A simple method for construction of artificial microRNA vector in plant

Artificial microRNA (amiRNA) is a powerful tool for silencing genes in many plant species. Here we provide an easy method to construct amiRNA vectors that reinvents the Golden Gate cloning approach and features a novel system called top speed amiRNA construction (TAC). This speedy approach accomplishes one restriction-ligation step in only 5 min, allowing easy and high-throughput vector construction. Three primers were annealed to be a specific adaptor, then digested and ligated on our novel vector pTAC. Importantly, this method allows the recombined amiRNA constructs to maintain the precursor of osa-miR528 with exception of the desired amiRNA/amiRNA* sequences. Using this method, our results showed the expected decrease of targeted genes in Nicotiana benthamiana and Oryza sativa.     

Improved method for constructing plant amiRNA vectors with blue-white screening and MAGIC

Artificial microRNA (amiRNA) technology is a novel tool in reverse genetic research for discovering or validating gene functions in plants. A convenient cloning strategy has been developed to construct plant amiRNA vectors based on lacO reconstruction and mating-assisted, genetically-integrated cloning (MAGIC). The amiRNA precursor fragment was generated by PCR and inserted into a small donor plasmid through reconstruction of integrated lacO sequence. Blue recombinants were selected on plates containing X-gal and the efficiency of successful clones was 100%. The amiRNA expression cassette was transferred from the donor plasmid to the recipient plasmid p1301-gfp through MAGIC and an amiRNA expression plasmid was created. More than 40 plant amiRNA vectors were generated through this method, one of which was transformed into Arabidopsis thaliana and the target gene was silenced efficiently. The approach will be useful for amiRNA expression vectors construction in plants.     

Two-step method for constructing Arabidopsis artificial microRNA vectors

Artificial microRNA (amiRNA) technology is used for gene silencing in Arabidopsis. We describe a method for constructing amiRNA vectors that requires only one PCR and one ligation reaction. Vectors produced by this method are the same as those from the method of Schwab et al. (Plant Cell 2006, 18:1121-1133). Transgenic plants created by this method can therefore be tested in the same way or compared with existing transgenic material without the risk of alteration to the amiRNA skeleton. With optimized parameters, 36-42 % colonies had the insertion in the expected orientation and 85-95 % of these had the correct sequence. Using this method, a transient gene knock-down analysis in Arabidopsis could be completed in 4-5 days.     

A novel approach for the construction of plant amiRNA expression vectors

Artificial microRNA (amiRNA) technology has been applied in Arabidopsis thaliana and other plants to efficiently silence target genes of interest. Here we described a novel approach to construct plant amiRNA expression vectors with seamless enzyme-free cloning (SEFC) and mating-assisted genetically integrated cloning (MAGIC). Two pairs of primers were designed when the loop of amiRNA precursor was longer than 60 bp while three oligonucleotides were used to amplify the linearized vector containing the amiRNA precursor whose loop was smaller than 60 bp. The PCR products were transformed into Escherichia coli to generate the donor plasmid containing the amiRNA expression cassette through homologous recombination in vivo. The amiRNA expression cassette was then transferred to the recipient plasmid via MAGIC and an amiRNA expression plasmid was created. More than 200 amiRNA expression vectors were generated with this approach, three of which have been transformed into A. thaliana and successfully silence the target genes. Given its low-cost and simplicity, this novel approach of plant amiRNA expression vectors construction will benefit the study of individual gene function and establishment of plant amiRNA libraries.     

Geographic and temporal distributions of four genotypes found in Erysiphe gracilis var. gracilis,a powdery mildew of evergreen oaks (Erysiphales)

A survey of 402 samples of Erysiphe gracilis var. gracilis on evergreen oaks collected from a wide area of western Japan showed that they were divided into four distinct genotypes each forming a separate clade with high bootstrap support, which were referred to as E. hiratae (genotype I), E. uncinuloides (genotype II), E. gracilis s. str. (genotype III), and E. pseudogracilis (genotype IV) in a separate taxonomic treatment. However, there are no clear differences in geographic distributions among these four genotypes. Quercus myrsinifolia was only infected by genotype II and Q. salicina only by genotype IV, whereas Q. glauca was infected by all four. These results strongly suggest an association between host species and speciation of these genotypes. A further 312 samples of Q. glauca with E. gracilis s. lat. colonies were collected from four locations in the Mie University campus to investigate frequency of genotypes I and II every month from May 2015 to January 2016. No temporal isolation was found in genotype frequencies. These genotypes frequently co-existed on a single leaf surface, especially at the locations disturbed by human activities. Two oak powdery mildews, E. gracilis s. lat. and Cystotheca wrightii, produced conidia only one month a year and their life cycle differed from most other powdery mildew species. This suggests that these oak mildews developed their unique life cycles to synchronize with the life cycle of evergreen oaks.     

针对乙肝病毒的串联序列amiRNA表达载体的构建

目的为优化RNA干扰研究方法,构建了针对乙肝病毒的串联序列amiRNA(artificial microRNA)质粒表达载体。方法设计针对乙型肝炎病毒S区的靶干扰序列,构建单一序列amiRNA质粒表达载体;将其中干扰效率好的两个序列串联起来,构建串联序列amiRNA质粒表达载体。结果经酶切及测序鉴定,插入序列与靶序列一致,载体构建正确。结论成功构建了新型针对乙肝病毒的串联序列amiRNA质粒表达载体,为进一步体外及体内实验奠定了基础。     

A Highly Efficient Method for Construction of Rice Artificial MicroRNA Vectors

Artificial microRNA (amiRNA) has become a powerful tool for gene silencing in plants. A new method for easy and rapid construction of rice artificial miRNA vector is described. The procedure involved modification of the pCAMBIA1300-UR vector by insertion of a ‘vector modification fragment’. This was prepared from the precursor of Os-amiR528 by eliminating the central miRNA-containing region while simultaneously creating an AfeI restriction site. The fragment was then introduced to the destination vector to produce a multipurpose ‘Highly Efficient gene Silencing Compatible vector’ (HESC vector). AfeI was used to produce linearized HESC vectors, and a blunt end PCR product that included amiRNA sequence was cloned into this site by a single ligation reaction to create the completed amiRNA vector. Tests showed that the method was highly efficient, and greatly reduced the time needed for vector construction and resulted in a DNA sequence identical to that of the current method, making it particularly suitable for use in a systems biology approach to functional genomic research.     

Construction and application of the vectors to identify genes encoding exported proteins of Escherichia coli

In order to clone genes having signal sequences of Escherichia coli, four vectors with or without Lac or Ara promoter were constructed using a leaderless β-lactamase as reporter. Fragments of tetracycline resistance gene (Tet) with or without promoter were used to confirm the vectors’ ability to clone and report signal sequences. The minimum inhibitory concentration of ampicillin of the transformants was measured to detect the expression and secretion efficiency of the vectors. The results showed that the β-lactamase could be co-expressed and secreted with Tet protein. The Lac or Ara promoter in the vectors could be regulated by different inducers, and the Ara promoter showed higher regulative efficiency than the Lac. The best induction dose of l-arabinose for the Ara promoter is 1.25 %. All the four vectors were stably maintained in host after being inoculated for 20 passages in antibiotics-free media. Genomic library of an avian pathogenic strain, E. coli O₂, was constructed using the pMB-Ara-T vector we developed. 318 clones were obtained from the genomic library of E. coli strain O₂, and the inserts in these clones represented 276 genes based on sequence analysis. Among the 276 cloned fragments, only 128 had complete promoter sequence. For the 128 fragments with promoter, only 27 could be expressed under LB culture condition without inducer, the other 101 were only expressed under induction. The results showed our constructed vectors could efficiently capture all kinds of exported protein genes in vitro, including the ones without promoter or with inactive promoter.     

Cloning and characterization of a tuberous root-specific promoter from cassava (Manihot esculenta Crantz)

In order to obtain a tuberous root-specific promoter to be used in the transformation of cassava, a 1,728 bp sequence containing the cassava granule-bound starch synthase (GBSSI) promoter was isolated. The sequence proved to contain light- and sugar-responsive cis elements. Part of this sequence (1,167 bp) was cloned into binary vectors to drive expression of the firefly luciferase gene. Cassava cultivar Adira 4 was transformed with this construct or a control construct in which the luciferase gene was cloned behind the 35S promoter. Luciferase activity was measured in leaves, stems, roots and tuberous roots. As expected, the 35S promoter induced luciferase activity in all organs at similar levels, whereas the GBSSI promoter showed very low expression in leaves, stems and roots, but very high expression in tuberous roots. These results show that the cassava GBSSI promoter is an excellent candidate to achieve tuberous root-specific expression in cassava.     

Multi‐targeting of viral RNAs with synthetic trans‐acting small interfering RNAs enhances plant antiviral resistance

RNA interference (RNAi)‐based tools are used in multiple organisms to induce antiviral resistance through the sequence‐specific degradation of target RNAs by complementary small RNAs. In plants, highly specific antiviral RNAi‐based tools include artificial microRNAs (amiRNAs) and synthetic trans‐acting small interfering RNAs (syn‐tasiRNAs). syn‐tasiRNAs have emerged as a promising antiviral tool allowing for the multi‐targeting of viral RNAs through the simultaneous expression of several syn‐tasiRNAs from a single precursor. Here, we compared in tomato plants the effects of an amiRNA construct expressing a single amiRNA and a syn‐tasiRNA construct expressing four different syn‐tasiRNAs against Tomato spotted wilt virus (TSWV), an economically important pathogen affecting tomato crops worldwide. Most of the syn‐tasiRNA lines were resistant to TSWV, whereas the majority of the amiRNA lines were susceptible and accumulated viral progenies with mutations in the amiRNA target site. Only the two amiRNA lines with higher amiRNA accumulation were resistant, whereas resistance in syn‐tasiRNA lines was not exclusive of lines with high syn‐tasiRNA accumulation. Collectively, these results suggest that syn‐tasiRNAs induce enhanced antiviral resistance because of the combined silencing effect of each individual syn‐tasiRNA, which minimizes the possibility that the virus simultaneously mutates all different target sites to fully escape each syn‐tasiRNA.     

Transient expression of artificial microRNAs targeting Grapevine fanleaf virus and evidence for RNA silencing in grapevine somatic embryos

Grapevines are affected worldwide by viruses that compromise fruit yield and quality. Grapevine fanleaf virus (GFLV) causes fanleaf degeneration disease, a major threat to grapevine production. Transgenic approaches exploiting the RNA silencing machinery have proven suitable for engineering viral resistance in several crop species. However, the artificial microRNA (amiRNA)-based strategy has not yet been reported in grapevine. We developed two amiRNA precursors (pre-amiRNAs) targeting the coat protein (CP) gene of GFLV and characterised their functionality in grapevine somatic embryos. To create these pre-amiRNAs, natural pre-miR319a of Arabidopsis thaliana was modified by overlapping PCR in order to replace miR319a with two amiRNAs targeting different regions of the CP gene: amiR^CP-1 or amiR^CP-2. Transient expression of these two pre-amiRNA constructs was tested in grapevine somatic embryos after co-cultivation with Agrobacterium tumefaciens. Expression of amiR^CP-1 and amiR^CP-2 was detected in plant tissues by an endpoint stem-loop RT-PCR as early as 1?day after a 48-h co-cultivation, indicating active processing of pre-amiRNAs by the plant machinery. In parallel, GUS-sensor constructs (G^CP-1 and G^CP-2) were obtained by fusing the target sequence of amiR^CP-1 or amiR^CP-2 to the 3?? terminus of the GUS gene. Co-transformation assays with GUS-sensors and the pre-amiRNA constructs provided evidence for in vivo recognition and cleavage of the 21-nt target sequence of GUS-sensors by the corresponding amiRNA. This is the first report of amiRNA ectopic expression in grapevine. The constructs we developed could be useful for engineering GFLV-resistant grapes in the future.     

Comparative Studies of Various Artificial microRNA Expression Vectors for RNAi in Mammalian Cells

Artificial microRNA (amiRNA) has recently become an important RNA interference (RNAi) technology for gene therapy and gene function studies. Here nine expression strategies were employed to construct plasmid vectors expressing amiRNA (amiR-Fluc) against firefly luciferase (Fluc). Our results indicate that all nine vectors can successfully produce mature amiR-Fluc and specifically suppress the expression of Fluc, although the RNAi efficiency in different mammalian cells displays obvious differences. Among these nine vectors, three can efficiently co-express DsRed reporter gene linked with amiR-Fluc cassette. Moreover, the recommended number of concatenated amiRNAs in a multi-amiRNA expression vector should not be more than four, and the relative position of an amiRNA in the multi-amiRNA expression vector has no apparent influence on its RNAi activity. In summary, all these results described here provide valuable information for the rational design and application of amiRNA expression vector.     

Calibration of a burrow count index for the Indian desert jird, Meriones hurrianae

Population estimates, often difficult to acquire, warrantee the use of an index as an economical substitute for rapid assessments of populations. We estimated population size of the little known social, semi-fossorial Indian desert jird (Meriones hurrianae) in Kachchh, Gujarat, India under closed population capture-mark-recapture (CMR) framework to calibrate a burrow count index for the species. A total of 147 individuals were trapped in 16 colonies using baited Sherman traps and the number of burrow entrances at each colony was recorded. Data from colonies with low number of captures were pooled to estimate capture probability using Huggins heterogeneity models with gender, site, body weight and age category as covariates in Program MARK. Colony sizes ranged from 2 to 46 individuals. The number of burrow entrances was calibrated against CMR-based population estimates using least squares regression (n = 16, adjusted R ² = 0.96, t = 18.18, P < 0.001). The index was further validated using Jackknife (JK) analysis where JK-predicted population estimates strongly correlated with CMR estimates (r = 0.96, P < 0.001). In habitats and climatic conditions similar to Kachchh and within the range of colony sizes sampled, our calibrated index can be a valuable and effective tool for large scale surveys of the desert jird, which occupies a keystone trophic level in the semi-arid ecosystem.     

The colonization of two Phaeocystis species (Prymnesiophyceae) by pennate diatoms and other protists: a significant contribution to colony biomass

The association of Phaeocystis spp. with small pennate diatoms during three Phaeocystis-dominated spring blooms were investigated in the Eastern English Channel (2003 and 2004) and in coastal waters of Western Norway during a mesocosm experiment (2005). In each of these studies, colonization of the surface of large Phaeocystis spp. colonies by small needle-shaped diatoms (Pseudo-nitzschia spp.) were observed. In the English Channel the diatom Pseudo-nitzschia delicatissima colonized the surface of large (>100 μm) Phaeocystis globosa colonies. The abundance of Pseudo-nitzschia delicatissima reached 130 cells per colony and formed up to 70% of the total carbon associated with Phaeocystis cells during late bloom stages. In Norwegian waters, the surface of large (>250 μm) Phaeocystis pouchetii colonies were colonized by Pseudo-nitzschia cf. granii var. curvata and to a lesser degree by other phytoplankton and protist species, although the abundance of these diatoms was never greater than 40 cells per colony. Based on these observations we suggest that diatoms utilize Phaeocystis colonies not only as habitat, but that they are able to utilize the colonial matrix as a growth substrate. Furthermore, these observations indicate that a considerable fraction of biomass (chlorophyll) associated with Phaeocystis colonies, especially large colonies concerned with intense and prolonged blooms, are due to co-occurring plankton species and not exclusively Phaeocystis cells.     

An effective method based on wet-heat treatment for the selective isolation of Micromonospora from estuarine sediments

Several methods for the isolation of Micromonospora from soil samples have been developed; however, it is unclear whether these methods are optimal for estuarine samples. In this study, we optimized the conditions of a wet-heat method for the selective isolation of Micromonospora from estuarine sediments. Sediments were collected from the Arakawa River (estuarine sediments) and Tokyo Bay (marine sediments). Sediment samples were wet-heated at 45, 55, or 65 °C for 30 min and then incubated at 27 °C for 3 weeks. After incubation, most of the actinomycete colonies were macroscopically determined to be of the genus Micromonospora or Streptomyces. In contrast to the treatment at 55 °C, treatment at 65 °C drastically reduced the number of Streptomyces colonies but increased the number of Micromonospora colonies from the estuarine sediments. This procedure allowed us to grow cultures that were composed of more than 90 % Micromonospora. In addition, treatment at 65 °C did not affect the diversity of Micromonospora species compared with treatment at 55 °C. These results indicate that the wet-heat method, which involves pre-treating the sediment at 65 °C for 30 min, is a very simple and effective method for the selective enrichment of a large number of diverse Micromonospora from estuarine sediments. Our results may lead to the isolation of new Micromonospora species, which produce novel bioactive compounds, from different estuarine sediments.     

Statistical evaluation of Ips typographus population density: a useful tool in protected areas and conservation-oriented forestry

Ips typographus (Col., Curculionidae, Scolytinae) occurring on Picea abies stems is a species characterised by large fluctuations in population numbers and causing frequent outbreaks. In protected areas, I. typographus is regarded as a sensitive bioindicator responsive to changes in forest health and vitality. In conservation-oriented forestry, attention is being paid to the ecological value of I. typographus beetles as ecosystem engineers and keystone species, driving forest natural regeneration and conversion. Despite many publications devoted to I. typographus, no accurate method for estimating the population density of this species has been developed. The objective of this study was to develop a statistical method for estimating I. typographus population density that enables calculation of estimation errors. The proposed method consists of two parts: tree-level analyses and stand-level analyses. Part one allows calculation of the total density of I. typographus infestation of each of P. abies selected stem (after selecting sample windfalls), part two allows estimation of the mean total infestation density of the stem in the area investigated. Linear regression functions were applied to part one and survey sampling to part two. The method is only marginally invasive because it involves debarking of one 0.5 m-long stem section on maximum 50 P. abies windfalls (trap trees can optionally be used). The developed method was employed to assess I. typographus population density in the ?wi?tokrzyskie Mountains in Central Poland in an area of ca. 4,000 ha. In 2010, in the area investigated, the mean total I. typographus infestation density of the P. abies stem was 440.6 maternal galleries/m² (from 358.7 to 522.6 maternal galleries/m² with ?? = 0.05; the relative error of estimation was 18.6%). The examined I. typographus population was in a progradation phase. The proposed method can be used in nature reserves, national parks and managed forests, mainly for scientific purposes.