云南保种山羊线粒体DNA限制性酶切研究

　采用碱变性法,提取来自云南省不同地区4个保种山羊的13个个体的线粒体DNA(mtDNA),并用ApaⅠ,AvaⅠ,BamHⅠ,BclⅠ,BcIⅠ，BglⅡ,ClaⅠ,DraⅠ, EcoRⅠ,EcoRⅤ,HaeⅠ,HindⅢ,KpnⅠ,PstⅠ,PvuⅡ,SacⅠ,SalⅠ,SmaⅠ,StuⅠ和XhoⅠ等20种限制性内切酶进行酶切分析。结果发现它们的线粒体DNA的分子量大 小约为15.8Kb；不同限制性内切酶的酶切位点分别为：DraⅠ有7个酶切位点，AvaⅢ有6个酶切位点，EcoRⅤ和StuⅠ共有5个酶切位点，HindⅡ和HaeⅡ有4个酶 切位点，BamHⅠ,BglⅡ,PstⅠ和PvuⅡ有3个酶切位点，ApaⅠ，ClaⅠ有两个酶切位点，其余有1个酶切位点。各保种山羊间未发现变异，说明云南的4个保种山 羊极可能来自于共同的母性祖先。

MITOCHONDRIAL-DNA POLYMORPHISM IN NATURAL-POPULATIONS OF DROSOPHILA-ALBOMICANS(I)

The technique of mtDNA restriction fragments length polymorphism (RFLP) was used to survey the population structure of D. albomicans. Remarkable mtDNA polymorphism has been observed in D. albomicans populations. A total of 34 nucleomorphs were detected from 82 isofemale lines assayed by only 8 restriction enzymes. The cause and the effect of this phenomenon were discussed. As a result, it is suggested that a mechanism which maintains mtDNA diversity exists in this fly, and that the high intra-populational polymorphism could numerically conceal the extent of differentiation between populations. In addition, on the base of restriction maps, it was found that the mtDNA molecule of D. albomicans might be impacted by the selection pressure during its evolution process both on the nucleotide composition and on the functional regions.

THE ORIGIN AND GENETIC DIFFERENTIATION OF NATIVE BREEDS OF PIGS IN SOUTHWEST CHINA - AN APPROACH FROM MITOCHONDRIAL-DNA POLYMORPHISM

Mitochondrial DNA (mtDNA) of six breeds of native domestic pigs from Yunnan province, southwest China, and two wild boars obtained from Sichuan, China, and Vietnam was analyzed using 20 restriction endonucleases that recognize six nucleotides. Restriction maps were made by double-digestion methods and polymorphic sites were located on the map. According to their mtDNA restriction types, all the breeds were classified into six groups. Genetic distances among groups were calculated to define their phylogenetic relationships. The relationship between the Sichuan wild boar and domestic pigs is close, while the Vietnamese wild boar is relatively far from them, so the domestic pigs in southwest China are likely to have originated from a wild pig which distributed in west China. We compare our results with previous reports in literature and discuss the relationship among Chinese pigs, Japanese pigs, and European pigs. The mtDNA cleavage pattern of the Mingguang pig digested by EcoRV was identical to that of Duroc; mutations at the EcoRI site, detected in the mtDNA of two Dahe pigs, are the same as in the Vietnamese wild boar, suggesting that mutational hot spots exist in the mtDNA of pigs.

Variation in mitochondrial DNA and phylogeny of six species of pikas (Ochotona)

Phylogenetic relationships of six species of Ochotona were investigated using mitochondrial DNA (mtDNA) restriction-site analysis. The phylogenetic tree constructed using PAUP was based on 62 phylogenetically informative sites with O. erythrotis designated as an outgroup. Two clades were evident. One contained O. curzoniae, O. huangensis, and O. thibetana. in the second, O. daurica was a sister taxon of O. cansus. The five species appear to come from different maternal lineages. The branching structure of the tree and sequence divergence confirm that huangensis and cansus are distinct species, and that mol-osa is a synonym of O. cansus rather than O. thibetana. Divergence time, estimated from genetic distances, indicates that the ancestors of the two maternal lineages diverged ca. 6.5 x 10(6) years ago. O. curzoniae diverged from O. huangensis, and O. daurica diverged from O. cansus, at about the same time (ca. 3.4 x 10(6) years ago). These data suggest a period of rapid radiation of the genus Ochotona in China, perhaps during the late Pliocene. These calculations correspond roughly to tectonic events and environmental changes in China throughout this period, and appear to be substantiated by the fossil record.

Diversity and abundance of ammonia-oxidizing bacteria in eutrophic and oligotrophic basins of a shallow Chinese lake (Lake Donghu)

Classical cultivation and molecular methods based on the ammonia monooxygenase gene (amoA) were used to study the abundance and diversity of beta-proteobacterial ammonia-oxidizing bacteria (AOB) in lake sediments. The eutrophic and oligotrophic basins of a Chinese shallow lake (Lake Donghu), in terms of ammonium (NH4+) concentrations, were sampled. The AOB number was significantly lower in the oligotrophic basin, but significantly higher in the eutrophic basin. In addition, using restriction fragment length polymorphism targeting the amoA, ten restriction patterns including six unique ones were found in the eutrophic basin, while five patterns were observed in the oligotrophic basin with only one unique restriction group. Phylogenetic analysis for AOB revealed that Nitrosomonas oligotropha- and Nitrosomonas ureae-related AOB and Nitrosospira-affiliated AOB were ubiquitous; the former dominated in the eutrophic basin (87.2%), while the latter dominated in the oligotrophic basin (65.5%). Furthermore, Nitrosomonas communis-related AOB was only detected in the eutrophic basin, at a small proportion (3.2%). These results indicate significant selection and adaptation of sediment AOB in lakes with differing trophic status. (C) 2009 Elsevier Masson SAS. All rights reserved.

Origin and evolution of the RIG-I like RNA helicase gene family

Background: The DExD/H domain containing RNA helicases such as retinoic acid-inducible gene I (RIG-I) and melanoma differentiation-associated gene 5 (MDA5) are key cytosolic pattern recognition receptors (PRRs) for detecting nucleotide pathogen associated molecular patterns (PAMPs) of invading viruses. The RIG-I and MDA5 proteins differentially recognise conserved PAMPs in double stranded or single stranded viral RNA molecules, leading to activation of the interferon system in vertebrates. They share three core protein domains including a RNA helicase domain near the C terminus (HELICc), one or more caspase activation and recruitment domains (CARDs) and an ATP dependent DExD/H domain. The RIG-I/MDA5 directed interferon response is negatively regulated by laboratory of genetics and physiology 2 (LGP2) and is believed to be controlled by the mitochondria antiviral signalling protein (MAVS), a CARD containing protein associated with mitochondria. Results: The DExD/H containing RNA helicases including RIG-I, MDA5 and LGP2 were analysed in silico in a wide spectrum of invertebrate and vertebrate genomes. The gene synteny of MDA5 and LGP2 is well conserved among vertebrates whilst conservation of the gene synteny of RIG-I is less apparent. Invertebrate homologues had a closer phylogenetic relationship with the vertebrate RIG-Is than the MDA5/LGP2 molecules, suggesting the RIG-I homologues may have emerged earlier in evolution, possibly prior to the appearance of vertebrates. Our data suggest that the RIG-I like helicases possibly originated from three distinct genes coding for the core domains including the HELICc, CARD and ATP dependent DExD/H domains through gene fusion and gene/domain duplication. Furthermore, presence of domains similar to a prokaryotic DNA restriction enzyme III domain (Res III), and a zinc finger domain of transcription factor (TF) IIS have been detected by bioinformatic analysis. Conclusion: The RIG-I/MDA5 viral surveillance system is conserved in vertebrates. The RIG-I like helicase family appears to have evolved from a common ancestor that originated from genes encoding different core functional domains. Diversification of core functional domains might be fundamental to their functional divergence in terms of recognition of different viral PAMPs.

First description of a novel Weissella species as an opportunistic pathogen for rainbow trout Oncorhynchus mykiss (Walbaum) in China

Six strains of Gram-positive, catalase-negative, non-motile, irregular short rod-shaped Weissella bacteria, with width and length of 0.5-0.6 and 1.2-2.7 mu m were isolated from diseased rainbow trout Oncorhynchus mykiss (Walbaum) in winter of 2007 at a commercial fishery in Jingmen, Hubei province, China. The diseased rainbow trout exhibited hemorrhage in eyes, anal region, intestine and abdomen wall, petechia of liver, some fish with hydrocele in stomach. Six isolates had identical biochemical reactions, phylogenetic analysis of 16S rDNA sequences, amplified ribosomal DNA restriction analysis (ARDRA), enzymatic profile analysis and antimicrobial susceptibility results, indicating as a single clonal outbreak. But all were different from any other validated twelve Weissella species in the term of physiological and biochemical characters. It is indicated that isolates are phylogenetically closer to Weissella halotolerans, Weissella viridescens and Weissella minor on 16S rDNA phylogenetic analysis result, than to W halotolerans and W viridescens on the result of ARDRA study and enzymatic profile analysis. Antimicrobial susceptibility testing was used to scan effective drugs for the therapy of this disease. Experimental infection assays with one isolate were conducted and pathogenicity (by intraperitoneal injection) was demonstrated in rainbow trout O. mykiss (Walbaum) and crucian carp (Carassius auratus gibelio) fingerlings. Because no Weissella was detected in fish feedstuffs and pond water, the source of this pathogen remains unknown, and Weissella isolates were regarded as an opportunistic pathogen for rainbow trout. This is the first report of Weissella strains which can cause disease of cultured fish in the world. (C) 2009 Elsevier B.V. All rights reserved.

Bacterial diversity in activated sludge from a consecutively aerated submerged membrane bioreactor treating domestic wastewater

The bacterial diversity of activated sludge from submerged membrane bioreactor (SMBR) was investigated. A 16S rDNA clone library was generated, and 150 clones were screened using restriction fragment length polymorphism (RFLP). Of the screened clones, almost full-length 16S rDNA sequences of 64 clones were sequenced. Phylogenetic tree was constructed with a database containing clone sequences from this study and bacterial rDNA sequences from NCB1 for identification purposes. The 90.6% of the clones were affiliated with the two phyla Bacteroidetes (50%) and Proteobacteria (40%), and beta-, -gamma-, and delta-Proteobacteria accounted for 7.8%, 28.1%, and 4.7%, respectively. Minor portions were affiliated with the Actinobacteria and Firmicutes (both 3.1%). Only 6 out of 64 16S rDNA sequences exhibited similarities of more than 97% to classified bacterial species, which indicated that a substantial fraction of the clone sequences were derived from unknown taxa. Rarefaction analysis of operational taxonomic units (orrUs) clusters demonstrated that 150 clones screened were still insufficient to describe the whole bacterial diversity. Measurement of water quality parameter demonstrated that performance of the SMBR maintained high level, and the SMBR system remained stable during this study.

Genetic structure and low-genetic diversity suggesting the necessity for conservation of the Chinese Longsnout catfish, Leiocassis longirostris (Pisces : Bagriidae)

Genetic variation and phylogenetic relationship of Leiocassis longirostris populations from the Yangtze River were investigated at mitochondrial DNA level. The samples were collected from the upstream and mid-downstream of the Yangtze River. Three mitochondrial DNA fragments, ND5/6, cytochrome b (Cyt b) and control region (D-loop), were amplified and then digested by 10 restriction endonucleases. Twenty-three D-loop fragments randomly selected were sequenced. Digestion patterns of ND5/6 by AluI and HaeIII, D-loop by HinfI and RsaI, and Cyt b by HaeIII were polymorphic. Ten and eighteen haplotypes were obtained from RFLP data and sequence data, respectively. The individuals from upstream and mid-downstream of the Yangtze River were apparently divided into two groups. The average genetic distance was 0.008 and 0.010 according to the two data. Low diversities and decreasing abundance indicated that Leiocassis longirostris may be in severe danger and reasonable measures of fishery management should be taken.

Molecular characterization of three Rana grylio virus (RGV) isolates and Paralichthys olivaceus lymphocystis disease virus (LCDV-C) in iridoviruses

Three Rana grylio virus (RGV) isolates and lymphocystis disease virus (LCDV-C) were molecularly characterized by antigenicity comparison, Western blot detection of viral polypeptides, restriction fragment length polymorphism analysis of viral genomes, and MCP sequence analysis. Significant antigenicity differences existed among the three RGV isolates and LCDV-C. Western blot detection indicated that the viral polypeptides of three RGV isolates could be recognized by the anti-RGV9807 serum, whereas no bands were observed in the LCDV-C, and significant differences exist among the band patterns of three RGV isolates. Restriction fragment length polymorphism (RFLP) analysis was performed by digesting genomic DNA of the four iridovirus isolates with restriction endonucleases HindIII, KpnI, XbaI and BamHI. On the whole, obvious discrepancies existed between LCDV-C and RGV isolates, and some significant band pattern differences were also revealed between RGV9808 and RGV9506 (or RGV9807) in the profiles of restriction endonucleases Xbal, Kpn I and BamHI. PCR amplification and sequence analysis of MCP gene sequence further revealed their phylogenetic relationship among the three RGV isolates, LCDV-C and other iridoviruses. RGV9506, RGV9807 and RGV9808 are clustered together with other ranaviruses, such as FV3, BIV, TFV and ENHV, although the RGV9808 is more close to EHNV than to other ranaviruses. Additionally, LCDV-C is clustered with LCDV-1, the type species of genus Lymphocystisvirus. The current study provides clear evidence that significant genetic difference exists among the three RGV isolates. Therefore, further work on comparative genomic studies will contribute significantly to understanding of their taxonomic position and pathological mechanism. (C) 2005 Elsevier B.V. All rights reserved.

Characterization of transgene integration pattern in F4 hGH-transgenic common carp (Cyprinus carpio L.)

The integration pattern and adjacent host sequences of the inserted pMThGH-transgene in the F4 hGH-transgenic common carp were extensively studied. Here we show that each F4 transgenic fish contained about 200 copies of the pMThGH-transgene and the transgenes were integrated into the host genome generally with concatemers in a head-to-tail arrangement at 4-5 insertion sites. By using a method of plasmid rescue, four hundred copies of transgenes from two individuals of F4 transgenic fish, A and B, were recovered and clarified into 6 classes. All classes of recovered transgenes contained either complete or partial pMThGH sequences. The class I, which comprised 83% and 84.5% respectively of the recovered transgene copies from fish A and B, had maintained the original configuration, indicating that most transgenes were faithfully inherited during the four generations of reproduction. The other five classes were different from the original configuration in both molecular weight and restriction map, indicating that a few transgenes had undergone mutation, rearrangement or deletion during integration and germline transmission. In the five types of aberrant transgenes, three flanking sequences of the host genome were analyzed. These sequences were common carp beta-actin gene, common carp DNA sequences homologous to mouse phosphoglycerate kinase-1 and human epidermal keratin 14, respectively.

Geographic distribution of Pelteobagrus fulvidraco and Pelteobagrus vachelli in the Yangtze River based on mitochondrial DNA markers

Three populations of Pelteobagrus vachelli and Pelteobagrus fulvidraco of the Yangtze River were examined by PCR-RFLP analysis of mitochondrial DNA fragments. ND5/6 and D-loop fragments were digested by 10 restriction endonucleases. Significant geographic variations between upstream and mid-downstream populations in the haplotype frequencies and restriction patterns were revealed. This suggested that the diversity of P. vachelli was high; 11 haplotypes were obtained from all the samples. The upstream population shared seven haplotypes and the middle and downstream populations shared another four haplotypes. Among all of the haplotypes, one haplotype was shared in 30 samples of the populations from middle and downstream, but it was not found in the upstream population. Any haplotype found in the upstream population was not detected in the middle and downstream populations. Genetic diversity of P. fulvidraco was low and only five haplotyes were detected from all 60 samples. Phylogenic relationships also indicated that the fishes from upstream and mid-downstream were apparently divided into two populations.

Broad host range plasmid-based gene transfer system in the cyanobacterium Gloeobacter violaceus which lacks thylakoids

Gloeobacter violaceus, a cyanobacterium lack of thylakoids, is refractory to genetic manipulations because its cells are enveloped by a thick gelatinous sheath and in colonial form. In this study, a large number of single cells were obtained by repeated pumping with a syringe with the gelatinous sheath removed. And an exogenous broad host range plasmid pKT210 was conjugatively transferred into G. violaceus. Analyses with dot-blot hybridization and restriction mapping showed that the exogenous plasmid pKT210 had been introduced into G. violaceus and stably maintained with no alteration in its structure. pKT210 extracted from G. violaceus exconjugants could be transformed into the mcr - mrr - E. coli strain DH10B but not the mcr(+) mrr(+) strain DH5alpha, which suggests that a methylase system may be present in G. violaceus.

Cloning of biologically active genomes from a Helicoverpa armigera single-nucleocapsid nucleopolyhedrovirus isolate by using a bacterial artificial chromosome

Purification of genotypes from baculovirus isolates provides understanding of the diversity of baculoviruses and may lead to the development of better pesticides. Here, we report the cloning of different genotypes from an isolate of Helicoverpa armigera single-nucleocapsid nucleopolyhedrovirus (HaSNPV) by using a bacterial artificial chromosome (BAC). A transfer vector (pHZB10) was constructed which contained an Escherichia coli mini-F replicon cassette within the upstream and downstream arms of HaSNPV polyhedrin gene. Hz2e5 cells were co-transfected with wild-type HaSNPV DNA and pHZB10 to generate recombinant viruses by homologous recombination. The DNA of budded viruses (BVs) was used to transform E. coli. One of the bacmid colonies, HaBacHZ8, has restriction enzyme digestion profiles similar to an in vivo cloned strain HaSNPV-G4, the genome of which has been completely sequenced. For testing the oral infectivity, the polyhedrin gene of HaSNPV was reintroduced into HaBacHZ8 to generate the recombinant bacmid HaBacDF6. The results of one-step growth curves, electron microscopic examination, protein expression analysis and bioassays indicated that HaBacDF6 replicated as well as HaSNPV-G4 in vitro and in vivo. The biologically functional HaSNPV bacmids obtained in this research will facilitate future studies on the function genomics and genetic modification of HaSNPV. (C) 2003 Elsevier B.V. All rights reserved.

Silver carp, Hypophthalmichthys molitrix, in the Poyang Lake belong to the Ganjiang River population rather than the Changjiang River population

The Poyang Lake is the largest lake and the main nursery area in the middle basin of the Changjiang (Yangtze) River. We compared molecular genetic markers of silver carp among populations of the Changjiang River, the Ganjiang River and the Poyang Lake using the ND5/6 region of mtDNA. Analysis of restriction fragment length polymorphisms (RFLPs) of this region revealed distinct variation between the Ganjiang River and the Changjiang River populations. The Poyang Lake is linked with the Ganjiang River and the Changjiang River. Shared RFLP fragments between the Ganjiang River population and the Poyang Lake population are as high as 61.4%. The value is 47.74% between the populations of the Changjiang River and that of the Poyang Lake. Frequencies of bands peculiar to the Ganjiang River population are the same as in the Poyang Lake population. We conclude that the Poyang Lake silver carp population consists mainly of the Ganjiang River population. The water level of the Poyang Lake outlet, which is higher than that of the Changjiang River in the silver carp spawning season, supports this conclusion.