甲胎蛋白特异性小干扰RNA表达载体的构建及鉴定

目的：构建特异性抑制甲胎蛋白（AFP）的小干扰RNA（siRNA）表达载体,为进一步研究AFP基因功能及AFP相关肿瘤的基因治疗奠定基础。方法：设计并合成AFP特异性的短链寡核苷酸,退火形成双链DNA片段,通过与RNAi-Ready pSIREN-DNR-DsRed-Express Donor Vector连接、转化大肠杆菌、扩增、纯化得到所需质粒,用琼脂糖凝胶电泳及基因测序鉴定其分子量及插入片段的序列。结果：琼脂糖凝胶电泳证实纯化后的质粒大小约为6740bp,测序证实插入序列与合成的寡核苷酸序列完全符合。结论：构建了AFP特异性的siRNA表达质粒pSIREN-DNR—DsRed-Express Donor Vector-AFP。     

斑马鱼中囊胚过渡调控机制

斑马鱼中囊胚过渡(MBT)始于受精卵的第10次卵裂,此时亦伴有细胞周期延长,分裂同步性丧失,合子型基因开始转录活化,胚胎细胞开始具备运动迁移能力等现象。斑马鱼MBT。的发生依赖于胚胎细胞的核质比,胚胎细胞周期中的G1时相则只有在合子型基因组开始被转录活化后才能出现。细胞周期检验点的激活可能也是受转录调控的,但中期检验点对DNA复制抑制状态的响应不仅在MBT前后、甚至在MBT前的不同阶段也可能有具体作用途径的差异。活化的P38蛋白在胚胎中的不对称分布是维持卵裂阶段细胞分裂同步性的关键因素。尽管大规模的合子型基因的表达发生在MBT开始后,也有少数与胚层分化有关的合子型基因是在MBT。前表达的,还有一些既有母型表达也有合子型表达的基因在MBT前后分别参与不同的信号途径来调控胚胎的发育与分化。     

激光诱变小麦后代材料籽粒贮藏蛋白编码基因变异的研究

本研究采用SDS—PAGE分析了冀麦七号、冀麦18和冀早15三个品种激光后代籽粒贮藏蛋白的变异情况。结果表明,高分子量麦谷蛋白和醇溶蛋白亚基变异较小,其控制位点为Glu—1和Gl—3或Gli—1;而低分子量部分变异较大,其控制位点多在Glu—3、Gli—2和Gli—3。这些变异具有一定的随机性。有关激光诱变对小麦面粉品质的影响有待进一步研究。     

Improving the Secretory Expression of an α-Galactosidase from Aspergillus niger in Pichia pastoris

α-Galactosidases are broadly used in feed, food, chemical, pulp, and pharmaceutical industries. However, there lacks a satisfactory microbial cell factory that is able to produce α-galactosidases efficiently and cost-effectively to date, which prevents these important enzymes from greater application. In this study, the secretory expression of an Aspergillus niger α-galactosidase (AGA) in Pichia pastoris was systematically investigated. Through codon optimization, signal peptide replacement, comparative selection of host strain, and saturation mutagenesis of the P1’ residue of Kex2 protease cleavage site for efficient signal peptide removal, a mutant P. pastoris KM71H (Mut^s) strain of AGA-I with the specific P1’ site substitution (Glu to Ile) demonstrated remarkable extracellular α-galactosidase activity of 1299 U/ml upon a 72 h methanol induction in 2.0 L fermenter. The engineered yeast strain AGA-I demonstrated approximately 12-fold higher extracellular activity compared to the initial P. pastoris strain. To the best of our knowledge, this represents the highest yield and productivity of a secreted α-galactosidase in P. pastoris, thus holding great potential for industrial application.     

黄曲霉菌原生质体的制备和再生技术优化

黄曲霉菌的遗传转化是研究黄曲霉菌致病相关功能基因的前提和基础,而原生质体是研究和建立真菌遗传转化系统的重要工具。本文分别以黄曲霉孢子和菌丝为材料,研究不同条件下黄曲霉原生质体的形成和再生,结果表明,黄曲霉孢子在酶液浓度为纤维素酶∶蜗牛酶∶溶壁酶=1.5%∶1.5%∶1.5%,30℃酶解3 h,原生质体制备率高达97.3%,再生率达89.2%;黄曲霉菌丝在菌龄为42 h,酶液浓度为纤维素酶∶蜗牛酶∶溶壁酶=1.5%∶1.5%∶1.5%,30℃酶解1 h,可获得最高原生质体产量为2.0×10^6个/m L,再生培养基中以1 mol/L蔗糖作为渗透压稳定剂时,原生质体再生率达5.5%。故本实验条件下,黄曲霉孢子原生质体的形成和再生优于菌丝。     

油菜菌核病危害损失的初步研究

本文就油菜菌核病病情、发病期及发病部位与油菜产量损失的相互关系进行了模似研究。结果表明：病斑绕茎度是影响产量的主要因素,其大小与产量损失成正相关,而病斑纵向长度与产量损失无明显相关性;发病期不同产量差异显著,以初花期发病产量损失最重,发病期与产量损失线性相关,可利用线性回归方程对产量损失进行预测;主茎发病部位愈近基部,枝条发病部位愈趋于主茎,单株产量损失率愈高。     

Optimization of ultrasound-assisted water extraction of flavonoids from Psidium guajava leaves by response surface analysis

Psidium guajava leaves are rich in health-promoting flavonoids compounds. For better utilization of the resource, the ultrasound-assisted aqueous extraction was investigated using Box-Behnken design under response surface methodology. A high coefficient of determination (R²?=?97.8%) indicated good agreement between the experimental and predicted values of flavonoids yield. The optimal extraction parameters to obtain the highest total flavonoids yield were ultrasonic power of 407.41?W, extraction time of 35.15?min, and extraction temperature of 72.69?°C. The average extraction rate of flavonoids could reach 5.12% under the optimum conditions. Besides, HPLC analysis and field emission scanning electron microscopy indicated that the ultrasonic treatment did not change the main component of flavonoids during extraction process and the higher flavonoids content was attributed by the disruption of the cell walls of guava particles. Thus, the extraction method could be applied successfully for large-scale extraction of total flavonoids from guava leaves.     

极端耐盐放线菌白色普氏菌YIM 90005T四氢嘧啶及5-羟基四氢嘧啶合成相关基因的克隆

摘要：【目的】 为了研究耐盐放线菌对高盐环境的适应机理。【方法】 用HPLC定量检测了极端耐盐、丝状产孢放线菌——白色普氏菌（Prauserella alba） YIM 90005T在不同盐浓度下胞内相容性溶质的种类和含量。【结果】 结果发现,四氢嘧啶和5-羟基四氢嘧啶是其主要的相容性溶质。在培养基NaCl浓度为10%时,四氢嘧啶在胞内累积浓度最大,为18.77 μg/mg干菌体重。之后随NaCl浓度的升高,胞内的四氢嘧啶含量逐渐减少,而5-羟基四氢嘧啶的含量逐渐增加,在该菌耐受的最高NaCl浓度下（24% w/v）,胞内5-羟基四氢嘧啶含量达到最大值,为22.98 μg/mg干菌体重。设计兼并引物,利用染色体步移,克隆得到四氢嘧啶及5-羟基四氢嘧啶合成相关基因ectABCD。序列分析表明,ectABCD位于一个操纵子中。进一步对不同NaCl浓度培养条件下ectB,D的表达量进行定量分析,结果表明该基因簇表达量随着培养基中NaCl浓度的增加而增大。【结论】 研究结果证实5-羟基四氢嘧啶是P. alba YIM 90005T在极高盐浓度条件下起渗透调节及保护的相容性溶质。     

三倍体湘云鲫繁殖季节的性腺结构观察

在繁殖季节解剖３７尾龄和１６尾声龄湘云鲫,用光镜和电镜观察了湘云鲫的性腺结构。在湘鲫的性腺部位有三种类型的结构：（１）清巢型;精巢小叶内分布有精子细胞,精子细胞呈退化现象,没有发现有正常成熟精子。（２）卵巢型,该类型性腺的大部分为体积较小,分化程度较低的呈囊状排列的细胞,在这些小细胞中镶嵌着少量初级卵母细胞和体积较大的退化的卵母细胞。（３）脂肪型;该类型的性腺部位仅有两条脂肪组织,即没有卵巢结构,