Cloning and Bioinformatics Analysis of CsFK111 Gene from Cucumbers

At the early stage,the transcriptome sequencing technique was used to detect the differentially expressed gene CsFK111 between vine cucumber and dwarf cucumber D0462.The gene was cloned,and bioinformatics software tools were used to analyze and predict the gene family and this gene.There were 30 members of the cucumber F-box gene family.The coding region of the cucumber CsFK111 gene was full-length 1314 bp,which encoded 437 amino acids and was predicted to be located in the nucleus.The protein encoded by this gene was a non-transmembrane protein,and the prediction of the secondary structure showed thatβ-lamellar structure and irregular crimp were dominant.A comparison of the phylogenetic tree showed that it was closest to cantaloupe and belonged to the same branch.The results provided a basis for future study on the regulation mechanism of the CsFK111 gene on cucumber dwarfing and also laid a foundation for further study of FBK family proteins.

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, Characterization and Chromosome Localization of Two Powdery Mildew Resistance-Related Gene Sequences from Wheat

Reverse_transcription Polymerase Chain Reaction (RT_PCR) was performed using cDNAs as templates from wheat_ Haynaldia villosa 6VS/6AL translocation line and 'Yangmai 5' induced with fungus Erysiphe graminis , and degenerate primers designed based on the conserved amino acid sequences of known plant disease_resistance genes. The cDNA sequences encoding cyclophilin_like and H +_ATPase_like genes were first isolated and characterized in wheat. The putative amino acid sequences of the two clones showed that they were highly homologous to those of cyclophilin proteins and H +_ATPases isolated from other plants. Thus they were designated as Ta_Cyp and Ta_MAH . The obvious expression differences could be observed between wheat_ H. villosa 6VS/6AL translocation line and susceptible wheat cultivar 'Yangmai 5', implying that the two genes may be related with the resistance of wheat_ H. villosa 6VS/6AL translocation line to disease. Southern blot indicated that the wheat genome contained 2-3 copies of Ta_Cyp gene and one copy of the Ta_MAH gene. Chinese Spring nulli_tetrasomic line analysis located the Ta_Cyp homologous genes on wheat chromosome 6A, 6B and 6D. Southern blot using Ta_Cyp clone as a probe showed that the polymorphic bands existed among the H. villosa , amphiploid of Triticum durum _ H. villosa , wheat_ H. villosa 6VS/6AL translocation line and 'Yangmai 5', suggesting that Ta_Cyp homologies exist in wheat genome as well as on the short arm of chromosome 6V in H. villosa .

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.

Cost-Effective Method of Gene Synthesis by Sequencing from Microchip-Derived Oligos for Droplet Cloning

Gene synthesis has provided important contributions in various fields including genomics and medicine. Current genes are 7 - 30 cents depending on the assembly and sequencing methods performed. Demand for gene synthesis has been increasing for the past few decades, yet available methods remain expensive. A solution to this problem involves microchip-derived oligonucleotides (oligos), an oligo pool with a substantial number of oligo fragments. Microchips have been proposed as a tool for gene synthesis, but this approach has been criticized for its high error rate during sequencing. This study tests a possible cost-effective method for gene synthesis utilizing fragment assembly and golden gate assembly, which can be employed for quicker manufacturing and efficient execution of genes in the near future. The droplet method was tested in two trials to determine the viability of the method through the accuracy of the oligos sequenced. A preliminary research experiment was performed to determine the efficacy of oligo lengths ranging from two to four overlapping oligos through Gibson assembly. Of the three oligo lengths tested, only two fragment oligos were correctly sequenced. Two fragment oligos were used for the second experiment, which determined the efficacy of the droplet method in reducing gene synthesis cost and speed. The first trial utilized a high-fidelity polymerase and resulted in 3% correctly sequenced oligos, so the second trial utilized a non-high-fidelity polymerase, resulting in 8% correctly sequenced oligos. After calculating, the cost of gene synthesis lowers down to 0.8 cents/base. The final calculated cost of 0.8 cents/base is significantly cheaper than other manufacturing costs of 7 - 30 cents/base. Reducing the cost of gene synthesis provides new insight into the cost-effectiveness of present technologies and protocols and has the potential to benefit the fields of bioengineering and gene therapy.

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.

Cloning of Potato POTHR-1 Gene and Its Expression in Response to Infection by Phytophthora infestans and Other Abiotic Stimuli

A complete cDNA of potato Phytophthora infestans-induced hypersensitive response-related protein gene (POTHR-1) was cloned using rapid amplification of cDNA ends (RACE) strategy according to a fragment sequence which we had cloned using suppression subtractive hybridization (SSH) technique. The potato POTHR-1 gene encodes a protein of 225 amino acids, which shares 81% identity with tobacco hin1 gene-enoded protein (harpin-induced protein). Southern blot revealed that there are two to three copies of POTHR-1 in potato genome. The POTHR-1 gene expression in potato leaves showed that its transcripts accumulated remarkably in leaves after 36 h inoculation with P. infestans. Mechanical wounding and jasmonic acid (JA) could induce the POTHR-1 gene expression and osmotic stress just induce a slight accumulation of POTHR-1 gene mRNA, while salicylic acid (SA) had no detectable function on the induction accumulation of POTHR-1 gene transcripts. The potato POTHR-1 gene may preferentially associate with hypersensitive response (HR) or biotic cell death during interaction between host and pathogen.

Molecular Cloning and Expression of RSSG58 Gene in Rice Sperm Cells

Myosins, a large family of structurally diverse mechanoenzymes, which, upon interaction with actin filaments, convert energy from ATP hydrolysis into mechanical force, play an important role in male reproductive processes. In this study we report the rice ( Oryza sativa L.) RSSG58 gene, which was cloned from the cDNA library of rice sperm cells by using sperm cell mainly expression subtractive clone as probe. This gene encodes a putative 66.7 W polypeptide, which shows similarity to the myosin heavy chain of Arabidopsis thaliana, and consists of 579 amino acids with an isoelectric point (pI) of 4.885. RSSG58, which is a member of a divergent gene family, generates transcripts of 2 278 bp and 2 437 bp that differ only in their polyadenylation sites. Southern hybridization showed that RSSG58 has only one copy in rice genome and RSSG58 transcripts are most abundant in sperm cells, with two distinct signals. The RT-PCR analysis indicated that the transcriptions of the RSSG58 gene were various in the different development stages and tissues. The greatest accumulation of RSSG58 mRNA was detected in sperm cells, while weaker expression was detected in leaves, microspore mother cells, unicellular microspore pollen stage, two-cell stage pollens, mature pollens and pollinated ovaries. These results suggest that RSSG58 is especially abundantly expressed in rice sperm cells.

Cloning and Analysis of WRKY Gene of Rice Induced by Rhizoctonia solani Kuhn

[Objective] The aim was to clone the up-regulated expression gene of rice induced by Rhizoctonia solani.[Method] The EST fragment K16 obtained by suppression subtraction hybridization（SSH）was cloned and confirmed by reverse transcription-polymerase chain reaction（RT-PCR）.Then RT-PCR products were cloned into the PMD18-T vector and sequenced.The functions of the sequence were predicted with bioinformatics method.[Result] A 1 079 bp gene was obtained.The gene encoded a protein with 236 amino acids.The protein contains many motif sites,two WRKY domains and a C2H2 zinc finger motif.The gene showed high identities with WRKY8,WRKY24 and WRKY30 gene of rice.[Conclusion] The up-regulated expression gene induced by R.solani was representative WRKY family gene.The gene could play an important role on rice sheath blight resistance.

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 Prokaryotic Expression of NS1 Gene of Porcine Parvovirus (PPV) SD1 Strain

[Objective] The research aimed to provide the theoretical basis for establishing a rapid diagnosis method for porcine parvovirus（PPV）. [ Method] One pair of primers were designed according to PPV genome sequences on GenBank website and the sequences of prokaryotic expression vector pET30a （ ＋ ） with multiple cloning sites. The whole sequence of NS1 gene in PPV SD1 strain was amplified by using PCR technology and the positive recombinant plasmid was analyzed by sequencing and homology comparison. The prokaryotic expression recombinant plasmid PET30a/NS1 was constructed to make its induction expression in Escherichia coll. [ Result] The target fragment with the length of 2 208 bp was obtained from PCR amplification. The nucleotide homologies between the cloned NS1 gene and the reported relevant PPV genes were from 97.3 % to 99.4 %, which indicated that NS1 gene had high conservation. But it had a 12-basepair successive deletion near the hydroxyl end. The cloned PPV NS1 gene was successfully expressed in prokaryotic cell, and its expression products existed mostly in inclusion bodies. [ Conclusion] The results of SDS-PAGE detection showed that the molecular weight of PPV NS1 protein was 86 KD.

Molecular Cloning and Bioinformatics Analysis of cyaA Gene of Vibrio alginolyticus Strain HY9901

[Objectives]This study was conducted to explore the biological functions of cyaA gene of Vibrio alginolyticus.[Methods]With DNA of V.alginolyticus HY 9901 as a template,primers were designed according to the sequence of cyaA gene,and the cyaA gene was amplified by PCR.Bioinformatics analysis was performed.[Results]The cyaA gene of V.alginolyticus HY9901 was 2529 bp in size,and encoded 842 amino acids.The molecular structure of CyaA protein was C_(4358)H_(6745)N_(1171)O_(1286)S_(35).Its theoretical molecular weight was 97.24167 kDa and the theoretical pI value was 5.56.It had no signal peptide and transmembrane domain.CyaA protein had three N-terminal glycosylation sites,one cAMP and cGMP-dependent protein kinase phosphorylation site,nine protein kinase C phosphorylation sites,nine casein kinase II phosphorylation sites,one tyrosine kinase phosphorylation site,seven N-terminal myristoylation sites,one pentenyl binding site and ten microbody C-terminal localization signal sites.Subcellular localization prediction showed that CyaA protein was mainly located in the nucleus and cytoplasm.Through multi-sequence alignment and phylogenetic tree construction,it was concluded that V.alginolyticus had high CyaA homology with other Vibrio species.cyaA of V.alginolyticus was clustered with Vibrio fluminensis and Vibrio marinisedimini,and they were closely related.The secondary structure of CyaA protein consisted ofα-helixes(43.11%),random coils(38.00%)and extended strands(14.49%).In protein network interaction,it was found that the proteins adjacent to CyaA protein were Crp-2,CpdA,Crr,PtsG-2,ANP67209.1,Crp-1,PykF,Pyk,RelA and Ndk.[Conclusions]This study provides a new idea for formulating strategies for the prevention and control of vibriosis.

Cloning and Expression of Conserved Sequences of cry Gene in E.coli Strain Rosetta(DE3)

[Objective] The aim was to clone the conserved sequences of cry gene and express them in Rosetta （DE3）. [Method] Specific primers were designed according to NCBI database information and the conserved sequences of cry gene were amplified by PCR from Bt transgenic cotton. Then recombinant plasmids were constructed and expressed in E. coil strain Rosetta （DE3）. Finally, the effects of different concentrations and inducing time of IPTG on the expression level of protein were investigated. [Result] Two conserved sequences （304 and 853 bp respectively） of cry gene were amplified. The result of SDS-PAGE confirmed that the recombinant plasmids pGEX-4t-I-304 and pGEX-4t-1-853 could express fusion proteins by IPTG induction and the molecular weight of protein products was 39 and 62.4 kDa respectively, which was in accordance with predicted result. The optimal protein ex- pression conditions were confirmed as induction with 0.15 mmol/L IPTG for 7 h. [Conclusion] This study prepared the ground for the further detection of Bt transgenic crops.

Cloning and Characterization of β-actin Gene in Dunaliella parva

[Objective] The aim was to obtain the full-length cDNA sequence of Dunaliella parva β-actin gene.[Method] Based on the highly conserved amino acid regions of known β-actin,a pair of degenerate primers was synthesized to amplify 533 bp cDNA sequences in Dunaliella parva.Then,the 5＇ genomic DNA and 3＇ cDNA sequences were obtained by Genome walking and 3＇-RACE technology based on the obtained sequence.According to the sequences of the 5＇-termini and 3＇-termini,specific primers were synthesized to obtain the full-length cDNA.[Result] The full-length β-actin cDNA included 1 137 bp open reading frame（ORF）,617 bp of 3＇ noncoding region.Similarity analysis indicated that the highest similarity was found between Dunaliella parva and Dunaliella salina.The Dunaliella parva β-actin also showed wide similarity with other algae.[Conclusion] The full-length cDNA sequence of D.parva was firstly obtained,which was highly conserved.

Cloning the Structure Genes and Expression the N Gene of Porcine Epidemic Diarrhea Virus DX

The structure genes spike(S) ,nucleocapsid(N) ,membrane(M) ,small membrane(sM) of a porcine epidemic diarrhea virus(PEDV) strain DX isolated in Gansu province,North-west of China,were cloned,sequenced and compared with published sequences of PEDV strains. The nucleotide sequences encoding the entire S,sM,M and N genes open reading frame(ORF) of DX were 4 152,231,681 and 1 326 bases long respectively. There were transcription regulatory sequences(TRSs) upstream of the initiator ATG of the S,N and M genes. The amino acids sequences of S,M and N contained 30,3 and 7 potential asparagine(N) -linked glycosylation sites. Homologous analysis and phylogenetic trees showed that DX had the closest relationship with strains LJB/06,JS-2004-2Z and CH/HLJH/06 that were also isolated from China and indicated the prevalence of some PEDV isolates in China were widespread since the JS-2004-2Z strain originated from the south of the China,and LJB/06 and CH/HLJH/06 were isolated from northeast China. The N gene was cloned using two primers which contained Nco I and BamH I restriction enzyme sites and subcloned into expression vector pET30a. The recombinant plasmid was then transformed into E.coli Rossta. SDS-PAGE showed there was a protein of about 55kDa as expected and Western blot indicated the N protein had biological activity.

Cloning and Expression of pilA Gene of Outer Membrane Protein of Haemophilus parasuis

[Objective] The aim was to clone and express the pilA gene of outer membrane protein of Haemophilus parasuis.[Method] The published pilA gene sequence of HPS was analyzed for primer synthesis,and the genome of serotype 5-type of HPS was used as template for PCR amplification of the pilA gene of HPS;the recombinant expression plasmid was constructed and transformed into E.coli BL21（DE3）after induced by IPTG.SDS-PAGE and Western blot analysis were then carried out.[Result] The molecular weight of expressed protein was consistent with the expected（43 kD）.[Conclusion] The results provided a foundation for the preparation of subunit vaccine and diagnostic reagents.

Cloning and Functional Analysis of HDR Gene from Ginkgo biloba L

[Objective] The aims were to obtain cloning of HDR gene from Ginkgo biloba.and study its function.[Method] The coding sequence of HDR gene was cloned from G.biloba by reversed transcription polymerase chain reaction,which was designated as GbHDR （GenBank accession No.：DQ364231）.The cDNA full-length of GbHDR is 1 827 bp containing a 1 425-bp open reading frame （ORF） encoding a 474-amino-acid polypeptide and constructed into the prokaryotic expression vector pTrcGbHDR.The β-carotene biosynthetic pathway in E.coli strain XL1-Blue was reconstructed by transforming with pAC-BETA.This engineered XL1-Blue was transformed with pTrcGbHDR.[Result] A 1 441 bp GbHDR was obtained containing a 1 425-bp ORF encoding a 474-amino-acid residues of protein,the predicted molecular weight was 53.2 kD,and predicted isoelectric point was 5.76.Functional complementation assay indicated that GbHDR could promote theβ-carotene accumulation in engineered XL1-Blue harboring pTrcGbHDR and pAC-BETA,and as a result,the engineered bacteria showed the brightly orange given by β-carotene.This suggested that GbHDR had the typical function of known HDR genes.[Conclusion] A engineered bacteria of E.coli which could highly accumulate β-carotene was obtained,which will provide candidate genes and targets for realizing β-carotene metabolic engineering.

Cloning of Acetyl-CoA Carboxylase accD Subunit Gene from Brassica campestris and Effect of Its Expression on Oil Content

[Objective] This study aimed to clone accD gene from Brassica campestris （Yunnan small rapeseed） with different oil content and analyze the effect of accD gene expression level on rapeseed oil content. [Method] The accD gene encoding β-CT Ⅱsubunit of acetyl-CoA carboxylase was cloned from Yunnan small rapeseeds with different oil content. Then, sequence alignment and the influence upon seed oil content by the expression of accD gene were analyzed. [Result] There were a few base pair substitutes among the accD genes from different Yunnan small rapeseeds, and their homology was up to 98.6%. The expression of accD gene was increased with reproduction development, and reached peak in late-stage siliques. The seed oil content was positively influenced by the expression of accD gene in middle-stage and late-stage siliques. [Conclusion] This study provides a certain theoretical basis for illustrating the molecular mechanism about rapeseed oil content and breeding new high-oil rapeseed cultivars.

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.