大肠杆菌基因无痕敲除技术及策略

基因敲除技术是大肠杆菌基因组减小和代谢途径改造的有效手段,其中基于同源重组原理的基因无痕敲除技术显现出其他技术所不具备的应用优势和发展潜力。该技术可以快速敲除大肠杆菌基因组中的目标基因,并且在基因组中不残留任何外源片段,所以不会干扰后续的基因操作。我们分类介绍了无痕敲除技术中所涉及载体的结构、功能及其相应的敲除策略,着重介绍了无痕敲除技术的原理及载体构建方法。     

1株拮抗真菌的生防细菌Xj11的分离与鉴定

从江西省井冈山自然保护区发病毛竹中分离到1株具有明显抑制植物病原真菌活性的生防细菌Xj11。其对小麦赤霉病菌、尖孢镰刀菌、甘蔗节菱孢菌、链格孢菌和炭角菌均有较强的抑制作用。通过形态特征和培养特征观察、生理生化实验及16S rDNA序列系统发育分析,初步鉴定该菌为唐菖蒲伯克霍尔德氏菌,命名为Burkholderia gladioli strain Xj11。     

醇母PHO81基因的克隆和表达

从酵母染色体ＤＮＡ中获得１个约３．０ｋｂ的ＢａｍＨＩ的克隆片段和１个约５．０ｋｂ的ＰｓｔＩ片段,前者包含１７４５ｂｐ的ＰＨＯ８１基因编码序列及１２４４ｂｐ的上游序列,后者包含２２３６ｂｐ的编码序列及约２．８ｋｂ的下游序列,经拼接得到完整的ＰＨＯ８１基因。以ＵＲＡ３基因取代部分ＰＨＯ８１的编码序列,通过体内同源重组,获得ＰＨＯ８１基因缺陷的酵母细胞株。构建ＰＨＯ８１－ＬａｃＺ融合基因,以β－半乳糖苷酶的活力表示ＰＨＯ８１基因的表达水平,研究了它的表达作用。ＰＨＯ８１基因为阻遏型表达,受无机磷浓度的控制,高磷使基因表达阻遏,低磷去阻遏。ＰＨＯ８１对其自身的表达有正调控作用,它与ＰＨＯ５和ＰＨＯ１１基因的调控模式相似,但ＰＨＯ８１上游调控序列和ＰＨＯ５及ＰＨＯ１１的同源性很低。     

松口蘑深层发酵工艺的研究

对松口蘑深层发酵工艺进行系统研究。通过正交试验初步确定松口蘑适宜的培养基组成 :玉米粉 30g,葡萄糖 1 0g ,豆饼粉 1 0g ,玉米浆 1 0g ,KH₂ PO₄1g ,定容至 1L。适宜发酵条件 :最适生长温度为 2 5℃ ,摇瓶转速为 1 60r/min ,最适pH为 5 0 ,最适接种量为 1 0 % ,装液量 1 2 0mL/ 5 0 0mL摇瓶培养 1 0d ,菌体生物量达 1 2 94g/L。     

青海湖岸带土壤与沉积物的地化特征与细菌群落对水位上升的响应

【目的】探究青海湖岸带土壤与沉积物的地化特征与细菌群落对水位扩张的响应。【方法】从岸上至岸下沿垂直青海湖岸带方向,采集距离湖面不同高度土壤(土壤：S1、S2)、岸边不同水深表层沉积物(过渡区：E0、E6、E17)及湖心表层沉积物(沉积物：D1、D2)样品,土壤与沉积物水深(土壤水深表示为负数)从小到大的变化表征岸边土壤被淹水转变为沉积物的过程。采用地球化学分析和16SrRNA基因高通量测序技术,探究岸带土壤与沉积物样品中的地化特征与微生物群落构成。【结果】青海湖水位上升导致的生境转变对岸带土壤与沉积物的理化性质、营养水平、有机碳类型等地化特征产生显著影响。具体表现为,随着水位升高,岸带土壤与沉积物的pH、矿物结合态有机碳含量显著升高,而碳氮比值、可溶性有机碳(dissolved organic carbon,DOC)、颗粒态有机碳含量显著下降。随着水位上升,青海湖岸带被淹没土壤的细菌群落多样性下降,且群落结构发生明显变化。这种变化与环境因子变化密切相关,具体表现为,细菌群落物种丰富度指数和香农多样性指数随着水位上升呈下降趋势;活性金属结合态有机碳含量与细菌群落多样性的变化密切相关;理化...     

酵母PHO2蛋白及其变异体与PHO5USA体外的相互作用

酵母ＰＨＯ２蛋白及其变异体与ＰＨＯ５ＵＳＡ体外的相互作用杨军,敖世洲（中国科学院上海生物化学研究所分子生物学国家重点实验室,２０００３１）关键词酵母;ＰＨＯ２;突变;ＤＮＡ结合ＰＨＯ２是酵母阻遏型酸性磷酸酯酶基因转录的正调控因子［１］,由５５９个氨基...     

Effects of different states of sheep fetal fibroblasts as donor cells on the early development in vitro of reconstructed sheep embryos

To investigate the effects of different states of donor cells on the development of reconstructed sheep embryos, we designed five treatments of donor cells, including cell passage, cell size, serum starvation,colchicine treatment and gene transfection. Results are as follows: ( Ⅰ ) Compared with 16-18 passage cells, the morula/blastocyst rate of 5-7 passage cells as donor nuclei was significantly higher (17.3%vs. 4.9%, P＜0.05), suggesting the advantage of short-time cultured cells in supporting the development of reconstructed embryos. (Ⅱ) The morula/blastocyst rate of reconstructed embryos derived from medium cells (15-25 μm) as donor nuclei was higher than that from large cells (25-33 μm) and small cells (8-15 μm)( 20.0% vs. 8.0%, 9.7%), indicating that reconstructed embryos from medium cells had a greater potentiality to develop into morula/blastocysts than those from small or large ones. (Ⅲ) The morula/blastocyst rate of reconstructed embryos from donor cells of SS (serum starvation) was lower than that from donor cells of NSS (non-serum starvation), but no significant difference was detected between SS and NSS( 11.8% vs. 18.6%, P＞0.05). (Ⅳ) Fetal fibroblasts treated with 0.05 μmol/L colchicine exhibited a higher morula/blastocyst rate of reconstructed embryos than those treated with 0.10 μmol/L colchicine and untreated ones (27.5% vs. 12.1%, 17.1%), however, no significant difference among the three treatments was detected (P＞0.05). (Ⅴ) The morula/blastocyst rate of reconstructed embryos from fetal fibroblasts transfected with GFP gene only was 3.1%, significantly lower than that from non-transgenic cells (3.1% vs. 20.4%, P＜0.05). In conclusion, our results demonstrated that fetal fibroblasts of fewer passages, medium size could ensure a higher morula/blastocyst rate of reconstructed embryos. Serum starvation of donor cells might be unnecessary to the development of reconstructed embryos. Donor cells treated with 0.05 μmol/L colchicine could facilitate the development of reconstructed embryos. Additionally, as cells transfected with GFP gene were used as donor nuclei, adverse effect on the development of reconstructed embryos was observed. Therefore, the developmental efficiency of reconstructed embryos could be improved if proper treatments to donor cells were used.     

蓝藻有机无机复混肥对几种作物的增效试验

研究了几种专用蓝藻有机无机复混肥,并对农作物进行了田间肥效试验。结果表明,施用蓝藻有机无机复混肥与普通N、P、K复合肥相比较,对烟草,西芹,韭菜,康乃馨等都有增产和改良土壤的效果。烟草试验表明,蓝藻复混肥的施用量以900kg/hm2效果最好,烟草产量、产值、上等烟比例分别比对照增加7.28%,5.04%,19.81%;西芹试验表明,施用蓝藻有机无机复混肥的各处理比对照高,其中以900kg/hm2和1200kg/hm2最高;其次是600kg/hm2;分别比对照高17.1%、16.3%和9.5%;而施用蓝藻有机无机复混肥900kg/hm2和1200kg/hm2,韭菜的株高、叶宽和产量比对照分别提高了12.21%和13.32%;康乃馨产花量则以施肥量为1200kg/hm2的最高,产花量为4865枝/100m2,比对照高11.6%。韭菜和土壤样品中未检测出藻毒素含量,表明使用蓝藻有机无机复混肥对植物和土壤是安全的。利用水华蓝藻生产有机无机复混肥,既解决了富营养化湖泊大量暴发的水华蓝藻的巨大生物量处置难题,又达到了改善环境、增加了经济和社会效益。       

Semi-automatic algorithm for construction of the left ventricular area variation curve over a complete cardiac cycle

Background  

Two-dimensional echocardiography (2D-echo) allows the evaluation of cardiac structures and their movements. A wide range of clinical diagnoses are based on the performance of the left ventricle. The evaluation of myocardial function is typically performed by manual segmentation of the ventricular cavity in a series of dynamic images. This process is laborious and operator dependent. The automatic segmentation of the left ventricle in 4-chamber long-axis images during diastole is troublesome, because of the opening of the mitral valve.     

甘草酸的提取及其抑菌活性研究

本文对甘草酸的各种提取方法进行了对比研究。结果表明,超声提取与传统搅拌提取、酶解提取及反复冻溶提取相比,是较适于从甘草中快速高效提取甘草酸的新方法,其最优条件为用20倍量0.7%氨水超声处理2次,每次20min。此外,探讨了提取的甘草酸的体外抑菌活性,其对金黄色葡萄球菌,大肠埃希氏杆菌,绿脓杆菌和枯草杆菌的最小抑菌浓度(MIC)分别为:0.0625,0.125,0.25,0.03125mg/mL,表明其具有较好的抑菌活性。