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

运用减法杂交、差异筛选与信使RNA差别显示聚合酶链式反应克隆冬小麦春化相关基因

对于某些一年生或二年生高等植物，春化作用是诱导其成花的一个重要的环境因子。冬小麦春化进程中存在着一个核酸代谢的关键期，利用分子生物学技术分离特异表达的基因是研究春化诱导成花机理的一个突破口。 利用TRIzol试剂快速提取冬小麦燕大1817(Triticum aestivum L. cv Yanda 1817)未春化、春化4d、春化20d、5d脱春化的胚芽中的总RNA，去除污染的DNA后，将引物P_1(5'TTTTTTTTTTTCA3')、P_2(5'TTTTTTTTTTCC3')与10个碱基的随机引物OPF_1-OPF_(20)、OPG_1-OPG_(20)组成80个引物对，对不同来源的RNA进行差别显示，共显示了大约10,000种mRNA，结果发现了两个仅在春化20d这一关键期表达而在未春化、春化4d、5d脱春化时不表达的春化相关基因(VRG)VRG49与VRG54。Northern分析进一步表明这两个基因仅与春化20d的冬小麦RNA有杂交信号。将VRG49与VRG54亚克隆于pGEM-4Z载体上，利用T_7测序系统获得了VRG49和VRG54的DNA序列，它们的长度分别为307bp与169bp。 春化21d的冬小麦京冬1号(T. aestivum L. cv Jingdong No. 1)胚芽的mRNA在逆转酶作用下反转录成sscDNA杂交，将过量的未春化、脱春化的mRNA与sscDNA杂交，运用磁珠法分离出未杂交上的sscDNA，以特异的sscDNA为模板，用DNA聚合酷I合成了dscDNA。通过对dscDNA内部EcoRI位点的甲基化、末端补平、EcoRI接头的安装、连接进入λgt10载体的EcoRI位置，以及运用包装系统进行体外包装，建立了库容为4 * 10~6pfu的富集低温诱导的冬小麦cDNA噬菌体文库。用来源于未春化、春化21d、脱春化的冬小麦mRNA合成3种cDNA探针，对噬菌斑进行原位杂交，结果筛选出了3个春化相关基因(VRG)VRG79、VRG111和VRG231。Dot blotting与Northern分析表明VRG79仅在冬小麦春化关键期21d表达。运用PCR方法从λgt10DNA中扩增出VRG79片断并亚克隆于PUC18载体上，通过T_7测序系统获得了VGR79的序列，其包括349个碱基。 通过Internet将VRG49、VRG54、VRG79与GenBank、EMBL、DDBJ、PBD中的序列进行同源性分析，结果发现这些基因至少是在植物中新发现的基因，对这些基因推测的一些功能也进行了讨论。

两种完熟相关基因反义RNA在转因番茄中的表达及其对果实完熟的仰制作用

植物激素乙烯作为一种信使分子调节控制果实完熟。ACC合成酶是植物体内乙烯生物合成途径的限速酶，其反义RNA的表达将能有效地抑制乙烯的生物合成而延缓果实完熟，利用反转录PCR技术克隆获得了ACC合成酶多基因家族成员之一LE-ACC2阅读框架约1.7kb的cDNA，经酶切图谱和序列分析鉴定无误后，反向连入植物表达载体pBin437中构建成组成型表达ACC合成酶反义RNA的双元载体。经农杆菌途径转化番茄“丽春”品种，获得了60株抗卡那再生杭株，PCR检测证明有6株为转基因植株，Southern杂交和Northern杂交分析进一步确证了外源基因的插入及其转录活性。反义番茄果实的乙烯释放受到明显抑制，表现出更好的耐储保鲜特性，并且与对照相比，在果实品质上没有明显差别。大田培育Fl和F2代转化番茄植株，反义番茄纯合品系的筛选工作正在进行之中。 同时，本研究利用已经获得的ACC合成酶和PG的cDNA克隆，构建了两个嵌合转化基因载体pPGACC1、pPGACC10，它包括1300bp的ACC合成酶cDNA编码序列，并分别含有反向与正向的250bp的5’端PG基因片断。酶切图谱和序列分析鉴定无误后，以pBin437为植物表达载体构建了双元载体pBPGACC1和pBPGACC10，分别表达PG正义RNA和反义RNA，并均表达ACC合成酶反义RNA。经农杆菌转化番茄子叶，植株的再生培育有待进行。通过对转基因植物的分析，我们期望阐明用单一嵌合基因表达载体通过反义抑制与抑制作用实现对内源两同源基因——PG和ACC合成酶下降调节的可能性，并可望得到具有更好耐储效果且品质优良的番茄品系。

Phylogenetic relationships of the Chinese brown frogs (Genus rana) inferred from partial mitochondrial 12S and 16S rRNA gene sequences

Based on partial sequences of the 12S and 16S ribosomal RNA genes, we estimated phylogenetic relationships among brown frogs of the Rana temporaria group from China. From the phylogenetic trees obtained, we propose to include Rana zhengi in the brown frog

Identification of a Giardia krr1 homolog gene and the secondarily anucleolate condition of Giaridia lamblia

Giaridia lamblia was long considered to be one of the most primitive eukaryotes and to lie close to the transition between prokaryotes and eukaryotes, but several supporting features, such as lack of mitochondrion and Golgi, have been challenged recently. It was also reported previously that G. lamblia lacked nucleolus, which is the site of pre-rRNA processing and ribosomal assembling in the other eukaryotic cells. Here, we report the identification of the yeast homolog gene, krr1, in the anucleolate eukaryote, G. lamblia. The krr1 gene, encoding one of the pre-rRNA processing proteins in yeast, is actively transcribed in G. lamblia. The deduced protein sequence of G. lamblia krr1 is highly similar to yeast KRR1p that contains a single-KH domain. Our database searches indicated that krr1 genes actually present in diverse eukaryotes and also seem to present in Archaea. However, only the eukaryotic homologs, including that of G. lamblia, have the single-KH domain, which contains the conserved motif KR(K)R. Fibrillarin, another important pre-rRNA processing protein has also been identified previously in G. lamblia. Moreover, our database search shows that nearly half of the other nucleolus-localized protein genes of eukaryotic cells also have their homologs in Giardia. Therefore, we suggest that a common mechanism of pre-RNA processing may operate in the anucleolate eukaryote G. lamblia and in the other eukaryotes and that like the case of "lack of mitochondrion," "lack of nucleolus" may not be a primitive feature, but a secondarily evolutionary condition of the parasite.

Transcriptome of embryonic and neonatal mouse cortex by high-throughput RNA sequencing

Brain structure and function experience dramatic changes from embryonic to postnatal development. Microarray analyses have detected differential gene expression at different stages and in disease models, but gene expression information during early brain development is limited. We have generated >27 million reads to identify mRNAs from the mouse cortex for>16,000 genes at either embryonic day 18 (E18) or postnatal day 7 (P7), a period of significant synapto-genesis for neural circuit formation. In addition, we devised strategies to detect alternative splice forms and uncovered more splice variants. We observed differential expression of 3,758 genes between the 2 stages, many with known functions or predicted to be important for neural development. Neurogenesis-related genes, such as those encoding Sox4, Sox11, and zinc-finger proteins, were more highly expressed at E18 than at P7. In contrast, the genes encoding synaptic proteins such as synaptotagmin, complexin 2, and syntaxin were up-regulated from E18 to P7. We also found that several neurological disorder-related genes were highly expressed at E18. Our transcriptome analysis may serve as a blueprint for gene expression pattern and provide functional clues of previously unknown genes and disease-related genes during early brain development.

Hybrid cytomegalovirus-U6 promoter-based plasmid vectors improve efficiency of RNA interference in zebrafish

Short hairpin RNA (shRNA) directed by RNA polymerase III (Pol III) or Pol II promoter was shown to be capable of silencing gene expression, which should permit analyses of gene functions or as a potential therapeutic tool. However, the inhibitory effect of shRNA remains problematic in fish. We demonstrated that silencing efficiency by shRNA produced from the hybrid construct composed of the CMV enhancer or entire CMV promoter placed immediately upstream of a U6 promoter. When tested the exogenous gene, silencing of an enhanced green fluorescent protein (EGFP) target gene was 89.18 +/- 5.06% for CMVE-U6 promoter group and 88.26 +/- 6.46% for CMV-U6 promoter group. To test the hybrid promoters driving shRNA efficiency against an endogenous gene, we used shRNA against no tail (NTL) gene. When vectorized in the zebrafish, the hybrid constructs strongly repressed NTL gene expression. The NTL phenotype occupied 52.09 +/- 3.06% and 51.56 +/- 3.68% for CMVE-U6 promoter and CMV-U6 promoter groups, respectively. The NTL gene expression reduced 82.17 +/- 2.96% for CMVE-U6 promoter group and 83.06 +/- 2.38% for CMV-U6 promoter group. We concluded that the CMV enhancer or entire CMV promoter locating upstream of the U6-promoter could significantly improve inhibitory effect induced by the shRNA for both exogenous and endogenous genes compared with the CMV promoter or U6 promoter alone. In contrast, the two hybrid promoter constructs had similar effects on driving shRNA.

Functional domains and the antiviral effect of the double-stranded RNA-dependent protein kinase PKR from Paralichthys olivaceus

The double-stranded RNA (dsRNA)-dependent protein kinase PKR is thought to mediate a conserved antiviral pathway by inhibiting viral protein synthesis via the phosphorylation of the alpha subunit of eukaryotic initiation factor 2 (eIF2 alpha). However, little is known about the data related to the lower vertebrates, including fish. Recently, the identification of PKR-like, or PKZ, has addressed the question of whether there is an orthologous PKR in fish. Here, we identify the first fish PKR gene from the Japanese flounder Paralichthys olivaceus (PoPKR). PoPKR encodes a protein that shows a conserved structure that is characteristic of mammalian PKRs, having both the N-terminal region for dsRNA binding and the C-terminal region for the inhibition of protein translation. The catalytic activity of PoPKR is further evidence that it is required for protein translation inhibition in vitro. PoPKR is constitutively transcribed at low levels and is highly induced after virus infection. Strikingly, PoPKR overexpression increases eIF2 alpha phosphorylation and inhibits the replication of Scophthalmus maximus rhabdovirus (SMRV) in flounder embryonic cells, whereas phosphorylation and antiviral effects are impaired in transfected cells expressing the catalytically inactive PKR-K421R variant, indicating that PoPKR inhibits virus replication by phosphorylating substrate eIF2 alpha. The interaction between PoPKR and eIF2 alpha is demonstrated by coimmunoprecipitation assays, and the transfection of PoPKR-specific short interfering RNA further reveals that the enhanced eIF2 alpha phosphorylation is catalyzed by PoPKR during SMRV infection. The current data provide significant evidence for the existence of a PKR-mediated antiviral pathway in fish and reveal considerable conservation in the functional domains and the antiviral effect of PKR proteins between fish and mammals.

Molecular cloning, characterization and expression analysis of the PKZ gene in rare minnow Gobiocypris rarus

Double-stranded RNA-activated protein kinase (PKR) plays an important rote in interferon-induced antiviral responses, and is also involved in intracellular signaling pathways, including the apoptosis, proliferation, and transcription pathways. In the present study, a PKR-like gene was cloned and characterized from rare minnow Gobiocypris rarus. The full length of the rare minnow PKR-like (GrPKZ) cDNA is 1946 bp in Length and encodes a polypeptide of 503 amino acids with an estimated molecular mass of 57,355 Da and a predicted isoelectric point of 5.83. Analysis of the deduced amino acid sequence indicated that the mature peptide contains two Zalpha domains and one S_TKc domain, and is most similar to the crucian carp (Carassius auratus) PKR-like amino acid sequence with an identity of 77%. Quantitative RT-PCR analysis showed that GrPKZ mRNA expression is at low levels in gill, heart, intestine, kidney, liver, muscle and spleen tissues in healthy animals and up-regulated by viruses and bacteria. After being infected by grass carp reovirus, GrPKZ expression was up-regulated from 24 h post-injection and lasted until the fish became moribund (P < 0.05). Following infection with Aeromonas hydrophila, GrPKZ transcripts were induced at 24 h post-injection (P < 0.05) and returned to control levels at 120 h post-injection. These data imply that GrPKZ is involved in antiviral defense and Toll-like receptor 4 signaling pathway in bacterial infection. (C) 2008 Elsevier Ltd. All rights reserved.

Variation patterns of the mitochondrial 16S rRNA gene with secondary structure constraints and their application to phylogeny of cyprinine fishes (Teleostei : Cypriniformes)

The mitochondrial 16S ribosomal RNA (rRNA) gene sequences from 93 cyprinid fishes were examined to reconstruct the phylogenetic relationships within the diverse and economically important subfamily Cyprininae. Within the subfamily a biased nucleotide composition (A > T, C > G) was observed in the loop regions of the gene, and in stem regions apparent selective pressures of base pairing showed a bias in favor of G over C and T over A. The bias may be associated with transition-transversion bias. Rates of nucleotide substitution were lower in stems than in loops. Analysis of compensatory substitutions across these taxa demonstrates 68% covariation in the gene and a logical weighting factor to account for dependence in mutations for phylogenetic inference should be 0.66. Comparisons of varied stem-loop weighting schemes indicate that the down-weightings for stem regions could improve the phylogenetic analysis and the degree of non-independence of stem substitutions was not as important as expected. Bayesian inference under four models of nucleotide substitution indicated that likelihood-based phylogenetic analyses were more effective in improving the phylogenetic performance than was weighted parsimony analysis. In Bayesian analyses, the resolution of phylogenies under the 16-state models for paired regions, incorporating GTR + G + I models for unpaired regions was better than those under other models. The subfamily Cyprininae was resolved as a monophyletic group, as well as tribe Labein and several genera. However, the monophyly of the currently recognized tribes, such as Schizothoracin, Barbin, Cyprinion + Onychostoma lineages, and some genera was rejected. Furthermore, comparisons of the parsimony and Bayesian analyses and results of variable length bootstrap analysis indicates that the mitochondrial 16S rRNA gene should contain important character variation to recover well-supported phylogeny of cyprinid taxa whose divergences occurred within the recent 8 MY, but could not provide resolution power for deep phylogenies spanning 10-19 MYA. (c) 2008 Published by Elsevier Inc.

Inhibition of no tail (ntl) gene expression in zebrafish by external guide sequence (EGS) technique

External guide sequence (EGS) technique, a branch of ribozyme strategy, can be enticed to cleave the target mRNA by forming a tRNA-like structure. In the present study, no tail gene (ntl), a key gene participating in the formation of normal tail, was used as a target for ribonuclease (RNase) P-mediated gene disruption in zebrafish in vivo. Transient expression of pH1-m3/4 ntl-EGS or pH1-3/4 ntl-EGS produced the full no tail phenotype at long-pec stage in proportion as 24 or 35%, respectively. As is expected that the full-length ntl mRNA of embryos at 50% epiboly stage decreased relative to control when injected the embryos with 3/4 EGS or m3/4 EGS RNA. Interestingly, ntl RNA transcripts, including the cleaved by EGS and the untouched, increased. Taken together, these results indicate that EGS strategy can work in zebrafish in vivo and becomes a potential tool for degradation of targeted mRNAs.

The cytomegalovirus promoter-driven short hairpin RNA constructs mediate effective RNA interference in zebrafish in vivo

The ability to utilize the RNA interference (RNAi) machinery for silencing target-gene expression has created a lot of excitement in the research community. In the present study, we used a cytomegalovirus (CMV) promoter-driven DNA template approach to induce short hairpin RNA (shRNA) triggered RNAi to block exogenous Enhanced Green Fluorescent Protein (EGFP) and endogenous No Tail (NTL) gene expressions. We constructed three plasmids, pCMV-EGFP-CMV-shGFP-SV40, pCMV-EGFP-CMV-shNTL-SV40, and pCMV-EGFP-CMV-shScrambled-SV40, each containing a CMV promoter driving an EGFP reporter cDNA and DNA coding for one shRNA under the control of another CMV promoter. The three shRNA-generating plasmids and pCMV-EGFP control plasmid were introduced into zebrafish embryos by microinjection. Samples were collected at 48 h after injection. Results were evaluated by phenotype observation and real-time fluorescent quantitative reverse-transcription polymerase chain reaction (Q-PCR). The shGFP-generating plasmid significantly inhibited the EGFP expression viewed under fluorescent microscope and reduced by 70.05 +/- 1.26% of exogenous EGFP gene mRNA levels compared with controls by Q-PCR. The shRNA targeting endogenous NTL gene resulted in obvious NTL phenotype of 30 +/- 4% and decreased the level of their corresponding mRNAs up to 54.52 +/- 2.05% compared with nontargeting control shRNA. These data proved the feasibility of the CMV promoter-driven shRNA expression technique to be used to inhibit exogenous and endogenous gene expressions in zebrafish in vivo.

Knock down of gfp and no tail expression in zebrafish embryo by in vivo-transcribed short hairpin RNA with T7 plasmid system

A short-hairpin RNA (shRNA) expression system, based on T7 RNA polymerase (T7RP) directed transcription machinery, has been developed and used to generate a knock down effect in zebrafish embryos by targeting green fluorescent protein (gfp) and no tail (ntl) mRNA. The vector pCMVT7R harboring T7RP driven by CMV promoter was introduced into zebrafish embryos and the germline transmitted transgenic individuals were screened out for subsequent RNAi application. The shRNA transcription vectors pT7shRNA were constructed and validated by in vivo transcription assay. When pT7shGFP vector was injected into the transgenic embryos stably expressing T7RP, gfp relative expression level showed a decrease of 68% by analysis of fluorescence real time RT-PCR. As a control, injection of chemical synthesized siRNA resulted in expression level of 40% lower than the control when the injection dose was as high as 2 mu g/mu l. More importantly, injection of pT7shNTL vector in zebrafish embryos expressing T7RP led to partial absence of endogenous ntl transcripts in 30% of the injected embryos when detected by whole mount in situ hybridization. Herein, the T7 transcription system could be used to drive the expression of shRNA in zebrafish embryos and result in gene knock down effect, suggesting a potential role for its application in RNAi studies in zebrafish embryos.

Identification of a gene, ccr-1 (sll1242), required for chill-light tolerance and growth at 15 degrees C in Synechocystis sp PCC 6803

Synechocystis sp. PCC 6803 exposed to chill (5 degrees C)-light (100 mu mol photons m(-2) s(-1)) stress loses its ability to reinitiate growth. From a random insertion mutant library of Synechocystis sp. PCC 6803, a sll1242 mutant showing increased sensitivity to chill plus light was isolated. Mutant reconstruction and complementation with the wild-type gene confirmed the role of sll1242 in maintaining chill-light tolerance. At 15 degrees C, the autotrophic and mixotrophic growth of the mutant were both inhibited, paralleled by decreased photosynthetic activity. The expression of sll1242 was upregulated in Synechocystis sp. PCC 6803 after transfer from 30 to 15 degrees C at a photosynthetic photon flux density of 30 mu mol photons m(-2) S-1. sll1242, named ccr (cyanobacterial cold resistance gene)- 1, may be required for cold acclimation of cyanobacteria in light.

Mutations stabilize small subunit ribosomal RNA in desiccation-tolerant cyanobacteria Nostoc

The ribosomal RNA molecule is an ideal model for evaluating the stability of a gene product under desiccation stress. We isolated 8 Nostoc strains that had the capacity to withstand desiccation in habitats and sequenced their 16S rRNA genes. The stabilities of 16S rRNAs secondary structures, indicated by free energy change of folding, were compared among Nostoc and other related species. The results suggested that 163 rRNA secondary structures of the desiccation-tolerant Nostoc strains were more stable than that of planktonic Nostocaceae species. The stabilizing mutations were divided into two categories: (1) those causing GC to replace other types of base pairs in stems and (2) those causing extension of stems. By mapping stabilizing mutations onto the Nostoc phylogenetic tree based on 16S rRNA gene, it was shown that most of stabilizing mutations had evolved during adaptive radiation among Nostoc spp. The evolution of 16S rRNA along the Nostoc lineage is suggested to be selectively advantageous under desiccation stress.