番茄抗TYLCV 基因新标记的开发及其在多抗聚合选择中的应用

根据已经克隆的番茄黄化曲叶病毒病（TYLCV）抗性基因Ty-1/Ty-3 和与基因Ty-4 紧密连锁的分子标记在抗感材料中的序列差异，开发出简单可靠的InDel 标记，建立了多重PCR 体系。采用分子标记辅助选择将TYLCV、斑点病、疮痂病和溃疡病中一个病害的多个抗性基因或不同病害的抗性基因聚合到一个材料中，培育出育种中间材料。     

黑龙江省西甜瓜栽培品种及种质资源对细菌性果斑病的抗性鉴定

采用室内苗期人工接种鉴定方法，对黑龙江省市售的16 份西、甜瓜栽培品种和84 份种质资源进行了细菌性果斑病的抗病性鉴定。10 份西瓜栽培品种中新红宝、京欣1 号、双抗大地雷、特大庆红宝、超丰F1、庆发7 号等6 个品种表现为中抗；景丰宝和齐红西瓜表现为中感；吉福2 号和庆农1 号表现为感病；6 份甜瓜栽培品种中齐甜1 号、永甜3 号和龙甜3 号3 个品种表现为中抗；日本甜宝和龙甜1 号2 个品种表现为中感；本甜3 号表现为感病；西、甜瓜栽培品种中均未发现高抗及免疫品种。49 份甜瓜种质资源中抗病材料占鉴定种质资源总数的57.1%，从中筛选出3 份高抗材料分别为X11-3、X11-1 和X11-8；在35 份西瓜种质资源中抗病材料占鉴定总数的48.6%，从中筛选出2 份高抗材料11W005 和11W003。     

终冷温度对加剂改性大庆原油蜡沉积的影响

为了分析终冷温度对加剂改性原油蜡沉积规律的影响,保持油壁温差相同,控制不同的终冷温度,采用搅拌槽蜡沉积装置、流变仪、差热扫描量热仪等试验仪器,对添加纳米降凝剂的大庆油、添加EVA降凝剂的大庆油以及大庆空白油进行静态蜡沉积对比试验与动态剪切对比试验。同时,结合分子扩散、胶凝等机理,分析不同试验条件下加剂改性大庆油的结蜡规律。结果表明:当终冷温度较低时,加剂油结蜡总量明显高于空白油,但结蜡总量中含凝油多、蜡晶结构较弱;空白油结蜡总量低,但蜡晶结构较强;随着终冷温度升高,加剂油与空白油的结蜡量逐渐降低并趋于一致。终冷温度较低且剪切剥离强度较弱时,添加降凝剂会增加管输蜡沉积量;一旦经过高剪切或提高终冷温度,加剂油的结蜡量将明显降低,管输安全性提高。     

波罗的海希瓦氏菌和杀鲑气单胞菌致腐性及TorA还原酶差异比较

为了研究冷藏海产品中腐败菌希瓦氏菌和气单胞菌的致腐性差异，本实验比较分析了大黄鱼源波罗的海希瓦氏菌和杀鲑气单胞菌在28℃和4℃下的生长及三甲胺（TMA）、生物胺和挥发性盐基氮（TVB-N）的生成；通过PCR技术扩增2种腐败菌的氧化三甲胺还原酶基因（torA），利用生物信息学比较TorA蛋白的相似性、理化特性和蛋白空间结构。结果显示，杀鲑气单胞菌在28℃生长较快，而波罗的海希瓦氏菌在4℃生长更快。相对于杀鲑气单胞菌形成较高的尸胺，波罗的海希瓦氏菌产生更多TMA和腐胺，在冷藏鱼汁中积累更高TVB-N。同时在波罗的海希瓦氏菌和杀鲑气单胞菌中分别扩增出2 490和1 959 bp的torA基因，2种TorA蛋白与同属菌相似性高于97%，而二者相似性仅为36.90%。波罗的海希瓦氏菌TorA蛋白的分子量和等电点分别为92.3 ku和6.52，甘氨酸含量最高，而杀鲑气单胞菌中TorA蛋白的分子量和等电点分别为90.6 ku和6.74，丙氨酸含量最高，蛋白结构差异明显。且希瓦氏菌中torA 和鸟氨酸脱羧酶（DOC）基因表达量分别为气单胞菌的1.26和19.04倍。可见，波罗的海希瓦氏菌和杀鲑气单胞菌为海产品嗜冷腐败菌，其中希瓦氏菌胺类代谢能力更强，与其TorA特定理化特性和高表达量相关。本研究为揭示海产品微生物的致腐机制提供理论支持。     

溶血性曼氏杆菌PCR检测方法的研究

溶血性曼氏杆菌是导致牛、羊呼吸道传染病的一种病原菌。为快速、准确诊断由本菌导致的疾病 ,从基因库中获得溶血性曼氏杆菌( M.haemolytica )的基因序列 ,再从其基因序列中获得与其它细菌包括 M.annheimiagranulomatis,M.varigena ,M.ruminalis,M.glucosidal ,M.annheimiaspp和多杀性巴氏杆菌等不同的基因片段 ,设计成引物 ,建立了特异性好、敏感性高的 M.haemolytica PCR检测方法。结果表明 ,除溶血性曼氏杆菌为阳性外 ,其余所有细菌均为阴性 ,特异性为 1 0 0 % ,最小检出量为 8× 1 0 2 cfu/ m L 曼氏杆菌或 1 / 1 0个单个菌落。检验人工感染牛 ,检出率为 75%。此PCR检测方法的建立 ,为 M.haemolytica提供了一个快速诊断方法     

肉牛饲养专家系统的设计

本文对肉牛饲养专家系统进行了构想,确定了系统目标,建立了肉牛专家系统的框架结构,确定和建立了相关的知识库。在此基础上,完成了肉牛饲养专家系统的设计。     

不同收获期玉米青贮干物质在奶牛瘤胃内降解率的研究

本文采用随机实验设计，利用瘤胃尼龙袋技术，测定全株玉米青贮分剐在乳熟、腊熟期收获时其干物质(DM)在奶牛瘤胃的降解率。结果表明：各期DM在奶牛瘤胃降解率依次为40．22％、53．24％。测试结果证明，在黑龙江省东部地区，腊熟期收获的全株玉米青贮对奶牛的饲用价值较高。     

Effect of adoptive transfer of CD4+ T cells with miR-7 knockdown on acute liver injury in mice

AIM:To study the effect of adoptive transfer of CD4⁺ T cells with microRNA-7 (miR-7) knockdown (KD) on mouse acute liver injury model and to investigate its significance. METHODS:CD4⁺ CD62L⁺ T cells were purified from the spleen of normal wild-type (WT) mice and miR-7KD mice by magnetic bead sorting, and were stained with CFSE. These 2×10⁶ CFSE-labeling cells were injected into normal mice via tail vein, and then the mouse acute liver injury model was induced by intraperitoneal injection of 30 mg/kg concanavalin A. After 72 h, the appearance, weight and weight index of the liver were investigated. The pathological change of the liver tissues was observed by HE staining. Real-time PCR was used to examine the mRNA expression of Bax and P53. The expression levels of CD62L, interleukin-4 (IL-4) and interferon-γ (IFN-γ) in the CD4⁺ T cells were analyzed by flow cytometry. RESULTS:We found that the liver tissue became lighter, and the weight (P<0.01) and weight index (P<0.05) were changed significantly in miR-7KD mice compared with control group. Moreover, HE staining showed that the liver cell damage was increased in the liver of miR-7KD mice. Meanwhile, the expression levels of Bax and P53 were significantly increased in miR-7KD group (P<0.05). The percentage of CD62L in CD4⁺ T cells was significantly decreased (P<0.01) in miR-7KD mice, with high expression of IFN-γ (P<0.05) and low expression of IL-4 (P<0.01) in CD4⁺T cells. CONCLUSION:These findings suggest that miR-7 knockdown significantly promotes the pathology of CD4⁺ T cell-mediated acute liver injury, which provides a preliminary experimental basis for further exploration on the mechanism of acute liver injury occurrence.     

ROS promotes adhesion of neutrophils to bone marrow stromal cells and its mechanism

AIM:To investigate the effect of reactive oxygen species (ROS) on the adhesion of neutrophils to bone marrow stromal cells (BMSCs) and its mechanism. METHODS:Murine bone marrow neutrophils were isolated from mouse tibia and femur by density gradient centrifugation. HL60 cells were induced into human mature neutrophils (dHL60 cells) by DMSO treatment. Murine bone marrow neutrophils and dHL60 cells were labeled with CFDA-SE. The adhesion of the CFDA-SE-labeled cells to BMSC monolayer was tested by microplate reader after H₂O₂ treatment. The level of glutaredoxin 1 (Grx1) in dHL60 cells infected by lentivirus carrying Grx1 expression vector was examined by fluorescence microscopy and Western blot. The genotype of Grx1^-/- mice was identified by PCR. RESULTS:Diff-Quick staining result displayed that the purity of murine bone marrow neutrophil was higher than 90%. The adhesion of H₂O₂-pretreated neutrophils to BMSCs was higher than that of the control cells (P<0.01). The expression of Grx1 in Grx1 stably transfected dHL60 cells was significantly higher than that in the control cells. After H₂O₂ treatment, the results of in vitro adhesion assay showed that the adhesion of dHL60 cells with Grx1 over-expression to BMSCs was lower than that of the control cells (P<0.01). The results of PCR showed no Grx1 was detected at the whole gene level in Grx1^-/- mice. Compared with the neutrophils from wild-type mice, the neutrophils from Grx1^-/- mice displayed increased adhesion to BMSCs after H₂O₂ treatment. Vascular cell adhesion molecule-1 (VCAM-1) antibody pretreatment induced the adhesion rate back to non-H₂O₂-treated condition. CONCLUSION:ROS promotes the adhesion of neutrophils to BMSCs in bone marrow, which might be regulated by VCAM-1 adhesion signaling-related S-glutathionylation.     

Effect of hypothalamic PVN exogenous orexin-A on food intake in obese rats and regulation by NPY receptor signaling pathway

AIM:To investigate whether neuropeptide Y (NPY) receptor signaling pathway is involved in the regulation of orexin-A on food intake and glucose-sensitive (GS) neuronal excitability in the hypothalamic paraventricular nucleus (PVN) of diet-induced obese (DIO) rats. METHODS:Fluorescence immunohistochemistry experiment was used to observe the expression of orexin-A receptor (orexin receptor 1, OX1R) and NPY receptor Y5 (NPY-5R) in the PVN. The effect of orexin-A on the excitability of GS neurons in PVN was observed by single cell discharge recording. The cannula was implanted into the PVN of SD rats and DIO rats. The orexin-A, OX1R antagonist SB-334867 and NPY-5R antagonist CGP-71683 were injected through the cannula to observe the 0~2 h and 0~4 h food intake of the rats. RESULTS:The expression of OX1R and NPY-5R in the PVN of DIO rats was significantly higher than that in the SD rats. Orexin-A inhibited glucose-inhibited (GI) neurons and excited glucose-excited (GE) neurons in the PVN. However, the effects of orexin-A on GS neurons were partially blocked by the NPY-5R antagonist CGP-71683. Compared with the SD rats, orexin-A had more pronounced excitatory and inhibitory effects on PVN GS neurons in the DIO rats. Injection of orexin-A in the PVN increased food intake in the SD rats and DIO rats. However, the orexin-A-induced feeding was partially blocked by the NPY-5R antagonist CGP-71683. The effect of orexin-A on feeding was stronger in the DIO rats than that in the SD rats. CONCLUSION:The hypothalamic PVN orexin-A regulates food intake and GS neuronal excitability mainly through the OX1R signaling pathway, and NPY-5R signaling is also involved in this process, in which the regulatory effect on DIO rats is more sensitive.