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 of Rabbit Bone Morphogenetic Protein 15 and Its Expression During in vitro Maturation of Rabbit Oocytes

Partial cDNA sequence of rabbit BMP15 was cloned by RT-PCR from rabbit ovaries, showing a similarity of 83%-90% with the BMP15 nucleotide sequences in humans, mice, ovine, sheep, cows and pigs. The expression of BMP15 in rabbit cumulus-oocyte complexs during oocytes in vitro maturation （IVM） was measured by fluorescent quantitative RT-PCR method. BMP 15 was expressed at low levels in immature oocytes and increased to the highest level at 16h of IVM, which coincides with the time of cumulus cell expansion, then declined slowly under IVM cultivation. The expression pattern of BMP 15 suggested that it might be important in cumulus expansion in rabbits.

Study on the Cloning and Isolation of sus scrofa GPX2 Gene by RACE Method

[Objective] Using molecular biotechnology to clone the sus scrofa GPX2 gene. [Method] Using total RNA of sus scrofa duodenum as template, degenerated primer pairs were designed according to the homology alignment analysis of GPX2 gene of human, rat, mouse, dog and cattle. A sus scrofa GPX2 gene sequence of 330 bp was obtained by RT-PCR application method. Primes were designed respectively according to the known sequence, sus scrofa GPX2 gene was isolated and cloned by 3-RACE and 5-RACE method and analyzed the gene sequence. [Result] A mRNA sequence of 924 bp was successfully cloned and isolated in this research. This sequence contained complete 3＇end and had higher sequence homology with human,mouse,cattle and dog GPX2 gene, and there was codon called TGA which encoding Sec on the position of No. 114-116 gene. [Conclusion] Sequence alignment analysis showed that the cloned gene was sus scrofa GPX2 gene （ NCBI GenBank database, the sequence number was D098982）.

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 Expression of Wheat Heat-shock Protein 60 (HSP60) Gene in E.coli

[Objective]The aim was to construct the wheat heat-shock protein 60 （HSP60） gene in prokaryotic expression vector and express HSP60 efficiently in E.coli. [Method]According to the wheat HSP60 gene sequence in GenBank,a pair of primers P1/P2 were designed and synthesized. The wheat HSP60 gene fragment was amplified from the wheat RNA by RT-PCR and inserted into bacterial expression vector of pGEX-4T-1. The construct of pGEX-4T-1-HSP60 was subsequently transformed into E.coli BL21. [Result]The construct of pGEX-4T-1-HSP60 was verified by restriction endonuclease digestion and sequenced. Compared with the sequences of wheat HSP60 genes in GenBank,homology accounted to 100%. Expression of the GST-HSP60 fusion protein was induced with IPTG. Its molecular weight was about 90 kD. The result was identified by electrophoresis of SDS-PAGE. Expression of the protein bands was consistent with the expected size. [Conclusion]The recombinant prokaryotic expression vector in pGEX-4T-1-HSP60 was constructed successfully and expressed stably in E.coli BL21. This will lay the foundation for further study on the functions of the protein and its mechanism.

Cloning of TRP Gene of Bombyx mori

[Objective]The aim was to clone Bombyx mori TRP gene.[Method]The total RNA of Bombyx mori was extracted during its pupal period with Trizol method,and then the TRP gene was cloned by RT-PCR.[Result]The full-length cDNA of TRP gene was successfully obtained,and the ORF fragment（858 bp） of TRP gene was cloned by PCR.[Conclusion]TRP gene of Bombyx mori was cloned for the first time,which could provide solid basis for the study on its function.

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 Genetic Transformation of OsOle1 Gene in Rice

[Objective] The cloning and transformation of rice OsOle1 gene were conducted in the research.[Method] OsOle1 gene was cloned by RT-PCR.The amplified OsOle1 was then ligased to pCAMBIA 1300 to construct GUS overexpression vector.Then the Agrobacterium-mediated method was used in rice callus transformation.[Result] The full-length of OsOle1 gene was 498 bp and it encoded 189 amino acids.The over-expression vector Ub：：OsOe1-GUS was prepared and transgenic plants were successfully obtained.[Conclusion] The transgenic lines laid the foundation for the function research of OsOle1.

Cloning of Atr MYB Transcription Factor Gene from Acer truncatum

[Objective] Cloning of the AtrMYB transcription factor gene from Acer truncatum was conducted to further explore the red leaf development mechanism and breed cultivars of colored-leaf maple. [Method] The Acer truncatum ‘Luhong No.1＇ cultivar was used as the material for cloning the MYB gene by mean of RTPCR and RACE-PCR. [Results] Sequence analysis showed that the fragment contained a full coding region of 831 bp encoding 276 amino acid residues with a molecular weight of 32.17 kD and a molecular formula C_（1430）H_（14052）N_（2247）O_（406）S_（14）. The gene was named as AtrMYB with a Gen Bank accession number of 1825712. This coded protein had apI of 9.44. The results showed that the AtrMYB exhibited typical features of the R2R3-MYB domain. The AtrMYB was highly homologous with the MYB of other species at nucleotide and amino acid levels. The AtrMYB had no signal peptide, but a nuclear localization signal. The phylogenetic tree showed that the AtrMYB was at the same clade as the MYB from Citrus sinensis. [Conclusion] The AtrMYB was cloned from Acer truncatum ‘Luhong No.1＇ cultivar. These results have provided a foundation for further purification and identification of target protein and function study of the AtrMYB.

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.

Identification of Senescence-associated Protein DpXTH1 and its Gene Cloning in Dahlia Petals

[Objective] This study aimed to explore the molecular mechanism of senescence in ethylene-insensitive flowers. [Method] The dahlia petals were used as matedal, and the senescence-associated proteins were isolated and identified using two-dimensional electrophoresis, mass spectrometry and an encoding gene was cloned using molecular biology techniques. [Result] In the two-dimensional elec- trophorogram of proteins from dahlia petals at building color, full flowering and flow- er senescence pedods, a total of 44 protein spots with differences in expression level more than two times were detected. From the 44 protein spots, xyloglucan （XTHs）, a senescence-associated protein, was iso- lated and identified and its expression level was increased continuously with the senescence process of dahlia petals. By using the total RNA of dahlia petals as matedal and a pair of degenerate pdmers, the cDNA sequence of XTH gene was cloned by RT-PCR. The encoding region of XTH gene has a full length of 882 bp, encoding 293 amino acid residues, and is named as DpXTH1 （Accession number： HM053613.1）. The cluster analysis showed that the amino acid sequence of DpXTH1 has high homology with those of XTHs in other plants. [Conclusion] The isolated and identified DpXTH1 from dahlia petals belonged to the XTH family in plants, and its biological function was associated with the senescence process and regulation of dahlia petals.

Cloning and expression analysis of the chloroplast fructose-1,6-bisphosphatase gene from Pyropia haitanensis

Fructose-1,6-bisphosphatase（FBPase） is one of the key enzymes in Calvin circle and starch biosynthesis. In this study, the full-length of cpFBPase gene from Pyropia haitanensis was cloned by using rapid amplification of cDNA ends（RACE） technology. The nucleotide sequence of PhcpFBPase consists of 1 400 bp, including a 5′ untranslated region（UTR） of 92 bp, a 3′？UTR of 69 bp, and an open reading frame（ORF） of 1 236 bp, which can be translated into a 412-amino-acid putative peptides with a molecular weight of 44.3 kDa and a theoretical pI of 5.23. Multiple sequence alignment indicated that the protein belonged to the chloroplast FBPase enzyme. Phylogenetic analysis showed that the protein assembled with the cpFBPase of a thermal tolerant unicellular red micro-algae Galdieria sulphuraria. Expression patterns analyzed by qRT-PCR revealed that the expression of PhcpFBPase gene in the thallus phage was 7-fold higher than in the conchocelis phage, which suggested the different mechanisms of inorganic carbon utilization among the different life phages of P. haitanensis. And the different response modes of PhcpFBPase mRNA levels to high temperature and desiccation stress indicated that PhcpFBPase played an important role in responsing to abiotic stress.

Identification of a marine agarolytic bacterium Agarivorans albus QM38 and cloning and sequencing its beta-agarase genes

A total of 117 agar-decomposing cultures were isolated from coastal seawater around Qingdao, China. The phenotypic and agarolytic features of an agarolytic isolate, QM38, were investigated. The strain was gram negative, strictly aerobic, curved rod and polar flagellum. On the basis of several phenotypic characters, biochemical and morphological characters and phylogenetic analysis of the gene coding for the 16S rRNA, the strain was identified as Agarivorans albus strain QM38. This strain can liquefy the agar on the solid agar plate. An excellular agarase activity was determined in liquid culture. The enzyme exhibited maximal activity at 40 ℃, pH 7.6. Its activity was greatly affected by different concentrations of agarose. The highest activity 32 U/ml was achieved in the culture supernatant. The hydrolytic product was analyzed by fluorophore-assisted carbohydrate electrophoresis （FACE）. After complete hydrolysis of agarose, a series of agaro-oligosaccharides were produced. The main products of the enzymes were oligosaccharides in the degree of polymerization （DP） of 2, 4, 6 and 8. Three genes agaD01, agaD02 and agaD03, encodingβ-agarases, had been cloned from genomic DNA of Agarivorans albus strain QM38. The open reading frame of agaDOl, consisted of 2 988 bp, and shared 95.5%-98.9% identity to the β-agarase genes of some strains of Vibrio and Agarivorans. Gene agaD02 comprised 2 868 bp and encoded a 955- amino-acid protein. It showed 97.4% and 98.7~0 identity to the β-agarase genes of strain Vibrio sp. PO-303 and strain Vibrio sp. JT0107, respectively. Only partial sequence of agaD03 gene has been cloned. It showed 96.5% identity to β-agarase gene （agaB） of Pseudoalteromonas sp. CY24, and shared 96.8% identity to β-agarase-c gene of Vibrio sp. PO-303.

Cloning and Characterization of a Pathogenesis-Related Protein Gene TaPR10 from Wheat Induced by Stripe Rust Pathogen

Pathogenesis-related proteins （PRs） play many important roles in plant defense response against pathogen attack. To better understand the molecular mechanism of PR genes involved in wheat adult plant resistance （APR） to stripe rust, based on a differentially expressed transcribed derived fragment （TDF）, a novel PR gene from wheat cv. Xingzi 9104 infected by the Puccinia striiformis Westend f. sp. tritici Erikss. pathotype CY32, which was highly similar to the maize ZmPRIO gene and designated as TaPRIO, was identified using in silico cloning and RT-PCR method. This novel TaPRIO gene was predicted to encode a 160-amino acid protein with a deduced molecular weight of 17.06 kDa and an isoelectronic point （pI） of 5.19. An amino acid sequence analysis of TaPR10 demonstrated the presence of a typical conserved domain of pathogenesis related protein Bet v I family. Multiple alignment analysis based on the amino acids encoded by 10 different PRIO genes from maize （Zea mays）, rice （Oryza sativa）, broomcorn （Sorghum bicolor）, and wheat （Triticum aestivum） indicated that PR proteins of class 10 was conserved among the 4 plant species with about 80% similarity. DNA sequence of TaPRIO suggested the presence of one 84-bp intron with the splicing sites of GT-AT bi-nucleotide sequence between 188 and 271 bp. Using a real-time quantitative RT-PCR （qRT-PCR）, expression profiles of TaPRIO revealed that at the adult-plant stage, TaPRIO transcript was up-regulated as early as 12 h post-inoculation （hpi）, with the occurrence of maximum induction at 24 hpi. At the seedling stage, TaPRIO was also slightly induced 18 hpi. However, the transcript amount was relatively lower than that of the adult-plant stage. Taken together, these results suggest that TaPRIO may participate in wheat defense response of APR to stripe rust.

Cloning and Phylogenetic Analysis of a Fatty Acid Elongase Gene from Nannochloropsis oculata CS179

Nannochloropsis oculata CS179, a unicellular marine microalga, is rich in long-chain polyunsaturated fatty acids （LCPUFAs）. Elongase and desaturase play a key role in the biosynthesis of PUFAs. A new elongase gene, which encodes 322 amino acids, was identified via RT-PCR and 5＇ and 3＇ RACE. The sequence of the elongase gene was blast-searched in the NCBI GenBank and showed a similarity to those of the coptosporidium. But the NJ-tree revealed that the N. oculata CS 179 elongase clustered with those of the microalgae Phaeodac^lum tricornutum, Ostreocoecus tauri and Thalassiosira pseudonana.

Cloning and Expression Analysis of an AP2/ERF Gene and Its Responses to Phytohormones and Abiotic Stresses in Rice

Ethylene response factors （ERFs） play important roles in response to plant biotic and abiotic stresses. In this study, a gene encoding a putative AP2/ERF domain-containing protein was isolated by screening a SSH cDNA library from rice and designated as Oryza sativa AP2/ERF-like protein （OsAP2LP） gene. OsAP2LP is 1491 bp in length, interrupted by seven introns, and encodes a putative protein of 348 amino acids. Temporal and spatial expression analysis showed that the OsAP2LP gene was preferentially expressed in roots, panicles, mature embryos and seeds in rice. Real-time quantitative PCR analysis indicated that the expression levels of the OsAP2LP gene were increased under the treatments of drought and gibberellin but decreased under the treatments of low temperature, salt, abscisic acid （ABA） and zeatin. Taken together, these results suggest that OsAP2LP might be involved in stress responses, and probably plays roles as a transcription regulator when plants response to cold, salt and drought stresses through ABA and gibberellin pathways.

Cloning and characterization of a stearoyl-ACP desaturase gene from Jatropha curcas

Using degenerate primers and RT-PCR, RACE techniques, a 1491 bp cDNA segment of stearoyl-acyl carrier protein desaturase （SAD） is cloned from developing seeds of Jatropha curcas L. The segment contains a 1191 bp of complete open reading frame （ORF）. Analysis in the BLAST on NCBI shows that Jatropha curcas SAD （JSAD） gene encodes a protein precursor composed of a signal peptide of 33 amino acids and a mature peptide of 363 amino acids. The homological analysis shows that JSAD has high level of homology both in nucleotide sequence and in amino acid sequence to other plants SADs. The nucleotide and peptide identity of JSAD to Ricinus communis SAD （RSAD） is up to 89% and 96.2% respectively. Molecular modeling of JSAD indicates that its three-dimensional structure strongly resembled the crystal structure of RSAD.

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 Characterization of WOX4 Gene from Vitis vinifera L. Involved in Stem Cell Regulation

Wuschel-related homeobox （WOX） genes play essential, specific, and sometimes redundant roles in plant embryo development, shoot and root meristem maintenance, and plant development. Though much information was quickly gained with members of the WOX gene family of Arabidopsis, monocotyledonous crops, and gymnospermous conifers, little is known about perennial woody plants. In this study, we isolated the first WOXgene family member from grape （Vitis vinifera L. cv. Cabernet Sauvignon）, and named it VvWOX4 based on its characteristic domains and phylogenetic analysis. The identity of VvWOX4 was validated by MALDI-TOF MS and Western blot with polyclonal antibody against Arabidopsis thaliana Wuschel. Functional analysis showed that VvWOX4 markedly increased shoot primordia structures when overexpressed under CaMV 35S promoter in tobacco. A different expression pattern was found for VvVOX4 compared with AtWUCHEL and its expression was detected in unique organs of grapevines. Besides the expression in the vegetative shoot apical meristem （SAM） of grape shoot tips, VvWOX4 is expressed in dormant winter buds, inflorescence, young leaves, and tendril tips, but not in root tips. In young leaves, the expression of VvWOX4 is strongly upregulated by wounding, and also by plant growth regulators such as 2 mg L-1 2,4-D, 1 mg L-1 NAA and 1 mg L-1 BAP treatments, while downregulation was monitored by 1 mg L^-1 IBA treatment, and there was no response to 0.5 mg L-1 GA3 treatment. Together, our results revealed the first member of grape WOX gene family and indicated different roles and regulation of VvWOX4 in the perennial woody crop grapevine.