巴西橡胶树HbICE1基因酵母双杂交诱饵载体的构建及互作蛋白的筛选

寒害是我国植胶区的主要自然灾害之一,不仅影响橡胶产量还威胁到橡胶树的生存。拟南芥中,ICE1是低温胁迫信号通路中的重要转录激活因子,但橡胶树冷胁迫信号途径中包括Hb ICE1在内的大部分关键基因尚未被克隆与鉴定,与Hb ICE1蛋白发生互作的蛋白也不清楚,成为严重阻碍橡胶树抗寒分子机理研究的瓶颈。为了筛选和鉴定与巴西橡胶树Hb ICE1蛋白发生互作的蛋白,阐明橡胶树抵御寒害胁迫的分子机理,本文首先通过PCR扩增出Hb ICE1基因的编码序列(约1400 bp),然后将目标序列插入p GBKT-7载体,构建酵母双杂交诱饵载体;将该载体转入酵母Y2H菌株感受态细胞中,并在缺陷型培养基上检测其自激活活性,发现Hb ICE1基因的自转录激活在含有30 mmol/L和更高浓度3-AT(3-氨基-1,2,4-三氮唑)的培养基上能得到有效抑制;进一步通过与橡胶树c DNA文库进行酵母双杂交,筛选出一些可能与Hb ICE1发生互作的蛋白,包括DNA结合蛋白、核糖体蛋白和功能未知蛋白。本研究结果为橡胶树抗寒机理研究提供了重要理论依据。     

Clinical evaluation and mitochondrial DNA sequence analysis in three Chinese families with Leber's hereditary optic neuropathy

We report here the clinical, genetic, and molecular characterization of three Chinese families (WZ4, WZ5, and WZ6) with Leber's hereditary optic neuropathy (LHON). Clinical and genetic evaluations revealed the variable severity and age-of-onset in visual impairment in these families. Penetrances of visual impairment in these Chinese families were 33.3%, 35.7%, and 35.5%, respectively, with an average 34.8%. Furthermore, the average age-at-onset in those Chinese families was 17, 20, and 18 years. In addition, the ratios between affected male and female matrilineal relatives in these Chinese families were 3:0, 1:1, and 1.2:1, respectively. Sequence analysis of the complete mitochondrial genomes in these pedigrees showed the distinct sets of mtDNA polymorphism, in addition to the identical G11778A mutation associated with LHON in many families. The fact that mtDNA of those pedigrees belonged to different haplogroups F1, D4, and M10 suggested that the G11778A mutation occurred sporadically and multiplied through evolution of the mtDNA in China. However, there was the absence of functionally significant mutations in tRNA and rRNAs or secondary LHON mutations in these Chinese families. The I187T mutation in the ND1, the S99A mutation in the A6, the V254I in CO3, and I58V in ND6 mutation, showing high evolutional conservation, may contribute to the phenotypic expression of the G11778A mutation in the WZ6 pedigree. By contrast, none of mtDNA variants are evolutionarily conserved and implicated to have significantly functional consequence in WZ4 and WZ5 pedigrees. Apparently, these variants do not have a potential modifying role in the development of visual impairment associated with G11778A mutation in those two families. Thus, nuclear modifier gene(s) or environmental factor(s) seem to account for the penetrance and expressivity of LHON in these three Chinese families carrying the G11778A mutation.     

脑星形胶质细胞生物学功能研究进展

脑星形胶质细胞是中枢神经系统（CNS）内在数目占绝对优势的一类大胶质细胞,被认为在神经元的整个发育过程中起重要作用。本文主要就参与星形胶质细胞调节神经元活动的主要功能分子,星形胶质细胞在中枢神经系统的生物学功能,及其与疾病的关系作一简要回顾。     

A new DGGE protocol targeting 2,4-diacetylphloroglucinol biosynthetic gene phlD from phylogenetically contrasted biocontrol pseudomonads for assessment of disease-suppressive soils

In the rhizosphere, biocontrol pseudomonads producing 2,4-diacetylphloroglucinol (Phl) can protect plants from soil-borne pathogens. DGGE of phlD has been proposed to monitor these bacteria, but two distinct protocols were needed for analysis of both the 'Pseudomonas fluorescens' species complex and the strains from rrs restriction group ARDRA-1. Here, a single DGGE protocol performed on 668-bp GC-clamp-containing phlD amplicons was effective with both types of pseudomonads, and 36 reference biocontrol strains from the 'P. fluorescens' complex or group ARDRA-1 gave a total of 11 distinct DGGE bands. phlD amplicons with at least two to seven nucleotidic differences could be discriminated, and the discrimination level was similar to that of phlD restriction analysis with four enzymes. Multiple phlD-DGGE bands were obtained when studying rhizosphere soil containing indigenous phlD+ pseudomonads, and phlD diversity was higher when DGGE was implemented after incubation of tobacco rhizosphere extracts in semi-selective medium (MPN approach) in comparison with approaches based on direct analysis of rhizosphere DNA extracts or assessment of phlD+colonies. phlD-DGGE profiles differed for a soil suppressive and a soil conducive to black root rot of tobacco, and each soil yielded new phlD sequences. In conclusion, this DGGE protocol was useful for monitoring indigenous rhizosphere consortia of phlD+ pseudomonads.     

板蓝根水提物S-03体外抑制甲、乙型流感病毒感染的实验研究

以水提法分离制备板蓝根水提物S-03分析其基本化学成分,并在狗肾细胞(MDCK)上分别接种人甲1、3型和乙型流感病毒标准株、临床分离株以及禽流感病毒,采用空斑减少和免疫荧光及血凝抑制实验方法,在预防、治疗和直接作用三种试验模式下探讨S-03体外对流感病毒的抑制作用。研究结果表明S-03的主要化学成分为糖类,多糖所占总重的比例最高。S-03体外抗病毒药效显示:①预防模式:对各型流感病毒均无抑制作用;②治疗和直接作用模式:对不同亚型流感病毒均有一定程度的抑制作用,且直接作用(SI=2.5~16)效果优于治疗模式(SI=2.2~5.8);③血凝抑制试验:对不同亚型的人流感病毒血凝素有不同程度抑制作用(最低抑制浓度为3.12~25 mg/mL),并且对禽流感病毒(H6N2、H7N3、H9N2)的血凝素也有一定的抑制作用(最低抑制浓度25~50 mg/mL),其作用机制可能为抑制流感病毒表面的血凝素(HA),从而阻止病毒感染。     

双向凝胶电泳技术及其应用

介绍了双向电泳的基本原理、操作过程及其在动植物和医学中的应用。同时对当前双向电泳技术面临的挑战和发展前景进行了介绍。