白色念珠菌RAPD反应体系优化的研究

建立白色念珠菌RAPD的最佳反应体系,并应用于其基因组DNA扩增。通过单因子试验分别研究了Mg^2＋、dNTPs、Taq酶、引物和模板DNA等浓度对RAPD反应的影响;同时,应用L16（4^5）正交试验研究了DNA模板、Mg^2＋、Taq酶、dNTPs和引物浓度对RAPD反应的影响。以条带稳定、丰富、清晰为标准,获得了白色念珠菌基因组DNA的RAPD扩增优化条件;对于白色念珠菌的最适RAPD反应体系为Mg^2＋1．5mmol／L、dNTPs250μmol／L、引物0．6μmol／L、模板100ng／25μL、TaqDNA聚合酶1．5U／25μL。     

狭边大叶藓（Rhodobryum ontariense）ISSR-PCR反应体系的建立与优化

研究目的是获得适用于狭边大叶藓（Rhodobryum ontariense）遗传多样性研究的ISSR—PCR反应标准化程序。通过单因素试验设计,对Mg^2＋、dNTPs、Taq DNA聚合酶、模板、引物浓度,以及退火温度、循环次数等影响ISSR扩增的主要因素进行了研究和优化。结果表明,UBC808、UBC811、UBC812、UBC825、UBC826、UBC841、UBC888及UBC891引物适用于该研究;20μL ISSR—PCR最适反应体系包括：6ngDNA模板、0．4μmol／L引物、2．25mmoL／LMg^2＋、0．6U Taq DNA聚合酶、0．4mmol／LdNTPs。扩增程序为：94℃预变性4min;然后94℃变性1min,48～50℃（根据不同引物确定）复性2min,72℃延伸1min,共进行40个循环;最后,72℃延伸7min,4℃保存。     

桂花SRAP—PCR体系的确立及验证

以桂花[Osmanthus fragrans (Thunb. ) Lour. ]品种'早银桂'的总DNA为模板,对SRAP-PCR扩增体系中的模板DNA用量,Mg2+、 dNTPs和引物浓度以及Taq DNA聚合酶用量进行单因子实验, 确立了适合桂花总DNA的 SRAP-PCR反应体系:反应体系总体积10 μL,包括30 ng模板DNA、 2.5 mmol·L-1 Mg2+ 、 0.20 mmol·L-1dNTPs、 0.4 μmol·L-1上下游引物和0.75 U Taq DNA聚合酶及1×PCR buffer.采用SRAP引物组合pm21-em8,以78个桂花品种的总DNA为样品,对优化的SRAP-PCR反应体系进行初步验证,共扩增出632条带,多态性条带百分率75.32%;扩增位点15个,多态性位点百分率为86.67%.运用优化的SRAP-PCR体系,使用筛选出的18对SRAP引物组合对88个桂花品种、1个桂花野生种以及2个外类群[柊树O. heterophyllus (G. Don) P. S. Green和华东木犀O. cooperi Hemsl. ]的总DNA进行扩增,共扩增出296个位点,其中多态性位点248个,多态性位点百分率为83.78%.实验结果显示,运用优化的SRAP-PCR反应体系获得的DNA条带清晰、扩增结果稳定、多态性较丰富;SRAP分子标记可用于桂花遗传多样性、品种资源鉴定、亲缘关系以及系统进化等方面的研究.     

益智ISSR-PCR反应体系建立与优化

目的：获得适合的益智ISSR-PCR扩增反应体系。方法：利用正交设计L46（4^5）和单因素试验对益智ISSR-PCR反应体系的5因素（Taq酶,Mg^2＋,模板DNA．dNTPs,引物）在4个水平上进行优化试验,将二者所得结果进行综合比较分析。结果：最终扶得益智ISSR-PCR反应的最优体系（20μl）为：模板DNA 100ng,Mg^2＋ 3．0mmol／L,引物0．8μmol／L,dNTPs0．25mmol／L,Taq DNA聚合酶1．0U。最后,对益智ISSR-PCR最佳反应体系进行梯度退火,得到最佳退火温度为50℃。结论：这一优化系统的建立为今后利用ISSR标记技术研究分析益智遗传多样性奠定基础。     

岩白菜ISSR-PCR反应体系的优化

目的:优化岩白菜ISSR-PCR反应体系,为利用ISSR标记进行岩白菜遗传多样性研究服务。方法:采用5因子4水平正交设计法优化岩白菜ISSR-PCR反应体系。结果:五个因子从大到小的影响力排序结果为:dNTPs>Mg2+>模板DNA>引物=Taq酶。岩白菜ISSR-PCR最佳反应体系为:总体积25μL,内含10×PCR缓冲液2.5μL、2.5 mmol.L-1Mg2+、2.0 U Taq酶、0.2 mmol.L-1dNTPs、0.48μmol.L-1ISSR引物、125 ng模板DNA。结论:研究获得的最佳反应体系具有标记位点清晰、反应系统稳定、检测多态性能力强、重复性好等特点,为利用ISSR标记技术研究岩白菜遗传多样性奠定了基础。     

广西野生桃金娘SRAP体系优化及建立北大核心CSCD

利用均匀设计方法优化了桃金娘SRAP反应体系。得出最佳反应体系,其总反应体积为25μL,包括2.5μL10×PCR buffer,1.0U Taq DNA聚合酶,20 ng模板DNA,0.2 mmol.L-1 dNTPs,0.3μmol.L-1引物,2.5 mmol.L-1Mg2+。采用优化的扩增体系,以Me1-Em11引物组合对8个参试种质进行SRAP扩增,扩增出的条带清晰、多态性好。所确立的体系稳定可靠,适于进行桃金娘的SRAP遗传分析。     

与番茄花柄脱落相关的多聚半乳糖醛酸酶的分离纯化和特性

为弄清番茄花柄脱落相关酶—哆聚半乳糖醛酸酶（polygalacturonase,PG）的性质,采用离体培养条件下经乙烯处理的番茄花柄,分离和纯化了多聚半乳糖醛酸酶并测定其性质。结果表明：用Sephadex G-75凝胶过滤层析和CM Sepharose CL-6B阳离子交换层析方法可分离纯化得到分子量约为30．2kDa的多聚半乳糖醛酸酶,纯化倍数为30．85;纯化的PG活性最适pH值为5．0,最适反应温度为40℃;Km值为26．14mg·mL^-1;1mmol·L^-1Ca^2＋、Cu^2＋、Ba^2＋、Co^2＋和Mn^2＋可抑制PG活性,而1mmol·L^-1的Mg^2＋、K^＋、Fe^2＋、Zn^2＋、Fe^2＋促进PG活性,20mmol·L^-1的Fe^2＋和Fe^3＋促进效果尤为显著。     

莴苣属蔬菜资源SRAP标记PCR体系的构建与优化

以莴苣为材料研究了影响SRAP标记PCR结果的因素,包括Mg^2＋、dNTP、引物、Taq酶的浓度以及退火温度等,建立了适用于莴苣属蔬菜SRAP分析的PCR反应体系：在20μl反应体系中,模板DNA为50ng,Mg^2＋浓度为2．0mmol／L,dNTP浓度为0．2mmol／L,引物浓度为0．75μmol／L,Taq酶用量为1U。适宜的扩增程序为94℃5min;94℃1min,35℃1min,72℃1min,5个循环;94℃1min,51℃lmin,72℃1min,30个循环;72℃7min。对体系的验证结果表明本研究建立的莴苣SRAP体系重复性好、分辨率高、多态性丰富,在莴苣属蔬菜种盾资源评价、分子标记辅助选择言种等骞方面躲有专耍作用。     

西花蓟马ISSR PCR反应体系的建立与优化

【背景】西花蓟马是近年来在我国局部地区暴发成灾的重要入侵害虫。【方法】本文通过改进的STE法提取西花蓟马的基因组DNA,对西花蓟马ISSR PCR反应体系中的DNA、引物、Taq聚合酶、buffer缓冲液、dNTPs、Mg2＋和去离子甲酰胺等7个影响因素进行了单因素试验,初步筛选出各因素的较优浓度;〖JP2〗然后对影响较大的4个因素进行了正交设计试验L16(44),建立了适合西花蓟马ISSR分子标记的最佳反应体系。【结果】在20 μL的反应体系中包含模板DNA 30 ng、10×Buffer 2.0 μL、引物1.05 μmol·L-1、Taq聚合酶2.0 U、dNTPs 212.5 μmol·L-1及MgSO4 2.25 mmol·L-1。【结论与意义】利用采自云南昆明、玉溪、楚雄、红河、保山、昭通、丽江、大理和普洱9个地方的西花蓟马样本对所建立的反应体系进行检验,结果表明优化后的体系适用于西花蓟马的遗传多样性分析,本研究可为进一步研究西花蓟马的种群结构奠定基础。     

SRAP、ISSR技术的优化及在甘蓝类植物种子鉴别中的应用

将SRAP与ISSR 2种分子标记技术应用于8种甘蓝类植物（Brassica oleracea L.）的种子鉴别中。先以甘蓝（Brassica oleracea var. capitata）基因组DNA为模板,通过对SRAP、ISSR反应体系中各影响因素的逐一筛选,优化了甘蓝类植物SRAP、ISSR反应体系。进而采用30个SRAP引物组合和15个ISSR引物对白甘蓝、皱叶甘蓝、红甘蓝、羽衣甘蓝、花椰菜、青花菜、抱子甘蓝、球茎甘蓝的基因组DNA进行了PCR扩增,结果表明：M3E5与M4E5两个SRAP引物组合可以在8种甘蓝类植物之间显示较高的多态性;844和888两个ISSR引物也可在8种甘蓝类植物之间产生很好的多态,特别是844引物单独应用即可区分所有材料。     

壳聚糖酶的克隆表达与壳寡糖的制备分析

目的:克隆壳聚糖酶基因于大肠杆菌中实现高表达,制备壳寡糖。方法:以枯草芽孢杆菌总DNA为模板扩增壳聚糖酶基因(CSN),克隆至载体pET23a(+)上,转化菌株BL21(DE3)。重组子经0.5 mmol/L IPTG诱导后,SDS-PAGE和质谱检测与鉴定重组酶。酶纯化后水解壳聚糖,薄层色谱分析其水解产物。结果:质谱证明壳聚糖酶(31.5kDa)成功表达,表达量占菌体总蛋白的45%左右。纯化后重组酶浓度为900 mg/L,纯度95%、回收率85%,酶活力为10 000 U/mg。壳聚糖降解产物为壳二糖至壳四糖。结论:原核表达载体pET23a(+)-CSN构建正确,壳聚糖酶表达量与活性高,适用于水解壳聚糖制备壳寡糖。     

国内外蝗害治理技术现状与展望

本文首先概述了国内外蝗虫发生与为害的态势,总结了现阶段我国蝗虫发生与为害的主要特点:即农田飞蝗暴发频繁而且严重,草原土蝗的发生时常造成严重的经济损失,而且侵入城市干扰市民生活,我国与周边国家之间蝗虫过境迁移频繁,使用化学农药污染环境和农产品;分析了国内外蝗虫防治对策与技术的发展现状,重点介绍了应急防治和可持续治理对策、...     

Molecular characterisation of species and genotypes of Cryptosporidium and Giardia and assessment of zoonotic transmission

The molecular characterisation of species and genotypes of Cryptosporidium and Giardia is essential for accurately identifying organisms and assessing zoonotic transmission. Results of recent molecular epidemiological studies strongly suggest that zoonotic transmission plays an important role in cryptosporidiosis epidemiology. In such cases the most prevalent zoonotic species is Cryptosporidium parvum. Genotyping and subtyping data suggest that zoonotic transmission is not as prevalent in the epidemiology of giardiasis. Molecular characterisation of Cryptosporidium and Giardia is a relatively recent application that is evolving as new genes are found that increase the accuracy of identification while discovering a greater diversity of species and yet unnamed taxa within these two important genera. As molecular data accumulate, our understanding of the role of zoonotic transmission in epidemiology and clinical manifestations is becoming clearer.     

Comparison of BMI and percentage of body fat of Indian and German children and adolescents

Today, serious health problems as overweight and obesity are not just constricted to the developed world, but also increase in the developing countries (Prentice 2006, Ramachandram et al. 2002). Focusing on this issue, BMI and percentage of body fat were compared in 2094 schoolchildren from two cross-sectional studies from India and Germany investigated in 2008 and 2009. The German children are in all age groups significantly taller, whereas the Indian children show higher values in BMI (e.g. 12 years: Indian: around 22 kg/m2; German: around 19 kg/m2) and in the percentage of body fat (e.g. 12 years: Indian: around 27%; German: around 18-20%) in most of the investigated age groups. The Indian children have significantly higher BMI between 10 and 13 (boys) respectively 14 years (girls). Indian children showed significant higher percentage of body fat between 10 and 15 years (boys) and between 8 and 16 years (girls). The difference in overweight between Indian and German children was strongest at 11 (boys) and 12 (girls) years: 70% of the Indian but 20% of the German children were classified as overweight. In countries such as India that undergo nutritional transition, a rapid increase in obesity and overweight is observed. In contrast to the industrialized countries, the risk of overweight in developing countries is associated with high socioeconomic status. Other reasons of the rapid increase of overweight in the developing countries caused by different environmental or genetic factors are discussed.     

Synthesis and turnover of cerebrosides and phosphatidylserine of myelin and microsomal fractions of adult and developing rat brain.

The synthesis and turnover of cerebrosides and phospholipids was followed in microsomal and myelin fractions of developing and adult rat brains after an intracerebral injection of [U-14C]serine. The kinetics of incorporation of radioactivity into microsomal and myelin cerebrosides indicate the possibility of a precursor-product relationship between cerebrosides of these membranes. The specific radioactivity of myelin cerebrosides was corrected for the deposition of newly formed cerebrosides in myelin. Multiphasic curves were obtained for the decline in specific radioactivity of myelin and microsomal cerebrosides, suggesting different cerebroside pools in these membranes. The half-life of the fast turning-over pool of cerebrosides of myelin was 7 and 22 days for the developing and adult rat brain respectively. The half-life of the slowly turning-over pool of myelin cerebrosides was about 145 days for both groups of animals. The half-life of the rapidly turning-over microsomal cerebrosides was calculated to be 20 and 40 h for the developing and adult animals respectively. The half-life of the intermediate and slowly turning-over microsomal cerebrosides was 11 and 60 days respectively, for both groups of animals. The amount of incorporation of radioactivity into microsomal cerebrosides from L-serine was greatly decreased in the adult animals, and greater amounts of the precursor were directed towards the synthesis of phosphatidylserine. In the developing animals, considerable amounts of cerebrosides were synthesized from L-serine, besides phosphatidylserine. The time-course of incorporation indicated that a precursor-product relationship exists between microsomal and myelin phosphatidylserine. The half-life of microsomal phosphatidylserine was calculated to be about 8 h for the fast turning-over pool in both groups of animals.     

Identification and composition of the tonsillar and anal enterococcal and streptococcal flora of dogs and cats

Enterococcus faecalis was the most frequently isolated enterococcal species from anal swabs and tonsils of dogs and cats, although in the anal samples from dogs Ent. hirae was found almost as often as Ent. faecalis. Most Ent.faecium strains from dog tonsils differed from those associated with humans and other animals in that they fermented sorbitol. Typical Ent. avium as well as atypical Ent. avium -like strains were seen in dogs, while the related species Ent. raffinosus was associated with cat tonsils. Enterococcus cecorum also occurred mainly in cats. Certain atypical strains, presumptively identified as Ent. cecorum , shared characteristics with Ent. columbae.
The most frequent streptococcal species in tonsils of cats and dogs were Streptococcus suis and Strep. canis. Streptococcus canis and Strep. bovis predominated in anal swabs. The canine Strep. suis differed from the common porcine strains in fermenting mannitol.
Forty-seven of the 288 isolates examined could not be identified or related to known species. The characteristics of two groups of these bacteria, provisionally called 'Ton 31 group' and 'O7 group' are described.