Gene Cloning and Bioinformatics Analysis of phoR Gene from Vibrio alginolyticus HY9901

PhoR is a histidine kinase in a two-component regulatory system that regulates phosphorus metabolic pathways and undertakes the key mission of information transmission in pathogenic bacteria.The full-length phoR gene was successfully cloned from the Vibrio alginolyticus HY9901 strain.A comprehensive analysis of the cloned gene was conducted using bioinformatics.Sequence analysis revealed that the total length of the phoR gene(GenBank accession No.:KJ958404.1)is 1299 bp,with the coding region containing a total of 432 amino acid residues.The phylogenetic tree of PhoR revealed that it belongs to the same subclade as V.diabolicus.The SMART program was employed for the purpose of functional domain prediction,which revealed that PhoR possesses three major functional domains:PAS(amino acids 98-166),HisKA(amino acids 205-272),and HATPase_c(amino acids 317-429).

Molecular Cloning and Bioinformatics Analysis of msrA Gene from Vibrio alginolyticus Strain HY9901

[Objectives]This study was conducted to understand the structure and function of MsrA protein.[Methods]With Vibrio alginolyticus HY9901 as the object of study,primers were designed to amplify the full-length gene of msrA,and its bioinformatics analysis was carried out.[Results]The full length of msrA gene was 639 bp,encoding 212 amino acids,and its theoretical molecular weight was about 23729.60 Da.The protein had a stable structure,and it was hydrophobic overall.The structure of signal peptides at the N terminal of the amino acid sequence was predicted,and it was found that there was no signal peptide cleavage site and no transmembrane region.The amino acid sequence of MsrA contained multiple signal binding sites.Protein subcellular localization showed that MsrA protein was most likely located in the cytoplasm.Homology analysis showed that MsrA of V.alginolyticus had high homology with other Vibrio species,and the highest homology with V.alginolyticus.In the prediction of functional domains,MsrA had the function of methionine sulfoxide reduction.In secondary structure prediction,MsrA contained random coils at a proportion of 46.70%,which was the highest.The similarity between the tertiary structure model of MsrA and template Q87SW6.1.A was 89.15%.PTM analysis showed that MsrA protein had many PTM modification sites such as phosphorylation and glycosylation sites.[Conclusions]This study provides some reference value for further study on the role of MsrA in bacterial antioxidant stress.

Molecular Cloning of sodB Gene from Vibrio alginolyticus HY9901 and Its Bioinformatics Analysis

Vibrio alginolyticus is a zoonotic bacterium.A pair of specific primers was designed using the sodB gene sequence of Vibrio alginolyticus HY9901 in order to amplify the full length of the gene by PCR.The results indicated that the total length of the sodB gene was 585 bp and that it could encode 194 amino acids.The predicted amino acid sequence derivation indicated that the molecular weight of the protein was approximately 21.56 kDa,with an isoelectric point of 4.95.Upon prediction of the N-terminal signal peptide structure of the protein,no significant signal peptide cleavage site was observed,indicating that the protein lacked both a signal peptide and a transmembrane region.The amino acid sequence contained an N-glycosylation site,a casein kinase II phosphorylation site,a microsomal C-terminal target signal site,and a manganese and iron superoxide dismutase signal site.The probability of intracytoplasmic localization of the SodB protein was 56.5%,which was analyzed according to the subcellular localization of the protein.The amino acid sequence of the sodB gene of V.alginolyticus exhibited 98%-100%homology to other Vibrio species,clustering into the same subfamily with V.parahaem,indicating a relatively close relationship between them.In the prediction of protein structure,the proportions ofα-helix,random coil,β-sheet,and extended strand were 48.45%,30.41%,5.67%,and 15.46%,respectively.The similarity to template 1dt0.1.A reached 71.58%.A PTM site analysis revealed the presence of phosphorylation,glycosylation,ubiquitination,sumoylation,acetylation,and methylation modification sites,as well as the absence of lactylation modification sites.

Cloning and expression analysis of a long type peptidoglycan recognition protein(PGRP-L) from Xenopus tropicalis

Peptidoglycan recognition proteins（PGRPs） are a family of pattern recognition receptors（PRRs） of the immune system,which bind and hydrolyze bacterial peptidoglycan.Here,a long type PGRP（PGRP-L） was first cloned in the lower vertebrate species Xenopus tropicalis（Xt）.The XtPGRP-L possessed a conserved genomic structure with five exons and four introns.The alignment and phylogenetic analysis indicated that XtPGRP-L might be a type of amidase-like PGRP.The 3-D model showed that XtPGRP-L possessed a conserved structure compared with the Drosophila PGRP-Lb.During embryonic development,XtPGRP-L was not expressed until the 72 h tadpole stage.In adult tissues,it was strongly expressed in the liver,lung,intestine,and stomach.Furthermore,after LPS stimulation,the expression of XtPGRP-L was up-regulated significantly in the liver,intestine and spleen,indicating that XtPGRP-L may play an important role in the innate immunity of Xenopus tropicalis.

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 Bioinformatic Analysis of Cinnamoyl-CoA Reductase Gene (CCR) from Pennisetum purpureum

[Objective] The aim was to clone the cDNA and DNA sequences of the CCR （Cinnamoyl-CoA reductase） gene which involves in lignin biosynthesis, from Pennisetum purpureum, and to make comprehensive analysis on these sequences. [Method] CCR sequences were cloned from P. purpureum by using conventional RT-PCR and RACE （Rapid Amplification of cDNA Ends） methods; and the bioinformatic analyses of the CCR were conducted by means of NCBI, ProtParam ProtScale, TMHMM, TargetP, SignalP, Pfam20.0, Prosite, Swiss-Model, ClustalW2, DNAman, DNAstar and MEGA5. [Result] The cloned PpCCR （P. purpureum CCR） cDNA sequence was 1 316 bp, including a 1 110 bp ORF and 206 bp 3’-UTR. The cloned DNA sequence from PpCCR was 6 133 bp in full-length, containing five exons and four introns. Bioinformatic analysis indicated that PpCCR encoded a polypeptide of 369 amino acids, the secondary structure of which was primarily composed of random coil and α-helix, belonging to NAD-dependent epimerase/dehydratase family, and its co-factor binding sites and substrate binding sites were highly conserved. [Conclusion] DNA and cDNA sequences of CCR gene were obtained from P. purpureum, which had the typical characteristics of other homologous genes. The obtained bioinformatic data provided theoretical references for the further analysis of CCR and better application of P. purpureum in the future.

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 and Expression Analysis of PtFATB Gene Encoding the Acyl-acyl Carrier Protein Thioesterase in Populus tomentosa Carr

Acyl-ACP thioesterases （FATs） terminates the fatty acid synthesis and allow the transport of fatty acids out of the plastids, which are the important determinants of cellular metabolism. FATB is a member of FAT enzymes that has been described previously in most of the plants. In silico cloning is a new method that utilizes the bioinformatics on the complete genome and available EST database. In this study, a full-length cDNA clone of PtFATB gene was isolated from Populus tomentosa using this approach. It is 1,450 bp in length and the open reading frame encodes a peptide of 421 amino acids. The predicted amino acid sequence shows significant homology with those from other plant species, which contain typical domains owned by FATB proteins. The transcripts of PtFATB were abundant in leaves, and less in roots detected by using semiquantitative RT-PCR. When the shoots were subjected to the stress treatments （cold, dry, NaC1） and ABA （Abscisic acid）, the expression of PtFATB was only slightly reduced under the treatment of low temperature. This suggests that the expression of PtFATB is in a constitutive fashion. This study provides the basis not only for the identification and characterization of this gene but also for the improvement of cold tolerance by controlling the expression of the PtFATB gene in trees in near future.

Cloning and Sequence Analysis of Interleukin 10 (IL-10) Full-length cDNA from Cyprinus carpio L.

[Objective] This study aimed to obtain IL-10 (interleukin 10) full-length cDNA of common carp (Cyprinus carpio L.) and conduct the sequence analysis. [Method] The differentially expressed cDNA fragment was obtained by DD-RTPCR (differential display RT-PCR). The cDNA library of peripheral blood leukocytes which were separated from common carp and stimulated by mitogen was screened with a probe labeled with DIG (digoxigenin). The IL-10 full-length cDNA was cloned from 0.8×104 pfu of recombinant phages, and the sequence analysis and homology comparison were carried out. [Result] Sequence analysis indicated that the IL-10 full-length cDNA of common carp was 1 117 bp long, containing a 55 bp 5’-UTR, a 522 bp 3’-UTR, and a 540 bp open reading frame(ORF) encoding 179 amino acids. In addition, there were three mRNA instability motifs (ATTTA) in the 3’-untranslated region. The deduced protein sequence shared typical sequence features of the IL-10 family. Homology comparison indicated that the obtained sequence shared 89.1% homology with the carp IL-10 gene from GenBank. [Conclusion] This study laid foundation for further study of the expression manner, functional characteristic and regulation mechanism of IL-10 in vivo and the interaction mechanism in the inflammatory reaction and immune response.

Cloning and Sequence Analysis of ATPase Beta Subunit Gene from Eleutherococcus senticosus

[Objective] This study aimed to clone and analyze the cDNA of ATPase β subunit gene from Eleutherococcus senticosus.[Method] A pair of homologous primers was designed according to the chloroplast ATPase β subunit gene sequences of the known species;then the gene cDNA of E.senticosus were amplified by RT-PCR and compared with that of the known species;its structure was predicted finally.[Result] 1 099 bp of ATPase beta subunit cDNA of E.senticosus which encodes 366 amino acids was amplified by RT-PCR.Sequence comparison and structure prediction showed that amino acids encoded by the ATPase beta subunit gene of E.senticosus shared the highest homology,up to 96.41% with that of Oryza sativa.In the secondary structure,the protein contained 171 alpha helixes accounting for 46.72%,53 extended strands accounting for 14.48%,27 beta sheets accounting for 7.38% and 115 random coils which took up 31.42%.The amino acids 262-271 were the symbolic site of ATPase β subunit.The whole peptide chain had no obvious hydrophobic region and was primarily confirmed as a hydrophilic protein.[Conclusion] The cNDA of ATPase β subunit gene cloned from E.senticosus in this study will provide reference for learning the effect of energy metabolism on secondary metabolism,structure and function of ATPase in E.senticosus.

Cloning and Sequence Analysis of Interferon γ-2β Full-length cDNA in Cyprinus carpio L.

[Objective] The research aimed to carry out the cloning, identification and sequence analysis of full-length cDNA of carp interferon γ-2β （IFNγ-2β）. [Method] The cDNA library of peripheral blood leucocytes which were separated from carp and stimulated with mitogen was screened by a probe labeled with DIG. The IFNγ- 2β EST sequence was picked out from the constructed cDNA library of peripheral blood leucocyte, and the full length of carp interferon γ-2β was cloned. In addition, the sequence analysis was carried out. [Result] Three positive clones were obtained. Sequence analysis indicated that the sequence had a 119 bp 5’-UTR and a 218 bp 3’-UTR, and the open reading frame （ORF）of this gene was 537 bp which putatively coded 178 amino acids and there were several instable motifs for mRNA （ATTTA） in the 3’-untranslated region. Its homology with IFN from GenBank was up to 97% . Analysis on protein sequence and structure showed that the predicted protein sequence was identified as an IFN family signature. [Conclusion] The research laid the foundation for further studying the expression manner, function characteristic and regulation mechanism of IFNγ-2β in vivo and the action mechanism in the inflammatory reaction, emergency reaction and immune response.

Cloning,Identification and Differential Expression Analysis of Full-length cDNA of Carp Interleukin-1β

[Objective] The research aimed to carry out the cloning,identification and differential expression analysis of carp interleukin-1β （IL-1β） cDNA. [Method] By using DD-RTPCR method,the differential expression cDNA fragments were gained. The cDNA library of carp peripheral blood leucocytes which was stimulated by the mitogen was screened,and the full length cDNA of carp IL-1β was cloned. Moreover,the sequence analysis and differential expression analysis were carried out. [Result] The positive clone which had a whole ORF that encoded 276 amino acids was obtained. The cluster analysis showed that the amino acid sequence of carp IL-1β and Japanese carp closely gathered as a branch,and the homoeology of amino acid sequence reached 95%. The clustering order was the carassius,zebra fish,pig,cattle,horse,human and mouse in turn. The differential expression analysis showed that the expression of IL-1β in the leucocytes significantly increased in the prior period （4 h） after the mitogen stimulated. But as the time went by （12 and 24 h）,it didn＇t increase in the same period. The total trend of expression amount presented the peak type. [Conclusion] The research laid the foundation for further studying the expression manner,function characteristic,regulation mechanism of IL-1β in vivo and its action mechanisms in the inflammatory reaction,emergency reaction and immune response.

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 ras Promoter from Auricuralia auricular Strain YBS-3

[Objective] The ras promoters were cloned from genomic DNA of Auricuralia auricular so as to provide promoters for breeding better species by genetic technology. [Method] PCR was used to clone promoters with the genomic DNA of A. auricular strain YBS-3 as template,and then the sequences of four ras promoters were obtained. The analysis of promoter sequences was made by three promoter analysis software：Promoter prediction,Place and TFSEARCH ver.1.3. [Result] Four fragments contained core elements of promoter including TATA-box and CAAT-box,and many other important cis-elements such as GATA BOX,GCGC BOX,CCAAT BOX1,etc. At the same time,there was at least one transcriptional start site in these sequences. And several transcription factor binding sites were detected in these sequences. [Conclusion] Four promoter fragments all had promoter function theoretically.

Molecular Cloning and Sequence Analysis ofBoPGIP2 Gene from Brassica oleracea L. var. alboglabra

PGIP gene was obtained from Brassica oleracea L. var. alboglabra, named BoPGIP2. The full length of BoPGIP2 gene is 1 102 bp and the exon is 993 bp which encodes a protein of 330 amino acids with a predicted molecular mass of 37.1 kDa, interrupted by one intron of 95 bp in, length. Sequence analysis revealed that it has five potential N-giycosylation sites, two protein kinase C phosphrylation sites, five casin kinase Ⅱ phosphrylation sites and four N-myristoylation sites. The amino acids sequences alignment confirmed that ^145 LRR stucture was highly conserved in all aligned PGIP sequences.

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.

Cloning and Sequence Analysis on cDNA of Growth Hormone Releasing Hormone Receptor Gene from Wuzhishan Miniature Pig

[Objective] cDNA of growth hormone releasing hormone （GHRH） receptor gene from Wuzhishan miniature pig was cloned and its sequence was also analyzed. [Method] Using genomic DNA extracted from porcine ear tissues of Wuzhishan miniature pig as the template, three pairs of primers were designed by the reported cDNA sequence of porcine GHRH, and cDNA was also amplified by RT-PCR. After being recovered and purified, PCR products were ligated to pMD18-T and then transformed into Escherichia coli DH5a. The transformation products were analyzed by PCR and double enzyme digestion to screen positive clones, and the positive clones were sequenced after identification in LB liquid medium. [ Result] cDNA of Wuzhishan miniature pig GHRH receptor gene was obtained successfully, and its length was 1 577 bp coding 423 amino acids. BLAST analysis showed that there were only 23 nuoleotides in difference between this fragment and pomine GHRH receptor gene, and its homology was 98%. However, both GHRH receptor genes were constituted by 423 amino acids with the sequence homology of 96%. [ Conclusion] This study provides theoretical basis for further studies on the dwarf mechanism of Wuzhishan miniature pig.

cDNA Cloning, Bioinformatic and Tissue-specific Expression Analysis of Porcine JARID1C Gene

Jumonji, AT-rich interactive domain 1C （JARID1C） protein belongs to the highly conserved ARID protein family, which is involved in chromatin remodeling and transcriptional regulation during cell growth, differentiation, and development. In humans, this gene plays a vital role in normal brain development and function. Using an in silico approach in combination with 5＇ rapid amplification of cDNA ends （5＇ RACE）, the full-length cDNA of JARIDIC （GenBank accession No. EF139241） from porcine ovary, which contains 5,908 bp nucleotides, with an open reading frame （ORF） of 4,548 bp, has been cloned. The putative porcine JARID 1C protein, which is located in the nucleus, encodes 1,516 amino acids with a molecular weight of 170 kDa and a pI of 5.44. Bioinformatic prediction indicates that the protein contains several conserved domains： a JmjN domain, an ARID domain, a JmjC domain, a C5HC2 zinc finger domain, and a PHD zinc finger domain. Similarity comparisons for nucleic and amino acid sequences reveal that the porcine JARID1C protein shares a high identity with its dog, mouse, rat, and human counterparts. The phylogenetic tree of the JARID1 subfamily proteins has been constructed to reveal the evolutionary relationship of various species. Real-time PCR analysis shows that the JARIDIC gene is expressed in various tissues, but at different levels. The expression levels of this gene are higher in the brain and gonad than in other tissues, suggesting that the JARID1C protein plays a role in porcine brain and gonad functions.

Cloning and Functional Analysis of Lycopene ε-Cyclase (IbLCYe) Gene from Sweetpotato, Ipomoea batatas (L.) Lam.

This paper reported firstly successful cloning of lycopene ε-cyclase （lbLCYe） gene from sweetpotato, lpomoea batatas （L.） Lam. Using rapid amplification of cDNA ends （RACE）, lbLCYe gene was cloned from sweetpotato cv. Nongdafu 14 with high carotenoid content. The 1 805 bp cDNA sequence oflbLCYe gene contained a 1236 bp open reading frame （ORF） encoding a 411 amino acids polypeptide with a molecular weight of 47 kDa and an isoelectric point （pI） of 6.95. IbLCYe protein contained one potential lycopene ε-cyclase domain and one potential FAD （flavinadenine dinucleotide）/NAD（P） （nicotinamide adenine dinucleotide phosphate）-binding domain, indicating that this protein shares the typical characteristics of LCYe proteins. The gDNA oflbLCYe gene was 4 029 bp and deduced to contain 5 introns and 6 exons. Real-time quantitative PCR analysis revealed that the expression level of IbLCYe gene was significantly higher in the storage roots of Nongdafu 14 than those in the leaves and stems. Transgenic tobacco （cv. Wisconsin 38） expressing [bLCYe gene accumulated significantly more ^-carotene compared to the untransformed control plants. These results showed that lbLCYe gene has an important function for the accumulation of carotenoids of sweetpotato.