A new Multilocus Sequence Analysis Scheme for Mycobacterium tuberculosis

Objective Tuberculosis remains one of the most serious infectious diseases in the world. In this study, a scheme of Mycobacterium tuberculosis （M. tuberculosis） multilocus sequence analysis （MLSA） was established for the phylogenetic and epidemiology analysis. Methods To establish the scheme of M. tuberculosis MLSA, the genome of H37Rv, CCDC5079 and CCDC5180 were compared, and some variable genes were chosen to be the MLSA typing scheme. 44 M. tuberculosis clinical isolates were typed by MLSA, IS6110-RFLP, and soligotyping, to evaluate the MLSA methods. Results After comparison of the genome, seven high discrimination gene loci （recX, rpsL, rmlC, rpmG1, mprA, gcvH, ideR） were chosen to be the MLSA typing scheme finally. 11 variable SNP sites of those seven genes were found among the 44 M. tuberculosis isolate strains and 11 sequence types （STs） were identified. Based on the Hunter-Gaston Index （HGI）, MLSA typing was not as good for discrimination at the strain level as IS6110-RFLP, but the HGI was much better than that of spoligotyping. In addition, the MEGA analysis result of MLSA data was similar to spoligotyping/PGG lineage, showing a strong phylogenetic signal in the modern strains of M. tuberculosis. The MLSA data analysis by eBURST revealed that 4 sequence types （ST） came into a main cluster, showing the major clonal complexes in those 44 strains. Conclusion MLSA genotyping not only can be used for molecular typing, but also is an ideal method for the phylogenetic analysis for M. tuberculosis.

Isolation and Sequence Analysis of HMW Glutenin Subunit 1Dy10.1 Ecoding Gene from Xinjiang Wheat (Triticum petropavlovskyi Udacz. et Migusch)

A novel HMW glutenin subunit gene 1Dy10. 1 was isolated and characterized from Xinjiang wheat （Triticum petropavlovskyi. Udacz. et Migusch） accession Daomai 2. The complete open reading frame （ORF） of 1Dy10. 1 was 1 965 bp, encoding 655 amino acids. The numbers and distribution of cysteines in 1Dy 10.1 were similar to those of 1Dy10 and other y-type subunits. In the N-terminal of 1Dy 10.1, an amino acid was changed from L （leucine） to P （proline） at position 55. The repetitive domain of 1Dy10.1 differed from those of known HMW subunits by substitutions, insertions or/and deletions involving single or more amino acid residues. In the repetitive domain of subunit 1Dyl 0.1, the deletion of tripeptide GQQ in the consensus unit PGQGQQ resulted in the appearance of the motif PGQ that have not been observed in other known y-type HMW subunits. In comparison with the subunit 1Dy 12, a deletion of dipeptide GQ, which occurred in subunit IDy10, was also observed in subunit 1Dy10.1. The cloned IDylO. 1 gene had been successfully expressed in Escherichia coli, and the expressed protein had the identical mobility with the endogenous subunit IDy10.1 from seed.

Vitis amuerensis CBF3 Gene Isolation,Sequence Analysis and Expression

The full length cDNA sequence of CBF3 （CRT/DRE-binding factor） was cloned from Vitis amurensis by reverse transcription polymerase chain reaction （RT-PCR） using the primers designed based on CBF genes available in GenBank. Sequence analysis showed that the gene had 854 bp long and its coding sequence contained 720 bp, encoding a protein with 239 amino acids and an AP2 structural domain. The molecular mass of CBF3 was predicted to be 25.9 kDa and its theoretical isoelectric point was 7.02 and aliphatic index was 59.29. The average hydropathicity of the protein was -0.551. The tertiary structures of CBF3 were also analyzed. The prokaryotic expression vector pGEX-4T-CBF3 containing CBF3 gene was constructed and CBF3 fusion protein （52 kDa） was produced in Escherichia coli after induction with 1 mmol L-1 IPTG. Further studies are needed to evaluate its potential application for improving plant resistance to cold and other stress condition such as drought and salinity.

Cloning and Sequence Analysis of a Novel Cold-Adapted Lipase Gene from Strain lip35 (Pseudomonas sp.)

A combination method of the usual-PCR and reverse-PCR for the cloning of a novel lipase gene directly from the total genomic DNA of strain lip35 （Pseudomonas sp.） is described, whereby a lipase gene （lip） was cloned directly from genomic DNA. The sequence data have been deposited in the GenBank and EMBL data bank with the accession number EU414288. The nucleotide sequence showed a major open reading frame encoding a 59-kDa protein of 566 amino acid residues, which contained a lipase consensus sequence GXSXG. The lipase lip had 74 and 70% homologies with the lipases of an uncultured bacterium and P. fluorescens PfO-1, respectively, but it did not show any overall homology with lipases from other origins. The functional lipase was obtained when the lip gene was expressed in Pichia pastoris GS115.

Cloning and Sequence Analysis of Expansin Genes from Litchi chinensis Fruit

Using PCR degenerate primers, designed with reference to the sequences of the conserved amino acids of known expansins, to amplify cDNA fragments in litchi fruit by RT-PCR, two different cDNA fragments , named as Lc-Exp1 and Lc-Exp2 , were cloned. Lc-Exp1 and Lc-Exp2 was respectively composed of 531 bp encoding 177 amino acids and 537 bp encoding 179 amino acids. Eight cysteine residues and three tryp-tophan residues, which is supposed to be the characteristics of expansins, are conserved in both Lc-Exp1 and c-Exp2. In addition, the homology between the two expansins is 71. 6% at nucleotide acid sequences and 76.3% at amino acid sequences. The homology of Lc-Exp1 with Fa-Exp2 or Pp-Exp1 was 92.7% or 92.1%, but that of Lc-Exp2 with Fa-Exp2 or Pp-Exp1 was only 77. 4% or 76.3% at amino acid sequences.

Cloning and sequence analysis of the partial sequence of the rbcL from Bryopsis hypnoides

The partial sequence of the rbcL from Bryopsis hypnoides, including the sequences of the upstream, extron and partial intron, was amplified by PCR and their sequences were determined. With Spinacia oleracea as the outgroup, neighbor-joining method and maximum parsimony method were used respectively to build phylogenetic trees according to the rbcL exon sequence among 13 species that were the typical species of six phyla. Two kinds of trees showed clearly that there were two groups among those species, the green lineage and the non-green lineage. And the relationships of algae in the green lineage were similar in the two trees but those in the non-green lineage were not consistent. Analysis of codon preference indicated that the codon preference of the rbcL exon of Bryopsis hypnoides distinctly differed from that of the relevant sequence of photosynthetic bacteria.

Aequorea taiwanensis n. sp. (Hydrozoa,Leptomedusae) and mtCOI sequence analysis for the genus Aequorea

Nine species of Prionospio complex are recorded from China＇s waters,including one new species and six newly recorded species.Prionospio（Prionospio） pacifica sp.nov.,is characterized by having first and forth pairs of branchiae pinnate,second and third pairs of apinnate,ventral crest on Setiger 9 and dorsal crests on Setigers 10-25.Apoprionospio kirrae（Wilson,1990）,Prionospio（Aquilaspio） convexa Imajima,1990,Prionospio（Minuspio） multibranchiata Berkeley,1927,Prionospio（Prionospio） bocki Sderstrm,1920,Prionospio（Prionospio） dubia Maciolek,1985 and Prionospio（Prionospio） paradisea Imajima,1990 are recorded for the first time from China＇s waters.

Molecular Cloning and cDNA Sequence Analysis of Two New Lepidopteran OR83b Orthologue Chemoreceptors

The aim of this study was to isolate the full-length cDNA sequences of an unusually highly conserved olfactory receptor （orthologue to the Drosophila melanogaster DOR83b） from the antennae of Spodoptera exigua and S. litura by using reverse transcription-polymerase chain reaction （RT-PCR） and rapid amplification of cDNA ends （RACE） methods. Bioinformatics methods were used to further analyze the cDNA sequences and putative amino acid sequences. The two full-length cDNA sequences from olfactory receptor （OR） of male S. exigua and S. litura were named as SexiOR2 and SlitOR2, respectively. SexiOR2 and SlitOR2 consisted of nucleotide sequence of 1 906 and 2 483 bp, respectively, and both with deduced amino acid sequences of 473 residues. The sequence analysis indicated that the deduced amino acid sequences of the cDNA shared the high homologies with OR83b orthologue chemoreceptor sequences from previously reported moths, implying that the cDNA sequences were of OR83b orthologue chemoreceptor genes.

Isolation and Sequence Analysis of NSP2, ORF3 and ORF5 of PRRSV XH-GD Strain from Guangdong Province

The diseased samples collected from Guangdong Province were inoculated to Marc-145 cells, PRRSV-specific cytopathogenic effects （CPE） such as cell aggregation, cell shrinkage and cell exfoliation were observed and a PRRSV strain XH-GD was obtained. By using primers designed in accordance with the pub- lished gene se^lences of PRRSV in GenBank, NSP2, ORF3 and ORF5 fragments were amplified by reverse transcription polymerase chain reaction and analyzed by sequence aligment. The results demonstrated that PRRSV strain XH-GD belonged to the North American genotype and the NSP2 gene contained discontinuous de- letions of 30 amino acids. NSP2, ORF3 and ORF5 genes of XH-GD shared 83.3% -98.9%, 88.6% -99.2% and 88.1% -99.2% nucleotide homology, and 76.8% -98.3%, 83.7% -98.8% and 88.1% -99.2% amino acid homology with HUB1, NX06, BJsy06, VR-2332, HB-1（sh）2002, HB-2（sh）2002, CH-1a and RespPRRS MLV, respectively; however, NSP2, ORb3 and ORF5 genes of XH-GD shared only 52.9%, 65.0% and 64. 3% nucleotide homology, and 31.3%, 57.5% and 58.9% amino acid homology with LV, respectively. Phylogenetic analysis revealed that XH-GD had a closer relationship with HUB1, NX06, BJsy06, VR-2332, HB-1 （sh） 2002, HB-2 （sh） 2002, CH-la and RespPRRS MLV strains compared with LV strain.

Sequence Analysis of Polypeptide by Mass Spectrometry

The method described in this work provides a sensitive and fast technique for investigating the primary structure of peptides with molecular weight up to 3340 amu. Usually, the metastable ion kinetic energy spectra (MIKES) and collisional activated decomposition (CAD) spectra provide complementary information for the FAB mass spectra, the MIKES and CAD spectra generally contain high-mass sequence ions.

Gene cloning and sequence analysis of the cold-adapted chaperones DnaK and DnaJ from deep-sea psychrotrophic bacterium Pseudoalteromonas sp. SM9913

Pseudoalteromonas sp. SM9913 is a phychrotmphic bacterium isolated from the deep-sea sediment. The genes encoding chaperones DnaJ and DnaK of P. sp. SM9913 were cloned by normal PCR and TAIL - PCR （GenBank accession Nos DQ640312, DQ504163 ）. The chaperones DnaJ and DnaK from the strain SM9913 contain such conserved domains as those of many other bacteria, and show some cold-adapted characteristics in their structures when compared with those from psychro-, meso-and themophilic bacteria. It is indicated that chaperones DnaJ and DnaK of P. sp. SM9913 may be adapted to low temperature in deep-sea and function well in assisting folding, assembling and translocation of proteins at low temperature. This research lays a foundation for the further study on the cold-adapted mechanism of chaperones DnaJ and DnaK of cold-adapted microorganisms.

Sequence Analysis on Interleukin-2 Gene and Interleukin-6 Gene in a Hybrid Pig

[ Objective] To understand the functions of interteukin-2 （IL-2） and interleukin-6 （IL-6） in immune response. [ Method] Peripheral lymphocytas were isolated from a hybrid pig （ Duroc x Landrace x Shandong local pig） and stimulated with canavalin A in vitro. With total RNA as templates, the IL-2 and IL-6 cDNA were amplified by the RT-PCR and sequenced, respectively. [Result] The sequence of IL-2 gene had 100.0% nucleotide homology to the published IL-2 sequences. The sequence of IL-6 gene had 99.8% -100.0% nucleotide homology to the published IL-6 sequences, and the amino acid homology was 99.1% -100.0%. [ Conclusion] NO great variation of the IL-2 gene and IL-6 gene is observed in the hybdd pig, and these results provide a basis for research about functions of IL-2 and IL-6 protein.

Cloning and Sequence Analysis of Partial Exons of Sex Hormone Binding Globulin Gene in Pig

[Objedive] To clone the partial exons of sex hormone binding globulin （SHBG） gene in pig and provide experimental references for ac- curate separation and allocation of SHBG gene in pig. [Method] According to the sequences of SHBG gene in human and cow, one pair of primers was designed to amplify the DNA sequences of SHBG gene in pig, and the partial exons of SHBG gene were determined according to the principles of comparative genomics. Then the sequences and homology were analyzed. [ Result] The amplified SHBG gene was 841 bp in size, and its se- quences had 100.00% nucleotide homology to the CH242-411 E2 fragment on pig chromosome 12. The determined exon 4, exon 5 and exon 6 of SHBG gene were 162, 159 and 156 bp in size, respectively, and the amino acids sequences encoded by these three exons had 74.51% homology to that of human and 73.86% homology to that of cow, respectively. [ Conclusionl The partial exons of SHBG gene were determined successfully, which provides a basis for further research on the structure, accurate determination and allocation of SHBG gene in pig.

Molecular Cloning and Sequence Analysis of the Toc33 cDNA in Brassica napus Line Cr3529

Two primers, designed according to the Arabidoposis thaliana Toc33 sequence, were used to amplify the full coding region of the Toc33 cDNAs in leaves of a chlorophyll-reduced (Cr) mutant Cr3529 and its wild type 3529, Brassica napus, by RT-PCR technique. The RT-PCR results showed that the fragment homologous to Toc33 was expressed in Cr3529 as well as 3529 seedlings. PCR fragments were inserted into the pMD18-T vector and transferred into E. coli, then two cDNA clones, BnToc33-c and BnToc33, were obtained. Sequence analysis showed that the two sequences were 894 bp and the nucleotide and the deduced amino acid sequences were highly homologous to those of A. thaliana. There were three diverged nucleotides between the Cr3529 and the 3529 Toc33 cDNAs, i.e., GGT/AGT, TTG/TTT, AGG/AGT, all of which belonged to missense mutation. The amino acid replacement ((Leu/Phe) caused by TTG/TTT mutation located in the membrane anchor domain may result in chlorophyll-reduced character in Cr3529.

Cloning, Sequence Analysis, and Prokaryotic Expression of the Porcine DECR1 Gene

2,4-dienoyl-CoA reductase 1 （ DECR1 ） is the key rate-limiting enzyme in the metabolism of polyunsaturated fatty acids. Although this protein has been studied in a variety of mammals, its role in por- cine is yet to be fully elucidated. However, it is a candidate determinant/indicator of meat quality, growth traits, and carcass quality. Here, we employed RT-PCR and rapid amplification of cDNA ends （RACE） analysis to amplify the full-length cDNA of DECR1 from Mashen pig liver, and cloned it into the expression vector pET-32a＋. After confirmation by sequencing and restriction analysis, the recombinant plasmid was transformed into E. coli BL21 cells. The cDNA of pig DECR1 contained 2,352 nucleotides, including a 987 bp open reading frame flanked by a 53 bp 5＇-untranslated region （UTR） and a 1,312 bp 3＇-UTR. The pig DECR1 coding sequence encoded 328 amino acid residues, which shared 99%, 88%, 87%, 87%, 87%, 87%, and 83% identity with those of Sus scrofa （predicted）, Bos taurus, Homo sapiens, Macaca mulatta, Pan troglodytes, Equus caballus, Canis, and Mus musculus, respectively. SDS-PAGE analysis revealed that the recombinant protein was expressed and that the expression level reached its highest level after 4 h induction. Western blot analysis indicated that the molecular weight of the expressed protein was the same as that predicted, ap- proximately 35 kDa. Collectively these data provide the basis for further studies into the physiological functions and molecular mechanisms of the pig DE- CR1 gene.

Cloning and Sequence Analysis of gyrB Gene of Fluoroquinolones-resistant Salmonella Isolated from Chickens

Nine strains resistant to five fluoroquinolones (Ciprofloxacin, Ofloxacin, Enrofloxacin, Danofloxacin, Sarafloxacin) were isolated from clinical samples and extracted the chromosomal DNA of these strains. Designed primers to amplify the Quinolone-resistance-determining region(QRDR) of gyrB gene, then the PCR products were cloned and the sequence was analyzed. In comparison with the standarded strain NCTC5776, no mutation was found in the QRDR of gyrB gene of all resistant strains. The result indicated that the QRDR of gyrB has little relationship with fluoroquinolone resistance to salmonella.

Cloning and Sequence Analysis of cDNA Encoding MRJP3 of Apis cerana cerana

By screening the worker （Apis cerana cerana） heads cDNA library using a fragment of the mrjp3 gene of Apis cerana as probe, 120 positive clones were obtained. The clone containing A. cerana cerana MRJP3 （AccMRJP3） cDNA was selected. Based on the sequencing of the inserts of the positive clone, a sequence of AccMRJP3 cDNA which is 1 887 bp long including a poly （A） tail was obtained. The AccMRJP3 cDNA encompassed an open-reading frame （ORF） with 1 779 bp encoding 593 amino acids. The un-translated regions （UTR） of the 5′ end and 3′end are 46 bp and 160 bp in length, respectively. Similar to AmMRJP3 and AdMRJP3, the putative AccMRJP3 also has a repetitive region. The comparison of the repetitive region of AccMRJP3, AmMRJP3 and AdMRJP3 shows some differences between them.

E Sequence Analysis of Persistently Infected Mutant Japanese Encephalitis Virus Strains

Summary： A persistent infection model was established after human hepatoma cells infected by Japanese encephalitis viruses were subcultured for several times. Viral titers of mutant viruses in persistently infected cells were examined by plaque methods using BHK cells. Nucleotides of the E coding region of two wild and two mutant viruses were amplified by RT-PCR. PCR products were sequenced by ABI-PRSM^TM310 sequencing system. Compared to JaGAr-01 wild strains, four amino acids were replaced （E61Tyr→Asp, E219His→Tyr, E384Val→Glu, E418Pro→Ala） in the E sequence of JaGAr-01 persistently-infected mutant strains. Eleven amino acid replacement （E51Arg →Ser, E61Tyr→Asp, E83Lys→Glu, E123Ser→Arg, E209Arg→Lys, E227Pro→Ser, E276Asp→er, E290Arg→Lys, E387Lys→Arg, E418Leu→Pro, E454Arg→Gly） was also noted when we compared the E sequence between persistently infected Nakayama and its wild strains. A lot of similarities of amino acid sequence between mutant strains JaGAr-01 and Nakayama were also noted. It was concluded that geno-variation existed in E region of mutant viruses and the mutant protein encoded by E region, especially the mutation of E61 （Tyr→Asp） may contribute to the maintenance of the persistent infection of Japanese encephalitis virus.

Molecular Cloning and Sequence Analysis of Chitin Synthase cDNA from Mamestra brassicae (L.) (Lepidoptera: Noctuidae) Cuticle

Chitin is the most widespread amino polysaccharide in nature. Chitin synthase （CHS） plays an important role in chitin formation in the cuticle and the peritrophic membrane （PM） lining the midgut. Total RNA was isolated from the cuticle of Mamestra brassicae （L.） fourth instar larva, cDNA sequence was cloned by RT-PCR and Rapid Amplification of cDNA Ends （RACE）. cDNA 5 220 bp in length, contained an open reading frame of 4 704 bp coding for a polypeptide of 1 567 amino acid residues with a predicted molecular weight of 178.3 ku and its pI was 6.42. The deduced amino acid sequence from Mi brassicae （L.） shared the high level of identity with chitin synthase sequences from other insects, especially lepidopteran insects, cDNA sequence has been deposited with GenBank under accession No. GQ281761