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 16S rRNA and COI Gene in Mitochondrial DNA of Scortum barcoo

[Objective] The aim was to provide molecular biological basis for the researches on the genetic resources,genetic relationship among species and phyletic evolution of S.barcoo.[Method] PCR amplification and sequencing were used to study the 16S rRNA and COI gene fragments.[Result] As for 16S rRNA gene fragments,nucleotide sequences of 791 bp were obtained,and the A,T,G and C contents in this fragment were 31.6%,21.4%,20.4% and 26.7%respectively.As for the COI gene fragments,the size was 631 bp and the A,T,G And C contents were 27.7%,23.6%,29.8% and 18.9% respectively.Among these two gene fragments,the content of GC was lower than AT,and AT/GC of these two fragments was 1.13 and 1.05 respectively.[Conclusion] The genetic characteristics of gene fragments of 16S rRNA and COI of S.barcoo suggested that the variation in the same species was relatively low.The sequences of 16S rRNA gene in three samples the same,while the sequences of COI gene was also the same,indicating that these two gene of S.barcoo were conservative.

Cloning and Sequence Analysis of Lactate Dehydrogenase C(LDH-C)Gene from Black-lipped Pika in Western Sichuan Plateau

[Objective] The aim was to lay a foundation for research of contraceptive rodenticide with LDH-C4 as target protein. [ Method] EST sequence of LDH-C gene from black-lipped pika was cloned by PCR with degenerate primers; then the full length open reading frame （ORF） and 3＇UTR sequence were cloned by RACE technique. [ Result] The full length cDNA was 1 498 bp containing an ORF of 996 bp and a 3＇UTR of 486 bp. The ORF encoded a polypeptide of 332 amino acids. The alignment of LDH-C gene ORF nucleotide sequences from different species showed that the gene was conserved even between large taxons. The phylogenic tree showed that black-lipped pika LDH-C was closer to prima- tes and artiodactyla than to rodents. [Conclusion] cDNA sequence of LDH-C gene from black-lipped pika was cloned successfully.

Molecular Cloning, Sequence Analysis and Prokaryotic Expression of Ovine Activin Receptor Type IIB(ActRIIB) Gene

Objective] This study aimed to clone ovine activin receptor type llB （Ac-tRIIB） gene, construct the prokaryotic expression vector and express the target gene in vitro, thus providing basis for further function verification. [Method] The template cDNA which was reversely transcribed from total RNA of sheep liver tissue, was subjected to polymerase chain reaction （PCR） using specific primers of ActRIIB. The ful-length cDNA of ovine ActRIIB was obtained by pMD18-T cloning and sequencing for bioinformatics analysis. Ovine ActRIIB encoding sequence was subcloned into prokaryotic expression vector pET41a with restriction sites BamHl/Notl, and then transformed into BL21 （DE3）. The induced products by lPTG were analyzed with SDS-PAGE and Western Blot. [Result] The amplified ful-length cDNA of ovine Ac-tRllB gene was 1 564 bp in length （Genbank accession number： JX422071.1） with an open reading frame of 1 539 bp, encoding 512 amino acides. Ovine ActRllB shared the highest homology （99.6%） with bovine ActRllB. ActRllB had highly ho-mologous C-terminal domains and belonged to the TGFβ family. After prokaryotic expression, an approximately 92 kD His-tagged ActRllB recombinant protein was obtained, which was consistent with the excepted result. [Conclusion] Cloning and successful expression of ovine ActRIIB laid solid foundation for further investigation of its biological function.

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.

Construction of Midgut Tissue-Specific cDNA Library of Bombyx mandarina M. and Isolation and Sequence Analysis of Serine Protease Gene Fragment

[Objective] The aim of the study is to construct cDNA library of midgut tissue of wild silkworm and isolate the serine protease gene. [Method] The midgut tissue-specific cDNA library of wild silkworm was constructed via cDNA Library Construction Kit (TaKaRa), then the serine protease gene was cloned via sequencing of the yielded cDNA library. [Result] The titer of cDNA library reached 6.2×105 pfu/ml, average insert size was about 1.2 kb. The serine protease gene cDNA fragment was obtained from colony sequencing (Accession No: EU672968). The nucleotide sequence of the cloned 854 bp fragment encodes 284 amino acid residues. Homology analyses showed some homology between putative amino acid sequence of the cloned fragment and amino acid sequences of serine proteases from other ten insects. [Conclusion] The results may avail to reveal the resistance of silkworm and wild silkworm to exotic intrusion.

Cloning and Sequence Analysis of Sheeppox Virus RPO30 Gene

[Objective] The study aimed to clone RPO30 gene from Sheeppox virus （SPPV） and predict the structure and function of the sequence. [Method] RPO30 gene of SPPV was cloned with PCR, linked into pMD18-T simple vector and then transformed into E. coli DH5a. In blue-white screen, the white colonies were selected to prepare plasmids. The positive plasmids were selected by double digestion and PCR, and then sequenced. Finally, the structure and function of the sequence obtained were predicted by bioinformatics methods. [Results] The RPO30 gene was successfully obtained; its ORF was 585 bp, encoding 193 amino acids and containing a recognition site for Hind III. Moreover, the SPPV RPO30 gene shared different homologies with the RPO30 gene sequences of other pox virus strains from GenBank database. Further analysis by biological software showed that in RPO30 protein, amino acids 4-12, 18-26, 50- 61, 68- 92 and 176-190 had a high possibility to form the active center, and acting to these regions was likely to inactivate the enzyme encoded by the sequence, thus to inhibit viral replication efficiently. [Conclusion] This study will lay foundation for further study on the structure and function of RPO30.

Cloning and Sequence Analysis on IGF-1 Gene of Hubei White Swine

[Objective] The study aimed at cloning and analyzing the insulin-like growth factor-1 （IGF-1） gene from liver of Hubei white swine. [Method] The total RNA was extracted by using Trizol from the liver of Hubei white swine and used as template to amplify IGF-1 gene cDNA by RT-PCR. The cDNA product was cloned into pCRII vector, screened with blue-white colonies, digested with double enzymes and sequenced. [Result] The sequencing result indicated that the IGF-1 gene consisted of 607 nucleotides, containing 5＇-untranslated region at nucleotides 1-145, a complete ORF at nucleotides 146-538 encoding 130 amino acids, and 3＇-untranslated region at nucleotides 539-607. It shared 100% homology with the porcine IGF-1 gene reported by Muller et al. [Conclusion] The successful cloning and sequencing of the Hubei white swine IGF-1 gene confirmed that IGF-I gene was highly conserved, which provided technical basis for the use of transgenic technology for breeding of Hubei white swine.

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 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.

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.

Cloning and sequence analysis of US1 gene in duck enteritis virus

In this paper,a 1,860 bp sequence in IRs region of duck enteritis virus(DEV) was amplified by single oligonucleotide nested PCR with a single primer designed according to partial sequence of US1 and then a pair of primers designed according to the 3' UTR of US8 gene and 5' end of the new getting sequence were used to amplify a 2,426 bp sequence toward the TRs region.Sequence analysis revealed that the both sequences contained an identical 990 bp open reading frame of DEV US1 gene.The two ORFs were in opposite transcription orientation.Sequence comparison of the nucleotide sequence and the deduced amino acid sequence of US1 gene showed relatively high identity to Mardivirus.Phylogenetic tree analysis showed that the eleven herpesviruses viruses were classified into three groups,and the duck enteritis virus was most closely related to Mardivirus.

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 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 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 the gp41 Gene of Clanis bilineata Nuclear Polyhedrosis Virus

Clanis bilineata Nucleo Polyhedro Virus （CbNPV） was purified from Clanis bilineata larva. To obtain the molecular information of the virus, the genomic DNA of CbNPV was extracted, and a DNA fragment library of the virus was constructed using shotgun. The positive clones were then sequenced and analyzed. An open-reading frame （ORF） that has high identity with the gp41 gene of most NPVs was found in the library. The gp41 gene of CbNPV is 933 base pair long and encodes a protein of 310 amino acids. The result of the amino acid sequence analysis showed that the CbNPV gp41 has 53-61 and 56-73% identities with Group 1 and II NPVs gp41 proteins, respectively. The result indicates that the isolated CbNPV is a novel baculovirus, and the CbNPV shares a much closer relationship Ⅱ NPVs.

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.