Amplification and function analysis of N6-adenine-specific DNA methyltransferase gene in Nilaparvata lugens

Methylation of the N6 position of adenine, termed N6-methyladenine, protects DNA from restriction endonucleases via the host-specific restriction-modification system. N6-methyladenine was discovered and has been well studied in bacteria. N6-adenine-specific DNA methyltransferase（N6AMT） is the main enzyme catalyzing the methylation of the adenine base and knowledge of this enzyme was mainly derived from work in prokaryotic models. However, large-scale gene discovery at the genome level in many model organisms indicated that the N6AMT gene also exists in eukaryotes, such as humans, mice, fruit flies and plants. Here, we cloned a N6AMT gene from Nilaparvata lugens（Nlu-N6AMT） and amplified its fulllength transcript. Then, we carried out a systematic investigation of N6AMT in 33 publically available insect genomes, indicating that all studied insects had N6AMT. Genomic structure analysis showed that insect N6AMT has short introns compared with the mammalian homologs. Domain and phylogenetic analysis indicated that insect N6AMT had a conserved N6-adenine Mlase domain that is specific to catalyze the adenine methylation. Nlu-N6AMT was highly expressed in the adult female. We knocked down Nlu-N6AMT by feeding ds RNA from the second instar nymph to adult female, inducing retard development of adult female. In all, we provide the first genome-wide analysis of N6AMT in insects and presented the experimental evidence that N6AMT might have important functions in reproductive development and ovary maturation.

Reprogramming Mycobacterium tuberculosis CRISPR System for Gene Editing and Genomewide RNA Interference Screening

Mycobacterium tuberculosis is the causative agent of tuberculosis(TB), which is still the leading cause of mortality from a single infectious disease worldwide. The development of novel anti-TB drugs and vaccines is severely hampered by the complicated and time-consuming genetic manipulation techniques for M. tuberculosis. Here, we harnessed an endogenous type Ⅲ-A CRISPR/Cas10 system of M. tuberculosis for efficient gene editing and RNA interference(RNAi).This simple and easy method only needs to transform a single mini-CRISPR array plasmid, thus avoiding the introduction of exogenous protein and minimizing proteotoxicity. We demonstrated that M. tuberculosis genes can be efficiently and specifically knocked in/out by this system as confirmed by DNA high-throughput sequencing. This system was further applied to single-and multiple-gene RNAi. Moreover, we successfully performed genome-wide RNAi screening to identify M. tuberculosis genes regulating in vitro and intracellular growth. This system can be extensively used for exploring the functional genomics of M. tuberculosis and facilitate the development of novel anti-TB drugs and vaccines.

Specific anti-viral effects of RNA interference on replication and expression of hepatitis B virus in mice

Background RNA interference （RNAi） is a powerful tool to silence gene expression post-transcriptionally. Our previous study has demonstrated that small interfering RNAs （siRNAs） have sufficiently inhibited hepatitis B virus （HBV） replication and expression in vitro. In this study we observed the RNAi-mediated inhibitory effects on HBV replication in mice models and accessed the specificity of these effects. Methods A mutant RNAi vector （pSI-C mut） with two base pairs different from the original target gene sequence at the RNAi vector （pSI-C） was constructed according to the method described in this study, A mouse model of acute hepatitis B virus infection was established by injecting naked plasmid pHBV1.3 via the tail vein with acute circulatory overload, pSI-C, pSI-C mut and the irrelevant RNAi control plasmid for green fluorescent protein （GFP） gene, pSIGFP were respectively delivered with pHBV1.3 by tail vein injection method. Six days post injection, enzyme-linked immunosorbent assay （ELISA） assay was used to measure the concentration of HBV surface antigen （HBsAg） in mouse serum, immunohistochemical straining method was used to visualize the expressin of HBV core protein （HBcAg） in liver tissues, and the transcriptional level of HBV C mRNA in liver tissues was detectedd by reverse transcriptase PCR （RT-PCR） analysis. Results Injection of pSI-C exerted magnificent and specific inhibitory effects on the replication and expression of HBV in the murine model. After 6-day post-injection （ p. i. ）, the OD values were shown to be 5.07 ± 1.07 in infecting group and 0.62 ± 0. 59 in pSI-C group. The concentration of HBsAg in pSI-C group was significantly lower than that in infecting group （ P 〈 0. 01 ）. Liver intracellular synthesis of viral core protein was sharply reduced to 0. 9% ±0. 1%, compared with 5.4% ± 1.2% of positive hepatocytes in infecting group （P 〈0. 01 ）, and the transcriptional level of HBV C mRNA was greatly reduced by 84. 7%. However, the irrelevant RNAi control plasmid （pSIGFP）, and the pSI-C mut did not show the same robust inhibitory effects as pSI-C. Conclusion pSI-C exert efficient and specific inhibitory effects on HBV replication and expression in mice models.

Potent and specific inhibition of SARS-CoV antigen expression by RNA interference

Background Severe acute respiratory syndrome (SARS) is an infectious disease caused by SARS-CoV. There are no effective antiviral drugs for SARS although the epidemic of SARS was controlled. The aim of this study was to develop an RNAi (RNA interference) approach that specifically targeted the N gene sequence of severe acute respiratory syndrome associated coronavirus (SARS-CoV) by synthesizing short hairpin RNA (shRNA) in vivo , and to assess the inhibitory effect of this shRNA on SARS-CoV N antigen expression. Methods The eukaryotic expression plasmid pEGFP-C1-N, containing SARS-CoV N gene, was co-transfected into 293 cells with either the RNAi plasmid pshRNA-N or unrelated control plasmid pshRNA-HBV-C4. At 24, 48 and 72 hours post transfection, the green fluorescence was observed through a fluorescence microscope. The RNA levels of SARS-CoV N were determined by reverse transcription polymerase chain reaction (RT-PCR). The expression of Green Fluorescent Protein (GFP) and protein N were detected using Western blot.Results The vector, pshRNA-N expressing shRNA which targeted the N gene of SARS-CoV, was successfully constructed. The introduction of RNAi plasmid efficiently and specifically inhibited the synthesis of protein N. RT-PCR showed that RNAs of N gene were clearly reduced when the pEGFP-C1-N was cotransfected with pshRNA-N, whereas the control vector did not exhibit inhibitory effect on N gene transcription.Conclusions Our results demonstrate that RNAi mediated silencing of SARS-CoV gene could effectively inhibit expression of SARS-CoV antigen, hence RNAi based strategy should be further explored as a more efficacious antiviral therapy of SARS-CoV infection.

RNA silencing movement in plants

Multicellular organisms, like higher plants, need to coordinate their growth and development and to cope with environmental cues. To achieve this, various signal molecules are transported between neighboring cells and distant organs to control the fate of the recipient cells and organs. RNA silencing produces cell non-autonomous signal molecules that can move over short or long distances leading to the sequence specific silencing of a target gene in a well defined area of cells or throughout the entire plant,respectively. The nature of these signal molecules, the route of silencing spread, and the genes involved in their production, movement and reception are discussed in this review. Additionally, a short section on features of silencing spread in animal models is presented at the end of this review.