Characterization of SjIrV1 of Schistosoma japonicum and the preliminary analysis of immunoprotective effect of SjIrV1 recombinant protein

钙结合蛋白是日本血吸虫生长发育不可或缺的蛋白，执行着非常广泛而重要的功能。本研究在课题组日本血吸虫体被表膜蛋白研究基础上，利用PCR技术克隆了中国大陆株日本血吸虫66kDa钙结合蛋白（SjIrV1）编码基因的cDNA序列，BLAST分析与菲律宾株日本血吸虫SjIrV1cDNA编码序列一致，荧光定量PCR分析表明该基因在童虫和成虫期不同发育阶段均有表达，其中在35d和42d成虫中表达量较高，在42d雌虫中该基因表达水平远高于42d雄虫。构建重组表达质粒pET28a(+)-SjIrV1，在大肠杆菌中成功诱导表达，重组蛋白主要以可溶性形式存在，通过高效液相色谱法（RP-HPLC）以及串联质谱法（MS...Calcium-binding protein is an indispensable protein which performs extensive and important functions in the development of Schistosoma japonicum. Based on our primary study on tegument surface proteins of S. japonicun, a cDNA encoding a 66 kDa calcium-binding protein of S. japonicum (Chinese strain) was cloned, sequence analysis revealed that it was identical with that of SjIrV1 of Philippines str...学位：理学硕士院系专业：生命科学学院_动物学学号：2162010115227

Prokaryotic Expression, Purification, Crystalization and Electron Paramagnetic Resonance of a Mutant T179A of Alternative Oxidase

交替氧化酶（AlternativeOxidase，AOX），广泛存在于高等植物中，是植物线粒体内膜上呼吸链中抗氰呼吸途径的末端氧化酶。AOX参与植物多种生理代谢调节，例如调节呼吸代谢、抑制活性氧的形成、抵抗逆境以及调节细胞凋亡等。然而，有关AOX的调控机理尚不清楚，对其结构也不完全清楚，目前仅有其二级结构模型的两种假说：SUM模型和AN模型，而AOX三级结构研究一直没有结果，这阻碍了对AOX结构与功能的研究。本研究利用DNA重组技术，克隆AOX基因及其第179位苏氨酸（T）突变为丙氨酸（A）的突变基因至原核表达载体pET-15b，运用原核表达系统BL21和FN102，经异丙基β-D-硫代半乳糖...Alternative Oxidase(AOX), widely existing in higher plants, is the terminal oxidase in the cyanide-resistant respiration pathway in plant mitochondria. AOX can regulate many physical metabolizable pathways in plant, for example regulation of respiration, restraining formation of reactive oxygen species, resistence of discipline and regulation of programmed cell death and so on. However, its regula...学位：理学博士院系专业：生命科学学院生物学系_植物学学号：2162006015328

Subcellular localization of serum and glucocorticoid inducible protein kinase(SGK) in renal cells and relationship between SGK and aldose reductase

血清和糖皮质激素诱导蛋白激酶（serumandglucocorticoidinducibleproteinkinase,SGK）是一种新发现的丝/苏氨酸蛋白激酶，与其他蛋白激酶主要在酶活性水平上受翻译后的磷酸化和去磷酸化调控显著不同的是，SGK1的转录、活性以及亚细胞定位受到不同胞内和胞外刺激因素的调节，是多种胞内信号途径的功能性交汇点，参与了细胞增殖、渗透调节、离子通道调节以及细胞生存和/或凋亡应答等过程，与糖尿病肾病、高血压等疾病密切相关。 然而，SGK1在信号转导通路中的分子机制还不是很清楚，其底物的发现也很有限。尤其SGK1的亚细胞定位研究仍存在很多争议，在细胞内表达外源的SGK1与...Serum and glucocorticoid inducible protein kinase, SGK is a novel member of the serine/threonine protein kinase gene family. Unlike the vast majority of protein kinases, which are predominantly regulated at the enzymatic level by posttranslational phosphorylation and dephosphorylation, the transcript expression, activity and subcellular localization of SGK1 can be acutely controlled by a diverse s...学位：理学硕士院系专业：生命科学学院生物学系_水生生物学学号：2005130200

Studies of Microzooplankton Grazing on Phytoplankton in Tieshan Harbor, Guangxi and Shenhu Bay, Fujian

2010年5月和8月，应用稀释法，研究了广西铁山港和福建深沪湾两个海域不同粒径浮游植物的生长率、微型浮游动物对浮游植物的摄食率，估算了微型浮游动物的日摄食量、微型浮游动物对浮游植物现存量和初级生产力的摄食压力。研究旨在揭示微型浮游动物的摄食对浮游植物群落结构的影响以及探讨不同海域微食物网的碳流通量和流向。 主要成果如下： 1.铁山港海域表层水体中，浮游植物的生长率和微型浮游动物的摄食率的变化范围都很大。4月份，浮游植物的生长率为0.36~1.12d-1，微型浮游动物的摄食率为0.25~0.68d-1，相当于每天摄食浮游植物现存量的32.35%~151.07%和初级生产力的73.47%~73...Microzooplankton grazing on coastal phytoplankton was determined by the dilution technique in April and August 2010 at three station located in Tieshan and Shenhu Harbor, respectively. The carbon flux consumed by microzooplankton and the secondary production of microzooplankton were estimated in order to examine the impact of microzooplankton grazing on phytoplankton communities. The major result...学位：理学硕士院系专业：海洋与环境学院海洋学系_海洋生物学学号：2242008115149

中澳经贸合作的回顾与建议

一、中澳经贸合作的简要回顾及存在问题对于中国和澳大利亚来说，加强合作既是保证亚太地区良好发展的要求，又是亚太地区发展趋势所必然导致的结果。APEC的贸易投资自由化和经济技术合作进程将会使包括中国和澳大利亚在内的成员更为紧密地联系在一起。中国和澳大利亚..

Distribution Feature of Polysaccharides and Lipids in the Developing Anthers of Morinda officinalis How

巴戟天花药发育中多糖和脂滴类物质的分布呈现一定的规律:减数分裂之前,花药壁的绒毡层细胞中有少量脂滴,其他细胞中脂滴和淀粉粒都很少。四分体时期,四分体小孢子中开始出现脂滴,绒毡层细胞中的脂滴较以前增加,其他细胞中的脂滴和淀粉粒仍然很少。小孢子早期,游离小孢子在其表面形成了花粉外壁,靠外壁下方有一层周缘分布的多糖物质。绒毡层细胞中的脂滴明显减少。发育晚期的小孢子中形成一个大液泡,细胞质中出现淀粉粒;同时在药壁和药隔组织中也出现了淀粉粒。此时绒毡层退化。在二胞花粉早期,花粉中积累了大量淀粉粒和一些脂滴。但在成熟的花粉中(二胞花粉晚期),淀粉粒消失,只有一定数量的脂滴保留。巴戟天成熟花粉中积累的营养物质主要为脂滴。Distribution of polysaccharides and lipids in the developing anthers of Morinda officinalis How was regular.There were a few lipid drops in the sporogenous cells of young anthers,while neither lipid drops nor starches were found in other anther cells.Before the meiosis of microspore mother cells,some lipids appeared in tapetal cells.The size of tapetal cells began to increase at this stage.At the stage of tetrad,the lipids in the tapetal cells increased,and many lipid drops accumulated in the cells.Some lipids also appeared in tetrad microspores at this time.There were still no starches in young anther cells,and only cell wall of the cells formed anther wall and callose wall in tetrads displayed the feature of polysaccharids.During microspore development,the lipids in tapetal cells decreased evidently.The lipids in the young microspore also disappeared.There were still no starches in anther.At late microspore stage,some starches accumulated in microspore and appeared in anther wall and connective cells.Tapetal cells degenerated at this stage and the lipid drops concentrated to form lipid block.At early stage of 2-celled pollen,the vegetative cell accumulated a large number of starches,which disappeared with pollen development.Lipids were the main nutritional material accumulated in mature pollen of M.officinalis.国家自然科学基金(30970275);福建省漳州市科技局课题(Z2008038

Change of DNA Content in Male and Female Gametes of Tobacco(Nicotiana tabacum)

:采用显微分光光度法测定了烟草(nICOTIAnA TAbACuM)精细胞和卵细胞的dnA含量。烟草是二胞花粉,花粉萌发后生殖细胞在花粉管中分裂形成精细胞。授粉后45H花粉管到达子房,在花粉管内的精细胞dnA含量为1C。当花粉管在退化助细胞中破裂,释放出的两个精细胞开始合成dnA。在与卵细胞融合前,两个精细胞dnA含量接近2C。随着精细胞的到达及合成dnA,卵细胞也开始合成dnA,融合前的卵细胞dnA含量也接近2C。精、卵细胞融合后,合子dnA含量为4C。烟草雌、雄配子是在细胞周期的g2期发生融合,属于g2型。The nuclear DNA content of male and female gametes of tobacco(Nicotiana tabacum) was measured using DAPI stain and microspectrofluorimetric measurement.Tobacco pollen is bicellular at anthesis,containing a vegetative cell and a generative cell,which divides to form two sperm cells in a pollen tube.The nuclear DNA content of generative cell in a pollen tube was at 2C level and that of two sperm cells in a pollen tube,which elongated in the style,at 1C level.Two sperm cells began to synthesize DNA after both were released in the degenerated synergid,and the quantity of nuclear DNA in both sperm cells approached 2C level before both fusing with egg and central cells.During this process,the nuclear DNA content of egg cell also began to increase and approached 2C level before fusing with sperm cell.After male and female gamete fused,the nuclear DNA content of zygote reached 4C level.Therefore,the fusion of male and female gametes of tobacco was at G2 of cell cycle,and this fusion of fertilization belonged to G2 type.This result displayed the fertilization multiformity in angiosperms.国家自然科学基金(30670126

细胞三维动态培养微器件的设计与制作

细胞培养是进行细胞研究的基础,为了在细胞体外培养时提供一种近似于体内的微环境,设计了一种可供细胞三维动态培养的微器件。首先设计了用于输运流体的微通道网络,培养池对称布置于微通道网络中,通过一系列\"多进多出\"型微通道分别与进样口和出样口相连。利用Comsol软件中的层流物理场和多孔介质物理场耦合对培养池内的流场进行仿真,通过比较流场的均一性和稳定性优化微通道网络结构。然后,采用静电直写技术在培养池内集成聚己内酯(PCL)三维支架,构建细胞三维培养空间。最后,封合微器件,检测微器件培养池内的流体流动情况,并进行细胞实验。实验结果表明,\"2×2\"型微器件培养池内的流体稳定性和均一性较好;PCL三维支架的纤维间距400μm,纤维直径80μm,孔隙率64%,细胞存活率达到90%以上。该细胞三维动态培养微器件更好地模拟了生物体内细胞生存所需的微环境,培养池内的细胞生长良好,满足设计要求。国家自然科学基金资助项目(No.51475079,No.51375076

Characterization and immunoprotective effect of SjIrV1,a 66 kDa calcium-binding protein from Schistosoma japonicum

钙结合蛋白是日本血吸虫生长发育不可缺少的蛋白,具有非常广泛而重要的功能。在课题组日本血吸虫体被表膜蛋白研究基础上,利用PCr技术克隆了中国大陆株日本血吸虫66 kdA钙结合蛋白(SJIrV1)编码基因的CdnA序列,blAST分析与菲律宾株日本血吸虫SJIrV1 CdnA编码序列一致,荧光定量PCr分析表明该基因在童虫和成虫期不同发育阶段均有表达,其中在35 d和42 d成虫中表达量较高,在42 d雌虫中该基因表达水平远高于42 d雄虫。构建重组表达质粒PET28A(+)-SJIrV1,在大肠杆菌中成功诱导表达,重组蛋白主要以可溶性形式存在,通过高效液相色谱法(rP-HPlC)以及串联质谱法(MS/MS)鉴定所获蛋白为目的蛋白SJIrV1。蛋白质印迹(WESTErn blOTTIng)分析结果显示重组蛋白能被感染日本血吸虫鼠血清和免疫鼠血清所识别,SJIrV1蛋白在虫体各发育阶段中均表达。免疫荧光染色实验观察表明SJIrV1主要分布在日本血吸虫成虫的表膜。应用重组蛋白免疫bAlb/C小鼠后,免疫鼠血清中检测到较高水平的特异性Igg、Igg1和Igg2A抗体。结果表明SJIrV1可能在日本血吸虫的生长发育过程中起着重要作用。Calcium-binding protein is an indispensable protein which performs extensive and important functions in the growth of Schistosoma japonicum.Based on our primary study on tegument surface proteins of S.japonicun,a cDNA encoding a 66 kDa calcium-binding protein of S.japonicum(Chinese strain) was cloned,sequence analysis revealed that it was identical with that of SjIrV1 of Philippines strains S.japonicum.The expression of SjIrV1 were detected by Real-time PCR,using cDNA templates isolated from 7,14,21,28,35 and 42 days worms and the results revealed that the gene was expressed in all investigated stages,and the mRNA level of SjIrV1 is much higher in 42 d female worms than that in 42 d male worms.The cDNA containing the open reading frame of IrV1 was subcloned into a pET28a(+) vector and transformed into competent Escherichia coli BL21 for expression.The recombinant protein was purified using a Ni-NTA purification system,and confirmed by high performance liquid chromatography(RP-HPLC) and tandem mass spectrometry(MS/MS).Western blotting analysis showed that recombinant SjIrV1(rSjIrV1) could be recognized by the S.japonicum infected mouse serum and the mouse serum specific to rSjIrV1,respectively.Immunofluorescence observation exhibited that SjIrV1 was mainly distributed on the tegument of the 35-day adult worms.ELISA test revealed that IgG,IgG1 and IgG2a antibodies are significantly increased in the serum of rSjIrV1 vaccinated mice.The study suggested that rSjIrV1 might play an important role in the development of S.japonicum.国家自然科学基金(No.31172315); 上海科技发展基金(No.12140902700); 中国博士后科学基金(No.2012M510630)资助~