接头连接介导的PCR 扩增马铃薯抗晚疫病基因侧翼序列研究

利用接头连接介导的PCR 技术，以前期NBS profiling 试验获得的1 条马铃薯抗晚疫病基因
片段为基础，设计巢式基因特异性引物，扩增获得两侧序列。结果表明：基因组DNA 经DraⅠ酶切后，
在已有536 bp 片段的左侧和右侧均得到序列延伸，右侧扩增得到2 301 bp，左侧扩增得到820 bp，去除
边界序列后，向左延伸789 bp，向右延伸2 273 bp，拼接后共长3 443 bp。采用GENSCAN 进行基因预测，
发现包含内含子在内的晚疫病抗性基因全长2 413 bp，在该预测基因 的UTR 区域设计特异性引物，在马
铃薯抗病和感病的基因型中扩增包含完整基因在内的2 571 bp 序列，发现该特异候选基因与晚疫病抗性相
连锁。     

Effect of simvastatin on function of mouse pancreatic beta cells and its mechanisms

AIM: To observe the influence of simvastatin on insulin secretion function of mouse pancreatic beta cell line MIN6 and to explore its possible mechanisms.
METHODS: MIN6 cells were randomly divided into normal control group and low-, middle-and high-concentration simvastatin treatment groups, which were cultured for 48 h with high-glucose DMEM containing 15% fetal bovine serum plus 0, 2, 5 and 10 μmol/L simvastatin, respectively. The insulin secretion of MIN6 cells was measured by radioimmunoassay. The content of ATP in MIN6 cells was measured by biochemiluminescence method. The mRNA and protein expression levels of inwardly rectifying potassium channel 6.2 (Kir6.2), voltage-dependent calcium channel 1.2 (CaV1.2) and glucose transporter 2 (GLUT2) were detected by real-time fluorescence quantitative PCR and Western blotting, respectively.
RESULTS: Compared with normal control group, middle-and high-concentration simvastatin treatment markedly decreased the synthesis and secretion of insulin in MIN6 cells (P<005). The content of ATP in MIN6 cells was markedly decreased in simvastatin treatment groups (P<005). The mRNA expression level of Kir6.2 in MIN6 cells was significantly up-regulated in simvastatin treatment groups (P<001), while the mRNA expression levels of CaV1.2 and GLUT2 were significantly down-regulated in middle-and high-concentration simvastatin treatment groups (P<001). The protein expression of Kir6.2 was significantly increased but that of CaV1.2 was significantly decreased in middle-and high-concentration simvastatin treatment groups (P<001), and the protein expression level of GLUT2 was markedly decreased in high-concentration simvastatin treatment group (P<001).
CONCLUSION: Simvastatin inhibits insulin synthesis and secretion in mouse pancreatic beta cell line MIN6 via suppressing ATP production, up-regulating the expression of Kir6.2 and down-regulating the expression of CaV1.2 and GLUT2.     

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。     

Promoter methylation status and demethylation-induced expression of SFRP genes in human acute leukemia cell lines

AIM: To investigate the relationship between promoter hypermethylation of secreted frizzled-related protein (SFRP) genes and acute leukemia (AL)，and to study the mechanism how 5-aza-2-deoxycytidine (5-Aza-CdR) reverses the hypermethylation of SFRP genes in human AL cell lines. METHODS：Methylation-specific PCR (MSP) was used to detect the methylation levels of SFRP1, SFRP2, SFRP4 and SFRP5 genes in different human AL cell lines (Molt-4, Jurkat, HL-60 and NB4). The methylation levels of these genes in Jurkat cell line before and after 5-Aza-CdR treatment were also analyzed by MSP. The expression of SFRP1, SFRP2, SFRP4 and SFRP5 mRNA was detected by real-time fluorescence quantitative RT-PCR. The mRNA levels of DNA methyltransferase (DNMT) 1, DNMT3A and DNMT3B were analyzed by semiquantitative RT-PCR. RESULTS：None of normal human blood or bone marrow mononuclear cells showed methylation of SFRP genes. Hypermethylation in the promoter regions of SFRP1, SFRP2 and SFRP5 genes was observed in all of the four AL cell lines. SFRP4 gene was totally methylated in NB4, Molt-4 and Jurkat cell lines but partially methylated in HL60 cell line. Treatment with 5-Aza-CdR for 72 h successfully reversed the hypermethylation of SFRP genes, and significantly increased the mRNA expression of SFRP. Moreover, the mRNA expression of DNMT1, DNMT3A and DNMT3B was down-regulated by 5-Aza-CdR in a concentration-dependent manner. CONCLUSION：Methylated SFRP genes may serve as potential independent biomarkers for early detection of AL. 5-Aza-CdR activates SFRP gene expression by demethylation of SFRP genes and down-regulation of DNMT1, DNMT3A and DNMT3B mRNA expression.     

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.     

Effects of stretch on transient outward potassium and inward rectifier potassium current in cultured neonatal rat atrial myocytes

AIM：To investigate the effects of mechanical stretch on transient outward potassium current (Ito), inward rectifier potassium current (IK1) and action potential duration(APD) of cultured neonatal rat atrial myocytes. METHODS：Neonatal rat atrial myocytes were isolated and cultured on silicone sheeting with or without stretch for 24 h. The silicone membrane area was increased by 12% in stretched group. The cells without stretch served as control. Ito, IK1 and APD were recorded by the technique of whole-cell patch clamp. RESULTS：Compared with control group, Ito density in stretched myocytes was significantly reduced ［(16±04) pA/pF vs (121±29) pA/pF, P<001］, whereas IK1 density was increased ［(-108±08) pA/pF vs (-88±09) pA/pF, P<001］. The APDs at 50% and 90% levels of repolarization (APD50 and APD90) in the stretched cells were obviously decreased than those in non-stretched cells ［(105±14) ms vs (155±24) ms, (300±28) ms vs (563±36) ms, P<001］. CONCLUSION：Stretch stimulation leads to the reduction of Ito density, the increase in IK1 density and the shortness of APD in cultured rat atrial neonatal myocytes, which may contribute to atrial electrical remodeling induced by pressure overload.     

Effect of DNA methylation of miR-30a-5p promoter region on hepatic injury induced by homocysteine

AIM: To explore the role of DNA methylation of microRNA-30a-5p(miR-30a-5p) promoter region in hepatic injury. METHODS: Four-week-old normal mice and cystathionine β-synthase (CBS) single gene knockout mice were used and divided into normal (CBS^+/+, n=12) group and single gene knockout (CBS^+/-, n=12) group, and the mice were fed with high methionine diet for 8 weeks. HL-7702 hepatic cells were routinely cultured in vitro and divided into control group, homocysteine (Hcy) group and Hcy+5-azacytidne (AZC) group. Serum Hcy, alanine aminotransferase (ALT) and aspartate aminotransferase (AST) levels were measured by automatic biochemical analyzer. The levels of ALT and AST in the cells culture medium were determined by the microplate method. Hepatic injury in the mice were observed with HE staining. Cell viability staining was used to measure the viability of hepatocytes. RT-qPCR was used to detect the expression of miR-30a-5p in the liver tissues and hepatocytes. The correlation between the expression of miR-30a-5p and serum ALT and AST levels was analyzed by Pearson correlation analysis. DNA methylation level of miR-30a-5p promoter region in the liver tissues and hepatocytes was detected by nested landing methylation-specific PCR (nMS-PCR). RESULTS: Compared with the CBS^+/+ mice, the serum levels of Hcy, ALT and AST in the CBS^+/- mice were significantly increased (P < 0.05). HE staining showed the hepatocyte swelling and nuclear fragmentation and dissolution. The expression level of miR-30a-5p in the liver tissues was decreased (P < 0.01). Besides, the expression level of miR-30a-5p in the mice was negatively correlated with serum ALT and AST levels (r²=0.4557, P=0.0003, r²=0.4626, P=0.0003), and the DNA methylation of miR-30a-5p promoter region was increased (P < 0.01). In the HL-7702 cells, compared with control group,the ALT and AST levels were increased in Hcy group (P < 0.05, P < 0.01), and the cell viability was remarkablely decreased. DNA methylation of miR-30a-5p promoter region was increased (P < 0.01), which decreased after treated the cells with AZC (P < 0.05), while the expression level of miR-30a-5p in the cells was increased (P < 0.05). CONCLUSION: Hypermethylation of miR-30a-5p promoter region may play an important role in hepatic injury.     

矮生观赏杉木DNA甲基化的水平与模式分析

为探讨杉木矮化变异与DNA甲基化的关系,以矮生观赏杉木与野生杉木为试验材料,采用基于DNA甲基化敏感扩增多态性分析（Methylation-sensitive amplification polymorphism,MSAP）方法,研究其DNA 序列CCGG 位点的甲基化水平及模式变化特征。应用20个引物组合,在矮生观赏杉木和野生杉木的叶片DNA中均检测出745个CCGG位点,其中甲基化位点数分别为508个和505个,分别占总扩增位点数的68.17%和67.83%;在矮生观赏杉木与野生杉木木质部DNA中分别检测到742个和737个CCGG位点,其中甲基化位点数分别为471个与498个,分别占总扩增位点数的63.52%和67.51%,差异达极显著水平（P < 0.01）。与野生型相比,矮生观赏杉木叶片和木质部DNA甲基化模式发生了一定变化,在叶片DNA中,去甲基化率为17.81%,明显高于超甲基化率15.44%;在木质部DNA中,去甲基化率17.25%,也明显高于超甲基化率14.65%。通过甲基化序列的初步克隆及比对分析发现,矮生观赏杉木中参与MAPK级联途径的蛋白磷酸酶IBR5基因启动子区域的甲基化水平上升。因此推测,植物激素信号转导及其调控基因的甲基化变化可能是矮生观赏杉木形成的原因之一。     

Preparation of histones and their effects on macrophages

AIM: To prepare histones with different methods and to observe their effects on cell viability and cytokine release by macrophages in vitro.
METHODS: Prokaryotic expression plasmids of histone H3 and histone H4 were constructed by cloning methods. The histones containing GST-tag or His-tag (GST-H3, GST-H3, His-H3, His-H4) were expressed and purified. The histones from eukaryotic cells (RAW264.7 and 293F cells) were extracted with the high salt method. RAW264.7 cells were treated with histones (7.5~50 mg/L) for 4 h and the cell vitality was examined by MTT assay and flow cytometry. The concentrations of IL-6 and TNF-α in the culture supernatants of RAW264.7 cells treated with histones were also detected.
RESULTS: His-H3/H4 and the histones from eukaryotic cells significantly reduced the viability of RAW264.7 cells and induced apoptosis. The effects of histones from different sources on the releases of IL-6 and TNF-α were different.
CONCLUSION: His-H3/His-H4 expressed by prokaryotic plasmids and the histones extracted from eukaryotic cells affect the vitality of macrophages as well as induce late apoptosis and necrosis. Histone may involve in the inflammatory process by promoting the release of inflammatory cytokines.     

Influence of siRNA-mediated MIF knockdown on inhibition of inflammatory lipid mediator release by glucocorticoids

AIM：To investigate the influence of siRNA-mediated macrophage migration inhibitory factor (MIF) knockdown on inhibition of inflammatory lipid mediator release by glucocorticoids.
METHODS：Mouse macrophage cell line RAW2647 was transiently transfected with MIF siRNA and control siRNA by liposome method. The transfection efficiency was assessed by immunofluorescence technique. The expression of MIF mRNA and protein was examined by RT-PCR and Western blotting, respectively. Prostaglandin E2 (PGE2) and leukotriene B4 (LTB4) production in cell culture supernatants was measured by ELISA, and the protein expression of Annexin 1, cytosolic phospholipase A2α (cPLA2α) and phospho-cPLA2α were evaluated by Western blotting.
RESULTS：MIF siRNA significantly inhibited MIF expression both at mRNA and protein levels in RAW2647 cells and subsequently enhanced the inhibitory effect of dexamethasone (Dex) on PGE2 and LTB4 production. MIF siRNA also increased Annexin 1 expression becreased by Dex, and strengthened the inhibitory effect of Dex on the phosphorylation of cPLA2α.
CONCLUSION：MIF siRNA enhances the inhibitory effect of Dex on PGE2 and LTB4 production from RAW2647 cells partly via increasing Annexin 1 expression and inhibiting cPLA2α phosphorylation. Intracellular MIF knockdown mediated by siRNA may enhance the sensitivity of RAW2647 cells to the anti-inflammatory effect of glucocorticoids.     

Correlation between hypermethylation and expression of Syk gene in colorectal cancer cell lines

AIM:To explore the relationship of methylation status and expression of spleen tyrosine kinase (Syk) gene in colorectal cancer cell lines. METHODS:Bisulfite sodium modification sequencing, methylation specific PCR and Western blotting were used to detect the methylation status and expression of Syk gene in 23 colorectal cancer (CRC) cell lines. Luciferase assay was applied to measure the activity of promoter with or without methylation in CpG islands. Meanwhile, methylation status and expression level were compared in Syk(-) HCT-15 cell line before or after 5-Aza-CdR administration. RESULTS:(1) Among 23 cell lines, loss expression of Syk gene in 9 cell lines due to hypermethylation in promoter, and 14 expression with unmethylation status were observed. The total methylation rate was 39.2%. (2) Microsatellite instability was found in 7 of 9 cell lines with promoter hypermethylation, 4 of 14 were wihtout hypermethylation. The difference between methylation and unmethylation group was significant (P<0.05). (3) 5-Aza-CdR restored methylated-Syk gene promoter activity. Compared to methylated-promoter, luciferase activities increased to 4.5 and 4.7 folds with Syk full size promoter and unmethylated-promoter, respectively. (4) 5-Aza-CdR restored methylated-Syk gene expression and the effect had time-course dependence. CONCLUSION:Hypermethylation of CpG islands in Syk gene promoter silences Syk expression in CRC cell lines, and 5-Aza-CdR restores Syk expression by demethylation.     

Effects of interferon-γ, tumor necrosis factor-α and interleukin-1β on apoptosis of human airway smooth muscle cellsin vitro

AIM:To clarify if interferon-γ(IFN-γ), tumor necrosis factor-α(TNF-α)and interleukin-1β(IL-1β)can induce apoptosis of human airway smooth muscle cells (ASMCs) in vitro.METHODS:Human ASMCs were isolated and cultured in DMEM containing 10% fetal bovine serum. Passage 4-6 cell was used in the experiment. IFN-γ,TNF-α and IL-1β, were used separately or together in the treatment of human ASMCs. The effects of IFN-γ,TNF-α and IL-1β on the growth of the cells was detected by MTT method at the hour 0,24,48 and 72. Light microscopy and electron microscopy were used to examine the morphological change. DNA fragmentation was analyzed by agarose gel electrophoresis. SP immunohistological staing method was performed to detect the change of expressions of p 53, bcl- 2 and bax gene. The apoptosis cell percentage were detected by in situ end labeling technique (TUNEL)of fragmental DNA. RESULTS:(1)IFN-γ or IFN-γ together with TNF-α and IL-1β decreased the number of viable cells in a time dependent manner. (2) Light and electron microscopic examination showed cell shrinkage, membrane blebbing, nuclear contraction, chromatin condensation and nuclear fragmentation in human ASMCs. (3) Agarose gel electrophoresis showed a characteristic"ladder"of DNA bands representing integer multiples of the internucleosomal fragments (about 180-200 bp) in cytokine cotreated human ASMCs. (4)The expression of p 53 and bax gene in cytokine cotreated group was significantly higher than in control group, but the expression of bcl-2 gene was lower than in control group. (5)Stimultaneous treatment with IFN-γ(4×10⁵ U/L),TNF-α(4×10⁵ U/L)and /or IL-1β (10×10⁴ U/L) induced apoptosis of human ASMCs. Apoptotic index of human ASMCs in cytokine co-treated group was significantly higher than in control group (P＜0.01).CONCLUSION:Stimultaneous treatment with IFN-γ,TNF-α and /or IL-1β induced apoptosis of human ASMCs. These immune cytokines may play an important role in airway remodeling of asthma and of chronic obstructive pulmonary disease.     

Effects of aflatoxin G1 on proliferation and TNF-αsecretion of human peripheral blood mononuclear cells in vitro

AIM: To explore the effects of aflatoxin G₁(AFG₁ )on proliferation and TNF-α secretion of human peripheral blood mononuclear cells(HPBM) in vitro. METHODS: The effects of AFG₁ on proliferation of HPBM were analysed with flow cytometric (FCM) DNA analysis and MTT bioassay, while that on TNF-α secretion was detected with ELISA.RESULTS: FCM analysis revealed that 6 h after treatment, proliferation index(PI) of 1000 μg/L AFG₁ treated HPBM was significantly higher than that of control. 24 h after AFG₁ treatment, stimulating effects on proliferation was found in HPBM treated with AFG₁ at 200 μg/L and 1 000μg/L.Regression analysis showed that PI was postively correlated with the concentrations of AFG₁ in the concentration range from 0 to 1 000μg/L( r=0. 5122 and 0.5119 respectively,P＜0.05).MTT bioassay showed that the A value of the cells treated with AFG₁ at 2 000 μg/L was higher than that of the control. Double antibody sandwich enzyme linked immunosorbent assay (ELISA) results showed that AFG₁ at a dose of 100 μg/L could significantly inhibit lipopolysaccharide-induced TNF-α secretion.CONCLUSION: AFG₁ could stimulate the proliferation of HPBM and could decrease TNF-α secretion at certain concentration.     

Role of TRAF6 in maturation of human PBMC-derived dendritic cells

AIM: To investigate the immunological effect of tumor necrosis factor receptor-associated factor 6 (TRAF6) on the maturation of dendritic cells in vitro.METHODS: The human peripheral blood mononuclear cell (PBMC)-derived dendritic cells (DCs), induced by recombined human granulocyte macrophage colony-stimulating factor (GM-CSF) and interleukin (IL)-4, were stimulated to mature with TNF-α, LPS or cocktail of cytokines (TNF-α, IL-6, IL-1β and PGE₂).The cell surface markers, T-cell stimulatory proliferation capacity and IL-12 p40 production of the DCs were determined by the methods of flow cytometry, ELISA and mixed lymphocyte reation (MLR), respectively.The mRNA expression of TRAF6 was detected by real-time PCR.RESULTS: The expression of TRAF6 was observed in all groups, which in cocktail group was the highest in the DCs with optimum state of maturation.Furthermore, TRAF6 enhanced the production of IL-12 and the ability of T-cell stimulation of mature DCs.CONCLUSION: TRAF6 might play an important role in inducing the maturation of human PBMC-derived DCs.     

Aberrant DNA methylation in promoter region of p53 gene in acute leukemia

AIM: To investigate the significance of aberrant p53 gene promoter methylation in acute leukemia by detecting the occurrence of p53 gene promoter methylation. METHODS: Genomic DNA was digested using restriction endonuclease MspⅠ, HpaⅡ, EcoRⅡ, BstNⅠ, respectively. PCR amplification was conducted and the products after digestion and genomic DNA were used as template. The PCR product was subjected to electrophoresis and the results were analyzed by gel imaging and analysis system. Parts of the separated DNA were sequenced after purification from gel. RESULTS: The prevalence of methylation in acute leukemia group was 38.7％, of which ALL was 45.5％ (5 of 11) and ANLL 35.0％ (7 of 20). No methylation was detected in normal control group. There was significant difference between the prevalence of methylation in acute leukemia group and the normal control group (Fisher′s exact test, P<0.05). However, the prevalence between ALL and ANLL was not significantly different (Fisher′s exact test, P>0.05). Compared the relationship between aberrant methylation of p53 gene and clinical data, statistical significance between aberrant methylation of p53 gene and enlargement of lymph nodes, liver or spleen(P<0.05) was observed. CONCLUSION: ①Aberrant DNA methylation in P1 promoter region of p53 gene exits in part of acute leukemic patients, but not in health people. ②The prevalence of aberrant DNA methylation between ALL and ANLL is not significantly different. ③The patients with aberrant methylation of p53 gene seem to show more frequently the manifestations of enlarged lymph nodes, liver or spleen than usual.     

甜瓜果实酸性性状的遗传分析

以果实口感无酸味的甜瓜材料60 和酸甜味的材料61 为亲本,构建P₁、P₂、F₁、BC₁、BC₂ 及F₂ 六世代群体,利用主基因+多基因混合遗传模型的六世代联合分析法,分析甜瓜果实柠檬酸含量、可滴定酸值（TA）和pH 值的遗传效应。结果表明：柠檬酸含量的遗传模型为1 对加性-显性主基因+加性-显
性-上位性多基因模型,F₂ 群体的主基因遗传率为31.06%,多基因遗传率为30.48%;TA 值的遗传模型为2 对加性-显性-上位性主基因+加性-显性-上位性多基因模型,F₂ 群体的主基因遗传率为78.06%,多基因遗传率为0;pH 值的遗传模型为1 对加性-显性主基因+加性-显性-上位性多基因模型,F₂ 群体的主基因
遗传率为84.07%,多基因遗传率为12.90%。