病毒诱导的基因沉默及其在植物基因功能研究中的应用

RNA介导的基因沉默是近年来在生物体中发现的一种基于核酸水平高度保守的特异性降解机制.病毒诱导的基因沉默（virus induced gene silencing, VIGS）是指携带植物功能基因cDNA的病毒在侵染植物体后,可诱导植物发生基因沉默而出现表型突变,进而可以研究该目的基因功能.至今,已经建立了以RNA病毒、DNA病毒、卫星病毒和DNA卫星分子为载体的VIGS体系,这些病毒载体能在多种寄主植物（包括拟南芥、番茄和大麦）上有效抑制功能基因的表达.VIGS已开始应用于N基因和Pto基因介导的抗性信号途径中关键基因的功能研究、抗病毒相关的寄主因子研究以及植物代谢和发育调控研究.在当前植物基因组或EST序列大量测定的情况下,VIGS为植物基因功能鉴定提供了有效的技术平台.     

从动物组织提取高纯度总RNA方法的改进及应用

从动物组织中提取总RNA是现代分子生物学研究中经常使用的重要实验技术,按常规方法获得的总RNA产品中常伴有大分子量染色体DNA污染。为此,我们摸索出了保证RNA制品纯度、质量和产量的DNA酶消化最佳反应条件。利用改良后的方法提取了小鼠脏器组织总RNA,进行了新基因mPC-1在小鼠脏器中表达的组织分布研究。     

vsiRNA18 derived from tobacco curly shoot virus can regulate virus infection in Nicotiana benthamiana

Virus-derived small interfering RNAs (vsiRNAs) play important roles in regulating host endogenous gene expression to promote virus infection and induce RNA silencing to suppress virus infection. However, to date, how vsiRNAs affect geminivirus infection in host plants has been less studied. In this study, we found that tobacco curly shoot virus (TbCSV)-derived vsiRNA18 (TvsiRNA18) can regulate TbCSV infection in Nicotiana benthamiana plants. The virus-mediated small RNA expression system and stable transformation technique were used to clarify the molecular role of TvsiRNA18 in TbCSV infection. The results indicate that TvsiRNA18 can aggravate disease symptoms in these plants and enhance viral DNA accumulation. ATP-dependent RNA helicase (ATP-dRH) was proven to be a target of TvsiRNA18, and down-regulation of ATP-dRH in plants was shown to induce virus-like leaf curling symptoms and increase TbCSV infection. These results suggest that TvsiRNA18 is an important regulator of TbCSV infection by suppressing ATP-dRH expression. This is the first report to demonstrate that TbCSV-derived vsiRNA can target host endogenous genes to affect symptom development, which helps to reveal the molecular mechanism of symptom occurrence after the virus infects the host.     

蚜虫中携带马铃薯卷叶病毒检测方法的改进

马铃薯卷叶病毒( Potato leafroll virus,PLRV)对马铃薯生产的危害极大,是一种极为重要的马铃薯病毒病。 RT－PCR是马铃薯卷叶病毒检测较为常用的方法,该方法检测准确率高、成本低、适用范围广。但在实际生产中其检测对象多为染病植株,对PLRV传播的主要介体桃蚜( Myzus persicae)的检测,则由于蚜虫体积小、RNA提取难度大、成本高、且不能复检,因而在生产中不能被广泛使用。该研究以马铃薯感病植株和带毒蚜虫为材料,利用改进的RNA提取方法从它们中提取到PLRV的RNA,并以CP 基因设计特异性引物,进行PCR检测。结果表明：该方法提取的RNA完整性好,可用于蚜虫中PLRV检测,且同样适用于对马铃薯感病植株的检测。另外,通过对田间有翅蚜和无翅蚜携带 PLRV 情况进行检测发现,无翅蚜 PLRV 检出率为100％,有翅蚜PLRV检出率也高达60％,证明该体系在生产中的实用性。该研究使用改进的RNA提取方法,提取蚜虫中RNA,并利用RT－PCR进行了PLRV检测,与以前的方法相比简单实用,可被应用于生产检测中。该研究结果为马铃薯生产中PLRV的防控提供了一种新的手段。     

Influence of RNA-Seq library construction,sampling methods,and tissue harvesting time on gene expression estimation

RNA sequencing (RNA-Seq) is popular for measuring gene expression in non-model organisms, including wild populations. While RNA-Seq can detect gene expression variation among wild-caught individuals and yield important insights into biological function, sampling methods can also affect gene expression estimates. We examined the influence of multiple technical variables on estimated gene expression in a non-model fish, the westslope cutthroat trout (Oncorhynchus clarkii lewisi), using two RNA-Seq library types: 3′ RNA-Seq (QuantSeq) and whole mRNA-Seq (NEB). We evaluated effects of dip netting versus electrofishing, and of harvesting tissue immediately versus 5 min after euthanasia on estimated gene expression in blood, gill, and muscle. We found no significant differences in gene expression between sampling methods or tissue collection times with either library type. When library types were compared using the same blood samples, 58% of genes detected by both NEB and QuantSeq showed significantly different expression between library types, and NEB detected 31% more genes than QuantSeq. Although the two library types recovered different numbers of genes and expression levels, results with NEB and QuantSeq were consistent in that neither library type showed differences in gene expression between sampling methods and tissue harvesting times. Our study suggests that researchers can safely rely on different fish sampling strategies in the field. In addition, while QuantSeq is more cost effective, NEB detects more expressed genes. Therefore, when it is crucial to detect as many genes as possible (especially low expressed genes), when alternative splicing is of interest, or when working with an organism lacking good genomic resources, whole mRNA-Seq is more powerful.     

Rapid, specific detection of alphaviruses from tissue cultures using a replicon-defective reporter gene assay

We established a rapid, specific technique for detecting alphaviruses using a replicon-defective reporter gene assay derived from the Sindbis virus XJ-160. The pVaXJ expression vector containing the XJ-160 genome was engineered to form the expression vectors pVaXJ-EGFP expressing enhanced green fluorescence protein (EGFP) or pVaXJ-GLuc expressing Gaussia luciferase (GLuc). The replicon-defective reporter plasmids pVaXJ-EGFPΔnsp4 and pVaXJ-GLucΔnsp4 were constructed by deleting 1139 bp in the non-structural protein 4 (nsP4) gene. The deletion in the nsP4 gene prevented the defective replicons from replicating and expressing reporter genes in transfected BHK-21 cells. However, when these transfected cells were infected with an alphavirus, the non-structural proteins expressed by the alphavirus could act on the defective replicons in trans and induce the expression of the reporter genes. The replicon-defective plasmids were used to visualize the presence of alphavirus qualitatively or detect it quantitatively. Specificity tests showed that this assay could detect a variety of alphaviruses from tissue cultures, while other RNA viruses, such as Japanese encephalitis virus and Tahyna virus, gave negative results with this system. Sensitivity tests showed that the limit of detection (LOD) of this replicon-defective assay is between 1 and 10 PFU for Sindbis viruses. These results indicate that, with the help of the replicon-defective alphavirus detection technique, we can specifically, sensitively, and rapidly detect alphaviruses in tissue cultures. The detection technique constructed here may be well suited for use in clinical examination and epidemiological surveillance, as well as for rapid screening of potential viral biological warfare agents.     

Establishing RNA virus resistance in plants by harnessing CRISPR immune system

Recently, CRISPR‐Cas (clustered, regularly interspaced short palindromic repeats–CRISPR‐associated proteins) system has been used to produce plants resistant to DNA virus infections. However, there is no RNA virus control method in plants that uses CRISPR‐Cas system to target the viral genome directly. Here, we reprogrammed the CRISPR‐Cas9 system from Francisella novicida to confer molecular immunity against RNA viruses in Nicotiana benthamiana and Arabidopsis plants. Plants expressing FnCas9 and sgRNA specific for the cucumber mosaic virus (CMV) or tobacco mosaic virus (TMV) exhibited significantly attenuated virus infection symptoms and reduced viral RNA accumulation. Furthermore, in the transgenic virus‐targeting plants, the resistance was inheritable and the progenies showed significantly less virus accumulation. These data reveal that the CRISPR/Cas9 system can be used to produce plant that stable resistant to RNA viruses, thereby broadening the use of such technology for virus control in agricultural field.     

Anin vitro procedure to eradicate potato viruses X,Y, and S from Russet Norkotah and two of its strains

Summary A tissue culture method was developed for the eradication of three important potato viruses, PVX, PVY, and PVS, from the Russet Norkotah variety and two of its strains (TXNS 112 and TXNS 278). The method combined the use of liquid medium, thermotherapy, and chemotherapy. Initially, virus-free plants were inoculated with PVX, PVY, and PVS and, after 10 d, tested quantitatively for virus titer by ELISA to determine the initial virus concentration. The apical tip and the roots were removed from the inoculated plants, and only stem sections from inoculated plants were cultured in liquid medium containing MS inorganic salts, vitamins, and ribavirin (40 μM or 80 μM). After 5 d, half of the plants were subjected to thermotherapy and half were kept at room temperature. At 6 wk, the uppermost node (5–7 mm) was removed and recultured, and plants were tested then and again after 6 wk using ELISA to identify the virus-free plants. Ribavirin alone eradicated the viruses from some plants; however, more virus-free plants were obtained when the treatments included heat. Additionally, thein vitro culture technique using liquid medium promoted rapid lateral shoot elongation and resulted in significantly faster plant production. Also this approach, which required less skilled labor, produced more plants than the meristem culture method for virus eradication. A detailed procedure for elimination of multiple viruses is described. This procedure resulted in production of more than 10% virus-free plants.     

Isolation of High-Quality RNA from <Emphasis Type="Italic">Reaumuria soongorica</Emphasis>, a Desert Plant Rich in Secondary Metabolites

RNA isolation is a prerequisite for the study of the molecular mechanisms of stress tolerance in the desert plant Reaumuria soongorica, an extreme xeric semi-shrub. However, R. soongorica that contains high levels of secondary metabolites that co-precipitate with RNA, making RNA isolation difficult. Here the authors propose a new protocol suitable for isolating high-quality RNA from the leaves of R. soongorica. Based on a CTAB method described by Liu et al., the protocol has been improved as follows: the samples were ground with PVPP to effectively inhibit the oxidation of phenolics, contaminating DNA was removed with DNase I, and NaAc was used along with ethanol for precipitation to enhance the RNA yield and shorten the precipitation time. Gel electrophoresis and spectrophotometric analysis indicated that this isolation method provides RNA with no DNA contamination. Moreover, the yield (183.79 ± 40.36 μg/g) and quality were superior to those using the method of Liu et al., which yields RNA with significant DNA contamination at 126.30 ± 29.43 μg/g. Gene amplification showed that the RNA obtained using this protocol is suitable for use in downstream molecular procedures. This method was found to work equally well for isolating RNA from other desert plants. Thus, it is likely to be widely applicable.     

Detection of bean yellow mosaic virus in gladioli corms by the polymerase chain reaction

The polymerase chain reaction (PCR) method readily detected bean yellow mosaic virus (BYMV) in gladioli leaves, but in initial tests PCR did not detect virus in corm tissue. Extracts of RN A from corm tissue were shown to inhibit the amplification of viral sequences when added to a PCR reaction. An additional purification step for the RNA extracts using a Sephadex G-50 column eliminated the inhibitory effect and enabled PCR to amplify and detect viral RNA in corm tissue preparations.     

Targeting of SPCSV-RNase3 via CRISPR-Cas13 confers resistance against sweet potato virus disease

Sweet potato (Ipomoea batatas) is one of the most important crops in the world, and its production rate is mainly decreased by the sweet potato virus disease (SPVD) caused by the co-infection of sweet potato chlorotic stunt virus (SPCSV) and sweet potato feathery mottle virus. However, methods for improving SPVD resistance have not been established. Thus, this study aimed to enhance SPVD resistance by targeting one of its important pathogenesis-related factors (i.e., SPCSV-RNase3) by using the CRISPR-Cas13 technique. First, the RNA targeting activity of four CRISPR-Cas13 variants were compared using a transient expression system in Nicotiana benthamiana. LwaCas13a and RfxCas13d had more efficient RNA and RNA virus targeting activity than PspCas13b and LshCas13a. Driven by the pCmYLCV promoter for the expression of gRNAs, RfxCas13d exhibited higher RNA targeting activity than that driven by the pAtU6 promoter. Furthermore, the targeting of SPCSV-RNase3 using the LwaCas13a system inhibited its RNA silencing suppressor activity and recovered the RNA silencing activity in N. benthamiana leaf cells. Compared with the wild type, transgenic N. benthamiana plants carrying an RNase3-targeted LwaCas13a system exhibited enhanced resistance against turnip mosaic virus TuMV-GFP and cucumber mosaic virus CMV-RNase3 co-infection. Moreover, transgenic sweet potato plants carrying an RNase3-targeted RfxCas13d system exhibited substantially improved SPVD resistance. This method may contribute to the development of SPVD immune germplasm and the enhancement of sweet potato production in SPVD-prevalent regions.     

Simultaneous detection of miRNA and mRNA at the single-cell level in plant tissues

Detecting the simultaneous presence of a microRNA (miRNA) and a mRNA in a specific tissue can provide support for the prediction that the miRNA regulates the mRNA. Although two such methods have been developed for mammalian tissues, they have a low signal-noise ratio and/or poor resolution at the single-cell level. To overcome these drawbacks, we develop a method that uses sequence-specific miRNA-locked nucleic acid (LNA) and mRNA-LNA probes. Moreover, it augments the detection signal by rolling circle amplification, achieving a high signal-noise ratio at the single-cell level. Dot signals are counted for determining the expression levels of mRNA and miRNA molecules in specific cells. We show a high sequence specificity of our miRNA-LNA probe, revealing that it can discriminate single-base mismatches. Numerical quantification by our method is tested in transgenic rice lines with different gene expression levels. We conduct several applications. First, the spatial expression profiling of osa-miR156 and OsSPL12 in rice leaves reveals their specific expression in mesophyll cells. Second, studying rice and its mutant lines with our method reveals opposite expression patterns of miRNA and its target mRNA in tissues. Third, the dynamic expression profiles of ZmGRF8 and zma-miR396 during maize leaf development provide evidence that zma-miR396 regulates the preferential spatial expression of ZmGRF8 in bundle sheath cells. Finally, our method can be scaled up to simultaneously detect multiple miRNAs and mRNAs in a tissue. Thus, it is a sensitive and versatile technique for studying miRNA regulation of plant tissue development.     

Transiently expressed short hairpin RNA targeting 126 kDa protein of tobacco mosaic virus interferes with virus infection

RNA-interference (RNAi) silences gene expression by'guiding mRNA degradation in asequence-specific fashion.Small interfering RNA (siRNA),an intermediate of the RNAi pathway,has beenshown to be very effective in inhibiting virus infection in mammalian cells and cultured plant cells.Here,wereport that Agrobacterium tumefaciens-mediated transient expression of short hairpin RNA (shRNA) couldinhibit tobacco mosaic virus (TMV) RNA accumulation by targeting the gene encoding the replication-asso-ciated 126 kDa protein in intact plant tissue.Our results indicate that transiently expressed shRNA efficientlyinterfered with TMV infection.The interference observed is sequence-specific,and time-and site-dependent.Transiently expressed shRNA corresponding to the TMV 126 kDa protein gene did not inhibit cucumbermosaic virus (CMV),an unrelated tobamovirus.In order to interfere with TMV accumulation in tobaccoleaves,it is essential for the shRNA constructs to be infiltrated into the same leaves as TMV inoculation.Ourresults support the view that RNAi opens the door for novel therapeutic procedures against virus diseases.We propose that a combination of the RNAi technique and Agrobacterium-mediated transient expressioncould be employed as a potent antiviral treatment in plants.     

Detection of mRNA degradation intermediates in tissues using the 3'-end poly(A)-tailing polymerase chain reaction method

It has become increasingly clear that mRNA stability is an important determinant of mRNA abundance in virtually all organisms. Although our understanding of prokaryotic lower eukaryotic mRNA stability mechanisms has progressed considerably, little is known about mammalian mRNA stability mechanisms, particularly at the tissue and animal levels. This is due largely to the lack of suitable methods to approach the problem. In this study, we have developed and refined the 3'-end poly(A)-tailing polymerase chain reaction (PCR) method to detect degradation intermediates in vivo. Using an in vitro transcribed RNA as a template, we found that the method could be used to detect a homogeneous pool of RNA down to 0.1 ng. The addition of 10 microg of total RNA from tissues decreased the sensitivity limit to 4 ng. Detection limits of the technique were determined precisely by varying the concentrations of in vitro transcribed RNA in a constant amount of total RNA and varying the concentration of total RNA while maintaining a constant amount of in vitro transcribed RNA. Our overall results showed that the poly(A)-tailing PCR method could be used to detect specific RNA species of approximately 1000 nt in a pool of heterogeneous RNA in the range of 1 in 2500 to 1 in 10,000. To our knowledge, this is the most sensitive method to date for identifying mRNA degradation intermediates. Employing sense strand gene-specific primers in this method, we have discovered the class II and class III P-glycoprotein (Pgp) mRNA degradation intermediates in normal rat tissues. This method should serve as an additional tool to help us understand mRNA decay mechanisms in tissues and at animal levels.