不同保存方法对武汉单极虫DNA提取和PCR扩增的影响

为探讨不同保存方法对武汉单极虫Thelohanellus wuhanensis DNA提取和PCR扩增的影响,将从患武汉单极虫病的异育银鲫Carassius auratus gibelio鱼苗上分离出含有孢子的孢囊,分别置于100%乙醇、70%乙醇、2.5%戊二醛、10%福尔马林、Bouin's液、Carnoy's液和Müller's液中保存50 d,经计数调整孢子等量后分别进行DNA提取、常规PCR和巢式PCR扩增。结果表明:100%乙醇和70%乙醇保存方法对武汉单极虫孢子DNA提取、常规PCR和巢式PCR扩增均无任何影响,等同于新鲜孢子,可作为孢子的保存方法;2.5%戊二醛和Bouin's液保存方法只对孢子DNA提取有影响,但均能在常规PCR和巢式PCR扩增后出现特异电泳条带,可作为用于常规PCR和巢式PCR扩增的孢子保存方法;10%福尔马林和Müller's液保存方法对孢子DNA提取和常规PCR扩增均有影响,但均能在巢式PCR扩增后出现特异电泳条带,故只能作为用于巢式PCR扩增的孢子保存方法;Carnoy's液保存方法无论在孢子DNA提取还是进行常规PCR和巢式PCR扩增均无阳性结果,故不能用作孢子核酸分子生物学研究的保存方法。研究表明,除100%乙醇和70%乙醇保存方法外,其他保存方法对孢子DNA提取、常规PCR和巢式PCR扩增均有不同程度的影响。     

CPA-核酸试纸条检测鸭肉源性成分方法的建立和优化

为建立简单、有效的动物源性成分定性检测方法,根据交叉引物恒温扩增技术(CPA)原理,以鸭属线粒体D-loop基因序列设计引物和探针,优化反应体系与恒温扩增条件,并配合核酸试纸条显色,建立鸭肉源性成分的快速检测方法。结果表明,建立的鸭D-loop基因CPA扩增体系中交叉引物、内引物、外引物分别为0.6、0.3、0.1μmol·L-1,Mg SO4为8 mmol·L-1。在63℃、60min恒温扩增条件下,单一鸭源性DNA成分检出限为0.1 ng·μL-1,对混合肉制品中鸭肉成分的检出比例为1%(相当于1ng·μL-1)。本研究建立的鸭源性成分检测方法为肉制品掺假鉴定提供了有效的手段。     

兰州大尾羊心脏型脂肪酸结合蛋白（H-FABP） 基因克隆及其同源性比较

【目的】克隆兰州大尾羊心脏型肪酸结合蛋白（H-FABP）基因全长cDNA序列,为研究绵羊H-FABP生物学作用和生产应用提供理论依据。【方法】根据已知哺乳动物H-FABP基因 cDNA 序列,设计5''和3''特异引物,运用cDNA 末端快速扩增（RACE）技术获得兰州大尾羊H-FABP基因全长 cDNA 序列。【结果】 扩增获得兰州大尾羊5''端425 bp、3''端231 bp片段和 177 bp中间片段,拼接获得748 bp兰州大尾羊H-FABP基因全长cDNA 序列（GenBank登录号：JQ780322）。 兰州大尾羊H-FABP基因ORF长 402 bp,编码 133 个氨基酸。核苷酸序列分析显示兰州大尾羊H-FABP基因序列与大多数哺乳动物相似,但其第66位发生的碱基转换（T←→G）引起所编码的第22位天门冬氨酸（N）不同于其它所有物种的赖氨酸（K）。构建的基因进化树分析结果显示兰州大尾羊与山羊亲缘关系最近。预测兰州大尾羊H-FABP蛋白质的空间结构与山羊和牛H-FABP类似,由2个α螺旋和10个反向平行的β折叠组成,10 个折叠片围成一个桶状结构,疏水性残基位于桶内,用于结合脂肪酸。【结论】克隆了兰州大尾羊H-FABP基因,为进一步研究该基因的功能奠定了基础。     

First report of Wolbachia from field populations of Culex mosquitoes in south-western Nigeria

Recent reports on finding Wolbachia-strain infections in field mosquito species in some West African countries and the potential for developing these as disease vector biocontrol tools have prompted a search for Wolbachia in mosquitoes within the study area. Using a completely randomised design, mosquito traps were set at different locations in a rural and an urbanised community. One hundred and eighty (180) mosquitoes were trapped and pooled on the basis of genus, sex and site of collection, because there have been no earlier reports of Wolbachia isolated from Nigeria. Twenty pools, made up of not more than ten mosquitoes per pool, were homogenised and analysed for Wolbachia-specific DNA. Mosquitoes were trapped within Ede (urbanised community) and Akoda (rural community). Genomic DNA was extracted from trapped mosquito samples and used as a template in a PCR reaction. The Wolbachia sp. specific 16S rRNA gene was amplified, sequence analysis of PCR products was performed and a chromatogram of the sequence was subjected to Basic Local Alignment Search Tool analysis to identify the Wolbachia sp. This sequence was subsequently submitted to GenBank with accession number MK127541. The first evidence of the presence of the endosymbiont, Wolbachia in field-caught mosquitoes is hereby documented. The homology of this strain of Wolbachia bears similarities to those reported recently from other parts of West Africa and forms a single clade with a Wolbachia sp. from Mali, with a strong bootstrap support of 99%. This finding of a Wolbachia strain in mosquitoes at Ede could form the basis for more searches for diverse strains of Wolbachia in Nigeria.     

罗汉果葡萄糖基转移酶基因的克隆及原核表达

 从罗汉果（Siraitia grosvenorii）转录组中获得一条与罗汉果甜苷Ⅴ生物合成相关的葡萄糖基转移酶（UDPG）的unigene片段,以罗汉果授粉后70 d的果实RNA为模板,利用RACE和RT-PCR技术克隆UDPG全长基因,将克隆得到的SgUDPG1基因连接到原核表达载体pEASY-E1上,构建融合表达载体,转化到大肠杆菌BL21（DE3）,通过IPTG诱导表达,重组蛋白纯化,SDS-PAGE检测表达产物以及Western-blotting和质谱鉴定蛋白产物。结果表明,获得了1条SgUDPG1,全长为1 959 bp,开放阅读框ORF为1 365 bp,编码1条454 aa的肽链,理论分子量为51.2 kD,等电点为5.39,具有植物中次生代谢产物糖基转移酶特有的保守结构域PSPG-box motif。SgUDPG1在授粉后50 d和70 d的果实中表达逐渐升高,是对照授粉后3 d的5.16倍和13.12倍,与果实中甜苷Ⅴ含量呈相同趋势。此基因的ORF可以在大肠杆菌中表达,并且可以纯化出比理论分子量大5.3 kD的融合蛋白,通过Western-blotting和质谱鉴定,确定该蛋白属于罗汉果葡萄糖基转移酶。     

青稞坚黑穗病LAMP快速检测技术的建立

由大麦坚黑粉菌(Ustilgo hordei)引起的坚黑穗病广泛分布于我国青稞主产区,病原侵染后形成病穗从而造成产量损失。基于前期初步建立的青稞黑穗病快速检测体系,通过对种子洗涤沉淀物的总基因组DNA进行提取,并进行环介导等温扩增技术(loop-mediated isothermal amplification,LAMP)检测人工模拟及实际种子带菌情况,以期建立完善的青稞坚黑穗病快速检测技术。结果表明,裂解液快速提取法可以对样品总DNA进行有效提取,完善后的LAMP快速检测技术可以有效开展青稞种子携带大麦坚黑粉菌的检测。     

Independent target region amplification polymorphism and single‐nucleotide polymorphism marker utility in genetic evaluation of sugarcane genotypes

The independent target region amplification polymorphism (TRAP) and single‐nucleotide polymorphism (SNP) marker s were used for genetic evaluation of different selected 47 sugarcane genotypes. A total of 23 pairs of TRAP markers generated 925 alleles, of which 74% alleles were polymorphic. Polymorphism was generally high (>50%), ranging from 54 to 98%. The polymorphism information content (PIC) values 0.20 varied among the primer combination ranging from 0.17 in SAI + Arbi 2 to 0.31 in GL 2+ Arbi 1 with an average of 0.24. However, the Pearson correlation between PIC and power of discrimination (PD) was found to be less significant. Single‐nucleotide polymorphisms were used first time for the assessment of genetic diversity among different species of Saccharum and cultivated sugarcane varieties. The SNPs were detected from 454 sequencing. A total of 245 SNP markers were assayed across the 47 genotypes, and 167 SNPs were found to be polymorphic. The PIC values ranged from 0.04 to 0.38 with an average of 0.21, and their respective PD varied from 0.58 to 0.04 with an average value of 0.31. The obtained results relatively significant were compared with the other marker systems through genetic similarity and the clusters formed in different unweighted pair group method with arithmetic mean clustering dendrogram. The clustering analysis established genetic relationship in the order of Erianthus > Sclerostachya > Narenga > Saccharum spontaneum > S. robustum > S. barberi > S. officinarum/cultivars. These results ratify TRAP and SNP marker systems for assessing genetic diversity studies, and more diversified Erianthus spp. can contribute substantially towards sugarcane varietal improvement through breeding with Saccharum spp. or hybrid cultivars.     

转基因作物及产品检测技术的研究进展

为了规范管理转基因产品,以建立转基因产品的检测技术标准为前提,通过分析转基因作物商业化的18a中转基因作物种植面积持续增长的态势,以核酸检测方法为主,介绍了基因芯片、PCR技术、变性高效液相色谱和环介导等温扩增技术在转基因检测领域的研究进展,并对今后转基因检测技术标准的发展进行了展望。     

二倍体及四倍体枳橙DNA甲基化变异的MSAP分析

为探讨染色体加倍对四倍体枳橙(Citrus sinensis (L.) Osb.×Poncirus trifoliate (L.) Raf.)叶片基因组DNA甲基化修饰的影响,明确四倍体枳橙基因组DNA甲基化水平及模式的变化特征。本研究以枳橙二倍体为对照,利用MSAP分子标记技术对同源四倍体枳橙叶片基因组DNA甲基化水平及模式变化进行分析。结果表明,10对选择性扩增引物共扩增出775条条带,二倍体和同源四倍体枳橙扩增带数分别为391条和384条,对应的总甲基化率分别为31.97%(含全甲基化率16.37%,半甲基化率15.6%)和31.25%(含全甲基化率18.49%,半甲基化率12.76%);MSAP扩增条带统计表明,同源四倍体的总甲基化率变化较小,全甲基化率高于二倍体,半甲基化率较二倍体均有所降低,相比二倍体对照,四倍体枳橙叶片基因组DNA主要发生了甲基化模式的变化。     

镁铝双氢氧化物和镁铁铝改性蒙脱土去除水体中磷的吸附效果研究

通过间歇式批实验和动力学实验研究了不同pH条件下磷酸盐在镁铝双氢氧化物（Mg-Al-LDH）、钠基膨润土（Na-Mt）及镁铁铝改性膨润土（Mg-Al-Mt、Fe-Mt和Fe-Al-Mt）的吸附特征。结果表明,在pH4.5～9.0范围内,随着pH的升高,Na-Mt,Fe-Mt和Fe-Al-Mt3种矿物对磷的吸附率相应减少,镁铝双氢氧化物对磷的吸附率有所增加;Mg-Al-LDH、Fe-Mt和Fe-Al-Mt的对磷吸附率约为95%,比Mg-Al-Mt高40%,比Na-Mt高80%。用Langmuir方程描述磷的等温吸附过程,最大吸附容量（Qm）大小顺序为Fe-Al-Mt〉Fe-Mt〉Mg-Al-LDH〉Mg-Al-Mt〉Na-Mt,b值大小依次为Mg-Al-LDH〉Fe-Mt〉Fe-Al-Mt〉Mg-Al-Mt〉Na-Mt,最大缓冲容量（Qmb）以Mg-Al-LDH的为最大,Na-Mt的为最小;Freundlich等温吸附方程参数KF代表相对吸附容量,以Mg-Al-LDH的KF值最高,依次是Fe-Mt、Fe-Al-Mt和Mg-Al-Mt,Na-Mt的KF值最小,这与Qmb的结果一致;决定系数（R2）表明,Langmuir等温吸附方程能更好地拟合铁改性蒙脱土和铁铝改性蒙脱土,而Freundlich方程对钠蒙脱土,双氢氧化物和镁铝改性蒙脱土的拟合效果要好。磷酸盐吸附过程分为快反应和慢反应,用动力学实验数据进行拟合,准二级动力学方程能够更好的拟合改性蒙脱土和Mg-Al-LDH对磷的动力学吸附数据,其决定系数为0.999;Elovich方程拟合改性蒙脱土磷酸盐的吸附数据也有很好的相关性（R2为0.89～0.94）。Na-Mt矿物磷酸盐的吸附用抛物线扩散方程描述最为合适,原因可能是磷酸盐镶嵌在矿物层间是一个扩散过程,不涉及化学吸附过程。