CaDHN3, a Pepper (Capsicum annuum L.) Dehydrin Gene Enhances the Tolerance against Salt and Drought Stresses by Reducing ROS Accumulation

Dehydrins (DHNs) play an important role in abiotic stress tolerance in a large number of plants, but very little is known about the function of DHNs in pepper plants. Here, we isolated a Y₁SK₂-type DHN gene “CaDHN3” from pepper. To authenticate the function of CaDHN3 in salt and drought stresses, it was overexpressed in Arabidopsis and silenced in pepper through virus-induced gene silencing (VIGS). Sub-cellular localization showed that CaDHN3 was located in the nucleus and cell membrane. It was found that CaDHN3-overexpressed (OE) in Arabidopsis plants showed salt and drought tolerance phenotypic characteristics, i.e., increased the initial rooting length and germination rate, enhanced chlorophyll content, lowered the relative electrolyte leakage (REL) and malondialdehyde (MDA) content than the wild-type (WT) plants. Moreover, a substantial increase in the activities of antioxidant enzymes; including the superoxide dismutase (SOD), peroxidase (POD), catalase (CAT), ascorbate peroxidase (APX), and lower hydrogen peroxide (H₂O₂) contents and higher O₂^•− contents in the transgenic Arabidopsis plants. Silencing of CaDHN3 in pepper decreased the salt- and drought-stress tolerance, through a higher REL and MDA content, and there was more accumulation of reactive oxygen species (ROS) in the CaDHN3-silenced pepper plants than the control plants. Based on the yeast two-hybrid (Y2H) screening and Bimolecular Fluorescence Complementation (BiFC) results, we found that CaDHN3 interacts with CaHIRD11 protein in the plasma membrane. Correspondingly, the expressions of four osmotic-related genes were significantly up-regulated in the CaDHN3-overexpressed lines. In brief, our results manifested that CaDHN3 may play an important role in regulating the relative osmotic stress responses in plants through the ROS signaling pathway. The results of this study will provide a basis for further analyses of the function of DHN genes in pepper.     

一株微生物采油菌的16S rDNA鉴定和绿色荧光蛋白分子标记

筛选到一株高效生产多糖的YX菌,油田先导试验证明该菌可以用于微生物调剖提高原油采收率（Microbial Enhanced Oil Recovery,MEOR）.经16SrDNA分析鉴定,YX菌属于肠内杆菌属（Enterobacter.）,为跟踪监测其动态信息,构建表达绿色荧光蛋白（green fluorescent protein,GFP）的重组质粒pET-30a（＋）-EGFP,利用CaCl2法将其转化至YX菌,利用荧光显微镜可以观察到重组YX（GFP）工程菌的绿色荧光.结果表明YX菌可以被绿色荧光蛋白基因标记,用于微生物采油研究.     

转基因树突状细胞激活细胞毒性T细胞特异性杀伤淋巴瘤的体外实验

目的 探讨转基因树突状细胞激活细胞毒性T细胞产生抗淋巴瘤的特异性细胞免疫反应。方法 采用人骨髓来源的髓系前体细胞,在人细胞因子IL-4、GM-CSF和INF-α诱导下,在体外生成大量树突状细胞。将制备好的含有IgVH1核酸质粒,用脂质体法转染树突状细胞。转染成功的树突状细胞与外周血T淋巴细胞共培养,激活特异性CTL细胞,与阳性表达IgVH1的人淋巴瘤Namalwa细胞反应,用3H-TdR掺入法观察CLLs对瘤细胞的特异性杀伤效应。结果 树突状细胞能够用脂质体方法转染IgVH1核酸质粒,并且有效递呈给外周血T细胞,对表达IgVH1的人淋巴瘤Namalwa细胞产生特异性免疫杀伤活性,与对照组相比差异有显著意义（P<0.01）。结论 体外诱导扩增的 DC能够转染 IgVH1核酸质粒,体外激活T淋巴细胞,产生特异性细胞毒效应。     

重组质粒pMG36e-nisI-gfp在乳酸菌中的应用

本研究将带有nisI和咖（绿色荧光蛋白）基因的乳酸菌表达载体pMG36e-nisI-gfp转化至乳酸菌中,观察其在宿主内的稳定性．对重组菌进行了菌体形态、传代培养、质粒验证等研究,考察了该质粒的稳定性．结果显示：重组菌在不含抗生素的培养基中连续传代20代后,质粒丢失率低;菌体大小和形态基本不变;质粒经HindⅢ酶切后大小不变;GFP在第0、5、10、15和20代宿主菌中都可以表达,表达量没有明显差别,在SDS-PAGE中的带型一致;初步的动物实验验证了该质粒作为遗传标记的应用效果．说明该质粒在宿主菌中具有良好的稳定性．     

细菌质粒DNA的快速提取及检测

介绍一种质粒DNA的快速提取方法,所分离出的DNA纯度较好,全部操作仅在两只Eppendorf管中进行,含质粒DNA的上清液可直接点样走琼脂糖凝胶电泳。整个提取过程在较短时间内完成。该方法简便、快捷、效果好。     

一种特异性检测单增李斯特菌的质粒DNA标准物质

研制了一种质粒DNA标准物质,包含目前单增李斯特菌检测常用的靶基因序列——内化素基因(Inl A)序列,可适用于扩增Inl A基因的单增李斯特菌PCR相关检测方法。采用紫外分光光度法(UV)和高分辨电感耦合等离子体质谱(HR-ICP-MS)两种方法,多家实验室联合定值的方法,对该套标准物质的均匀性、稳定性、量值以及不确定度进行详细的考察,质粒DNA标准物质的最终定值结果为94.9(±5.1)ng/μL。该质粒DNA标准物质可用于微生物实验室对单增李斯特菌PCR相关检测进行检测结果质量控制、开展方法比对和能力验证等。     

枯草芽孢杆菌脂肪酶表达质粒的构建及其表达

以枯草芽孢杆菌168(B.subtilis 168)染色体为模板PCR扩增出P43启动子,与大肠杆菌-枯草杆菌穿梭质粒pUBC19相连得到表达载体pUBC-P43,然后将枯草芽孢杆菌脂肪酶基因lipA克隆到载体pUBC-P43启动子下游,得到重组质粒pUBCPL并转化B.subtilis TZ10.经中性红油脂平板、酶切和PCR方法鉴定得到重组菌TZ10/pUBCPL.宿主菌TZ10是B.subtilis DB104染色体缺失了lipA基因后获得.重组菌经初步发酵,以橄榄油为底物测定发酵上清液最高脂肪酶活力为49.1 U·L-1,而相应菌株DB104发酵最高酶活力仅为11.4 U·L-1.     

CAMBRIDGE PRIZE LECTURE DEVELOPING NEW STRAINS OF YEAST*

Genetic engineering or recombinant DNA technology is now routinely applied to the construction and development of new strains of brewing yeast. This is a direct consequence of the power of the technology which facilitates the modification, introduction and stable maintenance of specific genes in brewing yeast, without compromising the intrinsic brewing properties of the yeast itself. The way in which gene technology has been applied to the development of new strains of Bass yeast is briefly illustrated by the provision of plasmid-based systems for ensuring the stable maintenance of recombinant genes, the construction of amylolytic and β-glucanolytic yeast and the design and development of genetic systems for enhancing the value of waste brewers yeast. Commercial and regulatory issues are discussed.     

Genetic Carriers and Genomic Distribution of cadA6—A Novel Variant of a Cadmium Resistance Determinant Identified in Listeria spp.

Listeria monocytogenes is a pathogen responsible for severe cases of food poisoning. Listeria spp. strains occurring in soil and water environments may serve as a reservoir of resistance determinants for pathogenic L. monocytogenes strains. A large collection of Listeria spp. strains (155) isolated from natural, agricultural, and urban areas was screened for resistance to heavy metals and metalloids, and the presence of resistance determinants and extrachromosomal replicons. Of the tested strains, 35% were resistant to cadmium and 17% to arsenic. Sequence analysis of resistance plasmids isolated from strains of Listeria seeligeri and Listeria ivanovii, and the chromosome of L. seeligeri strain Sr73, identified a novel variant of the cadAC cadmium resistance efflux system, cadA6, that was functional in L. monocytogenes cells. The cadA6 cassette was detected in four Listeria species, including strains of L. monocytogenes, isolated from various countries and sources—environmental, food-associated, and clinical samples. This resistance cassette is harbored by four novel composite or non-composite transposons, which increases its potential for horizontal transmission. Since some cadAC cassettes may influence virulence and biofilm formation, it is important to monitor their presence in Listeria spp. strains inhabiting different environments.