Potyvirus属成员基因组全序列的简并引物PCR和RACE扩增方法

Based on multi-alignment of complete polyprotein amino acid sequences of genus Potyvirus,five degenerated primers were designedThey were Sprimer(5′-GGX AAY AAY AGY GGX CAZ CC-3′),pNIa(+)(5′-TNY TGG AAM CAY TGG AT-3′),pCI2(+)(5′-GCX ACX AAX ATX ATX GAX AA-3′),pCI1(+)(5′-GTX GGX TCX GGX AAX TCX AC-3′)and pHC(+)(5′-TGY GAY AAY CAZ TTX GA-3′)(X=A,T,C or G:Y=T or C;Z=A or G;N=A or T;M=A,T or G)Using degenerated PCR and modified RACE methods,a protocol for determination of complete genome sequence of potyviruses was established and proved to be successful on five

Complete Sequence and Gene Organization of the Mitochondrial Genome of Tokay (Gekko gecko)

Long-PCR amplification, clone and primer-walking sequencing methods were employed in determine the complete sequence of mitochondrial genome of tokay (Gekko gecko). The genome is 16 435 bp in size, contains 13 protein-coding, 2 ribosomal and 22 transfer RNA genes. The mt genome of Gekko is similar to most of the vertebrates in gene components, order, orientation, tRNA structures, low percentage of guanine and high percentage of thymine, and skews of base GC and AT. Base A was preferred at third codon positions for protein genes is similar to amphibians and fishes rather than amnion vertebrates. The standard stop codes (TAA) present only in three protein genes, less than those of most vertebrates. Transfer RNA genes range in length from 63 to 76 nt, their planar structure present characteristic clover leaf, except for tRNA-Cys and tRNA-Ser (AGY) because of lacking the D arm.

Complete Genome Sequencing and Genetic Variation Analysis of Two H9N2 Subtype Avian Influenza Virus Strains

[Objective] The study aimed to investigate the genetic variation characters of entire sequences between two H9N2 subtype avian influenza virus strains and other reference strains.[Method] The entire sequences of 8 genes were obtained by using RT-PCR,and these sequences were analyzed with that of six H9N2 subtype avian influenza isolates in homology comparison and genetic evolution relation.[Result] The results showed that the nucleotide sequence of entire gene of the strain shared 91.1%-95.4% homology with other seven reference strains,and PG08 shared the highest homology 91.3% with C/BJ/1/94;ZD06 shared the highest homology 92.3% with D/HK/Y280/97.HA cleavage sites of two H9N2 subtype avian influenza virus isolated strains were PARSSR/GLF,typical of mildly pathogenic avian influenza virus.[Conclusion] Phylogenetic tree for entire gene of eight strains showed that the genetic relationship was the closest between ZD06 and C/Pak/2/99 strains,which belonged to the Eurasian lineage;PG08 shared the highest homology 91.3% with ZD06,it may be the product of gene rearrangements of other sub-lines.

Molecular Characterization of a Chinese Soybean Mosaic Virus Isolate by RT_PCR, cDNA Sequence Analysis and Direct Expression of PCR Products in Bacteria

Soybean mosaic virus (SMV) causes one of the most severe viral diseases in soybean ( Glycine max L.) and is known to contain many pathogenically and serologically related isolates. In the present study, the authors have obtained cDNAs to all cistrons of a Chinese SMV isolate, SMV_ZK, by RT_PCR. By analysing the nucleotide and amino acid sequence of the HC_PRO, NIb and CP cistrons, it was found that SMV_ZK was highly homologous to the G2 strain of SMV, thus confirming the existence of G2_like isolates in soybean crop in China. The amplified cDNAs were directly cloned into a bacterial expression vector. With the exception of the P3 cistron, expression of the cDNAs of all other cistrons in bacteria gave rise to polypeptides of expected molecular weight. The expressed viral proteins were subsequently purified by gel elution. The preparation of viral_specific cDNAs and gene products will be useful in future functional study of the SMV genome.

Isolation and Characterization of Microsatellites in Snap Bean

The objectives of this study were to isolate and characterize microsatellites from a heat tolerant variety of snap bean (Phaseolus vulgaris L.) in order to generate polymorphic genetic markers linked to quantitative trait loci for heat tolerance. A genomic library contained 400-800 bp inserts was constructed and screened for the presence of (GA/CT) n and (CA/GT) n repeats. The proportion of positive clones yielded estimated of 3.72×10 4 such dinucleotide repeats per genome, roughly comparable to the abundance reported in other eukaryotic genomes. Twenty_six positive clones were sequenced. In contrast to mammalian genomes, the (GA/CT) n motif was much more abundant than the (CA/GT) n motif in these clones. The (GA/CT) n repeats also showed longer average repeat length (mean n =10.4 versus 6.5), suggesting that they are better candidates for yielding polymorphic genetic markers in the snap bean genome.

PCR Detection and Sequence Analysis of Duck Circovirus in Sick Muscovy Ducks

The duck circovirus （DuCV） infection in sick ducks from Fujian Province was investigated. The liver samples of 43 sick Muscovy ducks with infectious serositis were collected from 12 duck farms in Fujian Province Based on the published sequences of DuCV, two primers were designed for the detection of DuCV and four pairs of primers were designed to amplify four overlapping fragments that cover the complete genome of DuCV. The specific PCR products were amplified from positive samples. The fragments were then cloned into pMD18-T vector and sequenced, and the full length genomic sequence of the FJ0601 isolate of DuCV was obtained. PCR analysis showed that the proportion of ducks which were positive for circovirus was 79% and 10 out of the 12 farms were positive. Sequence analysis showed that the complete genome of DuCV-FJ0601 was 1988 bp and possessed features common to the family Circoviridae which included a stem-loop structure and the Rep protein motifs. Homology analysis showed that FJ0601 isolate of DuCV had 97.3%-97.5% nucleotide sequence identity to all the four Taiwan isolates （TC1/2002, TC2/2002, TC3/2002, TC4/2002）, 82.9% identity to the America （33753-52） isolate and 82.3% identity to the Germany isolate. Phylogenetic analysis with Clustal W, however, showed that FJ0601 isolate of DuCV was on a common branch with Taiwan isolates, and Germany and America isolates belonged to the other branch.

Molecular Characterization of Segments S7 to S10 of a Southern Rice Black-streaked Dwarf Virus Isolate from Maize in Northern China

Southern rice black-streaked dwarf virus (SRBSDV) is a novel Fijivirus prevalent in rice in southern and central China,and northern Vietnam. Its genome has 10 segments of double-stranded RNA named S1 to S10 according to their size. An isolate of SRBSDV,JNi4,was obtained from naturally infected maize plants from Ji'ning,Shandong province,in the 2008 maize season. Segments S7 to S10 of JNi4 share nucleotide identities of 72.6%-73.1%,72.3%-73%,73.9%-74.5% and 77.3%-79%,respectively,with corresponding segments of Rice black-streaked dwarf virus isolates,and identities of 99.7%,99.1%-99.7%,98.9%-99.5%,and 98.6%-99.2% with those of SRBSDV isolates HN and GD. JNi4 forms a separate branch with GD and HN in the phylogenetic trees constructed with genomic sequences of S7 to S10. These results confirm the proposed taxonomic status of SRBSDV as a distinct species of the genus Fijivirus and indicate that JNi4 is an isolate of SRBSDV. Shandong is so far the northernmost region where SRBSDV is found in China.

Cloning and Analysis of Calmodulin Gene from Porphyra yezoensis Ueda (Bangiales, Rhodophyta)

In order to understand the mechanisms of signal transduction and anti-desiccation mechanisms of Porphyra yezoensis, cDNA and its genomic sequence of Calmodulin gene (CaM) was cloned by the technique of polymerase chain reaction (PCR) based on the analysis of P. yezoensis ESTs from dbEST database. The result shows that the full-length cDNA of CaM consists of 603 bps including an ORF encoding for 151 amino acids and a terminate codon UGA, while the length of genomic sequence is 1231 bps including 2 exons and 1 intron. The average GC content of the coding region is 58.77%, while the GC content of the third position of this gene is as high as 82.23%. Four Ca2+ binding sites (EF-hand) are found in this gene. The predicted molecular mass of the deduced peptide is 16688.72 Da and the pI is 4.222. By aligning with known CaM genes, the similarity of CaM gene sequence with homologous genes in Chlamydomonas incerta and Chlamydomonas reinhardtii is 72.7% and 72.2% respectively, and the similarity of the deduced amino acid sequence of CaM gene with homologous genes in C. incerta and C. reinhardtii are both 71.5%. This is the first report on CaM from a species of Rhodophyta.

CLONING AND ANALYSIS OF THE GENOMIC DNA SEQUENCE OF AUGMENTER OF LIVERR EGENERATION FROM RAT

Objective.To search for genomic DNA sequence of the augmenter of liver regeneration(ALR)of rat.Methods.Polymerase chain reaction(PCR)with specific primers was used to amp lify the sequence from the rat genome.Results.A piece of genomic DNA sequence and a p iece of pseudogene of rat ALR were identified.The lengths of the gene and pseudogene are 1508bp and 442bp,respectively.The ALR gene of rat includes 3exons and 2introns.The 442bp DNA sequence may represent a pseudogene or a ALR-related peptide.Predicted amino acid sequence analysis showed that there were 14different amino acid residues between the gene and pseu do-gene.ALR-related peptide is 84amin o acid residues in length and relates closely to ALR protein.Conclusion.There might be a multigene family of A LR in rat.

Isolation and rapid genetic characterization of a novel T4-like bacteriophage

Ubiquitous on earth,bacteriophages are the most abundant entities in every ecosystem,but human knowledge about them is still limited compared with that about other forms of organisms.To enrich human knowledge and promote the utilization of bacteriophages,it is necessary to isolate and characterize as many as possible different bacteriophages.Here we describe the isolation of a T4-like bacteriophage IME08 and a rapid method for its genetic characterization.With this method we easily cloned a few random fragments of the bacteriophage genome.Sequence analysis of these random clones showed that bacteriophage IME08 shared the highest sequence similarity with T4-like Enterobacteria phage T4（94%identity）,JS98（95% identity）,JS10（95%identity） and RB14（94%identity） respectively,which suggested that IME08 belonged to T4-like bacteriophage genus.

Complete Genome Sequence Analysis of Duck Circovirus Strains from Cherry Valley Duck

To investigate molecular epidemiology of DuCV in Cherry Valley ducks in China, the complete genomes of six DuCV strains, which were detected from Cherry Valley ducks in China between 2007 and 2008, were sequenced. Sequence and phylogenetic analysis were carried out to compare these six strains with another 27 DuCV strains from Mulard duck, Muscovy duck, Pekin ducks and Mule duck. The analysis showed that the six DuCV strains exhibited typical genetic features of the family of DuCV, such as a stem-loop structure, three major open reading frames (Rep, Cap and ORF3), four intergenic repeats and the conserved motifs for rolling circle replication and for the dNTP binding domain located in the Rep protein. Phylogenetic analysis of the nucleotide sequences of the complete genome and Cap gene of these strains together with those that have been previously published demonstrated two distinct DuCV genotypes. The DuCV strains with complete genomes containing 1988 and 1989 nucleotides clustered in genotype A, whereas the strains with complete genomes containing 1991, 1992, 1995 and 1996 nucleotides lay in genotype B. The six DuCV strains from Cherry Valley ducks were divided into the two groups. The results of the study provides some insight into the variation of DuCVs in Cherry Valley ducks.

The Clock gene clone and its circadian rhythms in Pelteobagrus vachelli

The Clock gene,a key molecule in circadian systems,is widely distributed in the animal kingdom. We isolated a 936-bp partial c DNA sequence of the C lock gene( Pva- clock) from the darkbarbel catfish P elteobagrus vachelli that exhibited high identity with C lock genes of other species of fish and animals(65%–88%). The putative domains included a basic helix-loop-helix(b HLH) domain and two period-ARNT-single-minded(PAS) domains,which were also similar to those in other species of fish and animals. P va- Clock was primarily expressed in the brain,and was detected in all of the peripheral tissues sampled. Additionally,the pattern of P va- Clock expression over a 24-h period exhibited a circadian rhythm in the brain,liver and intestine,with the acrophase at zeitgeber time 21:35,23:00,and 23:23,respectively. Our results provide insight into the function of the molecular C lock of P. vachelli.

Genetic Characteristics of 2009 Pandemic H1N1 Influenza A Viruses Isolated from China's Mainland

A total of 100 H1N1 flu real-time-PCR positive throat swabs collected from fever patients in Zhejiang, Hubei and Guangdong between June and November 2009, were provided by local CDC laboratories. After MDCK cell culture, 57 Influenza A Pandemic （H1N1） viruses were isolated and submitted for whole genome sequencing. A total of 39 HA sequences, 52 NA sequences, 36 PB2 sequences, 31 PB1 sequences, 40 PA sequences, 48 NP sequences, 51 MP sequences and 36 NS sequences were obtained, including 20 whole genome sequences. Sequence comparison revealed they shared a high degree of homology （96%-99%） with known epidemic strains （A/Califomia/04/2009（H1N1）. Phylogenetic analysis showed that although the sequences were highly conserved, they clustered into a small number of groups with only a few distinct strains. Site analysis revealed three substitutions at loop 220 （221-228） of the HA receptor binding site in the 39 HA sequences： A/Hubei/86/2009 PKVRDQEG→PKVRDQEA, A/Zhejiang/08/2009 PKVRDQEG→PKVRDQER, A/Hubei/75/2009 PKVRDQEG→PKVRDQGG, the A/Hubei/75/2009 was isolated from an acute case, while the other two were from patients with mild symptoms. Other key sites such as 119, 274, 292 and 294 amino acids of NA protein,627 of PB2 protein were conserved. Meanwhile, all the M2 protein sequences possessed the Ser32Asn mutation, suggesting that these viruses were resistant to adamantanes. Comparison of these sequences with other H1N1 viruses collected from the NCBI database provides insight into H1N1 transmission and circulation patterns.

Sequence analysis on drug-resistant rpoB gene of Mycobacterium tuberculosis L-form of isolated from pneumoconiosis workers

To study the characteristics of drug-resistant genetic mutation of rpoB on coal workers＇ pneumoconiosis complicated with L-form of Mycobacterium tuberculosis. Methods： A total of 42 clinical isolated strains of Mycobacterium tuberculosis L-forms were collected, including 31 drug-resistant strains. Their genomes DNA were extracted, the target genes were amplified by PCR, and the hot regions in the rpoB gene were analyzed by automated DNA sequenator. Results： No mutation of rpoB gene was identified in 11 rifampicin-sensitive strains while conformation changes were found in 31 rifampicin-resistant strains. The mutation rate was 93.55% （29/31） in resistant strains, mainly concentrated in codon 531 （51.6%, 16/31） and 526 （32.26%, 10/31）. Base substitutions happened, including 27 unit point mutation and 2 two point mutation. The mutation of codon 516 that new found wasn＇t reported by internal and overseas scholars. Conclusion： The substitution of highly conserved amino acids encoded by rpoB gene results in the molecular mechanism responsible for rifampicin resistance in Mycobacterium tuberculosis L-forms. It also proves that rpoB gene is diversiform.

Molecular cloning and characterization of SoxB2 gene from Zhikong scallop Chlamys farreri

The Sox proteins play critical roles during the development of animals, including sex determination and central nervous system development. In this study, the SoxB2 gene was cloned from a mollusk, the Zhikong scallop （Chlamysfarreri）, and characterized with respect to phylogeny and tissue distribution. The full-length cDNA and genomic DNA sequences of C. farreri SoxB2 （CflSoxB2） were obtained by rapid amplification of cDNA ends and genome walking, respectively, using a partial cDNA fragment from the highly conserved DNA-binding domain, i.e., the High Mobility Group （HMG） box. The full-length cDNA sequence of CJSoxB2 was 2 048 bp and encoded 268 amino acids protein. The genomic sequence was 5 551 bp in length with only one exon. Several conserved elements, such as the TATA-box, GC-box, CAAT-box, GATA-box, and Sox/sry-sex/testis-determining and related HMG box factors, were found in the promoter region. Furthermore, real-time quantitative reverse transcription PCR assays were carried out to assess the mRNA expression of CJSoxB2 in different tissues. SoxB2 was highly expressed in the mantle, moderately in the digestive gland and gill, and weakly expressed in the gonad, kidney and adductor muscle. In male and female gonads at different developmental stages of reproduction, the expression levels of CfS;oxB2 were similar. Considering the specific expression and roles of SoxB2 in other animals, in particular vertebrates, and the fact that there are many pallial nerves in the mantle, cerebral ganglia in the digestive gland and gill nerves in gill, we propose a possible essential role in nervous tissue function for SoxB2 in C.farreri.

Bioinformatic Analysis of zm ERECTA-LIKE2Gene from Zea mays

Zm ERECTA-LIKE2 sequences were isolated according to the Zea mays genome. The study indicates that zm ERECTALIKE2 is composed of 2 410 nucleotides and encodes a receptor-like kinase which consists of 774 amino acids by biological analysis. The results show that zm ERECTA-LIKE2 protein contains 13 domains of leucine-rich repeats and belongs to the PKclike family, it is a liposoluble protein, with a conserved transmembrane region.

The complete genomic sequence analysis of genotype 4 human astrovirus HASTVgz01 strain in Guangzhou

To analyze the genomic molecular structure and genotype of human astrovirus isolated from infant in Guangzhou of China, the primers were designed based on the genomic sequence of astrovirus from the C, enBank and the target sequence were amplified by RT-PCR. Then the PCR-products were cloned to T vector and sequenced. The genomic nucleotide sequences were analyzed by the programs CLUSTAL W and DNASTAR. It was found that the full genomic length of HASTVgz01 strain was 6721 bp and the ORFs were 6558 bp. The 5＇ and 3＇UTR were 82 and 81 nucleotides. The genome included 3 open reading frames （ORFs） ： ORFla, ORFlb and ORF2. The 5＇-terminal ORFla started at nueleotide 83 and extended to nucleotide 2845. ORFlb （nt 2785 to nt 4332） overlaped ORFla by 61 nueleotides. The 3＇-terminal ORF2 began at nucleotide 4325 and terminated at nucleotide 6640. ORF2 had 2316 nucleotides. Compared with other astrovirus sequences in GenBank, the homology of the amino acid sequence of ORF2 of HASTVgz01 strain with that of serotype 4 was 93%. Homology with other serotypes ranged from 61% to 70%. The complete nucleotide sequence of astrovirus HASTVgz01 strain isolated from Guangzhou in China was 6721 bp in length, GenBank accession NO. DQ344027. Comparing the ORF2 of astrovirus HASTVgz01 with the known sequences of types 1-8 the highest homology was serotype 4 （93%）. Comparative sequence analysis of the HASTVgz01 ORF2 with the reported human astrovirus sequences revealed that the isolated astrovirus belongs to genotype （serotype） 4.

Molecular Dissection of Bombyx mori Nucleopolyhedrovirus orf8 Gene

Viruses including baculoviruses are obligatory parasites, as their genomes do not encode all the proteins required for replication. Therefore, viruses have evolved to exploit the behavior and the physiology of their hosts and olden coevolved with their hosts over millions of years. Recent comparative analyses of complete genome sequences of baculoviruses revealed the patterns of gene acquisitions and losses that have occurred during baculovirus evolution. In addition, knowledge of virus genes has also provided understanding of the mechanism of baculovirus infection including replication, species-specific virulence and host range. The Bm8 gene of Bombyx mori nucleopolyhedrovirus （NPV） and its homologues are found only in group I NPV genomes. The Autographa californica NPV Ac 16 gene is a homologue of Bm8 and, encodes a viral structural protein. It has been shown that BmS/Ac 16 interacts with baculoviral and cellular proteins. BmS/Ac 16 interacts with baculoviral IE1 that is facilitated by coiled coil domains, and the interaction with IE1 is important for Bin8 function. Acl6 also forms a complex with viral FP25 and cellular actin and associates with membranes via palmitoylation. These data suggested that this gene family encodes a multifunctional protein that accomplishes specific needs of group I NPVs.

Progress in the Researches on Insect Mitochondrial Genome and Analysis of Gene Order

Insect mitochondrial genome is a double-stranded circular genomes which range from 14 503 bp to 19 571 bp in size.Nearly all the sequenced insect mitochondrial genomes encode 37 genes:two for rRNAs,13 for proteins and 22 for tRNAs.This review compares and summarizes the features of complete mitochondrial genomes from 175 sequenced species of insects in 22 orders.The genomic organization, contents,gene order,and rearrangements of gene order are analyzed.

Next generation sequencing under de novo genome assembly

The next generation sequencing （NGS） is an important process which assures inexpen- sive organization of vast size of raw sequence dataset over any traditional sequencing systems or methods. Various aspects of NGS such as template preparation, sequencing imaging and genome alignment and assembly outline the genome sequencing and align- ment. Consequently, de Bruijn graph （dBG） is an important mathematical tool that graphically analyzes how the orientations are constructed in groups of nucleotides. Basi- cally, dBG describes the formation of the genome segments in circular iterative fashions. Some pivotal dBG-based de novo algorithms and software packages such as T-IDBA, Oases, IDBA-tran, Euler, Velvet, ABYSS, AllPaths, SOAPde novo and SOAPde novo2 are illustrated in this paper. Consequently, overlap layout consensus （OLC） graph-based algorithms also play vital role in NGS assembly. Some important OLC-based algorithms such as MIRA3, CABOG, Newbler, Edena, Mosaik and SHORTY are portrayed in this paper. It has been experimented that greedy graph-based algorithms and software pack- ages are also vital for proper genome dataset assembly. A few algorithms named SSAKE, SHARCGS and VCAKE help to perform proper genome sequencing.