Studies on self-activation and self-interaction of circadian clock protein KaiC of Microcystis aeruginosa in yeast two hybridization

通过PCR扩增了铜绿微囊藻的生物钟主控基因KaiC,并将其分别克隆到酵母双杂交系统的诱饵质粒pGBKT7和猎物质粒pGADT7中,然后将重组质粒pGBKT7-kaiC/pGADT7-kaiC和pGBKT7-kaiC/pGADT7分别共转化酵母菌AH109,经营养缺陷生长和β-半乳糖苷酶印迹检测表明,KaiC蛋白不具有毒性不会影响酵母细胞的生长,也不具有自激活活性,不会激活报告基因的表达,并且KaiC蛋白自身存在较强的相互作用。诱饵质粒pGBKT7-kaiC可用于从基因组文库中筛选KaiC的相互作用蛋白。A clock system is universal among cyanobacteria.In this study,the clock gene kaiC was cloned from Microcystis aeruginosa by PCR.Through the study of the function and finding its interacting protein,the kaiC gene in Microcystis aeruginosa was subcloned into the bait vector pGBKT7 and prey vector pGADT7 of yeast two-hybrid system.After being verified by sequencing,the recombinant plamids pGBKT7-kaiC/pGADT7-kaiC and pGBKT7-kaiC/pGADT7 were co-transferred into yeast strain AH109 by polyethylene glycol/lithium acetate method respectively and its expression products were assayed whether it can affect the growth of yeast cells and activate the reporter(β-galactosidase) genes.The transformed cells were selected on SD dropout medium lacking tryptophan,leucine,histidine and adenine at 30℃ for 3~4 days.The β-galactosidase filter assay shows that KaiC protein was not toxic to AH109 and could not activate the reporter gene.The results also demonstrated homotypic interaction of KaiC in transformed yeast cells that expressed two types of fusion proteins.The constructed plasmid pGBKT7-kaiC may serve as bait vector of yeast two hybrid GAL4 system to fish KaiC interaction protein from genomic library of Microcystis aeruginosa.国家自然科学基

Effect of rich organic matter on Paraprionospio pinnata (Polychaeta:Spionidae) on Futian tidal flat in Shenzhen Bay

根据1999年1月至2002年10月在深圳湾福田潮滩A、D、E三个断面获得的大型底栖动物奇异稚齿虫(Paraprionospiopinnata)和有机质数据,分析了深圳湾福田潮滩奇异稚齿虫对有机质含量的效应特征。结果表明,深圳湾福田潮滩奇异稚齿虫有明显的季节变化,即3、4月密度高,形成全年的高峰期,9、10月密度低,形成全年低谷期。奇异稚齿虫密度随着有机质含量的增加逐渐降低,前者密度和生物量大小是:A>D>E,而有机质大小也是:AD>E.The distribution of organic matter content is also A<D<E.There is negative correlation between density of P.pinnata and organic matter content.The higher organic matter is,the lower density and biomass of P.pinnata are

Application of Mycobacterium Tuberculosis Specific Protein InterferonGamma Release Assay in Tuberculosis Diagnosis and Therapeutic Monitoring

目的:目的:探讨结核分枝杆菌相关γ-干扰素释放试验(Tb-IgrA)在结核病诊断及临床抗结核治疗效果评价中的应用价值。方法:对196例临床确诊结核患者进行相关病史收集,同时进行Tb-IgrA、结核菌素皮肤试验、结核杆菌培养、涂片镜检、结核抗体检测,结核患者的治疗过程中按用药疗程(初治患者第2、5、6个月,复治患者第2、5、8个月)对其进行随访,在治疗结束后6个月和1年后各随访一次,比较Tb-IgrA与结核菌素皮肤试验在治疗过程中的变化。结果:确诊结核病例196例,其中肺结核158例,肺外结核38例,Tb-IgrA在结核诊断中灵敏度为82.1%,特异性为91.7%,阳性预测值为93.1%,阴性预测值为79.3%,与传统实验室检查方法相比有显著性差异(P<0.01)。在结核患者的抗结核治疗过程中,Tb-IgrA阳性率及浓度水平治疗前与治疗后2、6月以及复治后2、5、8月比较显著下降(P<0.01),Tb-IgrA水平与结核分枝杆菌数量存在较好相关性,与传统的TST方法评估疗效相比有显著性差异(P<0.01)。结论:Tb-IgrA在结核诊断中敏感性和阳性预测值较高,可作为较好的结核诊断的依据,同时Tb-IgrA对肺外结核的灵敏度较高,为肺外结核患者的诊断提供了较为可靠的依据,通过治疗过程Tb-IgrA检测分析,外周血Tb-IgrA的变化随着抗结核治疗的进程下降的趋势明显,与结核涂片镜检结果符合率较高,是临床疗效评估较为理想的指标。Objective To investigate the applicative value of mycobacterium tuberculosis specific protein interferon gamma release test in early diagnosis of tuberculosis and the evaluation of clinical efficacy.Methods 196 clinical diagnosis of tuberculosis patients were related to the history collection,and detected by TB-IGRA simueltaneously,tuberculin skin test( purified protein derivative of tuberculin 5 units),tubercle bacillus culture,directly smear,tuberculosis antibody detection.Treatment of tuberculosis patients were followed up( 2,5,6 months,retreatment patients at 2,5,8 months),in 6 months and 1 years after the end of treatment followup once.Compared with difference between the IGRA and tuberculin skin test and other traditional laboratory results.Results Confirmed tuberculosis cases in 196 cases,including 158 cases of pulmonary tuberculosis,38 cases of extra-pulmonary tuberculosis.The sensitivity of TB-IGRA in the pulmonary tuberculosis and extra-pulmonary tuberculosis showed 81% and 86.8% respectively,at the same time,TB-IGRA in the diagnosis of tuberculosis,the negative predictive value express 86.3%,specificity was 91.7%,and the positive predictive value was 88.2%.In anti-tuberculosis treatment the positive rate of TB-IGRA and concentration levels before treatment and after treatment 2,5,6months,retreatment 2,5,8months decreased significantly( P < 0.01).There is better correlation TB-IGRA concentration level with number of mycobacterium tuberculosis.It is significant difference compared to TB-IGRA with the traditional TST method to the evaluation of clinical treatment( P < 0.01).Conclusion The sensitivity of TB-IGRA in the diagnosis of tuberculosis and the postive predictive value is higher; it can be used as a good basis for the diagnosis of tuberculosis.TB-IGRA provides a more reliable diagnosis method in patients with extra-pulmonary tuberculosis.Peripheral blood TB-IGRA decreased significantly in anti- tuberculosis treatment process.Coincidence rate between TB-IGRA concentration level and number of mycobacterium tuberculosis is higher.It is an ideal index to evaluate the clinical efficacy.浙江省医药卫生科技计划项目(2009A167

Compositional and structural characterization of marine organic colloids-an overview

国家自然科学基金项目(49976021);; 国家重点基础研究发展规划项目(G2000078500);; 杰出青年基金项目(49825162

以课题式实验教学提高学生科研兴趣和科研能力的探索——以“遗传学实验”为例

传统的实验教学多为验证性实验,实验结果可预测且大同小异,缺乏新意和挑战,对于拔尖学生来说如同鸡肋,因此在经典传统的实验教学中进行内容改革,提高难度势在必行。本文以遗传学实验中的经典实验模式生物果蝇为例,开展了课题式实验教学改革,以果蝇诱变为命题,大大激发了学生的科研兴趣,同时拓展了科学思维,培养了科研能力,体现了研究性学习的创造性和挑战性,是生物学实验教学改革的有益探索。“基础学科拔尖学生培养计划”研究课题(20180214,20170606

DNA加合物研究及在环境毒理学中的应用

致癌物在生物体内经解毒系统诱导与生物体内的DNA形成加合物,DNA加合物综合反映了生物体对致癌物的暴露、吸收、分布、代谢以及机体对DNA的修复能力,是致癌物有效作用的量度,与致癌作用直接相关。目前较多用于DNA加合物测定的方法是~(32)P后标记技术。DNA加合物作为一项反映人群暴露于致癌物的指标在肿瘤分子流行病学研究中得到了较好的应用,鱼与哺乳动物一样可活化致癌物形成DNA加合物,由于解毒系统不完善及DNA修复能力差而对致癌物特别敏感,因此鱼及其他水生动物的DNA加合物研究受到重视并且一直引人注目

DNA加合物研究及在环境毒理学中的应用

致癌物在生物体内经解毒系统诱导与生物体内的ＤＮＡ形成加合物，ＤＡＮ加合物综合反映了生物体对致癌的暴露，吸收，分布，代谢以及机体对ＤＮＡ的修复能力，是致癌物有效作用的量度，与致癌作用直接相关，目前较多钐于ＤＮＡ加合物测定的方法是＾３２Ｐ后标记技术，ＤＮＡ加合物作为一项反映人群暴露于致癌物的指标在肿瘤分子流行病学研究中得到了较好的应用，鱼与哺乳动物一样在活化致癌物形成ＤＮＡ加合物，由于解毒系统不完善

用环境毒物对草鱼组织ATPase的影响作为生态毒理学指标的研究

本实验报导了测定ATPase活力的快速方法。对化学法则ATPase活力所用的几种测磷方法进行了比较，通过实验研究了匀浆方法、缓冲液、酶纯化步骤。对酶浓度-活力曲线、测定方法的精密度也进行了研究。用此方法测定了草鱼鳃、肾Na~+ K~+ Mg~(2+)&mdash;&mdash;ATPase活力并对结果进行单因素方差分析，证实了在正常情况下，不同批鱼在温度16 &plusmn; 4 ℃驯化三天以上后，鳃、肾Na~+ K~+ Mg~(++)&mdash;&mdash;ATPase活力没有显著差异(P &lt; 0.05)。在此基础上，对Hg~(2+)、Cr~(6+)，三氯联苯(TCB),直链烷基苯磺酸钠(LAS)对草鱼组织ATPase的影响进行了离体实验。结果表明：Hg~(2+), TCB, LAS对草鱼组织Na~+k~+Mg~(++)-ATPase有较强的抑制作用，浓度对数对抑制百分数的几率单位作图有较好的线性关系。它们对草鱼组织Na~+K~+Mg~(++)&mdash;&mdash;ATPase的半抑制浓度分别为1.8ppm (肾), 18.44ppm (鳃), 3.92ppm (鳃)。酶实验还表明，肾Na~+K~+Mg~(++)-ATPase比鳃Na~+K~+Mg Mg~(++)&mdash;&mdash;ATPase对Hg~(2+)的敏感性大，Hg~(2+),LAS对ATPase的作用有失活作用存在。Hg~(2+)主要作用于Na~+K~+-ATPase。在毒物作用水平上，对本实验的方法进行了验证。总结了毒物作用规律，讨论了ATPase实验的意义，提出了可用毒物对ATPase的影响作为毒理学实验的一项指标并对这一指标应用的可行性及前景进行了讨论