Studies on Transgenic Porphyra haitanensis and Allophycocyanin Expression

本文从电转化条件、外源基因导入方法、同源重组序列和MAR序列、启动子、载体构型等方面，系统研究了外源基因导入坛紫菜原生质体的遗传转化体系。在此基础上，将由质体编码的别藻蓝蛋白基因导入到染色体DNA中。同时构建了别藻蓝蛋白共表达载体，建立了原核表达、纯化的工艺技术，并对其体外清除自由基和胃癌细胞SGC-7901生长的作用进行了初步的分析，主要研究结果如下：1.以野生坛紫菜为材料，利用自制的海螺酶酶解获得原生质体，以CAT作为报告基因，探讨了不同方法将外源基因导入坛紫菜原生质体的可行性。实验结果表明，用电转化法进行外源基因导入时，可选择电压强度为1kV/cm，电转化缓冲液中含0.8mol/L甘露醇...The system of transformation foreign gene into protoplasts of Porphyra haitanensis including condition of eletroporation, methods of introduction foreign gene into protoplasts, homologous recombinant sequence and matrix attachment regions, promoter and form of vector were studied. On this basis, the allophycocyanin gene which coded by chloroplast was transferred into chromosome DNA. At the same ti...学位：工学博士院系专业：海洋与环境学院环境科学研究中心_环境科学学号：B20023402

Allophycocyanin Purification from Porphyra haitanensis and Studies on Spectrum of Eluting Fractions

以福建省平潭县坛紫菜(Porphyra haitanensis)为材料,采用硫酸铵分级分离和柱层析法纯化别藻蓝蛋白(APC),并对纯化的条件进行了详细的探讨。研究结果表明:采用30%~35%饱和硫酸铵沉淀、Tris-HC l(pH=8.0)作为洗脱缓冲液、DEAE-Sephadex-A-50作为层析介质,所获得的APC的纯度和回收率分别为3.50和70.2%;SDS-PAGE表明APC有α和β两个亚基,分子量分别为18.9 kD和17.7 kD。因此该方法对于从坛紫菜中快速纯化APC是适合的。采用光谱分析研究柱洗脱组分,结果表明:坛紫菜中含有分子结构为(αβ)6γ的“双峰型”R-PE,含有结构为(αβ)3的R-PC,含有结构为(αβ)3的APC-Ⅱ。Allophycocyanin from Porphyra haitanensis collected in Pingtan County of Fujian Province was purified using ammonium sulfate precipitation and column chromatography.Meanwhile,factors of purification were studied in detail.The results showed that using 30%-35% of the saturation ammonium sulfate precipitation,Tris-HCl(pH=8.0)as eluting buffering and DEAE-Sephadex-A-50 as(chromatogra-)(phic) adsorbent;the purity and rate of recovery of allophycocyanin obtained were 3.50 and 70.2%,(respectively).SDS-PAGE demonstrated the presence of two subunits,α and β,with Mr(18.9 kD) and(17.7 kD),respectively.Therefore the method was suitable for quick purification of allophycocyanin from P.haitanensis.At the same time,the fractions collected from column were researched by analysis of spectrum.The findings indicated that P.haitanensis contained the R-phycoerythrin with two absorption peaks which had the polypeptide composition(αβ)_6γ,the R-phycocyanin which had the polypeptide composition(αβ)_3 and allophycocyanin-II which had the polypeptide composition(αβ)_3.国家海洋863项目(2002A603023)资

Advances in research methods for gene function

[中文文摘]随着生命科学的发展,研究领域的不断开拓,越来越多的未知新基因和基因的新功能被科学家们发现,研究这些未知新基因的功能和已知基因的新功能成为了极其重要的一项内容。本文对基因功能研究的最新方法进行了介绍。 [英文文摘]With the development of the research areas of life sciences,more and more new genes and new functions of genes were discovered.It would be the main task to study the function of these genes in the next few years.This review introduces several new methods and procedures on the gene function research.国家自然科学基金项目(40606027

书院制国际化办学模式的实践探索——以博伊特勒书院为例

新形势下加快我国现代书院的国际化进程,不仅具有十分重要的意义,且已成为其发展战略的必然选择。文章对我国书院国际化的重要意义及国内书院制的国际化经验进行了探讨,并以博伊特勒书院为例,指出国际化办学思路、国际化师资、国际化教学氛围是推进我国现代书院国际化进程的重要抓手,提出了进一步提升书院国际化办学的建议,以期为我国高校书院制的国际化发展提供参考。2017年“基础学科拔尖学生培养试验计划”研究课题“本科生全方位导师制新模式的探索与研究”（20170402

Development of a Loop-mediated Isothermal Amplification Method for Analysis of Heat Shock Cognate Protein HSC70) Gene in Liver of Sebastiscus marmoratus and its Application in Studying the Dose-response Effect of Oil Pollution

从褐菖鲉肝脏中克隆了热休克蛋白HSC70基因,利用环介导等温扩增技术(lOOP-MEdIATEd ISOTHErMAl AMPlIfICATIOn,lAMP)建立HSC70基因的定量检测方法。为检测该方法的可行性,将褐菖鲉分别暴露于石油水溶性成分(WATEr-SOlublE frACTIOn,WSf)20、60、180μg·l-11 d后,利用rEAl-TIME PCr及lAMP技术同时测定褐菖鲉肝HSC70 MrnA表达量,两种方法测定结果基本一致,证实lAMP技术可用于褐菖鲉肝HSC70基因的定量。为更细致了解石油WSf影响褐菖鲉肝脏HSC70基因表达的剂量-效应关系,将褐菖鲉分别暴露于25、50、75、100、125、150、175μg·l-1WSf中,5 d后采样,用lAMP技术定量检测HSC70 MrnA。结果表明,HSC70 MrnA表达量在50μg·l-1浓度组即被显著诱导,在75μg·l-1浓度下达到最大值,这说明褐菖鲉肝HSC70基因对石油污染较敏感,有潜力作为海洋石油污染的生物标志物。Heat shock cognate protein(HSC70) gene was cloned from the liver of Sebastiscus marmoratus.A quantitative loop-mediated isothermal amplification(LAMP) method was established to analyze the mRNA level of HSC70 in S.marmoratus.S.marmoratus had been exposed to different concentrations(20, 60, 180 μg·L-1) of the water-soluble fraction(WSF) of crude oil for 1 day.Then, the contents of HSC70 mRNA in the liver of S.marmoratus weremeasured using real-time PCR and LAMP.The change patterns of HSC70 mRNA levels measured by LAMP were consistent with those measured by real-time PCR, indicating that the LAMP method was feasible for quantify HSC70 mRNA.To clearly understand the dose-effect relationship between WSF and HSC70, S.marmoratus had been exposed to WSF of seven different concentrations(25, 50, 75, 100, 125, 150, 175 μg·L-1) for 5 days, and the mRNA levels of HSC70 in liver were examined by LAMP.The results showed that the HSC70 mRNA levels were significantly increased in 50 μg·L-1group and reached the peak in 75 μg·L-1group, indicating that HSC70 in the liver of S.marmoratus was sensitive to WSF, and could be used as a potential biomarker to oil pollution.海洋公益性项目"海洋污染生物效应快速监测与评价技术应用示范"子任务-海洋污染效应生物标志物技术集成与研发II(201005016

The Study on Molecular Phylogenetic and Molecular Marker of Species in Silk Insects

本文对家蚕、野桑蚕、蓖麻蚕、柞蚕和天蚕等 5种绢丝昆虫进行了随机扩增多态DNA(RAPD)分析。 40个引物中有 2 7个引物能扩增出 5 36个清晰且重复性强的条带 ,其中可变条带数为 5 2 0个 ,单个引物扩增的条带数在11～ 2 8之间 ,平均为 19 9,各片段分子量大小在 0 2 9～ 2 6 7kb之间。每个样本都能找出其独特的分子标记。家蚕与野桑蚕的遗传距离 (D)最小 ,为 0 376 0 ;家蚕与蓖麻蚕的遗传距离 (D)最大 ,为 0 7488。根据遗传距离 ,用UPG MA聚类分析方法构建了它们的分子树。Five species of silk insects including Bombyx mori, B. manolarina, Philosamia cynthia, Autheraea pernyi and A. yamamai were analyzed by RAPD method using 40 arbitrary primers. In these primers, 27 of them could amplify clear and repeating bands. 536 fragments were obtained and the variable bands were 520. Each primer gave 11～28 bands and the average was 19.9. The length of the fragments is 0.29～2.67 kb. Some distinctive bands were found in every species. The genetic distance( D ) between bombyx mori and B. manolarina is 0.3760, which is the lowest. The highest D value is 0.7488, which between Bombyx mori and Philosamia cynhia. The D value was then used to construct a dendrogram by unweighted pair-group method with arithmetical averages(UPGMA).国家自然科学基金!资助项目 (批准号 39870 410

Application of RAPD Technique in Genetic Relationship Study of Silk Insect Ⅰ. Genetic Variance in Eri Silkworm

用RAPD技术对蓖麻蚕基因组DNA进行多态性研究，分析了5个蓖麻蚕品种间的遗传差异。结果表明，所采用的40个随机引物中，有27个引物扩增谱带清晰且重复性较好，扩增总片段数达243个，单个引物的扩增片段数在4～17之间，平均为9条，片段大小在033～30kb之间。不同蓖麻蚕品种间的遗传距离(Ｄ)在00683～01603之间，根据Ｄ值，由UPGMA聚类分析软件绘制了它们的聚类分子树。Random amplified Polymorphic DNA (RAPD) was used to analyze the genetic diversity among eri sickworm. The genetic variance of five erisickworm was studied. The result showed that: 27 of 40 arbitrary primers could amplify clearly with repeatable bands.243 fragments were obtained.Each primer gave 4～17 bands and the average was 9.The length of the band was 0.33～ 3.0kb. The genetic distance (Ｄ) value between different breeds of Eri Silkworm was 0.0683～0.1603. The D value was used to construct a dendrogram by UPGMA.国家自然科学基金资助项目!（批准号39870410

Comparison of four promoters for transient expression of RFP reporter gene in cultured Bombyx mori cells (Bm-e-HNU5)

以红色荧光蛋白基因(RFP)为报告基因,构建含4种不同启动子的重组表达质粒,用脂质体介导法转染家蚕Bombyxmori细胞(Bm-e-HNU5),观察家蚕细胞质肌动蛋白4基因启动子(A4)、α微管蛋白基因启动子(α-tub)、蚕丝心蛋白重链基因启动子(Fib)和家蚕核型多角体病毒早期即刻蛋白基因启动子(IE)4种启动子调控RFP报告基因在家蚕细胞内的瞬时表达情况。构建的重组表达质粒pDsRed-α-tub、pDsRed-A4、pDsRed-IE和pDsRed-Fib经双酶切和PCR鉴定正确无误。转染和转录实验结果表明,除了pDsRed-A4外,其他3种重组质粒在Bm-e-HNU5细胞中都得到高转染率,α-tub、IE和Fib可依次增强RFP报告基因在家蚕细胞内的瞬时表达活性。The red fluorescent protein reporter gene (RFP) was used to construct recombinant plasmids containing four different promoters, i. e., the cytoplasmic actin4 promoter (A4), α-tubulin promoter (α-tub)from silkworm, the Bombyx mori nuclear polyhedrosis virus immediate early protein promoter (IE) and the fibroin heavy chain gene promoter (Fib), respectively. These recombinant plasmids, i. e., pDsRed-A4,pDsRed-α-tub,pDsRed-Fib and pDsRed-IE, had been constructed successfully by restriction enzyme digestion and PCR analysis, and then were transfected into B. mori cell lines (Bm-e-HNU5) by lipid-mediated method to observe the ability of the four promoters to drive RFP reporter gene transient expression in cells. Transfection and transcription experiments indicated that except pDsRed-A4, the other three kinds of recombinant plasmids all transfected Bm-e-HNU5 obviously. The promoters of α-tub, IE and Fib enhanced the transient expression activity of RFP reporter gene in the Bm-e-HNU5, and their activity strengthened sequentially.国家自然科学基金项目(39870410

我国部分家鸭品种遗传多样性及与野鸭亲缘关系的探讨

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