The mRNA Expression Profiles of Five Heat Shock Protein Genes from Frankliniella occidentalis at Different Stages and Their Responses to Temperatures and Insecticides

The westem flower thrips, Frankliniella occidental＆ （Pergande） is a highly invasive pest that is able to exploit many crops across a wide range of environmental conditions. Five full-length cDNAs of heat shock protein （HSP） genes （Fo-HSP90, Fo-HSP70, Fo-HSP60, Fo-HSP40 and Fo-HSP28.9） were cloned from F. occidentalis, and their expression profiles were investigated under conditions of thermal stress and insecticide exposure, and at different stages during development, using real-time quantitative PCR. All five gene sequences showed high similarity to homologs in other species, indicating the conserved fimction of this gene family. HSP60 represents an informative phylogenetic marker at the ordinal taxonomic level within Insecta, but HSP90, which has two homologous copies in Hymenoptera, was not informative. The expression of Fo-HSPs under thermal stress suggests that Fo-HSP90, Fo-HSP70, and Fo-HSP28.9 are inducible by both cold and heat stress, Fo-HSP40 is only heat-inducible, and Fo-HSP60 is thermally insensitive. There were two patterns of cold induction of Fo-HSPs： one is from 0 to 4℃ and the other is around -8℃. All five Fo-HSPs genes were induced by exposure to sublethal concentrations of the insecticide avermectin. The expression of the five Fo-HSPs during different developmental stages suggests that they all play a role in development of F. occidentalis.

Preliminary Study on Function of Calcineurin B-Like Protein Gene OsCBL8 in Rice

The homozygous T3 transgenic lines with sense OsCBL8 gene and antisense OsCBL8 gene obtained by agro-transformation were used to investigate the function of OsCBL8 in rice. Semi-quantitative RT-PCR showed that the expression of OsCBL8 extremely increased in sense transgenic lines, and decreased to some extents in antisense transgenic lines. Such up- and down-regulation of the OsCBL8 gene in these transgenic lines had little effects on main agronomic traits, but significantly decreased the number of filled grains per panicle and seed setting rate in some of transgenic lines. By evaluation of the tolerance to 150 mmol/L NaCl, 20% PEG6000 and low temperature treatments, and relevant physiological indices, 8F12, a sense transgenic line with high salt tolerance, and 8R14, an antisense transgenic line with high drought tolerance, were obtained, which suggests that the OsCBL8 gene is involved in the response of rice to abiotic stresses.

Pediatric restrictive cardiomyopathy due to a heterozygous mutation of the TNNI3 gene

Pediatric restrictive cardiomyopathy is rare and most commonly idiopathic in origin. Here, we applied a candi- date gene approach and identified a missense mutation in the cardiac troponin I gene in a 12-year-old Chinese girl with restrictive cardiomyopathy. This study indicates that mutation in sarcomere protein genes may play an im- portant role in idiopathic pediatric restrictive cardiomyopathy.

Bioinformatics analysis of structure and function in the MRP gene family and its expression in response to various drugs in Bursaphelenchus xylophilus

Genes homologous to members of the MRP gene family in Caenorhabditis elegans are important in drug resistance.To further explore the molecular mechanism of drug resistance in pine wood nematode(Bursaphelenchus xylophilus),we used bioinformatics approaches to analyze genomic data for B.xylophilus and identified Bx-MRP genes.We predicted the structure and function of the genes and encoded proteins.Using bioinformatics programs to predict and analyze various properties of the predicted proteins,including hydrophobicity,transmembrane regions,phosphorylation sites,and topologically isomeric structures,of these Bx-MRP genes,we determined that they function in transmembrane transport.From the results of RT-qPCR,the Bx-MRP family members confer significant differential resistance to different drug treatments.After treatment with different concentrations of emamectin benzoate,avermectin and matrine,the expression of each gene increased with increasing drug concentrations,indicating that the family members play a positive role in the regulation of multidrug resistance.

Association of gene and protein expression and genetic polymorphism of CC chemokine ligand 4 in colorectal cancer

BACKGROUND Leukocytes,such as T cells and macrophages,play an important role in tumorigenesis.CC chemokine ligand(CCL)4,which is produced by lymphocytes and macrophages,has been found to be expressed in the mucosa of the gastrointestinal tract and is a potent chemoattractant for various leukocytes.AIM To examine CCL4 expression and its genetic polymorphism rs10491121 in patients with colorectal cancer(CRC)and evaluate their prognostic significance.METHODS Luminex technology was used to determine CCL4 Levels in CRC tissue(n=98),compared with paired normal tissue,and in plasma from patients with CRC(n=103),compared with healthy controls(n=97).Included patients had undergone surgical resection for primary colorectal adenocarcinomas between 1996 and 2019 at the Department of Surgery,Ryhov County Hospital,Jönköping,Sweden.Reverse transcription quantitative PCR was used to investigate the CCL4 gene expression in CRC tissue(n=101).Paired normal tissue and TaqMan single nucleotide polymorphism assays were used for the CCL4 rs10491121 polymorphism in 610 CRC patients and 409 healthy controls.RESULTS The CCL4 protein and messenger RNA expression levels were higher in CRC tissue than in normal paired tissue(90%,P<0.001 and 45%,P<0.05,respectively).CRC tissue from patients with localized disease had 2.8-fold higher protein expression levels than that from patients with disseminated disease.Low CCL4 protein expression levels in CRC tissue were associated with a 30%lower cancer-specific survival rate in patients(P<0.01).The level of plasma CCL4 was 11%higher in CRC patients than in healthy controls(P<0.05)and was positively correlated(r=0.56,P<0.01)with the CCL4 protein level in CRC tissue.The analysis of CCL4 gene polymorphism rs10491121 showed a difference(P<0.05)between localized disease and disseminated disease in the right colon,with a dominance of allele A in localized disease.Moreover,the rate of the A allele was higher among CRC patients with mucinous cancer than among those with nonmucinous cancer.CONCLUSION The present study indicates that the CRC tissue levels of CCL4 and CCL4 gene polymorphism rs10491121,particularly in the right colon,are associated with clinical outcome in CRC patients.

Relationship Between Combined Genotypes of UCP Gene and Growth Traits in Chickens

The uncoupling protein （UCP） is a member of the mitochondrial membrane transporter family, which plays an important role in energy metabolism. In the present study, the UCP gene was considered as a candidate gene for chicken growth traits, and the association of UCP gene SNPs with growth rate was investigated in the eighth generation of NEAUHLF broiler lines. Two SNPs were found in chicken UCP gene, and the association analysis results showed that both the individual and combination of chicken UCPgene SNPs were significantly associated with body weight of 7 weeks （BW7） and carcass weight （CW） （P〈0.05）, and the combination had much significant effects than the single SNP. This research suggested that the UCP gene could be a candidate gene or linked to a major gene which affected growth traits in chicken

Transformation of Upland Cotton (Gossypium hirsutum L.) with gfp Gene as a Visual Marker

The green-fluorescent protein （gfp） gene was evaluated as a screening marker during cotton （Gossypium hirsutum L.） transforming and plant regeneration. High expression of GFP （green-fluorescent protein） was observed in transgenic cells as early as 42 h after co-culture with Agrobacterium. Most of the stable transformation events were detected in the cells of primary vascular tissue. GFP transient expression could be detected on all the explants after co-culturing for 4 d, however, the highest GFP stable expression was recorded when the explants were co-cultured for 3 d. We believe the transient and stable expression of a foreign gene in genetic transformation were two relative but different events, because high transient expression did not surely lead to high stable transformation. Under the same conditions of in vitro culture, transgenic and non-transgenic calli exhibited different morphological characters on different stages of development. High concentration of plant growth regulators （PGRs） was efficient for somatic embryogenesis of the transgenic calli, which means that the transgenic calli need relatively higher dose of hormone for further growth and somatic embryogenesis than non-transgenic ones. Strong GFP-expression was observed in leaf, stem, petioles, floral tissues, and seedlings of T~ progeny. Segregation ratios of eight transgenic lines were scored for expression of GFP in the T~ progeny that providing further evidence of stable transformation. These results proved that GFP is a powerful reporter gene for protocol optimization, selection, and monitioring in whole transformation events.

Pattern of expression of the CREG gene and CREG protein in the mouse embryo

Background The cellular repressor of ElA-stimulated genes(CREG) is a secreted glycoprotein that inhibits cell proliferation and/or enhances differentiation.CREG is widely expressed in adult tissues such as the brain,heart, lungs,liver,intestines and kidneys in mice.We investigated the level of CREG expression during mouse embryogenesis and its distribution at 18.5 days post coitus(dpc).Methods Immunohistochemical staining with diaminobenzidine,western blotting and reverse transcription-polymerase chain reaction were used.Results CREG expression was rst detected in mouse embryos at 4.5 dpc.It was expressed at almost all stages up to 18.5 dpc.The level of CREG was found to increase gradually and was highest at 18.5 dpc.Western blotting showed that the CREG protein was expressed at higher levels in the brain,heart,intestines and kidneys than in the lungs and liver at 18.5 dpc.In 9.5 dpc embryos,CREG was expressed only in the endothelial cells of blood vessels,after the vascular lumen had formed.With advanced differentiation, vascular smooth muscle cells developed in the embryonic vascular structures;the expression of smooth muscle a-actin protein and CREG were positive and increased gradually in 10.5 dpc embryonic vessels.CREG expression in the embryonic blood vessels peaked at 15.5 dpc and was reduced slightly at 18.5 dpc.Conclusions These results indicate that CREG is expressed during mouse embryogenesis and might participate in the differentiation of these organs during embryogenesis.

MAPT as a predisposing gene for sporadic amyotrophic lateral sclerosis in the Chinese Han population

A previous study of European Caucasian patients with sporadic amyotrophic lateral sclerosis demonstrated that a polymorphism in the microtubule-associated protein Tau （MAPT） gene was significantly associated with sporadic amyotrophic lateral sclerosis pathogenesis. Here, we tested this association in 107 sporadic amyotrophic lateral sclerosis patients and 100 healthy controls from the Chinese Han population. We screened the mutation-susceptible regions of MAPT- the 3＇ and 5＇ untranslated regions as well as introns 9, 10, 11, and 12 - by direct sequencing, and identified 33 genetic variations. Two of these, 105788 A 〉 G in intron 9 and 123972 T 〉 A in intron 11, were not present in the control group. The age of onset in patients with the 105788 A 〉 G and/or the 123972 T 〉 A variant was younger than that in patients without either genetic variation. Moreover, the pa- tients with a genetic variation were more prone to bulbar palsy and breathing difficulties than those with the wild-type genotype. This led to a shorter survival period in patients with a MAPT genetic variant. Our study suggests that the MAPT gene is a potential risk gene for sporadic amyotrophic lateral sclerosis in the Chinese Han population.

Beta-nerve growth factor promotes neurogenesis and angiogenesis during the repair of bone defects

We previously showed that the repair of bone defects is regulated by neural and vascular signals. In the present study, we examined the effect of topically applied β-nerve growth factor（β-NGF） on neurogenesis and angiogenesis in critical-sized bone defects filled with collagen bone substitute. We created two symmetrical defects, 2.5 mm in diameter, on either side of the parietal bone of the skull, and filled them with bone substitute. Subcutaneously implanted osmotic pumps were used to infuse 10 μgβ-NGF in PBS（β-NGF ＋ PBS） into the right-hand side defect, and PBS into the left（control） defect, over the 7 days following surgery. Immunohistochemical staining and hematoxylin-eosin staining were carried out at 3, 7, 14, 21 and 28 days postoperatively. On day 7, expression of β III-tubulin was lower on the β-NGF ＋ PBS side than on the control side, and that of neurofilament 160 was greater. On day 14, β III-tubulin and protein gene product 9.5 were greater on the β-NGF ＋ PBS side than on the control side. Vascular endothelial growth factor expression was greater on the experimental side than the control side at 7 days, and vascular endothelial growth factor receptor 2 expression was elevated on days 14 and 21, but lower than control levels on day 28. However, no difference in the number of blood vessels was observed between sides. Our results indicate that topical application of β-NGF promoted neurogenesis, and may modulate angiogenesis by promoting nerve regeneration in collagen bone substitute-filled defects.

Genome-wide identification,phylogeny and expression analysis of the SBP-box gene family in maize(Zea mays)

The SQUAMOSA promoter binding protein （SBP）-box genes encode a kind of plant-specific transcription factors （TFs） and play important roles in the regulation of plant development. In this study, a genome-wide characterization of this family was conducted in maize （Zea mays）. Thirty-one SBP-box genes were identified to be distributed in nine chromosomes and 16 of them were complementary to the mature ZmmiR156 sequences. All the Z. mays SBP （ZmSBP） genes were classified into two clusters with eight subgroups according to the phylogenetic analysis of proteins, which were consistent with the pattern of exon-intron structures. The phylogenetic tree of the ZmSBP, Oryza sativa SBP-like （OsSPL） and Arabidopsis thaliana SBP-like （AtSPL） genes were constructed and all the SBP-box genes were divided into eight groups, which was the same as the classification of ZmSBP genes. The comparision of the expression profiles of all SBP-box genes in these three species indicated that most orthologous genes had similar expression patterns. The results from this study provided a basic understanding of the ZmSBP genes and might facilitate future researches for elucidating the SBP-box genes function in maize.

Cloning and Expression of the vp39 Gene of Bombyx mori Nuclear Polyhedrosis Virus in E.coli

The nuclear capsid protein gene (vp39) ofBombyx mori nuclear polyhedrosis virus (BmNPV) was amplified successfully by PCR technique and inserted into pGEM 3zf(+). The 5′ and 3′ terminal area of the amplified vp39 gene were sequenced with silver-staining dideoxy method. Bmvp39 gene was sub-cloned into the expression vector pRSET-A, and transformed intoE. coli BL21. This gene was highly expressed by IPTG induction. SDS-PAGE analysis showed that the expressed protein is about 38 kd, and the expressed amount reached maxium in 4 h with IPTG induction.

SNP Detection of High Density Lipoprotein Binding Protein Gene (HBP) and Its Correlations with Growth Traits in Largemouth Bass(Micropterus salmoides)

High density lipoprotein binding protein （HBP） plays an important role in lipid metabolism of animals. Lipids are indispensable energy materials for fi- shes, especially for carnivorous fishes with low utilization efficiency of carbohydrates. The single nucleotide polymorphism （SNP） of HBP gene may affect the fat metabolism, thereby exerting an effect on the growth traits of largemouth bass （Micropterus salmoides）. Investigating the correlations between SNP and growth traits can provide candidate markers for molecular marker-assisted selection. In this study, partial genomic fraganents of HBP gene （ GenBank accession number： KF652241 ） were amplified based on the sequences of an available contig in the EST-SNP database of largemouth bass. Three SNP mutation loci were identified in the 3＇ non-ceding region of HBP gene by direct sequencing, including H1 （G ＋ 2782T）, 142 （T ＋ 2817C） and H3 （G ＋ 2857A）. Three SNP loci of 165 randomly selected largemouth bass individuals were detected and genotyped by SnaPshot assay. Genetic structure was analyzed by POPGENE32 software. By using spssl7.0 software, a general linear model （GLM） was established for correlation analysis between different genotypes at SNP loci of HBP gene and various growth traits. The results showed that three SNP loci were in Hardy-Weinberg equilibrium state. To be specific, loci H1 and H2 formed two haplotypes （ A and B）, and three geno- types （AA, AB, and BB） were observed; loci H1, H2 and H3 formed six diplotypes （DI, I）2, D3, D4, D5 and D6）. According to the correlations between dif- ferent genotypes and various growth traits, the body weight and total length of largemouth bass individuals with genotype BB were significantly higher than those of individuals with genotype AB （ P 〈 0.05 ） ; the body weight and total length of largemouth bass individuals with diplotype D6 were significantly higher than those of individuals with diplotype D4 （P 〈0.05）. In this study, SNP markers correlated with growth traits were obtained in the 3＇ non-coding region ofHBP gene in large-mouth bass, which could be used as candidate genetic markers for subsequent molecular marker-assisted selection breeding of largemouth bass.

Expression of a Lysine-rich Gene in Bacillus subtilis 168

Lysine-rich protein gene （lys） was cloned from winged bean （Psophocarpus tetragonolobus （L.） DC）, and cloned into prokaryotie expression vector pHT43, the recombinant plasmid pHT43/lys were constructed and then transferred into Bacillus subtilis168, upon IPTG induction, the recombinant protein was expressed, and the content of lysine was detected by HPLC. The result showed that lysine content increased by 9.85%. It was suggested that introducing lys gene into Bacillus subtilis 168 was an effective way to improve its nutrition quality.

Expression of a Carrot 36 kD Antifreeze Protein Gene Improves ColdStress Tolerance in Transgenic Tobacco

Antifreeze proteins （AFPs） enable organisms to survive under cold conditions, and have great potential in improving cold tolerance of cold-sensitive plants, In order to determine whether expression of the carrot 36 kD antifreeze protein gene confers improved cold-resistant properties to plant tissues, we tried to obtain transgenic tobacco plants which expressed the antifreeze protein. Cold, salt, and drought induced promoter Prd29A was cloned using PCR from Arabidopsis. Two plant expression vectors based on pBI121 were constructed with CaMV35S：AFP and Prd29A：AFP. Tobacco plantlets were transformed by Agrobacterium-medicated transformation. PCR and Southern blotting demonstrated that the carrot 36 kD afp gene was successfully integrated into the genomes of transformed plantlets. The expression of the afp gene in transgenic plants led to improved tolerance to cold stress. However, the use of the strong constitutive 35S cauliflower mosaic virus （CaMV） promoter to drive expression of afp also resulted in growth retardation under normal growing conditions. In contrast, the expression of afp driven by the stress-inducible Prd29A promoter from Arabidopsis gave rise to minimal effects on plant growth while providing an increased tolerance to cold stress condition （2℃）. The results demonstrated the prospect of using Prd29A-AFP transgenic plants in cold-stressed conditions that will in turn benefit agriculture.

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.

Contruction of the Genetic Engineering Strain Expressed Nontoxic ST_1-LT_B Fusion Protein Against Enterotoxigenic Eschenichia coli

Thermostable enterotoxinⅠ(ST1) mutant genes and thermolabile enterotoxin B subunit (LTB)genes were amplified by PCR from plasmids of Eschenichia coli C83902. The recombinantexpression plasmid pZST3LTB containing ST1-LTB fusion gene was constructed by recombinantDNA technique and then transformed into Escherichia coli BL21(DE3). The ST1-LTB fusionprotein was highly expressed in recombinant strain BL21(DE3)(pZST3LTB) and the fusionprotein was about 38.53% of total cellular protein by SDS-PAGE and thin-layer gelscanning analysis. More important, mice immunized with crude preparation containing thefusion protein inclusion bodies or inactivated recombinant strain produced antibodiesthat were able to recognize ST1 in vitro. These sera antibodies were able to neutralizethe biological activity of native ST1 in the suckling mouse assay. Hence the ST1-LTBfusion protein was nontoxic and immunogenic, the constructed recombinant strain BL21(DE3)(pZST3LTB) could be used as a candidate of vaccine strain.

Expression Pattern Analysis of Zinc Finger Protein Genes in Wheat(Triticum aestivum L.) Under Phosphorus Deprivation

Zinc finger protein（ZFP） genes comprise a large and diverse gene family, and are involved in biotic and abiotic stress responses in plants. In this study, a total of 126 ZFP genes classified into various types in wheat were characterized and subjected to expression pattern analysis under inorganic phosphate（Pi） deprivation. The wheat ZFP genes and their corresponding GenBank numbers were obtained from the information of a 4×44K wheat gene expression microarray chip. They were confirmed by sequence similarity analysis and named based on their homologs in Brachypodium distachyon or Oriza sativa. Expression analysis based on the microarray chip revealed that these ZFP genes are categorized into 11 classes according to their gene expression patterns in a 24-h of Pi deprivation regime. Among them, ten genes were differentially up-regulated, ten genes differentially downregulated, and two genes both differentially up- and down-regulated by Pi deprivation. The differentially up- or down-regulated genes exhibited significantly more or less transcripts at one, two, or all of the checking time points（1, 6, and 24 h） of Pi stress in comparison with those of normal growth, respectively. The both differentially up- and down-regulated genes exhibited contrasting expression patterns, of these, TaWRKY70;5 showed significantly up-regulated at 1 and 6 h and down-regulated at 24 h whereas TaAN1AN20-8;2 displayed significantly upregulated at 1 h and downregulated at 6 h under deprivation Pi condition. Real time PCR analysis confirmed the expression patterns of the differentially expressed genes obtained by the microarray chip. Our results indicate that numerous ZFP genes in wheat respond to Pi deprivation and have provided further insight into the molecular basis that plants respond to Pi deprivation mediated by the ZFP gene family.

Expression of the Capsid Precursor Protein gene of Foot-and-mouth Disease Virus and Green Fluorescent Protein Gene in BHK-21 Cells Mediated by Retroviral Vector

We have constructed a retroviral vector mediated mammalian cell expression system of the capsid precursor protein of foot-and-mouth disease virus(FMDV).The recombinant retroviral vector pBABEpuro-P1-2A-EGFP was constructed by sequentially inserting capsid precursor protein gene(P1) of FMDV and enhanced green fluorescent protein gene(EGFP) into pBABEpuro.The recombinant retroviral vector and the pVSV-G plasmid were co-transfected into packaging cells(GP2-293) by liposomemediated transduction to produce the pseudovirus.The pseudovirus was used to infect BHK-21 cells and resistant cells were screened with puromycin.Green fluorescent proteins were observed by fluorescence microscopy and expression of the capsid precursor protein gene of FMDV was detected by indirect immunofluorescence.The recombinant retroviral vector pBABEpuro-P1-2A-EGFP was constructed successfully.The capsid precursor protein of FMDV and green fluorescent protein were expressed in BHK-21 cells.The mammalian cell expression system for the capsid precursor protein of FMDV has been constructed successfully,which lays the foundation of development of a FMDV subunit vaccine.