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.

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,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.

Identification and expression analysis of group Ⅲ WRKY transcription factors in cotton

The WRKY proteins constitute a large family of transcription factors in plants containing highly conserved WRKYGQK sequences and zinc-finger-like motifs. To comprehensively study WRKY III genes in cotton, we analyzed the genome sequences of Gossypium hirsutum, G. raimondii and G. arboreum. According to the three genome sequences, 18 group III Gh WRKY genes were identified in G. hirsutum, 12 both in G. raimondii and G. arboreum. Phylogenetic and motif analysis showed that proteins with high similarities could be clustered together and had the same motif components. The ratios of non-synonymous（Ka） to synonymous（Ks） of the Gh WRKY to Gr WRKY or Ga WRKY were lower than 1, which indicated that group III WRKY genes in Gossypium species are under purifying selection. Expression analysis revealed that group III Gh WRKY genes expressed during fiber development and leaf senescence, and most of them could be induced by salicylic acid（SA）, jasmonic acid（JA）, ethylene, abscisic acid（ABA）, mannitol, and Na Cl both in roots and cotyledons. Our study gives a briefly introduction on cotton group III WRKY genes and implicates their potential function in cotton fiber development, leaf senescence and abiotic stresses.

Transcriptome-based identification and expression analysis of the glutathione S-transferase(GST)family in tree peony reveals a likely role in anthocyanin transport

For red-flowered cultivars of tree peony(Paeonia suffruticosa),anthocyanin content is a critical factor determining the different petal pigmentations.Glutathione S-transferases(GSTs)are ubiquitous and multifunctional conjugating proteins that may be responsible for the transport of anthocyanin pigments from the cytoplasm to vacuole.The underlying function of the GST family in tree peony,however,remains unclear.In this study,we systematically isolated and identified a total of 54 putative full-length Ps GST genes through a combination of bioinformatics approaches from transcriptome databases.Intraspecific phylogenetic analyses revealed extensive differentiation in their coding sequences and divided them into 10 of the 14 known classes of plant GSTs.The phylogenetic relationships,evolutionary characteristics,protein domain,and motif organization were clearly conserved among the different phylogenetic subclasses.The results of the RNA-seq and quantitative real-time polymerase chain reaction experiments exhibited extensive variation in gene expression profiles among different developmental stages and varieties.Furthermore,the phylogenetic relationships,expression profiles,protein interactions,weighted gene co-expression network analysis,and correlation analysis results suggested that PsGSTF3(Unigene 0064200)is a candidate participant in anthocyanin transport and the promotion of pigment accumulation,exhibiting a strong positive correlation with anthocyanin content among different tissues(r=0.908**)and an increasing rate of anthocyanin content during the flower developmental process(r=0.961*).These results furthered our understanding of the transport and accumulation functions of the GST family as well as the enhancement of tree peony breeding through molecular biology techniques.

Genome-wide Identification,Classification and Expression Analysis of Lhc Supergene Family in Castor Bean(Ricinus communis L.)

Light-harvesting chlorophyll a/b-binding （LHC） proteins are a group of nuclear-encoded thylakoid proteins that play a key role in plant photosynthesis and are widely involved in light harvesting, energy transfer to the reaction center, maintenance of thylakoid membrane structure, photoprotection and response to en- vironmental conditions, etc. Although/dw supergene family is well characterized in model plants such as Arabidopsis, rice and poplar, little information is available in castor bean （Ricinus communis L. ）. In this study, a genome-wide search was carried out for the first time to identify castor bean L/w genes and analyze the gene structures, biochemical properties, evolutionary relationships and expression characteristics based on the published data of castor bean genome and ESTs. According to the results, a total of 28 Rclhcs genes representing 13 gene families （ l_hca , l_hcb , Elip , Ohpl , Ohp2 , SEP1, SEP2 , SEP3 , SEP4 , SEP5 , PsbS , Rieske and FCII） and 25 subgene families were identified in castor bean genome; to be specific, 25 and 5 genes were found to have corresponding ESTs in NCBI and have al- ternative splicing isoforlns, respectively. These RcLhcs contain 0 to 9 introns and distribute on 26 of the 25 878 released scaffolds. All RcLhcs genes were found to be expressed in all examined tissues, i.e. leaf, flower, II/III stage endosperm, V/VI stage endosperm and seed, with the highest expression level in leaf tissue.

Molecular cloning and expression analysis of Crustin-like gene from Chinese shrimp Fenneropenaeus chinensis

A new member of antimicrobial protein genes of the Crustin family was cloned from haemocytes of the Chinese shrimp Fennero- penaeus chinensis by 3 ′and 5′ RACE. The full-length cDNA of Crustin-like gene contains a 390 bp open reading frame, encoding 130 amino acids. The deduced peptide contains a putative signal peptide of 17 amino acids and mature peptide of 113 amino acids. The molecular mass of the deduced mature peptide is 12. 3 ku. It is highly cationic with a theoretical isoelectric point of 8.5. The deduced amino acids sequence of this Crustin showed high homology with those of Penaeus （ Litopenaeus ） setferus. Northern blotting showed that the cloned Crustin gene was mainly expressed in haemocytes, gill, intestine, and RNA in situ hybridization indicated that the Crustin gene was constitutively expressed exclusively in haemocytes of these tissues. Capillary elec- trophoresis RT-PCR analysis showed that Crustin was up-regulated dramatically from 12 to 48 h after a brief decrease of mRNA during first 6 h in response to microbe infection. The level of Crustin mRNA began to restore at 72 h post-challenge. This indicated that Crustin gene might play an important role when shrimps are infected by bacterial pathogen.

Expression Analysis of the Insulin-Like Growth Factors Ⅰ and Ⅱ During Embryonic and Early Larval Development of Turbot(Scophthalmus maximus)

The insulin-like growth factors Ⅰ and Ⅱ （IGF-Ⅰ and IGF-Ⅱ） are important proteins involved in fish growth and develop- ment. Here, we report the isolation of IGF-Ⅱ and expression analysis of IGFs in turbot Scophthalmus maximus, aiming to clarify their function in embryonic and larval development of fish. The deduced IGF-Ⅱ gene is 808 bp in full length, which encodes a protein of 219 amino acids and is 93% similar with that ofParalichthys olicaceus in amino acid sequence. The tissue abundance and the ex- pression pattern of IGFs in a turbot at early development stages were investigated via reverse transcription-polymer chain reaction. Result showed that the IGF-Ⅰ and IGF-Ⅱ genes were widely expressed in tissues of S. maximus. IGF-Ⅰ was detected in all tissues ex- cept intestines with the highest level in liver, while IGF-Ⅱ transcript presented in all tissues except muscle. At the stages of embry- onic and larval development, the mRNA levels of IGFs sharply increased from the stage of unfertilized egg to post larva, followed by a decrease with larval development. However, there was an increase in IGF-Ⅰ at the embryonic stage and IGF-Ⅱ at the gastrula stage, respectively. These results suggested that IGFs play important roles in cell growth and division of the turbot. Our study provides reference data for further investigation of growth regulation in turbot, which can guarantee better understanding of the physiological role that IGFs play in fish.

Novel structural annotation and functional expression analysis of GTP_EFTU conserved genes in pepper based on the PacBio sequencing data

Genes containing GTP_EFTU domain mainly express elongation factors(EF),Small GTPases,and GTP-binding proteins,which are closely related to protein synthesis,extension and ATP synthesis.In this study,we identified 39 genes containing GTP_EFTU domains from peppers.The evolutionary trees constructed from capsicum,Arabidopsis,rice,and tomato are mainly divided into 7 subfamilies.Using PacBio(Pacific Biosciences)sequencing and assembly data,we extracted these 39 gene sequences,fromwhich 25 genes had alternative splicing.Particularly,the Capana08g000545 had 16 alternative splicing processes.Accordingly,we performed promoter sequence analysis,subcellular location prediction,the expression analysis of different tissues and periods,and also the GO(Gene ontology)analysis of co-expressed genes.Lastly we did the qRTPCR analysis in 5 stages of pepper fruit development.These analyses revealed important structural and functional information for the identified 39 genes that contain GTP_EFTU domains,providing important references for further follow-up experiments to verify the genes function on plants or their unique roles in peppers.

Cloning and Expression Analysis of a Prion Protein Encoding Gene in Guppy (Poecilia reticulata)

The full length cDNA of a prion protein (PrP) encoding gene of guppy (Poecilia reticulata) and the corresponding ge-nomic DNA were cloned. The cDNA was 2245 bp in length and contained an open reading frame (ORF) of 1545 bp encoding a pro-tein of 515 amino acids,which held all typical structural characteristics of the functional PrP. The cloned genomic DNA fragment corresponding to the cDNA was 3720 bp in length,consisting of 2 introns and 2 exons. The 5’ untranslated region of cDNA origi-nated from the 2 exons,while the ORF originated from the second exon. Although the gene was transcribed in diverse tissues in-cluding brain,eye,liver,intestine,muscle and tail,its transcript was most abundant in the brain. In addition,the transcription of the gene was enhanced by 5 salinity,implying that it was associated with the response of guppy to saline stress.

Cloning and Expression Analysis of a Mitogen-Activated Protein Kinase Gene OsMPK14 in Rice

Mitogen activated-protein kinases （MAPKs） are important components in signal transduction pathways responding to various biotic and abiotic stresses. An MAPK gene, OsMPK14 （GenBank Accession No. GQ265780） from rice （Oryza sativa L.）, was cloned by RT-PCR. The full-length cDNA of OsMPK14 consists of 1660 bp in size, containing an open reading frame of 1629 bp, which encodes a 542-amino-acid polypeptide and has a typical protein kinase domain and a phosphorylation activation motif TDY. Sequence alignment and analysis revealed that OsMPK14 was located on rice chromosome 5, and composed of nine exons and eight introns in the coding region. Semi-quantitative RT-PCR was performed to detect the expression patterns of OsMPK14 in rice shoots and roots under darkness, drought, high salinity, low temperature and abscisic acid treatments. The OsMPK14 mRNA was induced by abscisic acid, low temperature and high salinity, but weakly inhibited by drought. In addition, the expression of OsMPK14 was up-regulated in roots, but down-regulated in shoots by light. The results indicate that OsMPK14 could be implicated in diverse rice stimuli-responsive signaling cascades, and its expression might be regulated by multiple factors.

Cloning and Expression Analysis of FaSRP Gene in Festuca arundinacea under Abiotic Stresses

[Objective] This study aimed to study the structure and functions of SRP gene and variation in its expression under abiotic stresses. [Method] Using the SRP sequence obtained from transcriptome sequencing as the template, the full-length cDNA sequence of SRP gene in Festuca arundinacea was amplified using the 3＇RACE and 5＇RACE methods. [Result] The cDNA sequence of SRP gene has a full length of 1 165 bp, and it contains an open reading frame in full length of 855 bp. The encoded protein by SRP gene is composed of 284 amino acids, and contains a REF domain. The bioinformatic researches on structures and functions of SRPs show that the SRP gene in Festuca arundinacea （FaSRP） has relatively high ho- mologies with SRPs in monocots. Under low nitrogen, drought, high temperature and high salt stresses, the variations in expression of FaSRP gene were studied using fluorescence quantitative PCR. The results showed that FaSRP gene makes responses to low nitrogen, drought and high temperature stresses, but the relevant response mechanisms are not the same, indicating different pathways regulating re- sistance of plants. The expression of FaSRP gene is insensitive to high salt stress. [Conclusion] This study will provide certain candidate gene and technical reserve for breeding of drought- and high temperature-tolerant, nutritious and highly efficient Festuca arundinacea cultivars.

Expression Analysis of 14-3-3 Gene in Tall Fescue under Several Abiotic Stresses

To study the functions of 14-3-3 gene family in tall fescue, the potential functions of 13 14-3-3 proteins in Arabidopsis were investigated by bioinformatic analysis. Based on the sequences of 14-3-3 genes in tall fescue by transcriptome and proteomic sequencing, the full-length cDNA sequences of 4 14-3-3 genes in tall fescue were obtained. Their sequences were aligned by Clustal W2. The results showed that the genetic relationships between 14-3-3A and 14-3-3D, 14-3-3B and 14-3-3C are closer, and their main structures are very conservative. The changes in expression levels of 14-3-3 genes under low nitrogen, drought, high temperature and high salt stresses were investigated by fluorescence quantitative PCR. The expres- sion level of 14-3-3A makes responses to low nitrogen, drought, high temperature and high salt stresses; the expression levels of other genes also make responses to abiotic stresses in varying degrees, but the relevant response mechanisms are not exactly the same. Therefore, it is speculated that the 14-3-3 gene family regu- lates stress resistance of plants through different pathways, and functional differenti- ation occurs during its evolution.

Molecular cloning, characterization and expression analysis of a catalase gene in Paphia textile

Catalase is an important antioxidant protein that can protect organisms against various forms of oxidative damage by eliminating hydrogen peroxide. In this study, the catalase c DNA of Paphia textile（Pt CAT） was cloned using RTPCR and rapid amplification of c DNA ends（RACE）. Pt CAT is 1 921 bp long and consists of a 5′-UTR of 50 bp, a 3′-UTR of 349 bp, and an ORF of 1 542 bp that encodes 513 amino acids with a molecular weight of 58.4 k D and an estimated isoelectric point of 8.2. Sequence alignment indicated that Pt CAT contained a highly conserved catalytic signature motif（^（61）FNRERIPERVVHAKGAG^（77））, a proximal heme-ligand signature sequence（^（352）RLFSYSDP^（359））, and three catalytic amino acid residues（H^（72）, N^（145）, and Y^（356））. Pt CAT also contains two putative N-glycosylation sites（^（34）NKT^（36） and ^（437）NFT^（439）） and a peroxisome-targeting signal（^（511）AQL^（513））. Furthermore, Pt CAT shares 53%–88% identity and 29%–89% similarity with other catalase amino acid sequences. Pt CAT m RNA was present in all tested organs, including the heart, digestive gland, adductor muscle, gonad, gill, and mantle, but its expression was highest in the digestive gland. High-temperature-induced stress produced two expression patterns of Pt CAT m RNA： first, an initial up-regulation followed by a down-regulation in the heart, digestive gland, and gonad and, second, consistent down-regulation in all other organs. These results demonstrate that Pt CAT is a typical member of the catalase family and might be involved in the responses to harmful environmental factors.

Genome-wide identification and expression analysis of the GhIQD gene family in upland cotton(Gossypium hirsutum L.)

Background:Calmodulin(CaM)is one of the most important Ca^(2+)signaling receptors because it regulates diverse physiological and biochemical reactions in plants.CaM functions by interacting with CaM-binding proteins(CaMBPs)to modulate Ca^(2+)signaling.IQ domain(IQD)proteins are plant-specific CaMBPs that bind to CaM by their specific CaM binding sites.Results:In this study,we identified 102 GhIQD genes in the Gossypium hirsutum L.genome.The GhIQD gene family was classified into four clusters(Ⅰ,Ⅱ,Ⅲ,andⅣ),and we then mapped the GhIQD genes to the G.hirsutum L.chromosomes.Moreover,we found that 100 of the 102 GhIQD genes resulted from segmental duplication events,indicating that segmental duplication is the main force driving GhIQD gene expansion.Gene expression pattern analysis showed that a total of 89 GhIQD genes expressed in the elongation stage and second cell wall biosynthesis stage of the fiber cells,suggesting that GhIQD genes may contribute to fiber cell development in cotton.In addition,we found that 20 selected GhIQD genes were highly expressed in various tissues.Exogenous application of MeJA significantly enhanced the expression levels of GhIQD genes.Conclusions:Our study shows that GhIQD genes are involved in fiber cell development in cotton and are also widely induced by MeJA.Thw results provide bases to systematically characterize the evolution and biological functions of GhIQD genes,as well as clues to breed better cotton varieties in the future.

Cloning and Expression Analysis of Sucrose Non-fermenting-1 Related Protein Kinase (SnRK) in Cucumis sativus L. Under Low Nitrogen Conditions

The sucrose non-fermenting-1 related protein kinase（SnRK）, whose expression is induced by kinds of hyperosmotic stresses, plays a key role in improving stress resistance of plants. In order to investigate the molecular mechanism of low nitrogen resistance in cucumber, the full-length cDNA of SnRK gene was cloned in this study. The result showed that SnRK gene was 1 548 bp in length, encoded 515 amino acids, and had more than 80% homology with other crops. The protein encoded by this gene was an unstable and hydrophilic protein with no transmembrane structure and no signal peptide. Under nitrogen-free conditions and low nitrogen conditions, the expression pattern analysis of SnRK gene showed that this gene was up-regulated and its expression increased and was significantly higher than the normal level as the nitrogen concentration decreased. In addition, the expression of SnRK gene was also inhibited in the high nitrogen level and was significantly lower than the normal level. The result of this study would help us understand the molecular mechanism of low nitrogen resistance in cucumber.

Alternative RNA splicing in stem cells and cancer stem cells:Importance of transcript-based expression analysis

Alternative ribonucleic acid(RNA)splicing can lead to the assembly of different protein isoforms with distinctive functions.The outcome of alternative splicing(AS)can result in a complete loss of function or the acquisition of new functions.There is a gap in knowledge of abnormal RNA splice variants promoting cancer stem cells(CSCs),and their prospective contribution in cancer progression.AS directly regulates the self-renewal features of stem cells(SCs)and stem-like cancer cells.Notably,octamer-binding transcription factor 4A spliced variant of octamerbinding transcription factor 4 contributes to maintaining stemness properties in both SCs and CSCs.The epithelial to mesenchymal transition pathway regulates the AS events in CSCs to maintain stemness.The alternative spliced variants of CSCs markers,including cluster of differentiation 44,aldehyde dehydrogenase,and doublecortin-like kinase,α6β1 integrin,have pivotal roles in increasing selfrenewal properties and maintaining the pluripotency of CSCs.Various splicing analysis tools are considered in this study.LeafCutter software can be considered as the best tool for differential splicing analysis and identification of the type of splicing events.Additionally,LeafCutter can be used for efficient mapping splicing quantitative trait loci.Altogether,the accumulating evidence re-enforces the fact that gene and protein expression need to be investigated in parallel with alternative splice variants.

Genome-Wide Identification of the F-box Gene Family and Expression Analysis under Drought and Salt Stress in Barley

The F-box protein-encoding gene family plays an essential role in plant stress resistance.In present study,126 non-redundant F-box genes were identified in barley(Hordeum vulgare L.,Hv).The corresponding proteins contained 165–887 amino acid residues and all were amphiphilic,except 5 proteins.Phylogenetic analysis of F-box protein sequences in barley and stress-related F-box protein sequences in wheat and Arabidopsis thaliana(At)was used to classify barley F-box genes are divided into 9 subfamilies(A–I).A structure-based sequence alignment demonstrated that F-box proteins were highly conserved with a total of 10 conserved motifs.In total,124 F-box genes were unevenly distributed on 7 chromosomes;another 2 genes have not been anchored yet.The gene structure analysis revealed high variability in the number of exons and introns in F-box genes.Comprehensive analysis of expression profiles and phylogenetic tree analysis,a total of 12 F-box genes that may be related to stress tolerance in barley were screened.Of the 12 detected F-box genes,8 and 10 were upregulated after drought and salt stress treatments,respectively,using quantitative real-time polymerase chain reaction(qRT-PCR).This study is the first systematic analysis conducted on the F-box gene family in barley,which is of great importance for clarifying this family’s bioinformatic characteristics and elucidating its function in barley stress resistance.These results will serve as a theoretical reference for subsequent research on molecular regulation mechanisms,genetic breeding,and improvement.

Characterization and Expression Analysis of Peroxiredoxin Genes in NNK-induced V79 Cells

4-（Methylnitrosamino）-1-（3-pyridyl）-1-butanone （NNK） is a potent and prevalent nitrosamine procarcinogen found in cigarette smoke. The aim of this work is to study alterations in peroxiredoxin （Prx） expression induced by NNK during carcinogenesis. Characterization of Prx genes from hamster was performed using bioinformatics.

Cloning and Expression Analysis of Mlo Gene from Pericallis hybrida B. Nord.

The full-length Mlo gene was obtained by reverse transcription polymerase chain reaction （RT-PCR） and RACE. The result of sequence analysis indicated that M/o gene from Pericallis hybrida B. Nord. contained about 1296bp open reading frame and encoded 431 amino acids. According to the comparison of the exogenous gene sequences by BLAST analysis and phylogenetic analysis, Mlo gene shared over 85% nucleotide homology and 98% amino acid homology. Finally, through semi-quantitative-PCR and fluorescence quantitative analysis, we found that Mlo gene showed the highest expression levels in leaves and the lowest in roots after inoculated with powdery mildew pathogen for different days.