土壤中Frankia菌RNA提取

土壤中Ｆｒａｎｋｉａ菌ＲＮＡ提取王育英，林建群，郭秀荣，李彤，张忠泽（中国科学院沈阳应用生态研究所，１１００１５）Ｆｒａｎｋｉａ非亚科共生固氮是一类重要的固氮资源。对Ｆｒａｎｋｉａ共生生物学、生态学等特性曾进行过广泛的研究。８ｔ０年代末，Ｆｒａｎｋｉ...

Self-assembly of single-stranded RNA on carbon nanotube: Polyadenylic acid to form a duplex structure

All messenger-RNA (mRNA) molecules in eukaryotic cells have a polyadenylic acid [poly (rA)] tail at the 3'-end and human poly (rA) polymerase (PAP) has been considered as a tumor-specific target. A ligand that is capable of recognizing and binding to the poly(M) tail of mRNA might interfere with the full processing of mRNA by PAP and can be a potential therapeutic agent. We report here for the first time that single-walled carbon nanotubes (SWNTs) can cause single-stranded poly (M) to self-structure and form a duplex structure, which is studied by UV melting, atomic force microscopy, circular dichroism spectroscopy, and NMR spectrometry.

Targeted RNA interference of cyclin A(2) mediated by functionalized single-walled carbon nanotubes induces proliferation arrest and apoptosis in chronic myelogenous leukemia K562 cells

Cyclin A(2) plays critical role in DNA replication, transcription, and cell cycle regulation. Its overexpression has been detected and related to many types of cancers including leukemia, suggesting that suppression of cyclin A(2) would be an attractive strategy to prevent tumor progression. Herein, we apply functionalized single wall carbon nanotubes (f-SWNTs) to carry small interfering RNA (siRNA) into K562 cells and determine whether inhibition of cyclin A(2) would be a potential therapeutic target for chronic myelogenous leukemia.

Changes in RNA, DNA, protein contents and growth of turbot Scophthalmus maximus larvae and juveniles

The growth potential of turbot Scophthalmus maximus larvae and juveniles was studied using nucleic acid-based indices and protein variables. The experiment was carried out from 4 to 60 days post hatching (dph). A significant increase in instantaneous growth rate during metamorphosis and retarded growth rate during post-metamorphic phase were observed. Ontogenetic patterns of DNA, RNA and protein all showed developmental stage-specific traits. The RNA:DNA ratio decreased up to 12 dph, then increased rapidly till 19 dph and fluctuated until 35 dph followed by a decline to the end. The RNA:DNA ratio was positively correlated with growth rate of juveniles during the post-metamorphic phase, whereas this ratio was not a sensitive indicator of growth during the pre-metamorphic phase and metamorphosis. The protein:DNA ratio showed a similar tendency to the RNA:DNA ratio. Changes of DNA content and protein:DNA ratio revealed that growth of S. maximus performed mainly by hyperplasia from 4 to 12 dph and hypertrophy until 21 dph during the pre-metamorphic larval phase. Growth was dominantly hypertrophical from the early- to mid-metamorphosing phase and hyperplastic thereafter. The results show that the DNA content and protein:DNA ratio can evaluate growth rates of larval and juvenile S. maximus on a cellular level.

The immune responses in Chinese mitten crab Eriocheir sinensis challenged with double-stranded RNA

Double-stranded RNA (dsRNA) is a virus-associated molecular pattern which induces antiviral innate immune responses and RNA interference (RNAi) in mammals. In invertebrates, RNAi phenomenon has been widely studied, but dsRNA-induced innate immune response is seldom reported. In the present study, two different dsRNAs specific for green fluorescent protein (GFP) and the putative D1 protein of photosystem II (NoPSD) from Nannochloropsis oculata, were employed to challenge Chinese mitten crab Eriocheir sinensis. The temporal changes of phenoloxidase (PO), acid phosphatase (ACP), superoxide dismutase (SOD) and malondialdehyde (MDA) content, as well as the mRNA expression of some immune-related genes were examined in order to estimate the effect of dsRNAs on the innate immunity of E. sinensis. The activities of PO, ACP and SOD significantly increased after dsRNA treatment, whereas malondialdehyde (MDA) content did not change significantly. Among the examined genes, only the mRNA expression of EsALF, an antibacterial peptide in E. sinensis, was significantly up-regulated (about 5 fold, P < 0.05) at 12 h after dsRNA treatment, while no significant expression changes were observed among the other immune genes. The increase of PO, ACP and SOD activities, and mRNA expression level of EsALF after dsRNA stimulation indicate that phenoloxidase, hydrolytic enzyme, antioxidation and EsALF were involved in dsRNA-induced innate immunity, suggesting that broad-spectrum immune responses could be induced by dsRNA in E. sinensis. (C) 2009 Elsevier Ltd. All rights reserved.

Using the ribosomal internal transcribed spacer (ITS) as a complement marker for species identification of red macroalgae

Barcodes based on mitochondrial cytochrome oxidase (mtDNA CO1) sequences are being used for broad taxonomic groups of animals with demonstrated success in species identification and cryptic species discovery, but it has become clear that complementation by a nuclear marker system is necessary, in particular for the barcoding of plants. Here, we propose the nuclear internal transcribed spacer (ITS) as a potentially usable and complementary marker for species identification of red macroalgae, as well as present a primary workflow for species barcoding. Data show that for most red macroalgal genera (except members of the family Delesseriaceae), the size of ITS region ranges from 600 to 1200 bp, and contains enough variation to generate unique identifiers at either the species or genus levels. Consistent with previous studies, we found that the ITS sequence can resolve closely related species with the same fidelity as mtDNA CO1. Significantly, we confirmed that length polymorphism in the ITS region (including 5.8S rRNA gene) can be utilized as a character to discriminate red macroalgal species. As a complementary marker, the verifiable nuclear ITS region can speed routine identification and the detection of species, advance ecological and taxonomic inquiry, and permit rapid and accurate analysis of red macroalgae.

Molecular characterization and effect of RNA interference of retinoid X receptor (RXR) on E75 and chitinase gene expression in Chinese shrimp Fenneropenaeus chinensis

Retinoid X receptor (RXR)/ultraspiracle (USP) is the heterodimeric partner of ecdysteroid receptor and is required for the molting process of arthropods. To better understand the molecular aspects governing the process of molting in shrimp, the full-length cDNA of two RXRs, named as FcRXR-1 and FcRXR-2 were obtained from Chinese shrimp Fenneropenaeus chinensis which were of 1715 and 1700 bp long, revealed a 1315 and 1300 bp open reading frame (ORF) respectively. Quantitative Real time PCR analysis showed a marked tissue-specific difference in the expression of FcRXR transcript, which revealed that the expression of FcRXR Could be regulated in a tissue-specific manner. Moreover, high expression of FcRXR mRNAs was observed in late pre-molt period (D3) and post molt stages (A-B) of shrimp. Among the two isoforms, FcRXR-2 appeared in a considerably high level in all the stages compared to the FcRXR-1. In addition, we examined the temporal expression of two chitinase genes: FcChitinase (FcChi) and FcChitinase-1 (FcChi-1) during the molt cycle of F chinensis. Both the FcChi and FcChi-1 transcripts were detected in all stages of molting, although considerable fluctuations observed through the molt cycle. Injection of double stranded RXR (dsRXR) into juvenile shrimp resulted in a maximum silencing effect at 48 h post injection. We analyzed the expression levels of FcChi, FcChi-1 and the ecdysone inducible gene E75 (FcE75) in samples of dsRXR injected shrimp. Significant reduction in levels of both FcE75, FcChi and FcChi-1 transcripts Occurred in the silenced shrimp. This correlation suggested that RXR might involve in the downstream regulation of E75 and chitinase gene transcription in the ecdysone signaling pathway of decapod crustaceans. (C) 2009 Published by Elsevier Inc.

Identification of peptides that bind with thymidylate synthase RNA using mRNA display technique

Using in vitro selection method to isolate nucleic acids, peptides and proteins has been studied intensively in recent years. In vitro mRNA display is a new and effective technique for peptides selection, and the rationale of this technique is that a synthetic mRNA with puromycin could covalently link with the protein that it encodes, thus an mRNA-protein fusion is formed. This approach has been used in identification of many functional peptides. The peptides binding with thymidylate synthase RNA were isolated using mRNA display technique from a large peptide library (>10(13) different sequences). The selection scheme was constructed, and the experimental conditions, including library synthesis, formation of RNA-peptide fusion and RNA immobilization were optimized. Eight cycles have been processed and the results confirmed that the selected peptides could bind with thymidylate synthase mRNA specifically. Compared the amino acid sequences of the selected peptides with those from the initial random library, the basic and aromatic residues in selected peptides were enriched significantly, suggesting these peptide regions may be important in the peptide-TS mRNA interaction. As a novel in vitro selection approach, mRNA display technique would be developed as a powerful tool for isolation of functional peptides and proteins that could interact with immobilized targets with high affinity and specificity.

A key gene of the RNA interference pathway in the black tiger shrimp, Penaeus monodon: Identification and functional characterisation of Dicer-1

RNA interference (RNAi) is an evolutionarily conserved mechanism by which double-stranded RNA (dsRNA) initiates post-transcriptional silencing of homologous genes. Here we report the amplification and characterisation of a full length cDNA from black tiger shrimp (Penaeus monodon) that encodes the bidentate RNAase III Dicer, a key component of the RNAi pathway. The full length of the shrimp Dicer (Pm Dcr1) cDNA is 7629 bp in length, including a 51 untranslated region (UTR) of 130 bp, a 3' UTR of 77 bp, and an open reading frame of 7422 bp encoding a polypeptide of 2473 amino acids with an estimated molecular mass of 277.895 kDa and a predicted isoelectric point of 4.86. Analysis of the deduced amino acid sequence indicated that the mature peptide contains all the seven recognised functional domains and is most similar to the mosquito (Aedes aegypti) Dicer-1 sequence with a similarity of 34.6%. Quantitative RT-PCR analysis showed that Pm Dcr1 mRNA is most highly expressed in haemolymph and lymphoid organ tissues (P 0.05). However, there was no correlation between Pm Dcr1 mRNA levels in lymphoid organ and the viral genetic loads in shrimp naturally infected with gill-associated virus (GAV) and Mourilyan virus (P > 0.05). Treatment with synthetic dsRNA corresponding to Pm Dcr1 sequence resulted in knock-down of Pm Dcr1 mRNA expression in both uninfected shrimp and shrimp infected experimentally with GAV. Knock-down of Pm Dcr1 expression resulted in more rapid mortalities and higher viral loads. These data demonstrated that Dicer is involved in antiviral defence in shrimp. (c) 2007 Elsevier Ltd. All rights reserved.

Molecular characterization of an ecdysone inducible gene E75 of Chinese shrimp Fenneropenaeus chinensis and elucidation of its role in molting by RNA interference

Ecdysone inducible gene. E75 is a primary target of ecdysone receptor (EcR). and is found to play a critical role in the molting process of arthropods In this study, a cDNA encoding the E75 of Chinese shrimp Fenneropenaeus chinensis (FcE75) was cloned using RT-PCR and RACE techniques FcE75 cDNA was 3611 bp in length with an ORF of 2394 bp. The deduced amino acid sequence of FcE75 had the highest sequence identity to E75 from a land crab Gecarcinus lateral's and E75 of the shrimp Metapenaeus crisis Quantitative real-time PCR revealed a prominently high expression of FcE75 mRNA in the whole body RNA extract of late premolt period (D3) juvenile shrimp. The role of E75 in the process of shrimp molting was investigated using the RNA interference technique Long double-stranded RNA corresponding to the FcE75 (dsE75) efficiently silenced the FcE75 transcript levels in juvenile F. chinensis. Further, injection with dsE75 completely arrested the molting process in experimental shrimp which eventually caused death Setogenic analysis of the uropods from molt-arrested shrimp, showed defective epidermal retraction, poor development of setae and new cuticle. These results indicate that E75 might be related to the molting process and is essential for proper molting and survival of shrimp This is the first report demonstrating the use of double stranded RNA to elucidate the possible role of E75 in the molting of decapod crustaceans (C) 2010 Elsevier Inc All rights reserved

Characterization and mapping of expressed sequence tags isolated from a subtracted cDNA library of Litopenaeus vannamei injected with white spot syndrome virus

A large number of polymorphic simple sequence repeats (SSRs) or microsatellites are needed to develop a genetic map for shrimp. However, developing an SSR map is very time-consuming, expensive, and most SSRs are not specifically linked to gene loci of immediate interest. We report here on our strategy to develop polymorphic markers using expressed sequence tags (ESTs) by designing primers flanking single or multiple SSRs with three or more repeats. A subtracted cDNA library was prepared using RNA from specific pathogen-free (SPF) Litopenaeus vannamei juveniles (similar to 1 g) collected before (0) and after (48 h) inoculation with the China isolate of white spot syndrome virus (WSSV). A total of 224 clones were sequenced, 194 of which were useful for homology comparisons against annotated genes in NCBI nonredundant (nr) and protein databases, providing 179 sequences encoded by nuclear DNA, 4 mitochondrial DNA, and 11 were similar to portions of WSSV genome. The nuclear sequences clustered in 43 groups, 11 of which were homologous to various ESTs of unknown function, 4 had no homology to any sequence, and 28 showed similarities to known genes of invertebrates and vertebrates, representatives of cellular metabolic processes such as calcium ion balance, cytoskeleton mRNAs, and protein synthesis. A few sequences were homologous to immune system-related (allergens) genes and two were similar to motifs of the sex-lethal gene of Drosophila. A large number of EST sequences were similar to domains of the EF-hand superfamily (Ca2+ binding motif and FRQ protein domain of myosin light chains). Single or multiple SSRs with three or more repeats were found in approximately 61 % of the 179 nuclear sequences. Primer sets were designed from 28 sequences representing 19 known or putative genes and tested for polymorphism (EST-SSR marker) in a small test panel containing 16 individuals. Ten (53%) of the 19 putative or unknown function genes were polymorphic, 4 monomorphic, and 3 either failed to satisfactorily amplify genomic DNA or the allele amplification conditions need to be further optimized. Five polymorphic ESTs were genotyped with the entire reference mapping family, two of them (actin, accession #CX535973 and shrimp allergen arginine kinase, accession #CX535999) did not amplify with all offspring of the IRMF panel suggesting presence of null alleles, and three of them amplified in most of the IRM F offspring and were used for linkage analysis. EF-hand motif of myosin light chain (accession #CX535935) was placed in ShrimpMap's linkage group 7, whereas ribosomal protein S5 (accession #CX535957) and troponin I (accession #CX535976) remained unassigned. Results indicate that (a) a large number of ESTs isolated from this cDNA library are similar to cytoskeleton mRNAs and may reflect a normal pathway of the cellular response after im infection with WSSV, and (b) primers flanking single or multiple SSRs with three or more repeats from shrimp ESTs could be an efficient approach to develop polymorphic markers useful for linkage mapping. Work is underway to map additional SSR-containing ESTs from this and other cDNA libraries as a plausible strategy to increase marker density in ShrimpMap.

cDNA cloning and overexpression of acidic ribosomal phosphoprotein P1 gene (RPLP1) from the Giant Panda

RPLP1 is one of acidic ribosomal phosphoproteins encoded by RPLP1 gene, which plays an important role in the elongation step of protein synthesis. The cDNA of RPLP1 was cloned successfully for the first time from the Giant Panda (Ailuropoda melanoleuca) using RT-PCR technology, which was also sequenced, analyzed preliminarily and expressed in E. coli. The cDNA fragment cloned is 449bp in size, containing an open reading frame of 344bp encoding 114 amino acids. Alignment analysis indicated that the nucleotide sequence and the deduced amino acid sequence are highly conserved to other five species studied, including Homo sapiens, Mus musculus, Rattus norvegicus, Bos Taurus and Sus scrofa. The homologies for nucleotide sequences of Giant Panda PPLP1 to that of these species are 92.4%, 89.8%, 89.0%, 91.3% and 87.5%, while the homologies for amino acid sequences are 96.5%, 94.7%, 95.6%, 96.5% and 88.6%. Topology prediction showed there are three Casein kinase II phosphorylation sites and two N-myristoylation sites in the RPLP1 protein of the Giant Panda (Ailuropoda melanoleuca). The RPLP1 gene was overexpressed in E. coli and the result indicated that RPLP1 fusion with the N-terminally His-tagged form gave rise to the accumulation of an expected 18kDa polypeptide, which was in accordance with the predicted protein and could also be used to purify the protein and study its function.

Molecular systematics and biogeography of Crawfurdia, Metagentiana and Tripterospermum (Gentianaceae) based on nuclear ribosomal and plastid DNA sequences

Background and Aims The systematic position of the genus Metagentiana and its phylogenetic relationships with Crawfurdia, Gentiana and Tripterospermum have not been explicitly addressed. These four genera belong to one of two subtribes (Gentianinae) of Gentianeae. The aim of this paper is to examine the systematic position of Crawfurdia, Metagentiana and Tripterospermum and to clarify their phylogenetic affinities more clearly using ITS and trnL intron sequences.Methods Nucleotide sequences from the internal transcribed spacers (ITS) of nuclear ribosomal DNA and the plastid DNA trnL (UAA) intron were analysed phylogenetically. Ten of fourteen Metagentiana species were sampled, together with 40 species of other genera in the subtribe Gentianinae.Key Results The data support several previously published conclusions relating to the separation of Metagentiana from Gentiana and its closer relationships to Crawfurdia and Tripterospermum based on studies of gross morphology, floral anatomy, chromosomes, palynology, embryology and previous molecular data. The molecular clock hypothesis for the tested sequences in subtribe Gentianinae was not supported by the data (P < 0.05), so the clock-independent non-parametric rate smoothing method was used to estimate divergence time. This indicates that the separation of Crawfurdia, Metagentiana and Tripterospermum from Gentiana occurred about 11.4-21.4 Mya (million years ago), and the current species of these three genera diverged at times ranging from 0.4 to 6.2 Mya.Conclusions The molecular analyses revealed that Crawfurdia, Metagentiana and Tripterospermum do not merit status as three separate genera, because sampled species of Crawfurdia and Tripterospermum are embedded within Metagentiana. The speciation and rapid radiation of these three genera is likely to have occurred in western China as a result of upthrust of the Himalayas during the late Miocene and the Pleistocene.