秀丽纤杆线虫在寄生性线虫抗原研究中的应用

秀丽纤杆线虫 (Caenorhabditis elegans)是一种模式线虫 ,目前广泛应用于寄生性线虫基因的功能研究 ,并且还被用于捻转血矛线虫等寄生性线虫疫苗侯选抗原基因的表达和耐药性产生机制方面的研究     

二化螟dsRNA降解酶基因的克隆与表达分析

为解析二化螟Chilo suppressalis体内参与降解双链RNA(double-stranded RNA,dsRNA)的关键核酸酶的功能,克隆二化螟不同的非专一性核酸酶（non-specific nuclease,NUC）基因,并对这些基因进行生物信息学分析和组织定量表达分析,同时对dsRNA降解酶（dsRNA degrading nuclease,dsRNase）活力的组织分布进行研究。结果显示,共克隆获得5个NUC基因,其中有4个编码dsRNase亚家族基因（CsdsRNase1～CsdsRNase4）和1个编码Endonuclease G亚家族基因（CsEndoG）。5个NUC基因的开放阅读框核苷酸序列长度范围为828～1 338 bp,编码275～445个氨基酸残基,其分子量大小为31.68～49.57 kD,预测等电点为5.48～9.42。CsdsRNase1和CsdsRNase2含有信号肽序列,两者相似度极高,且与家蚕Bombyx mori和斜纹夜蛾Spodoptera litura中具有dsRNA降解酶活力的dsRNase同源聚类;CsdsRNase1和CsdsRN...     

PTGS/ RNAi的作用机制及其应用

论述了双链RNA对同源基因的表达进行特异性的封闭而引起的转录后的基因沉默（PTGS/ RNAi）现象, PTGS/ RNAi的作用机理，作用模型，参与这一过程的酶、基因、起始因子和作用因子，介绍了PTGS/ RNAi在基因组功能研究、基因的时空表达调控以及在作物改良等方面的应用。     

利用shRNA真核表达载体干涉雌性鸡胚性腺中芳香化酶基因(CYP19A1)的表达

本研究通过把构建的短发夹结构RNA(shRNA)真核表达载体导入鸡胚,检测鸡胚性腺分化期质粒载体在鸡胚体内代谢情况及雌性鸡胚性腺中芳香化酶基因(CYP19A1)mRNA表达效率,进而探讨利用该方法在鸡胚体内进行特定基因干涉的可行性.实验针对CYP19A1基因构建了4条特异性表达载体,一条非特异性表达载体.每个实验组选取45个新鲜种蛋作为实验材料进行胚盘下腔注射,并设立空白对照组.鸡胚发育12d时,检测各处理组鸡胚肝脏组织中质粒存在情况,并取其左侧性腺组织进行目标基因的荧光定量分析.研究结果表明,导入组在12日胚龄时所有鸡胚基因组中均可检测到绿色荧光蛋白基因(EGFP);荧光定量结果显示,导入特异性表达载体cyp-580、cyp-1083和cyp-1295后,对应雌性鸡胚性腺CYP19A1 mRNA表达效率显著低于空白对照组,干涉效率分别为:73％、52％和85％;特异性表达载体cyp-1403组CYP19A1mRNA表达效率与空白对照组相比有所降低但无显著性差异.本实验为诱导鸡胚性反转提供了新方法并建立了鸡胚发育期特定基因体内干涉新平台.     

猪生长抑素Ⅱ型受体基因(SSTR2)shRNA的构建与筛选

生长激素(GH)是调控动物生长的重要激素,GH分泌水平的高低主要受正性调控因子生长激素释放因子(GRF)和负性调控因子生长抑素(SS)的双向调节。SS通过生长抑素受体(somatostatin receptor,SSTR)发挥作用,减少GH的分泌水平。本研究设计了3条SSTR2的短发夹RNA(shRNA),构建猪(Sus scrofa)SSTR2真核表达质粒(pcDNA3.1(-)-SSTR2)和SSTR2慢病毒RNA干扰质粒(pshRNA-copGFP lentivector,LV-shRNA),并共转染中国仓鼠(Cricetulus griseus)卵巢细胞系(CHO),通过对转染细胞的荧光观察、Real-time PCR和酶联免疫法(ELISA)检测,结果表明,CHO转染细胞中猪SSTR2 mRNA表达水平不同程度的抑制,分别为90.4%、28.3%和86.3%,(P<0.05)。其中,LV-shRNA1表现出最高的沉默效率:转染效率在80%左右时,猪SSTR2mRNA水平沉默效率为90.4%,蛋白质水平降低了33.3%。本研究成功地筛选到了降低猪SSTR2基因表达的shRNA,为利用shRNA技术下调动物基因表达,构建转基因模型提供思路。     

玉米大斑病菌StSte12基因对H_2O_2氧化胁迫的应激调节

通过比较野生型菌株Wt01-23与StSte12基因RNAi沉默突变体菌株StRNAi9-10和StRNAi3-6在H_2O_2胁迫下生长和发育方面的差异,分析转录因子基因StSte12对玉米大斑病菌氧化胁迫的调节能力。在不同浓度H_2O_2胁迫条件下,测定野生型菌株和突变体菌株的菌落生长速度、菌丝形态、产孢量和菌丝萌发率。结果表明,随着H_2O_2浓度的增加,玉米大斑病菌野生型菌株和突变体菌株的菌落生长速度、产孢量和菌丝萌发率均显著降低,但突变体菌株的降低程度显著高于野生型菌株,表明StSte12基因对玉米大斑病菌的氧化应激调节具有重要的调控功能。     

Breeding for increased grain protein and micronutrient content in wheat: Ten years of the GPC-B1 gene

To provide food and nutrition security for a growing world population, continued improvements in the yield and nutritional quality of agricultural crops will be required. Wheat is an important source of calories, protein and micronutrients and is thus a priority to breed for improvements in these traits. The GRAIN PROTEIN CONTENT-B1 (GPC-B1) gene is a positive regulator of nutrient translocation which increases protein, iron and zinc concentration in the wheat grain. In the ten years since it was cloned, the impacts of GPC-B1 allelic variation on quality and yield traits have been extensively analyzed in diverse genetic backgrounds in field studies spanning forty environments and seven countries. In this review, we compile data from twenty-five studies to summarize the impact of GPC-B1 allelic variation on fifty different traits. Taken together, the results demonstrate that the functional copy of the GPC-B1 gene is associated with consistent positive effects on grain protein, Fe and Zn content with only marginally negative impacts on yield. We conclude that the GPC-B1 gene has the potential to increase nutritional and end use quality in a wide range of modern cultivars and environments and discuss the possibilities for its application in wheat breeding.     

Transgenic sorghum with suppressed synthesis of kafirin subclasses: Effects on flour and dough rheological characteristics

Arising from work showing that conventionally bred high protein digestibility sorghum types have improved flour and dough functionality, the flour and dough properties of transgenic biofortified sorghum lines with increased protein digestibility and high lysine content (TG-HD) resulting from suppressed synthesis of several kafirin subclasses, especially the cysteine-rich γ-kafirin, were studied. TG-HD sorghums had higher flour water solubility at 30 °C (p < 0.05) and much higher paste viscosity (41% higher) than their null controls (NC). TG-HD doughs were twice as strong as their NC and dynamic rheological analysis indicated that the TG doughs were somewhat more elastic up to 90 °C. CLSM of doughs and pastes indicated that TG-HD had a less compact endosperm protein matrix surround the starch compared to their NC. The improved flour and dough functional properties of the TG-HD sorghums seem to be caused by reduced endosperm compactness resulting from suppression of synthesis of several kafirin subclasses which modifies protein body and protein matrix structure, and to improved protein-starch interaction through hydrogen bonding specifically caused by reduction in the level of the hydrophobic γ-kafirin. The improved flour functionality of these transgenic biofortified sorghums can increase their commercial utility by complementing their improved nutritional quality.