Molecular Cloning and Sequence Analysis of Class Ⅱ Chitinase Gene in Leymus chinensis

[ Objective] The aim of this study was to clone Class Ⅱ chitinase gene in Leymus chinensis grown in saline land in Heilongjiang Province and analyze its sequence, which provided a foundation for further study on the biological function and application of chitinasa gene. [ Method] cDNA library of Leymus chinensis leaves were constructed, and its DNA sequence was determined or analyzed, while the homology of chitinasa gene and amino acid sequence was compared with that in GenBank. [ Result] One full length cDNA fragment with length of 996 bp was cloned from cDNA library of Leymus chinensis leaves. The length of ORF was 768 bp encoding 225 amino acids （GenBank accession number： EU344908）. The encoding products lacked CBD and C-terminal extension region from the view of structure, but had structural characters of Class Ⅱ chitinase gene, which indicated that amino acid sequence had high homology compared with Class Ⅱ chitinase gene of rye and wheat. The constructed recombinant vector pQE-LcChi2 could express a protein of 27 kD through induction, which was consistent with the deduced encoding product of pQE-LcChi2 gene. [ Conclusion] LcChl2 gene is an expression gene, which can express in E. coll.

Cloning and Sequence Analysis of HN and F Protein Genes from a Strain of Goose Paramyxovirus

[ Objective] The study was to clone HN and F genes from GX1 strain of goose paramyxovirus and analyze their sequences. [ Method] According to the full nucleotide sequence of GPV-SF02 strain of goose paramyxovirus, two pairs of pdmers were designed to amplify the HN and F genes from GX1 strain of goose paramyxovirus isolated from diseased goose in Guangxi Zhuang Autonomous Region; the amplified products were ligated into pMD18-T vector and sequenced. [ Result ] HN and F genes of this strain tested were 1 716 and 1 662 bp in full nucleotide length, respectively; both showed the homologues of about 97.3% with GPV- SF02 strain, of 80.3% -97.5% with strains LaSota, F48E9 and JS, of just 84.8% with Miyadera strain. [ Conclusion] The results show that isolated strain BX1 matches to virulent APMV-1 strain, belonging to genotype Ⅶ of APMV-1 strain.

Cloning,Sequence Analysis and Amino Acid Structure Prediction of TLR9 Gene from Wild Ovis ammon in Xinjiang

[Objective] The study aimed at cloning and identifying the toll receptor gene 9（TLR9） of wild Ovis ammon in Xinjiang,and predicting its structure and function.[Method] The TLR9 complete sequence of wild Ovis ammon was cloned from its peripheral blood by PCR technology.Then the PCR products were purified by agarose gel electrophoresis and then sequenced.Finally,the structure and function of TLR9 sequence were predicted by molecular biological software.[Result] The complete sequence of TLR9 gene was 3 192 bp in length,encoding 1 064 amino acids with a signal peptide composed of 30 amino acids;the leucine percentage reached as high as 18.5%.The TLR9 amino acid possibly contained three hydrophobic regions,at amino acids 455-475,740-760 and 780-800.The 3-D structure of TLR9 was constructed by the extracellular LRR domain and intracellular TIL domain（Toll/IL IR）.[Conclusion] The characteristics of TLR9 provided theoretical basis for further study on the TLR9 gene of wild Ovis ammon in Xinjiang.

Cloning and Sequence Analysis of 5′ Flanking Region and Exon of Inhibin α Precursor Gene in Goats

[Objective] The aim of this study was to reveal the relationship between inihibin （INH） α precursor gene and seasonal reproduction of goats, and investigate the evolutionary conservation of INHα precursor gene. [ Method] Cloning and sequence analysis of 5＇ flanking region and exon of inihibinα （INHE） precursor gene in twenty ewes between non-seasonal estrous breed （Haimen goats） and seasonal estrous breed （Anhui white goats） was analyzed in this study. [ Result] Compared with Anhui white goats, INHα precursor gene in Haimen goats had three SNP but no amino acid change, while its nucleotide homology was 99.7% and amino acid homology was 100%. The nucleotide homology of INHα precursor gene in goat, cattle, pig, person, chicken, horse, rat and dog ranged from 12.7% to 96.5%. [ Conclusion] INHα precursor gene tends to be highly conserved in species, and any change of nucleotide and amino acid maybe directly influence the function of the whole gene coding and regulation.

Cloning and Sequence Analysis of CmNAC Gene from Cucurbita moschata

[Objective] The aim was to clone the CmNAC gene from Cucurbita moschata and analyze the sequence characteristics. [Method] A pair of degenerate primers was designed based on the conserved sequences of NAC gene from Brassica napus, Lycopersicon esculentum and Capsicum annuum. NAC transcription factor gene was amplified by RT-PCR from Cucurbita moschata leaves and cloned into pMD-19T vector; then the recombinant clones were sequenced. Finally, the sequences of nucleic acid and amino acid were analyzed using BLAST and DNAMAN software. [Result] The NAC transcription factor gene cloned from C. moschata included 442 bp encoding 147 amino acids, named CmNAC. The NAC gene fragment contained a conserved region like other plant NAC genes and belonged to the NAC family ATAF1/2 subfamily. [Conclusion] The stress resistance related gene NAC cloned from C. moschata is a foundation for further study on the biological function of the gene and plant genetic engineering.

Isolation and Analysis of α-Gliadin Gene Coding Sequences from Triticum durum

Three coding sequences of gliadins genes, designed as Gli2_Dul, Gli2_Du2 and Gli2_Du3, were isolated from the genomic DNA of Triticum durum accessions CItr5083. Gli2_Dul and Gli2_Du2 contain 945 and 864 bp, encoding the mature proteins with 314 and 287 amino acid residues, respectively. Gli2_Du3 is recognized as a pseudogene due to the stop codon occurring in the coding region. The pseudogenes, commonly occurring in gliadins family, are attributed to the single base change C→T. The amino acid sequences deduced from these gene sequences were characterized with the typical structure of α-gliadin proteins, including the toxic sequences （PSQQQP）. The peptide fraction PF（Y）PP（Q）is thought to be an extra unit of repetitive domain, slightly diverging from the previous report. Six cysteine residues were observed within two unique domains. Phylogenetic analysis showed Gli2_Du2 and Gli2_Du3 were closely related to the genes on chromosome 6A, whereas Gli2_Dul seems to be more homologous with the genes on chromosome 6B.

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.

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.

Analysis of LMW-GS,α-and γ-Gliadin Gene Coding Sequences from Triticum macha

Novel LMW-GS （low molecular weight glutenin subunit）,α- and γ-gliadin from Triticum macha accessions were characterized via genomic PCR, which can do favor to improve the wheat quality. The complete coding regions of two α-gliadin, two γ-gliadin and two LMW-GS gene sequences, which designed as Gli-Mal, Gli-Ma2, Gli-Mrl, Gli-Mr2, Glu-LM1 and Glu-LM2, encoded the mature proteins with 307, 241, 348, 302, 474 and 377 amino acid residues, respectively. Gli-Mal and Gli-Ma2 were recognized as pseudogenes due to the in-frame stop codons. The amino acid sequences deduced from these gene sequences were characterized with the typical structure of α- or γ-gliadin or LMW- m type proteins with the exception of Gli-Ma2. Phylogenetic analysis showed Gli-Mal was closely related to those from T. aestivum, whereas Gli-Ma2 seemed to be more homologous with the gene sequences from Dasypyrum breviaristatum. Gli-Mrl was closely related to those from T. turgidum ssp. dicoccoides, while Gli-Mr2 was the nearest to those from T. aestivum. Glu-LM1 was closely related to those from Aegilops tauschii, whereas GIu-LM2 seemed to be more homologous with those from T. durum.

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 Actin Gene Fragment from Iris lactea var.chinensis Fisch.Koidz

Degenerate primers were designed based on the conserved sequences of the Actin gene from other plants. Total RNA was extracted from the leaves of lris lacteal var.chinensis Fisch.Koidz. Actin gene fragment was obtained by reverse transcription polymerase chain reaction （RT-PCR） and cloned into pMD18-T vector. The positive clone identified by PCR was sequenced. The sequencing result showed that the Actin gene fragment from lris lacteal var.chinensis Fisch.Koidz contained about 598 bp, encoding 199 amino acids. Homology comparison with Actin gene sequences of other plants in the GenBank showed that it shared over 82% nueleotide sequence homology and 90% amino acid sequence homology. It indicated that this was the Actin gene. Because of the stability expression ofActin gene, it usually cited as the internal reference to study the expression and regulation of foundation in other genes of lris lacteal var.chinensis Fisch.Koidz well.

Cloning and Sequence Analysis of Porcine IRGC Gene

[ Objective] This study was to analyze porcine IRGC gene, so as to lay the foundation of further revealing its function. [ Method] IRGC gene was cloned by using EST information together with sequencing, then aligned with the IRGC sequences of human, cattle, dog and orangutan for obtaining their similarities. [Result] Porcine IRGC cDNA obtained in the present study was 1 558 bp in length（GenBank accession number EU703776）, which shared high similarity with human （86%）, cattle （90%）, dog （91%）, orangutan （84%）. Sequence analysis shows that porcine IRGC gene encodes 464 amino acid residues which share high homologies with the counterparts from human （90%）, cattle （93%）, dog （95%）, orangutan （90%）. [Condusion] Cloning and sequence analysis of porcine IRGC gene would be helpful for studying its functions.

Cloning and Sequence Analysis of Glycoprotein D Gene of Bovine Herpesvirus-1 Strain Luojing

By means of PCR,the gene encoding gD of bovine herpesvirus-1 (BHV-1) strain Luojing was amplified,cloned and sequenced.The nucleotide sequence of this gD gene was (1 251 bp,)encoding 417 amino acids.Comparied with the published P8-2 strain,the homology of the necleotide sequence is 99.92%,and that of the deduced amino acid sequence is 100%.The results indicated that gD of BHV-1 was highly conservative.

Cloning and Sequence Analysis of IGFBP-7 Gene in Sheep

In this study, full-length CDS sequence of IGFBP-7 gene was cloned from Liangshan semi-fine wool sheep with RT-PCR method and analyzed with bioinformatics methods. The results showed that the full-length CDS sequence of IGFBP-7 gene in Liangshan semi-fine wool sheep was 846 bp in length, encoding 282 amino acids. The CDS sequence shared 99%, 95% and 90% homology with bovine, human and rat, respectively; the amino acid sequence shared 98%, 93% and 89%, respectively. The GenBank accession number was FJ589640.1. The amino acid molecular weight of IGFBP-7 was 29.0 kD, and the theoretical isoelec- tric point （pl） was 8.25. The result of phylogenetic analysis showed that IGFBP-7 gone in Liangshan semi-fine wool sheep exhibited close phylogenetic relationships with bovine, goat and other mammals, and distant phylogenetic relationships with Danio rerio and Haliotis diversicolor. IGFBP-7 gene had uniformly distributed hy- drophobic and hydrophilic regions, harboring one signal peptide, two transmembrane regions, 16 phosphorylation sites, four N-glycosylation sites and one O-glyco- sylation site. The result of secondary structure analysis showed that the random coil, or-helix and β-sheet regions accounted for 64.89%, 19.86% and 15.25%, respectively. The result of tertiary structure analysis showed that IGFBP-7 harbors an IGFBP_N domain and an Ig-like domain. This study provided scientific basis for further investigating the function of IGFBP-7 gene in sheep.

Cloning and Expression Analysis of Downy Mildew Resistance-Related cDNA Sequences in Melon

Melon downy mildew caused by Pseudoperonospora cubensis leads to significant losses in melon yields worldwide. Reverse-transcription Polymerase Chain Reaction （RT-PCR） was performed using cDNAs as templates from melon-Huangdanzi induced with fungus Pseudoperonospora cubensis, and degenerate primers designed based on the conserved amino acid sequences of known plant disease-resistance genes. A polymorphic cDNA fragment which we named rap-19 was cloned and sequenced. The Open Reading Frame （ORF） of this product comprised of 510 base pairs which encodes DNA or RNA-binding protein with 170 amino acids. The putative amino acid sequence of mp-19 appeared highly homologous with those of NBS-type resistant-genes isolated from other plants. Southern blot indicated that the melon genome contained more than 3 copies of rap-19. The obvious expression differences detected by semi-quantitative RT- PCR could be observed between resistant-line Huangdanzi and susceptible-line Jiashi after Pseudoperonospora cubensis infection, which implied that mp-19 gene may be related to the resistance of downy mildew in melon.

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.

Cloning and Sequence Analysis of Carbonic Anhydrase-related Protein 10-like in Apis mellifera

[ Objective ] This study aimed to clone and analyze the gene sequence encoding carbonic anhydrase-related protein lO-like （ CARP X） from Apis mellif era. [Method] The cDNA sequence of CARPX gene was cloned through RT-PCR, and then analyzed with bioinformatic method. [Result] The full-length cDNA sequence of CARPX was 972 bp long and encoded 324 amino acid residues, including a signal peptide and two transmembrane domains. The predicted molecular mass was 37.1 kDa and the predicted isoeleetric point was 7.458. The CARP X from A. mellifera shared close relationship with proteins from Apisflorae, Bombtas impatiens, Bombus terrestris, Nasonia vitripennis and Acyrthosiphon pisum. The insect CARP X family may include two subfamilies. [ Conclusion] The results pro- vide basis for studying CARPs family.

Characteristics of nucleotides and amino acids in cDNA sequence of xyloglucan endotransglycosylase cloned from Anthocephalus chinensis

Xyloglucan endotransglycosylase is an identified relaxation factor with functions of easing and extending of plant cell walls.Its activities are directly related to plant growth and elongation of organisms.Anthocephalus chinensis is a tall and fast growing evergreen tree species.We cloned the full cDNA sequence of AcEXT genes which is abundantly expressed in the cambium of A.chinensis.The sequence analysis of nucleotides and amino acids revealed the presence of a 1396 bp full cDNA sequence,including a 960 bp complete open reading frame（ORF） encoding a 320 amino acid protein.The deduced amino acid sequence of AcXET was homologous to the other known XET proteins and contained the conserved EIDFE catalytic site which was specific to all the XETs.Our data should serve as a foundation for further insight into AcXET gene molecular mechanisms during wood formation and cell wall engineering of woody plants.