RNA干扰的生物学功能

RNA干扰(RNA interference,RNAi)是一种保守的真核生物基因调控机制,它利用小的非编码RNA介导转录/转录后的基因沉默。虽然部分真菌丢失了RNAi系统,但随着对真菌RNAi机制研究的增加,越来越多的证据表明,真菌的RNAi系统不但参与维持基因组完整性,其在调节真菌生长发育、介导异染色质组装、促进着丝粒进化、调节真菌耐药性与毒力等方面也具有重要作用。本文主要对真菌中RNAi的生物学功能进行综述,以期为进一步深入研究真菌RNA干扰机制提供一定的理论与研究基础。