木尔坦棉花曲叶病毒编码蛋白的亚细胞定位分析

 木尔坦棉花曲叶病毒(Cotton leaf curl Multan virus,CLCuMV)是典型的单组分双生病毒,并伴随β卫星分子,是棉花曲叶病的主要病原之一。本研究利用农杆菌介导的瞬时表达系统,将CLCuMV及其卫星分子编码的7个病毒蛋白在本氏烟表皮细胞中表达。通过激光共聚焦显微镜观察发现:V1、C2和C3定位于细胞核;C1和βC1定位在细胞核以及细胞质或细胞膜上,并且在细胞质中形成丝状结构;V2和C4主要定位在细胞质或细胞膜上,细胞核也有微量表达,V2可形成大小不一的颗粒状聚集体结构,C4在细胞膜上可见点状聚集体结构。同时,利用RT-PCR和Western blot对病毒各基因的转录和表达水平进行了分析。CLCuMV编码蛋白的亚细胞定位为蛋白功能的进一步研究提供重要理论依据。     

病程相关蛋白在采后果蔬诱导抗病性中的研究进展

果蔬受到真菌、细菌或病毒侵染时,自身能诱导产生病程相关蛋白(Pathogenesis-related proteins,PRs)。根据它们的结构亲缘关系和生物活性,PRs被分为17个功能家族。在控制果蔬采后病害的研究中,利用激发子诱导产生果蔬抗性已逐渐成为果蔬采后病害防治中一种安全、高效的保鲜方法,这也成为果蔬采后抗病研究的热点和发展趋势。本文综述了果蔬病程相关蛋白的分类、功能、诱导表达,以及蛋白组学技术在果蔬采后PRs表达水平上的应用,以期为果蔬病程相关蛋白的研究提供借鉴和参考。     

木薯(Manihot esculenta)AKT1基因的克隆及生物信息学分析

植物细胞中的K^+通道蛋白AKT1(Arabidopsis K^+transpoter 1)主要负责吸收K^+。本研究基于木薯基因组数据库信息,利用PCR技术从木薯中克隆一个MeAKT1基因,该基因全长2634 bp,编码877个氨基酸,生物信息学分析表明MeAKT1含有10个外显子和9个内含子,编码的氨基酸分子量为99.02 kD,等电点为8.43,属于稳定蛋白。MeAKT1包含有5个跨膜区域,N端含有1个Ion_trans结构域,C端有1个KHA结构域,其二级结构以α-螺旋和无规则卷曲为主,进化树分析发现MeAKT1与蓖麻RcAKT1的亲缘关系最近。通过对MeAKT1蛋白的生物信息学分析有助于下一步深入研究MeAKT1在木薯耐贫瘠过程中的功能。     

MicroRNA-145 inhibits epithelial-mesenchymal transition in human renal cancer A-498 cells by ADAM28

AIM: To investigate the inhibitory effect of microRNA-145 (miR-145) on epithelial-mesenchymal transition (EMT) in renal cancer A-498 cells. METHODS: The A-498 cells were transfected with miR-145 mimics (M145) and mimic negative control(MNC), which served as M145 group and MNC group, respectively. Mock control (MC) group was set up using untreated A-498 cells. The expression level of miR-145 in each group was detected by RT-qPCR. Transwell assay was used to detect the invasion ability of the cells. The protein expression of vimentin, E-cadherin and ADAM28 was determined by Western blot. Bioinformatic method was used to predict the target genes of miR-145. Antagonistic effect of ADAM28 over-expression on the inhibition of EMT by miR-145 was detected by Western blot. The relationship between miR-145 and ADAM28 was analyzed by dual-luciferase reporter assay. RESULTS: The expression level of miR-145 in M145 group was significantly up-regulated than that in MC group (P<0.05). The number of invasive cells in M145 group was 12.78±3.37, which was significantly lower than that in MC group (P<0.05). ADAM28 may be the target gene of miR-145. Compared with MC group, the protein expression of vimentin and ADAM28 in M145 group was significantly decreased (P<0.05), while the protein expression of E-cadherin was significantly increased (P<0.05).After ADAM28 over-expression, the protein expression of vimentin in the A-498 cells of M145 group was significantly increased (P<0.05), and the protein expression of E-cadherin was significantly decreased (P<0.05). The results of dual-lucife-irasei reporter assay showed that ADAM28 was a downstream target gene of miR-145. CONCLUSION: miR-145 may inhibit the expression of EMT-related proteins through the downstream target gene ADAM28 and inhibit the EMT process of renal cancer A-498 cells.     

Notch1 promotes renal tubulointerstitial fibrosis in renal tissues of diabetic mice by inhibiting autophagy through Akt/mTOR signaling pathway

AIM: To observe the changes of Notch1 expression and autophagy in the renal tissues of diabetic mice, and to explore the regulatory effect of Notch1 on tubulointerstitial fibrosis by inhibiting autophagy in diabetic nephro-pathy. METHODS: The mice were randomly divided into normal control group (db/m mice) and diabetes group (db/db mice), with 8 rats in each group. After 12 weeks of feeding, the mice were sacrificed and the corresponding biochemical indexes were measured. The protein expression of Notch1 in the renal tubular epithelial cells was observed by immunohistochemical staining. The protein levels of Notch1, PTEN, p-Akt (Thr308), Akt, p-mTOR (Ser2448), mTOR, LC3, P62, collagen type Ⅰ (Col-Ⅰ) and collagen type Ⅲ (Col-Ⅲ) were determined by Western blot. RESULTS: Compared with the db/m mice, the blood glucose, glycosylated hemoglobin, serum creatinine, triglyceride and total cholesterol were increased in the db/db mice (P<0.01). Renal tubular epithelial cell vacuolar degeneration, renal tubular expansion and interstitial inflammatory cell infiltration in db/db mouse renal tissues with HE staining were observed. The images of Masson staining showed collagenous fiber-like substance deposition in the glomerular capillaries and renal interstitium, and disarrangement of tubular structure in the renal tissues of db/db mice. The protein expression levels of PTEN and LC3-Ⅱ were decreased (P<0.01 or P<0.05), while the protein levels of Notch1, P62, p-mTOR (Ser2448), p-Akt (Thr308), Col-I and Col-III were increased in the db/db mice as compared with the db/m mice (P<0.01). However, no significant change of total mTOR and Akt proteins between the 2 groups was found. CONCLUSION: Notch1 protein expression was increased, PTEN expression was significantly reduced, Akt/mTOR pathway was activated, autophagy was inhibited, and fibrosis was aggravated in the renal tissues of the diabetic mice.     

miR-204 inhibits proliferation of Hodgkin lymphoma cells by targeting Sirt1/p53 signaling pathway

AIM: To investigate the effect of microRNA-204 (miR-204) on the proliferation of Hodgkin lymphoma cells and the underlying mechanism. METHODS: The expression of miR-204 and Sirt1 mRNA in Hodgkin lymphoma tissues was detected by RT-qPCR. After transfection with miR-204 mimic, Sirt1 siRNA and miR-204 mimic+pcDNA3.1-Sirt1 into the L428 cells, the cell viability and BrdU incorporation were measured by CCK-8 assay and BrdU assay, respectively. The protein levels of Sirt1 and acetylated p53 (ac-p53) were determined by Western blot.The targeting relationship between miR-204 and Sirt1 was verified by double luciferase reporter assay. RESULTS: The low expression of miR-204 and the high mRNA expression of Sirt1 were found in the Hodgkin lymphoma tissues. Compared with control group, the cell viability, BrdU incorporation and the protein levels of Sirt1 and ac-p53 were significantly decreased after L428 cells were transfected with miR-204 mimic or Sirt1 siRNA (P<0.05). Compared with miR-204 mimic alone group, the cell viability, BrdU incorporation and the protein levels of Sirt1 and ac-p53 were increased after L428 cells were co-transfected with miR-204 mimic and pcDNA3.1-Sirt1 (P<0.05). The results of double luciferase reporter assay confiermed that Sirt1 was the target gene of miR-204. CONCLUSION: The inhibitory effect of miR-204 on the proliferation of L428 cells may be achieved by inhibiting the expression of Sirt1 and promoting the up-regulation of ac-p53.     

高效氯氰菊酯对小鼠卵巢生殖功能的影响及维生素E的干预作用

为探究高效氯氰菊酯 (beta-cypermethrin, β-CP) 对雌性小鼠卵巢生殖功能的影响及维生素E (vitamin E, VE) 的干预作用，将雌性昆白小鼠随机分为6 组:空白对照组 (花生油处理)、β-CP不同剂量 (10、20、40 mg/kg) 处理组、VE保护组 (20 mg/kg β-CP+20 mg/kg VE) 和VE组 (20 mg/kg VE)，连续灌胃10 d。灌胃结束后取小鼠卵巢组织，观察组织结构的病理变化，采用免疫组化法、蛋白免疫印迹试验及RT-PCR方法检测卵巢中StAR蛋白含量及casp-3、casp-8、INHα和INHβB 基因mRNA表达的变化。结果显示:与对照相比，10、20和40 mg/kg的β-CP处理均使小鼠卵巢组织结构发生了损伤，使组织中StAR蛋白的浓度分别降低了18.8%、36.3%和40.3%，casp-3基因的表达分别升高了16.0%、26.7%和52.9%，INHα基因的表达分别升高了34.5%、83.6%和228.7%，INHβB基因的表达分别升高了7.5%、39.2%和52.7%；20和40 mg/kg的β-CP处理使得casp-8基因的表达分别升高了27.1%和36.7%。上述处理组与对照组的差异均达显著水平 (P＜0.05)。与 20 mg/kg β-CP处理组相比，VE 保护组的StAR蛋白含量也显著增多 (P＜0.05)。研究表明，β-CP对小鼠卵巢具有毒性作用，这与β-CP抑制StAR蛋白的合成，上调casp-3、casp-8、INHα及INHβB基因的表达有关；添加VE对小鼠卵巢有一定的保护作用，这与VE可减弱β-CP对StAR蛋白合成的抑制作用有关。     

Comparative effect of advanced soy products or corn protein concentrate with porcine meal on growth,body composition,and distal intestine histology of Florida pompano,Trachinotus carolinus

The present study was designed to investigate the effects of diets containing advanced soy products (enzyme‐treated soy and fermented soy) or corn protein concentrate (CPC) in combination with porcine meal (PM) to completely replace poultry byproduct meal (PBM) on growth performance, body composition, and distal intestine histology of Florida pompano, Trachinotus carolinus. Four experimental diets were formulated to be isonitrogenous and isolipidic, to contain 400 g/kg crude protein and 80 g/kg lipid. A reference diet (PBM diet [PBMD]) contained 150 g/kg PBM and 495 g/kg soybean meal (SBM), and three test diets were formulated replacing PBM with 15 g/kg of CPC (CPC diet [CPCD]) or replacing all SBM and PBM with 535 g/kg fermented soy (fermented soybean meal diet [FSBMD]) or 451.3 g/kg enzyme‐treated soy (enzyme‐treated soybean meal diet [ESBMD]). All three test diets were supplemented with 38 g/kg of PM. Diets were fed based on a percentage of bodyweight adjusted after sampling the fish every 2 weeks to triplicate groups of Florida pompano juveniles (mean weight 8.06 ± 0.22 g). After 8 weeks of feeding, fish fed CPCD and ESBMD performed equally well in terms of final body weight, thermal growth coefficient, and percentage weight gain in comparison to fish fed PBMD. In all cases, feeding FSBMD resulted in poor feed conversion and lower feed intake compared to other treatments. Protein retention efficiency, whole‐body proximate composition, phosphorus, sulfur, potassium, magnesium, calcium, sodium, and zinc contents were not significantly influenced by the dietary treatments. The results obtained in the present histological study showed no significant differences in the thickness of serous layer, muscular layer, and submucosal layer of the intestine among treatments. Fish fed CPCD showed a significant widening of the lamina propria with an increase of cellular infiltration and higher presence of goblet cells compared to other dietary treatment. Based on these results, 451 g/kg ESBM or combination of 150 g/kg of CPC and 495 g/kg SBM supplemented with 38 g/kg PM can be utilized to develop a practical diet for juvenile Florida pompano without impacting growth, nutritive parameters, and several distal intestine health parameters.     

利用酵母双杂交系统筛选与草莓镶脉病毒移动蛋白P1互作的寄主因子

 构建感染草莓镶脉病毒(SVBV)森林草莓的酵母cDNA文库,利用酵母双杂交系统,筛选出与SVBV P1蛋白互作的15种寄主因子。生物信息学分析发现,这15种寄主因子参与茉莉酸途径、泛素化、光合作用、抗病抗逆、蛋白修饰、蛋白运输和氧化还原等多种生物过程。另外,这些寄主因子还具有其他分子功能,包括氧化还原酶活性、蛋白二硫化物异构酶活性和金属离子结合活性等。本研究初步探讨了P1与寄主因子的互作机理,为揭示SVBV侵染森林草莓以及SVBV在寄主中扩展的分子机制提供理论依据。     

品种间大豆脂肪氧化酶活性差异及β-胡萝卜素漂白性研究

为了解东北地区大豆品种间大豆脂肪氧化酶(LOX)活性差异规律,整理适于东北地区种植的大豆种质资源307份(6份半野生品种,17份地方品种及4份国外品种,其余为栽培品种),统一种植,秋季收获后,测定各品种成熟籽粒脂肪氧化酶总活性。分析品种间脂肪氧化酶总活性分布规律、大豆脂肪氧化酶总活性与β-胡萝卜素漂白性之间关系。结果表明,307份大豆品种间脂肪氧化酶总活性差异极显著,平均值为21.68 U·mL~(-1),最小值为2.51 U·mL~(-1),最大值为42.38 U·mL~(-1)。根据脂肪氧化酶总活性差异,通过聚类分析将307份大豆品种分为3类。低活性品种占总量43.65%,脂肪氧化酶总活性平均值为15.25 U·mL~(-1);中等活性品种占总量43.97%,脂肪氧化酶活性平均值为24.64 U·mL~(-1);高活性品种占总量12.38%,脂肪氧化酶总活性平均值为33.87 U·mL~(-1)。脂肪氧化酶总活性与油分含量呈正相关,与蛋白含量呈负相关,与漂白性呈正相关。