Study on HSP70 Gene Expression in Different Tissue of Cyprinus carpio

[ Objective] The aim of this study was to investigate whether HSP70 can be used as a stress monitoring indicator in Cypnnus carpio breeding. [Method] Based on HSP70 sequence of Cyprinus carpio （AY120894）, one pair of primers was designed and synthesized, while the total RNA of liver tissues in Cyprinus carpio was extracted. Some cDNA fragments of Cyprinus carpio HSP70 were cloned by RT-PCR, and its differential expression in various tissues such as heart, intestine, mucus, gonad, swim bladder, gill and fin in Cyprinus carpio was also studied. [Result] The cDNA sequence of 480 bp was obtained from Cypdnus carpio HSPTO gene by RT-PCR amplification. Homology comparison between the deduced amino acid sequence after sequencing and that of other types of fish showed that the homology among Cyprinus carpio, Danio rerio, Ohcorhynehus mylciss, Paralichthys olivaceus, Xiphophoorus maculates and Carassius auratus was 96%, 98%, 98%, 96%, 98% and 96% respectively. The expression of HSP70 was detected in eight tissues of Cypnnus carpio. The expression was the highest in heart, followed by swim bladder and fin, but there was no significant difference between them （ P 〉 0.05 ）. There was no significant difference among the ex- pression in three tissues of intestine, mucus and fat （ P〉0.05）, but their expression was significantly higher than those in gonad and gill （ P〈 0.05）. [ Conclusion] HSPTO gene expression is a suitable criterion for monitoring the stress degree, stress capacity and healthy conditions in Cyprinus carpio breeding.

Comparison of Newly Synthetic Hexaploid Wheat with Its Donors on SSR Products

Microsatellites or SSRs as powerful genetic markers have widely been used in genetics and evolutionary biology in common wheat. Because of the high polymorphism, newly synthesized hexaploid wheat has been used in the construction of genetic segregation population for SSR markers, However, data on the evolution of microsatellites during the polyploidization event of hexaploid wheat are limited. In this study, 66 pairs of specific to A/B genome SSR patterns among newly synthesized hexaploid wheat, the donor tetraploid wheat and Aegilops tauschii were compared. The results indicated that most SSR markers were conserved during the polyploidization events of newly synthetic hexaploid wheat, from Triticum turgidum and Ae. tauschii. Over 70% A/B genome specific SSR markers could amplify the SSR sequences from the D genome ofAe. tauschii. Most amplified fragments from Ae, tauschii were detected in synthetic hexaploid at corresponding positions with the same sizes and patterns as in its parental Ae. tauschii. This suggested that these SSR markers, specific for A/B genome in common wheat, could amplify SSR products of D genome besides A/B genome in the newly synthesized hexaploid wheat, that is, these SSR primers specific for A/B genome in common wheat were nonspecific for the A/B genome in the synthetic hexaploid wheat. In addition, one amplified Ae. tauschii product was not detected in the newly synthetic hexaploid wheat. An extra-amplified product was found in the newly synthetic hexaploid wheat. These results suggested that caution should be taken when using SSR marker to genotype newly synthetic hexaploid wheat.

Isolation of a Genomic DNA for Gastrodia Antifungal Protein and Analyses of Its Promoter in Transgenic Tobacco

A genomic DNA containing 5'-upstream region and complete open reading frame of a Gastrodia antifungal protein was isolated by screening of a genomic library from Gastrodia elata B1. To investigate the promoter activity, the 5'-flanking region - 1 157 lip upstream from the putative transcription start site was fused to the coding sequence of beta-glucuronidase (GUS) gene and transformed into Nicotiana tabacum. The strongest GUS activity was detected in the roots of transgenic tobacco, followed by stems. The leaves only showed a low GUS activity. Furthermore, the promoter established inducible expression pattern in transgenic tobacco upon fungus Trichoderma viride inoculation and jasmonic acid and salicylic acid treatments.

Cytological and Molecular Identification of Alien Chromatin in Giant Spike Wheat Germplasm

Alien chromosomes of twelve giant spike wheat germplasm lines were identified by C-banding, genomic in situ hybridization (GISH), sequence characterized amplified region (SCAR), and random amplified polymorphic DNA (RAPD). All lines showed a chromosome number of 2n = 42, five of them carried both a pair of wheat-rye (Triticum aestivum-Secale cereal) 1BL/1RS translocation chromosomes and a pair of Agropyron intermedium (Ai) chromosomes, three carried a pair of Ai chromosomes only, three others carried a pair of 1BL/1RS chromosomes only, and one carried neither 1BL/1BS nor Ai chromosome. Further identification revealed that the identical Ai chromosome in these germplasm lines substituted the chromosome 2D of common wheat (T aestivum L.), designated as 2Ai. The genetic implication and further utilization of 2Ai in wheat improvement were also discussed.

Sequence and organization of the mitochondrial genome of an urostylidid bug,Urochela quadrinotata Reuter(Hemiptera:Urostylididae)

The complete mitochondrial genome of Urochela quadrinotata Reuter was sequenced and analyzed. This represents the first sequence completed for an insect in the family Urostylididae. The genome is 16585 bp in size and contains 35 genes （including 13 protein-coding genes, 20 transfer RNA genes, two ribosomal RNAs and a control region） with an A＋T content of 75.4%. Two tRNA genes, tRNATle and tRNAGln, are not detected in this genome. The gene order is similar to that most commonly found in insects. All tRNAs possess the typical clover-leaf structure, except tRNASer（AGN）, in which the dihydrouridine （DHU） arm forms a simple loop. The secondary structure of rrnL consists of six structural domains and 43 helices, and the rrnS includes three structural domains and 27 helices. An obvious feature of the control region is the presence of 1652 bp long tandem repeat sequences comprising 16 tandem repeat units.

Associations between interleukin-1 polymorphisms and gastric cancers among three ethnicities

AIM:To investigate the associations between interleukin(IL)-1B and IL-1RN polymorphisms and gastric cancers among the Tibet,Hui and Han ethnicities.METHODS:Genomic DNA was extracted from peripheral blood of 210,205,and 202 healthy volunteers and from 155,158,and 197 gastric cancer patients from the Tibet,Hui,and Han populations,respectively.Polymorphisms in IL-1B and IL-1RN were analyzed by denaturing high-performance liquid chromatography.RESULTS:Carriers of the IL-1B-31 CC genotype had an increased risk of intestinal type gastric cancer [odds ratio(OR) = 2.17,P = 0.037] in the Tibet ethnicity.Carriers of the IL-1B 2/L genotype had an increased risk of both intestinal and diffuse types of gastric cancer(OR = 2.08,2.31,P = 0.007,0.016,respectively) in the Hui ethnicity.In the Han population,carriers of the IL-1B-31 CC,IL-1B-511CT,TT genotypes had increased risk of intestinal type gastric cancer(OR = 2.51,2.74,5.66,P = 0.005,0.002,0.000,respectively).CONCLUSION:IL-1B and IL-RN genotypes may differentially contribute to gastric cancer among the Tibet,Hui,and Han ethnicities in the Qinghai area of China.

Comparative genome analysis on intraspecific evolution and nitrogen fixation of Leptospirillum ferriphilum isolates

To reveal the intraspecific evolution of Leptospirillum ferriphilum isolates which thrived in industrial bioleaching ecosystems and acid mine drainages,genome sequences of L.ferriphilum YSK,L.ferriphilum DX and L.ferriphilum ZJ were determined to compare with complete genome of L.ferriphilum ML-04.The genome comparisons reveal that extensive intraspecific variation occurs in their genomes,and that the loss and insertion of novel gene blocks of probable phage origin may mostly contribute to heterogeneity of gene content among L.ferriphilum genomes.Surprisingly,a nif gene cluster is identified in L.ferriphilum YSK and L.ferriphilum ZJ genomes.Intensive analysis and further experiments indicate that the nif gene cluster in L.ferriphilum YSK inherits from ancestor rather than lateral gene transfer.Overall,results suggest that the population of L.ferriphilum undergoes frequent genetic recombination,resulting in many closely related genome types in recent evolution.The combinatorial processes profoundly shape their physiologies and provide the basis for adaptation to different niches.

Understanding biological functions through molecular networks

The completion of genome sequences and subsequent high-throughput mapping of molecular networks have allowed us to study biology from the network perspective. Experimental, statistical and mathematical modeling approaches have been employed to study the structure, function and dynamics of molecular networks, and begin to reveal important links of various network properties to the functions of the biological systems. In agreement with these functional links, evolutionary selection of a network is apparently based on the function, rather than directly on the structure of the network. Dynamic modularity is one of the prominent features of molecular networks. Taking advantage of such a feature may simplify network-based biological studies through construction of process-specific modular networks and provide functional and mechanistic insights linking genotypic variations to complex traits or diseases, which is likely to be a key approach in the next wave of understanding complex human diseases. With the development of ready-to-use network analysis and modeling tools the networks approaches will be infused into everyday biological research in the near future.

Mesenchymal stem cells derived from human placenta suppress allogeneic umbilical cord blood lymphocyte proliferation

Human placenta-derived mononuclear cells （MNC） were isolated by a Percoll density gradient and cultured in mesenchymal stem cell （MSC） maintenance medium. The homogenous layer of adherent cells exhibited a typical fibroblastlike morphology, a large expansive potential, and cell cycle characteristics including a subset of quiescent cells. In vitro differentiation assays showed the tripotential differentiation capacity of these cells toward adipogenic, osteogenic and chondrogenic lineages. Flow cytometry analyses and immunocytochemistry stain showed that placental MSC was a homogeneous cell population devoid of hematopoietic cells, which uniformly expressed CD29, CD44, CD73, CD105, CD166, laminin, fibronectin and vimentin while being negative for expression of CD31, CD34, CD45 and m-smooth muscle actin. Most importantly, immuno-phenotypic analyses demonstrated that these cells expressed class Ⅰ major histocompatibility complex （MHC-I）, but they did not express MHC-Ⅱ molecules. Additionally these cells could suppress umbilical cord blood （UCB） lymphocytes proliferation induced by cellular or nonspecific mitogenic stimuli. This strongly implies that they may have potential application in allograft transplantation. Since placenta and UCB are homogeneous, the MSC derived from human placenta can be transplanted combined with hematopoietic stem cells （HSC） from UCB to reduce the potential graft-versus-host disease （GVHD） in recipients.

Transcription analysis of peloric mutants of Phalaenopsis orchids derived from tissue culture

Tissue culture has been widely used for mass propagation of Phalaenopsis. However, somaclonal variation occurred during micropropagation process posed a severe problem by affecting product quality. In this study, wild type and peloric flower buds of Phalaenopsis hybrids derived from flower stalk nodal culture were used for cDNA-RAPD and cDNA suppression subtractive hybridization analyses in order to study their genetic difference in terms of expressed sequence tags. A total of 209 ESTs from normal flower buds and 230 from mutants were sequenced. These ESTs sequences can be grouped into several functional categories involved in different cellular processes including metabolism, signal transduction, transcription, cell growth and division, protein synthesis, and protein localization, and into a subcat- egory of proteins with unknown function. Cymbidium mosaic virus transcript was surprisingly found expressed fre- quently in the peloric mutant of P. Little Mary. Real-time RT-PCR analysis on selected ESTs showed that in mutant flower buds, a bZIP transcription factor (TGA1a-like protein) was down-regulated, while up-regulated genes include auxin-regulated protein kinase, cyclophilin, and TCP-like genes. A retroelement clone was also preferentially expressed in the peloric mutant flowers. On the other hand, ESTs involved in DNA methylation, chromatin remodeling and post- transcriptional regulation, such as DNA methyltransferase, histone acetyltransferase, ERECTA, and DEAD/DEAH RNA helicase, were enriched in normal flower buds than the mutants. The enriched transcripts in the wild type indicate the down regulation of these transcripts in the mutants, and vice versa. The potential roles of the analyzed transcripts in the development of Phalaenopsis flowers are discussed.

The molecular characterization of maize B chromosome specific AFLPs

The origin and evolution of B chromosomes could be explained by the specific DNA sequence on them. But the specific sequences known were quite limited. To investigate maize B chromosome sqicific DNA sequeces, maize genomes with and without B chromosomes were analyzed by AFLP. Only 5 markers were found specific to genomes with B chromosomes among about 2000 AFLP markers. Southern hybridization and sequence analysis revealed that only the sequence of M8-2D was a B chromosome specific sequence. This sequence contained the telomeric repeat unit AGGGTTT conserved in plant chromosome telomeres. In addition, the sequence of M8-2D shared low homology to clones from maize chromosome 4 centromere as well. M8-2D were localized to B chromosome centrorneric and telomeric regions.

Significantly reduced function of T cells in patients with acute arterial thrombosis

Objectives To explore the intrinsic factors related to the pathogenesis of acute arterial thrombosis （AAT） and to elucidate the patho- genesis of AAT on the basis of differentially expressed genes. Methods Patients with acute myocardial infarction （AMI）, stable angina （SA） and healthy controls （n = 20 per group） were recruited, and the whole human genome microarray analysis was performed to detect the differentially expressed genes among these subjects. Results Patients with AMI had disease-specific gene expression pattern. Biological functional analysis showed the function of T cells was significantly reduced, the mitochondrial metabolism significantly decreased, the ion metabolism was abnormal, the cell apoptosis and inflammatory reaction increased, the phagocytosis elevated, the neutrophil-mediated immunity increased and the post-traumatic repair of cells and tissues increased in AMI patients. The biological function in SA group and healthy controis remained stable and was comparable. Conclusions The reduced function ofT cell gene models in AAT showed the dysfunction of the immune system. The pathogenesis of AAT may be related to the inflammatory reaction after arterial intima infection caused by potential pathogenic microorganisms.

Joint Resource Allocation Using Evolutionary Algorithms in Heterogeneous Mobile Cloud Computing Networks

The problem of joint radio and cloud resources allocation is studied for heterogeneous mobile cloud computing networks. The objective of the proposed joint resource allocation schemes is to maximize the total utility of users as well as satisfy the required quality of service(QoS) such as the end-to-end response latency experienced by each user. We formulate the problem of joint resource allocation as a combinatorial optimization problem. Three evolutionary approaches are considered to solve the problem: genetic algorithm(GA), ant colony optimization with genetic algorithm(ACO-GA), and quantum genetic algorithm(QGA). To decrease the time complexity, we propose a mapping process between the resource allocation matrix and the chromosome of GA, ACO-GA, and QGA, search the available radio and cloud resource pairs based on the resource availability matrixes for ACOGA, and encode the difference value between the allocated resources and the minimum resource requirement for QGA. Extensive simulation results show that our proposed methods greatly outperform the existing algorithms in terms of running time, the accuracy of final results, the total utility, resource utilization and the end-to-end response latency guaranteeing.

Isolation of a pollen-specific promoter in tritordeum

The promoter is a cis-acting element in regulating gene expression. A promoterless plasmid containing UidA gene was transformed into tritordeum by barmbadment. Histochemical analysis of various tissues in transgenic tritordeum was carried to examine tissue-specific expression of GUS(beta-glucuronidase) activity. The pollen-specific promoter was trapped and identified successfully in a transformant line. PCR(polymerase chain reaction) method was used to isolate this pollen-specific promoter. By sequencing and analyzing the amplified fragment from PCR, a part of UidA gene and a flanking sequence were obtained. Some essential elements of plant promoters were found in the sequence. To determine the function of it, the cloned fragment was fused with UidA gene, then cloned and transformed into Triticum durum. The transgenic plant transformed by this vector showed GUS expression only in pollen. Therefore a pollen-specific promoter was isolated successfully.

MicroRNAs and cancer

MicroRNAs (miRNAs) belong to a class of noncoding, regulatory RNAs that are involved in oncogenesis and show remarkable tissue specificity.miRNAs are approximately 22 nt non-coding RNAs, which regulate gene expression in a sequence-specific manner via translational inhibition or messenger RNA (mRNA) degradation, thus affecting various cellular processes.Since the discovery of their fundamental mechanisms of action, the field of miRNAs has opened a new era in the understanding of small noncoding RNAs.Recent evidence has shown that miRNA controls cell growth, apoptosis, and differentiation.Cancer is a complex genetic disease caused by abnormalities in gene structure and expression, moreover, miRNA expression correlates with cancers and could have a crucial function in tumor progression.Bioinformatic data indicate that each miRNA can control hundreds of target genes, but identification of the accurate miRNA targets will be crucial to exploit the emerging knowledge of miRNA contribution to cancer process.

A real-time PCR targeted to the upstream regions of HlyB for specific detection of Edwardsiella tarda

Edwardsiella tarda has become one of the most important emerging pathogens in aquaculture industry. Therefore, a rapid, reproducible, and sensitive method for detection and quantification of this pathogen is needed urgently. To achieve this purpose, we developed a TaqMan-based real-time PCR assay for detection and quantification orE. tarda. The assay targets the hemolysin activator HlyB domain protein of E. tarda. Our optimized TaqMan assay is capable of detecting as little as 40 fg of genomic DNA per reaction. A standard curve was generated from the threshold cycle values （y） against log10 （E. tarda genomic DNA concentration） as x. The intra- and inter-assay coefficient of variation （CV） values were less than 2.06% and 1.05% respectively, indicating that the assay had good reproducibility. This method is highly specific to E. tarda strains, as it shows no cross-reactivity to Edwardsiella ictaluri, a member of the same genus, or to nine other fish-pathogenic bacteria species belonging to three other genera. This sensitive and specific real-time PCR assay provides a valuable tool for diagnostic quantitation of E. tarda in clinical samples.

Development of a Real-Time PCR Method (Taqman) for Rapid Identification and Quantification of Prorocentrum donghaiense

Prorocentrum donghaiense is a dinoflagellate that is widely distributed in the East China Sea and has become increasingly involved in Harmful Algal Blooms (HABs). Therefore, it is necessary to study this dinoflagellate to monitor HABs. In this study, 13 pairs of primers specific to P. donghaiense (within its internal transcribed spacer (ITS) regions) were designed for SYBR Green I real-time PCR. As the SYBR Green I real-time PCR could not identify P. donghaiense in a specific manner, a Taqman real-time PCR method was developed by designing a set of specific primers and a Taqman probe. A 10-fold serial dilution of recombinant plasmid containing ITS regions of P. donghaiense was prepared as standard samples and the standard curve was established. Additionally, we quantified the genomic DNA in P. donghaiense cells and utilized this DNA to prepare another 10-fold serial dilution of standard sample and accordingly set up the standard curve. The mathematic correlation between the cell number and its corresponding plasmid copy number was also established. In order to test the efficiency of the real-time PCR method, laboratory samples and P. donghaiense HAB field samples were employed for identification and quantitative analysis. As to laboratory samples, as few as 102 cells of P. donghaiense could be quantified precisely utilizing both centrifugation and filtration techniques. The quantification results from field samples by real-time PCR were highly similar to those by light microscopy. In conclusion, the real-time PCR could be applied to identify and quantify P. donghaiense in HABs.

Affinity chromatography-dependent selection (ACDS)of genomic DNA fragments bound specifically to bacterial synthesized Myc/Myn proteins

This paper describes an approach to seek for mouse c-Myc/Myn proteins-bound specific sequences among ge-nomic DNA. cDNA fragment of myn gene was obtained through RT-PCR technique from RNA of NIH3T3 cells. DNA fragments encoding BR/HLH/LZ structure of Myc and Myn proteins were cloned in frame into pGEX-2T vec-tor respectively Fusion GST-Myc and GST-Myn synthe-sized in E.coli hosts showed affinity to CACGTG E-boxDNA and subsequently interacted with genomic fragments prepared through whole-genome-PCR. A PCR-assisted procedure which combines protein-DNA interaction and affinity chromatography was designed to enrich Myc/Myn bound DNA. At least two genomic DNA fragments ob- tained exhibit specifical binding capacity to Myc/Myn complex but not to GST alone. Significance of the work and of the technique itself as well as identification of the DNAs are discussed.

Variation of DRB1 Gene in Tibetan Sheep

It reveals that the MHC （major histocompatibility complex） gene product always involved in the control of immune response and disease resistance. Nowadays many studies have indicated the OLA （ovine lymphocyte antigen） DRB1 gene is associated with some sheep diseases. Tibetan sheep is one of the three major shag sheep breeds in China, and also have the largest number of China＇s sheep breeds. But till now no report has been seen on studying DRB1 gene in Tibetan sheep of China. To understand the evolution and provide the basis for sheep disease resistance, polymorphism in the exon2 ofDRB1 gene in Tibetan sheep was analyzed. The PCR-SSCP, cloning and sequencing were used to analyse DRB1 gene variation in 600 Tibetan sheep of China. And the genetic relationship and evolutionary significance of the alleles had also been analyzed. Total of 31 alleles were identified, in which 15 alleles had not been reported before. And there were 70 SNPs （single nucleotide polymorphisms） sites in 31 sheep DRB1 gene haplotypes, the proportion was 29.5% to the whole exort2 sequence. All of this indicated that DRB1 exon2 is highly polymorphic in Tibetan sheep. The variation identified here might have an impact on both the function and level of expression of the OLA-DRB1.

Genomic and proteomic analysis of soybean heritable variations induced by space flight

To analyze the biological effects of space environment,the diversity of genomic DNA between the space flight soybean 194(4126) with phenotype of good yield and good fruit quality induced by space flight and the soybean with ground control was studied by amplified fragment length polymorphism(AFLP) method,and the polymorphism of space flight soybean 194(4126) was 3.56%.The differences of protein expression of seeds and leaves between the two kinds of soybeans were analysed by two-dimensional electrophoresis,PDQuest software and MALDI-TOF mass spectrometry.Results show that the loss and decrease of protein expression in 194(4126) soybean are subjected to the space fight of seeds,and three special proteins including Dehydrin,MAT1 and ceQORH are identified.It is concluded that the space environment changes the phenotype and genotype of soybeans due to the space flight of seeds.