庆祝《园艺学报》创刊50 周年

今年是《园艺学报》创刊发行50周年。50年来,《园艺学报》坚持为学术交流服务,为促进学科发展作贡献的办刊原则,以"科学性;创新性;对生产和科研有参考启迪作用"的标准,收录和发表了大量高水平的论文,记载了几代科技工作者呕心沥血创新之作,反映了中国园艺科学技术和园艺产业的发展历程。     

Repression of SFRP5 promoter activity by hepatitis B virus X protein

AIM: To investigate the molecular mechanism that hepatitis B virus X protein (HBx) inhibits the promoter activity of secreted frizzled-related protein 5 (SFRP5). METHODS: Serial truncated fragments of SFRP5 promoter were amplified and cloned into luciferase reporter vector pGL3-Basic. LO2 cells were transfected with recombinants, and then infected with the adenoviral vector expressing HBx protein (Ad-HBx) or the analogous adenovirus expressing green fluorescent protein (Ad-GFP) for control. The infection efficiency was examined under a fluorescence microscope and the activity of SFRP5 promoter was measured by luciferase assay. RESULTS: Truncated fragments of SFRP5 promoter were successfully constructed. Compared with the pGL3-Basic control, the luciferase activity was 6.32±0.04 of the -478 bp~+47 bp fragment, 5.79±0.32 of the -811 bp~+47 bp fragment, 3.59±0.34 of the -1 235 bp~+47 bp fragment, 3.86±0.39 of the -1 677 bp~+47 bp fragment and 3.26±0.42 of the -2 072 bp~+47 bp fragment, respectively. Overexpression of HBx inhibited the activity of SFRP5 promoter. The average inhibitory rates were 44% for -478 bp~+47b, 46% for -811 bp~+47 bp, 28% for -1 235 bp~+47 bp, 24% for -1 677 bp~+47 bp and 40% for -2 072 bp~+47 bp, respectively. CONCLUSION: HBx suppresses the activity of SFRP5 promoter, leading to down-regulation of SFRP5 expression.     

TNF-α exacerbates cholesterol accumulation via down-regulating LXRα promoter activity in HepG2 cells

AIM: To investigate the exacerbating effect of tumor necrosis factor alpha (TNF-α) on lipid accumulation in HepG2 cells by inhibiting liver X receptor alpha (LXRα) signaling pathway. METHODS: Luciferase reporter plasmid driven by the LXRα promoter (pGL3-Basic-LXRα-P) was constructed and transfected into HepG2 cells to detect the LXRα promoter activity. HepG2 cells were incubated with serum-free medium (control), 20 μg/L TNF-α (TNF-α), 100 mg/L LDL (LDL) and 20 μg/L TNF-α plus 100 mg/L LDL (LDL+TNF-α), respectively. The effects of TNF-α on cholesterol accumulation were examined by oil red O staining and quantitative intracellular cholesterol assay. The expression of LXRα, ABCA1 and ABCG1 at mRNA and protein levels was examined by real-time PCR and Western blotting. RESULTS: The pGL3-Basic-LXRα-P was constructed successfully. TNF-α decreased the activity of LXRα promoter in the absence or presence of LDL. Inflammatory stress inhibited the expression of LXRα, ABCA1and ABCG1 at mRNA and protein levels. The cholesterol efflux was increased after loading of LDL, while TNF-α decreased intracellular cholesterol efflux. The results of oil red O staining and quantitative intracellular cholesterol assay demonstrated that inflammatory stress increased cholesterol levels in HepG2 cells. CONCLUSION: TNF-α exacerbates the cholesterol accumulation in hepatic cells via inhibiting LXRα promoter activity and gene expression.     

Cholesterol-sensitive action sites in promoter of prohibitin gene

AIM: To verify the cholesterol-sensitive sites in the promoter of prohibitin (PHB) gene. METHODS: A series of successive PHB promoter truncated segments were amplified by PCR and cloned into pGL3-Basic luciferase reporter vector. Human prostate cancer PC-3 cells were transfected with the recombinant plasmids and then cultured in normal medium (NM) or cholesterol-depleted medium (CDM). The activity of PHB promoter was detected by luciferase activity analysis to find the cholesterol-sensitive region of approximately 200 bp in PHB promoter. Point mutations in sterol regulatory element (SRE) homologous loci of this 200　bp region were made to confirm the cholesterol-sensitive role of this SRE site. RESULTS: Plasmids with PHB promoter segments were successfully constructed. Luciferase activity in PC-3 cells transfected with PHB promoter segment pPHB-179 (-35/+138) plasmid was significantly decreased compared with the cells transfected with the whole PHB promoter pPHB-1192 plasmid (P<0.05). After point mutation was made in the SRE homologous locus located at -117 to -108 bp and this plasmid was transfected into PC-3 cells, the luciferase activity in these cells was significant decreased compared with the cells transfected with pPHB-1192 plasmid (P<0.05). CONCLUSION: Cholesterol regulates the activity of PHB promoter in human prostate cancer PC-3 cells. The cholesterol-sensitive site in PHB promoter is located at -117 to -108 bp of SRE homologous locus. PHB may become a new target of gene therapy for prostate cancer.     

NF-κB site of promotor mediates nicotine-induced ICAM-1 expression in human umbilical vein endothelial cells

AIM:To study the mechanisms of nicotine-induced expression of intercellular adhesion molecule-1(ICAM-1).METHODS:Related luciferase reporter gene plasmids were constructed with molecular cloning techniques;above plasmids and intracontrol plasmid pSV-β-gal were co-transfected into human umbilical vein endothelial cells(HUVECs) with eukaryotic gene transfection techniques; the relative luciferase activities were detected in the transfected HUVECs.RESULTS:Series of luciferase reporter gene containing different sequences of human ICAM-1 promotor and site-directed mutants of NF-κB and Sp-1 in promotor were successfully constructed; Nicotine could increase the expression of luciferase reporter gene plasmid containing-579 bp(pGL3E-579/+36),-230 bp(pGL3E-230/+36) and mutated Sp-1 version(pGL3E-Sp-1-MU)(P＜0.05 vs control) of ICAM-1 promotor in the transfected HUVECs, whereas deletion derivative (pGL3E-134/+36) and mutation (pGL3E- NF-κB -MU) of downstream NF-κB site of ICAM-1 promotor prevent nicotine-induced increase in expression of luciferase reporter gene plasmid.CONCLUSION:NF-κB site of promotor mediates nicotine-induced ICAM-1 expression in human umbilical vein endothelial cells.     

Cloning of P1 and P3 promoters of human insulin-like growth factorⅡ gene

AIM:To clone P1 and P3 promoters of the human insulin-like growth factor Ⅱ(IGF-Ⅱ) gene. METHODS:According to the complete DNA sequence of IGF-Ⅱ gene, the nested primer PCR was performed for amplifying P1 and P3 promoter fragments of the gene from human L-02 cell line.These PCR products were analyzed by agarose gel electrophoresis, and cloned by using TOPO TA Cloning kit. The positive clones containing P1 and P3 fragments were selected and confirmed by sequencing.RESULTS:The DNA sequences of P1 and P3 promoters cloned were accordant with GenBank data. CONCLUSION:In this study P1 and P3 promoters of the IGF-II gene were cloned successfully.     

Construction of human prostate-specific membrane antigen expression vector and identification of tissue specificity

AIM: To construct a prostate-specific expression vector of promoter and enhancer of human prostate-specific membrane antigen (PSMA).METHODS: The promoter and enhancer of PSMA were amplified by PCR separately. The two segments were cloned into the expression vector pGL3-Basic, a prostate-specific expression vector pGL3-PSMP-PSME was constructed. The vector was transfected into prostatic carcinoma cell PC-3M and four kinds of non-prostatic carcinoma cells by lipofectamine. The activity of luciferase of transfected cells and tissue specificity of vector was examined at 48 hours after transfection. RESULTS: With DNA sequence of the vector pGL3-PSMP-PSME, the segment of clone was proved correct. The activity of luciferase of the pGL3-PSMP-PSME was expressed distinctly in PC-3M and was not expressed in other nonprostate cell lines. CONCLUSION: The prostate-specific expression vector was constructed successfully. It lays foundation for studying target gene therapy of the prostate cancer.     

Effects of rs5418 site variation in SLC2A4 promoter region on gene expression

AIM: To investigate the effect of G/A mutation in rs5418 site of solute carrier family 2,facilitated glucose transporter,member 4 protein(SLC2A4) promoter region on gene expression. METHODS: The core promoter region of SLC2A4 gene was amplified by PCR. Mutant and wild-type recombinant expression vectors containing promoter of SLC2A4 gene were constructed by recombinant gene technique and the strategy of site-directed mutagenesis. Recombinant vectors were transfected into HEK293T cells by lipofectamine and the expression activity of the reporter gene in the recombinant expression vectors with different alleles was detected by a dual-luciferase reporter assay system. RESULTS: The 716-bp SLC2A4 promoter was amplified and the recombinant expression vectors pGL3-SLC2A4-prom(A) and pGL3-SLC2A4-prom(G) were successfully constructed. The luciferase reporter vector containing SLC2A4 promoter with rs5418-A alleles produced significantly higher relative luciferase activity (19.49±4.41) than that with rs5418-G allele (13.04±4.45; P<0.05). CONCLUSION: The G→A variation of rs5418 site in SLC2A4 promoter region increases the expression of SLC2A4 gene,thereby affecting the gene function.     

Construction of human anti-HBc ScFv eukaryotic expression vector and expression of anti-HBc ScFv in HepG2 cells

AIM: To construct an eukaryotic expression vector of human single-chain variable fragment against hepatitis B virus core protein (anti-HBc ScFv) and detect its expression in HepG2 cells. METHODS: Anti-HBc ScFv genes were amplified from the plasmids abstracted from positive clone and inserted into pEGFP-c1 vector that contained green fluorescent protein gene. The recombinant plasmids were transfected into HepG2 cells, and resistant clones were obtained by G418 selection. The expression of the gene of fusion protein was determined by fluorescent invert microscope and ELISA. RESULTS: Recombinant plasmids were successfully constructed. The plasmid transfected HepG2 cells were obtained by G418 selection. Specific fluorescence was observed in HepG2 cells 48 hours after transfection. ELISA analysis confirmed the expression of anti-HBc ScFv in the cells. CONCLUSION: The construction of human anti-HBc ScFv eukaryotic expression vector and its expression in HepG2 cells lay the foundation for advanced research of intracellular anti-HBc ScFv.     

Effects of rs35100176 polymorphism in IPO8 promoter on gene expression

AIM: To investigate the effect of rs35100176 CCT insertion/deletion polymorphism in the promoter region of importin 8 (IPO8) gene on its mRNA expression. METHODS: A 342-bp fragment of IPO8 gene promoter containing the rs35100176 polymorphism was amplified from 49 DNA samples and sequenced. The IPO8 promoter fragments containing CCT 3-nucleotide insertion or deletion were amplified using the corresponding homozygote DNA samples. The PCR products were sequenced and inserted into the luciferase reporter vector pGL3-Basic. Recombinant vectors were transfected into the cells by Fugene 6.0 and the expression of the reporter gene was detected by a dual-luciferase reporter assay system. The mRNA expression level of IPO8 was detected by real-time PCR in 3-nucleotide insertion or deletion homozygote cells. RESULTS：The sequencing results showed that there were 3 kinds of genotypes in the rs35100176 polymorphism, CCT/CCT,CCT/- and -/-, and the gene frequencies were 1837%, 5510% and 2653%, respectively. The recombinant expression vectors pGL3-3N Insertion and pGL3-3N Deletion were successfully constructed. The luciferase assay showed that pGL3-3N Insertion produced significantly lower luciferase activity than that by pGL3-3N Deletion. Real-time PCR showed that HEK293 cells with 3-nucleotide insertion homozygote expressed relative lower IPO8 mRNA than Saos-2 cells with 3-nucleotide deletion homozygote. CONCLUSION：The CCT 3-nucleotide insertion variant decreases the promoter activity of IPO8, thus affecting the gene expression.     

Construction of eukaryotic expression vector and expression of outer membrane lipoprotein LipL41 gene of Leptospiria lai

AIM:To construct a eukaryotic expression vector expressing outer membrane lipoprotein LipL41 of Leptospira lai and express it in mammalian cell. METHODS:LipL41 gene was amplified by PCR from genome of Leptospira lai 017 strain, and was subcloned into vector pGEX-4T-1. After sequencing, LipL41 gene digested by restriction endonuclease and cloned into vector pcDNA3. After confirming the correctness of the eukaryotic recombinant vector by restrication enzyme digestion, it was transfected into COS7 cells by liposome. Its expression was analyzed by RT-PCR. RESULTS:A fragment of 1 011 bp was amplified, and sequence analysis showed it had a 98% homology with Leptospira kirschneri. The analysis of restriction enzyme indicated that the eukaryotic recombinant vector was correctly constructed. A specific amplified fragment was showed in the cells transfected with recombinant plasmid by RT-PCR, but the cell transfected with blank plasmid did not show this band. CONCLUSIONS:The LipL41 gene of Leptospira lai was successfully inserted into eukaryotic expression plasmid and the recombinant plasmid expressed the LipL41 mRNA.