乙烯参与ALA-ABA/黑暗调控的苹果叶片气孔运动

以‘富士’苹果组培苗离体叶片下表皮为材料,运用不同试剂结合气相色谱、激光共聚焦扫描显微镜、光学显微镜和qRT-PCR等技术,从气孔开度、乙烯释放、保卫细胞H₂O₂及黄酮醇水平和基因表达等方面,研究了乙烯在5–氨基乙酰丙酸(ALA)抑制脱落酸(ABA)或黑暗诱导的气孔关闭过程中的作用。结果表明,ALA促进苹果叶片乙烯释放,而乙烯生物合成抑制剂氨氧乙基乙烯基甘氨酸(AVG)及乙烯受体抑制剂1–甲基环丙烯(1-MCP)削弱ALA对ABA或黑暗诱导气孔关闭的抑制效应。ALA上调乙烯生物合成及信号转导相关基因的表达。AVG削弱ALA抑制ABA或黑暗诱导的H₂O₂上升效应,也削弱ALA对保卫细胞黄酮醇积累的促进效应。据此认为,外源ALA通过上调基因表达,促进叶片保卫细胞中乙烯生物合成和信号转导,诱导黄酮醇积累,清除ABA或黑暗诱导增加的H₂O₂,抑制气孔关闭,从而为光合作用所需的气体交换打开通道。     

苹果蛋白磷酸酶基因MdPP2C-Like的克隆及其对ABA的响应分析

以‘嘎拉’苹果（Malus × domestica Borkh.）为试材,克隆了ABA信号转导过程中的响应基因蛋白磷酸酶基因MdPP2C-Like（基因序列号 MDP0000126636）。基因结构分析显示,MdPP2C-Like定位于苹果8号染色体上,含有5个外显子与4个内含子;该基因全长1 035 bp,编码344个氨基酸,预测其蛋白质分子量为37.84 kD,等电点（pI）为5.72。蛋白质结构分析发现,其存在1个蛋白磷酸酶2C（PP2C）保守结构域,并与拟南芥中的A类PP2C保守结构域高度同源。构建的系统进化树表明MdPP2C-Like与At4G32950位于同一进化支,同源性较高。亚细胞定位结果表明,MdPP2C-Like主要定位于细胞核,少数分布在细胞质。定量分析显示,MdPP2C-Like在苹果不同组织中表达量不同,根中表达量较高。对获得的转基因拟南芥株系和苹果愈伤组织进行ABA响应分析,结果表明该基因的过量表达降低了植株和愈伤组织对ABA的敏感性。以上结果表明,克隆得到的MdPP2C-Like在响应ABA信号过程中起着重要作用。     

铁皮石斛蛋白磷酸酶PP2C家族基因鉴定及其表达分析

利用生物信息学方法对铁皮石斛(Dendrobium catenatum Lindl.)2C型蛋白磷酸酶(protein phosphatase 2C,PP2C)家族基因进行鉴定分析,采用实时荧光定量PCR技术分析PP2C基因的组织表达特性,以及在不同非生物胁迫和植物生长调节剂处理下的表达模式.共鉴定出67个家族成员,其...     

苹果酰基辅酶A结合蛋白2 编码基因MdACBP2的克隆和表达分析

 酰基辅酶A结合蛋白（ACBP）是一个保守的蛋白家族,在酰基酯辅酶A的转运过程中发挥重要作用。在水稻和拟南芥中鉴定的ACBP除了参与长链酰基辅酶A的转运以外,还参与了植物对生物和非生物胁迫的反应。为了探讨在苹果树中是否也存在参与植物对逆境反应的ACBP,并可用于改良苹果品种,以提高抗逆能力,从苹果品种‘鸡冠’中克隆、鉴定了一个ACBP基因,命名为MdACBP2。MdACBP2包含一个378 bp的开放阅读框,编码包含125个氨基酸残基的蛋白质;推定的氨基酸序列中包含有一个ACB结构域,无信号肽序列。序列和结构特征表明该基因是ACBP家族ClassⅠ亚族的成员。利用荧光定量PCR技术检测MdACBP2的表达,发现在苹果的根、叶、花、幼果、枝皮、芽等组织中均有表达,在叶中的表达量最高,在幼果中的表达量最低,这一表达模式暗示MdACBP2可能参与多种生理途径。不同胁迫处理的时序表达分析表明,MdACBP2的表达能被轮纹病病原菌Botryosphaeria dothidea侵染和10% PEG渗透胁迫所抑制,低温胁迫则能诱导MdACBP2的表达,1 mmol·L^-1 Pb(NO₃)₂处理对MdACBP2的表达没有显著影响。推测MdACBP2可能参与了苹果对低温胁迫的防御反应。     

乙肝表面抗原大蛋白(S1S2S)基因在转基因苹果中表达

构建了乙肝表面抗原大蛋白基因PRS-S1S2S的表达载体pYF9616,通过根癌农杆菌介导法首次将该基因导入苹果品种红爱佳中,得到了抗卡那霉素的GUS阳性转化植株。随机取3株GUS染色阳性的转基因苹果植株经PCR扩增及RT-PCR检测证实该基因已整合入转基因苹果的基因组,并在转录水平得到了表达,ELISA检测证明在苹果植株中正确表达了乙肝表面抗原大蛋白基因。     

Neuroprotective effect of astrocyte protein phosphatase 2A up-regulation on APP/PS1 double transgenic mice

AIM: To investigate the protective effects of astrocyte protein phosphatase 2A(PP2A) up-regulation on APP/PS1 double transgenic mice.METHODS: An eGFP-wtPP2A lentivirus with glial fiber acidic protein promoter was constructed to specifically increase PP2A expression in the astrocytes. The mice were divided into wild -type mice+vector virus group(Con), APP/PS1 transgenic mice+vector virus group(APP/PS1) and APP/PS1 transgenic mice+eGFP-wtPP2A lentivirus group(PP2A) by lateral ventricular injection of the lentivirus. Four weeks after injection of the virus, the immunofluorescence of brain slices were used to detect the level of β-amyloid protein(Aβ). Golgi staining was used to detect the changes of dendritic spine density and morphology. Electron microscopy was applied to detect the thickness of postsynaptic density(PSD). The Morris water maze test was applied to examine the learning and memory abilities of the mice.RESULTS: Up-regulation of PP2A in the astrocytes attenuated Aβ level increasing in APP/PS1 group. Up-regulation of PP2A in the astrocytes significantly attenuated both decreases in the dendritic spine density and the percentage of mushroom-like dendritic spines in the hippocampal CA3 region of APP/PS1 mice. Up-regulation of PP2A in the astrocytes significantly attenuated the reduced thickness of PSD in APP/PS1 group. Up-regulation of PP2A in the astrocytes attenuated the escape latency extending in APP/PS1 group.CONCLUSION: Up-regulation of PP2A in the astrocytes reduces AD-like pathological changes, and attenuates synaptic impairment, synaptic plasticity deficits and cognitive impairment in the APP/PS1 double transgenic mice.     

Role of ghrelin in cell protection by up-regulating HSP70 through ERK1/2 signaling pathway in PC12 cells

AIM:To study the role of ghrelin in cell protection by up-regulating heat shock protein 70 (HSP70) and inhibiting apoptosis induced by oxidative stress through extracellular regulated protein kinases 1/2 (ERK1/2) signaling pathway in the PC12 cells. METHODS:Sodium nitoprusside (SNP) was used to induce oxidative stress injury in the PC12 cells. The cultured PC12 cells were divided into SNP-injured group (incubated with SNP at 0.5 mmol/L for 6, 12, 18 and 24 h), ghrelin pretreatment group (ghrelin at 100 nmol/L was given 30 min before adding SNP); HSP70 inhibitor group (quercetin at 10 μmol/L was added 60 min before ghrelin treatment), ERK inhibitor group (ERK 1/2 inhibitor PD98059 was added 60 min before ghrelin treatment) and control group (added same amount of culture medium only). The apoptotic rate was detected by flow cytometry. The protein expression was determined by Western blot and immunocytochemistry. RESULTS:Compared with control group, the apoptotic rate of PC12 cells in SNP-injured group was significantly increased (P<0.05). Compared with SNP-injured group, ghrelin (100 nmol/L) pretreatment significantly inhibited SNP-induced apoptosis of PC12 cells (P<0.05), and significantly up-regulated the protein expression of HSP70 (P<0.05). Time-effect analysis showed that ghrelin had the most significant effect at 18 h after SNP injury. Quercetin, an inhibitor of HSP 70, significantly reduced the anti-apoptotic effect of ghrelin (P<0.05). Ghrelin pretreatment promoted the phosphorylation of ERK1/2. ERK1/2 inhibitor PD98059 significantly inhibited the effects of ghrelin on up-regulation of HSP70 expression (P<0.05). CONCLUSION:Ghrelin upregulates the expression of HSP70 and inhibits the apoptosis in the PC12 cells induced by oxidative stress by promoting the phosphorylation of ERK1/2.     

Role of renal preS1/S2-Ag and other HBV antigen determination in diagnosis of HBV-associated glomerulonephritis

AIM：To detect the expression of preS1/S2 antigen (preS1/S2-Ag) and other antigens of hepatitis B virus (HBV) in renal tissues of patients with HBV-associated glomerulonephritis (HBV-GN), and to analyze their roles in the diagnosis of HBV-GN.
METHODS：Patients hospitalized in our department from January in 2003 to January in 2013 were retrospectively studied. A total of 49 patients with positive HBV surface antigen (HBsAg) serology, clinical manifestations of hematuria and/or proteinuria, and pathological diagnosis of glomerulonephritis, without systemic lupus erythematosus, anaphylactic purpura, diabetes or hepatitis C, were selected. PreS1/S2-Ag, HBV e antigen (HBeAg), HBsAg and HBV core antigen (HBcAg) in the renal tissues were examined. Five cases of glomerular minimal-change disease (MCD) with negative HBsAg and 5 cases of non-glomerulonephritis with positive HBsAg served as controls. RESULTS：The positive rates of preS1/S2-Ag, HBeAg, HBsAg and HBcAg in the renal tissues from the 49 patients of glomerulonephritis with HBV infection were 32.7% (16 cases), 38.8% (19 cases), 14.3% (7 cases) and 46.9% (23 cases), respectively. Total antigen positive rate was 70.2% (36 cases). The expression of preS1/S2-Ag was located in the cytoplasm of renal tubular epithelial cells, glomerular epithelial cells, endothelial cells and mesangial cells, and positively correlated with the expression of HBcAg (r=0.459, P<001). The 4 antigens were not detected in the 5 cases of HBsAg-negative patients with glomerular MCD. In the 5 cases of HBsAg-positive patients with non-glomerulonephritis, there were 2 cases expressing HBeAg and 1 case expressing HBcAg, but no cases expressing preS1/S2-Ag or HBsAg. CONCLUSION：The expression of preS1/S2-Ag in renal tissues suggests that HBV may invade the cells of renal tissue. Combined detection of the 4 antigens could elevate the rate of diagnosis of HBV-GN.     

Role of Slit2/Robo1 signaling in development of neural tube and somites in early chick embryos

AIM: To investigate the effects of Slit2/Robo1 signaling on the development of neural tube and somites in early chick embryos. METHODS: Plasmid DNA was injected into the lumen of the neural tube from dorsal side of HH10 chick embryo using microinjection, and then in ovo electroporation was performed at half-side of neural tube while another side served as control. Subsequent 10-hour incubation was carried on after transfection until the development of neural tube and neural crest cells migrating to somites were investigated using the methods of immunofluorescence and in situ hybridization. RESULTS: Blocking Slit2/Robo1 signaling resulted in abnormal development of neural tube, while the expression of Slug and neural crest cells migrating to somites pathway were abnormal as well.CONCLUSION: Slit2/Robo1 signaling can affect the expression of Slug and play an important role in the fusion of neural fold, the trajectory of generation and migration of neural crest cells, and the differentiation of somites in early chick embryos.     

Artesunate negatively controls expression of cyclin D1 and activity of AP-1 by inhibiting the activation of ERK1/2 protein in HSC-T6 cells

AIM: To investigate the effects of artesunate(Art) on the expression of ERK1/2, AP-1 and cyclin D1 in rat hepatic stellate cells (HSCs), and to elucidate the molecular mechanism of Art against hepatic fibrosis. METHODS: HSC-T6 cells were treated with platelet-derived growth factor BB(PDGF-BB) to induce cell proliferation. The cells were divided into control group, PDGF-BB group, PDGF-BB+Art groups (with 6.25 mg稬^-1, 25 mg稬^-1or 50 mg稬^-1 of Art) and PDGF-BB+PD98059 group. The level of collagen type I in the supernatant was detected by enzyme-linked immunosorbent assay (ELISA). The mRNA expression levels of ERK1/2 and cyclin D1 were measured by RT-PCR. The protein levels of p-ERK1/2 and cyclin D1 in HSC-T6 cells were detected by Western blotting. The activity of AP-1 was analyzed by electrophoretic mobility shift assay. RESULTS: The concentration of collagen type I was significantly higher in PDGF-BB group than that in control group (P<0.05), and decreased in PDGF-BB+Art group and PDGF-BB+PD98059 group in comparison with that in PDGF-BB group (P<0.05, P<0.01). The protein level of ERK1/2 in PDGF-BB+Art group (50 mg稬^-1) was lower than that in PDGF-BB group (P<0.05), and was even lower in PDGF-BB+PD98059 group (P<0.01). The mRNA expression of cyclin D1 in PDGF-BB+Art groups (25 mg稬^-1and 50 mg稬^-1) and PDGF-BB+PD98059 group were significantly lower than that in PDGF-BB group (P<0.05). The protein levels of p-ERK1/2 and cyclinD1 were the highest in PDGF-BB group, and significantly lower in PDGF-BB+Art groups (6.25 mg稬^-1, 25 mg稬^-1 and 50 mg稬^-1) and PDGF-BB+PD98059 group (P<0.05, P<0.01). The AP-1 binding activity in HSC-T6 cells was down-regulated by Art. CONCLUSION: Artesunate inhibits the proliferation of HSC-T6 cells in vitro by inhibiting the activation of ERK1/2, thus down-regulating the activity of AP-1 and expression of cyclin D1.     

Effect of curcumin on p-ERK1/2-AP-1 cascade and diabetic neuropathic pain in rats

AIM: To evaluate the role of p-ERK1/2-AP-1 cascade in the process of curcumin against diabetic neuropathic pain (DNP) in rats.METHODS: Ninety-six male Sprague-Dawley rats were randomly divided into 4 groups (n=24): normal control group, DNP group, DNP with solvent group and DNP with curcumin (100 mg/kg) group. The rat model of diabetes was induced by a single intraperitoneal injection of streptozotocin (STZ, 75 mg/kg). Mechanical allodynia and thermal hyperalgesia were tested by mechanical withdrawal threshold (MWT) and thermal withdrawal latency (TWL) 2 weeks after induction,respectively. The diabetic rats were treated with curcumin (100 mg·kg^-1·d^-1, ip) for 2 weeks. The conditions of hyperalgesia and allodynia were determined 2 d before STZ injection, 14 d after STZ injection, and 3 d, 7 d, 14 d after administered with curcumin. The change of p-ERK1/2 was measured by the methods of Western blotting and immunohistochemistry. The expression of AP-1 in spinal cord dorsal horn and dorsal root ganglion (DRG) was detected by electromobility shift assay (EMSA).RESULTS: Compared with normal control group, the rats in DNP group developed hyperglycemia and a decrease in MWT and TWL associated with an increase in the activity of p-ERK1/2 and AP-1 in dorsal horn and DRG(P<0.05). Compared with DNP group, 7-day treatment with curcumin significantly attenuated mechanical allodynia and thermal hyperalgesia, and these effects were correlated with inhibiting the hyper-activation of p-ERK1/2 and AP-1 14 days after treatment with curcumin (P<0.05).CONCLUSION: Curcumin has beneficial effects on hyperalgesia in STZ-induced peripheral neuropathic pain. Activation of p-ERK1/2 and AP-1 may be the key mechanism of DNP in spinal cord and DRG.     

Role of TGFβ1/Smad3 signaling pathway-mediated lysine hydroxylase 2 activity in collagen deposition of pulmonary fibrosis

AIM: To investigate the effect of TGFβ1/Smad3 signaling pathway on the changes of lysyl hydro-xylase2 (LH2) activity, and to study the role in the relationship between LH2 and collagen deposition of pulmonary fibrosis. METHODS: Human lung fibroblast cell line HFL1 was cultured in F12 medium with 10% fetal bovine serum. The cells were divided into control group, TGFβ1 (10 μg/L) stimulation group, and minoxidil (5 μmol/L) intervention group. The cells in control group were treated with the equivalent volume of medium. The RNA and protein were collected after 48 h. The mRNA levels of PLOD2, α-SMA and COLⅠ were detected by RT-qPCR. The protein levels of LH2, total Smad3, phosphorylated Smad3, α-SMA, COLⅠ and COL Ⅳ were determined by Western blot. Hydroxylysylpyridinoline (HP) content was detected by ELISA. RESULTS: After stimulation with TGFβ1, the mRNA expression of PLOD2, α-SMA and COLⅠ was increased (P<0.01), and the protein levels of LH2, p-Smad3, α-SMA, COLⅠ and COL Ⅳ were also up-regulated, but the total Smad3 protein did not change. Treatment with minoxidil decreased the levels of above indexes (P<0.01). Compared with control group, stimulation with TGFβ1 increased the content of HP. However, treatment with minoxidil decreased the synthesis of HP (P<0.05). CONCLUTION: Activation of TGFβ1/Samd3 signaling pathway enhances LH2 expression. Minoxidil inhibits the TGFβ1/Samd3 signaling transduction, thereby reducing the expression of LH2 and the synthesis of hydroxylysyl collagen pyridine chain, and reducing pulmonary fibrosis.     

Stable reversal of multidrug resistance in A549/DDP cells by shRNA targeting MRP1 gene

AIM: To reverse multidrug resistance (MDR) of A549/DDP cells with short hairpin RNA (shRNA) expression vectors. METHODS: Two multidrug resistance-associated protein 1( MRP1 ) gene-specific shRNA expression plasmids pSilencer 2.1-U6 neo-MRP1 were constructed and introduced into A549/DDP cells. MRP1 mRNA was assayed by real-time fluorescent quantitative PCR. The MRP1 function was determined by rhodamine 123(Rho123) retention and the protein expression of MRP1 was detected by immunofluorescent staining. The viability of A549/DDP cells was evaluated by MTT method. RESULTS: MRP1 shRNA expression plasmids were successfully constructed. The expression of MRP1 at mRNA and protein levels was significantly decreased after sh-MRP1-2.1-1 and sh-MRP1-2.1-2 were transfected into A549/DDP cells. The intracellular accumulation of Rho123 significantly increased from(16.93±0.58)% to (89.02±0.59)% and (82.56±1.37)%. IC₅₀ of cisplatin were decreased from (101.45±0.64) μmol/L to (38.06±0.05) μmol/L and (53.72±0.36) μmol/L. IC₅₀ of 5-fluorouracil were decreased from (263.20±2.00) μmol/L to (98.82±1.16) μmol/L and (141.81±0.49) μmol/L. CONCLUSION: The shRNA expression plasmid pSilencer 2.1-U6 neo-MRP1 can stably and permanently inhibit MRP1 gene. The sensitivity of A549/DDP cells to drug is reversed.