用随机扩增多态性DNA产物做探针产生鸡的DNA指纹图

我们用12个随机扩增多态性DNA(RAPD)引物对来自不同品系的4只鸡进行了RAPD分析,在扩增出的共99条带中,表现多态性的带为38条,占总带数的38％.回收了4个表现个体特异性的RAPD产物,当用鸡的基因组总DNA探针与它们杂交时,其中3个表现阳性,说明RAPD方法扩增出的高变异产物含有重复序列.用含重复序列的个体特异性RAPD产物作探针,与无关个体鸡基因组DNA的HaeⅢ酶切产物进行DNA印迹,获得了变异性较高的DNA指纹图谱.因此,高变异的RAPD产物可以有效地用作DNA指纹探针.     

纵纹腹小鸮DNA指纹谱探针的筛选和初步应用

以人工合成的微卫星序列 (GTG) 5,(GT) 8,(CAC) 5和人源小卫星 33 1 5作引物 ,扩增纵纹腹小的基因组DNA ,产生多态性DNA片段 ,回收了 8个表现个体特异性的片段。当用小的基因组总DNA探针与它们杂交时 ,其中 2个表现阳性 ,说明PCR方法扩增出的高变异产物含有重复序列。用含重复序列的个体特异性PCR产物作探针 ,与无关个体小基因组DNA的HaeⅢ酶切产物进行DNA印迹 ,获得了变异性较高的DNA指纹图谱。且通过对京白鸡家系分析表明 ,用小基因组DNA的PCR产物分离制备的探针所获得的DNA指纹图带能够稳定的遗传。因此 ,高变异的PCR产物可以有效地用作DNA指纹探针。     

银杏雌雄基因组DNA间的差异性分析

本研究应用RAPD技术,应用300个10bp随机单引物及200对随机双引物组合,检测了雌雄异株银杏基因组DNA的多态性。结果表明:雌雄基因组间具有极高的相似性,在检测到的3450个标记中,仅获得1个与银杏雄性基因组相关的RAPD标记。以该标记为探针,与雌雄银杏基因组DNA的Southern杂交分析,其杂交信号在两性之间表现为限制性片段长度多态性,该结果为寻找银杏早期性别鉴定的探针以及在细胞和分子水平进一步研究其性别问题奠定了基础。     

纵纹腹小HaoDNA指纹谱探针的筛选和初步应用

以人工合成的微卫星序列（GTG）5,（GT）8,（CAC）5和人源小卫星33.15作引物,扩增纵纹腹小Hao的基因组DNA,产生多态性DNA片段,回收了8个表现个体特异性的片段,当用小Hao的基因组总DNA探针与它们杂交时,其中2个表现阳性,说明PCR方法扩增出的高变异产物含有重复序列,用含重复序列的个体特异性PCR产物作探针,与无关个体小Hao基因组DNA的HaeⅢ酶切产物进行DNA印迹,获得了变性性较高的DNA指纹图谱,且通过对京白鸡家系分析表明,用小Hao基因组DNA 的PCR产物分离制备的探针所获得的DNA指纹图带能够稳定的遗传,因此,高变异的PCR产物可以有效地用作DNA指纹探针。     

一种基于寡核苷酸微阵列芯片的多重可扩增探针杂交技术

多重可扩增探针杂交技术(multiplex amplifiable probe hybridization,MAPH)是近年来发展起来的一种用于基因组中DNA拷贝数检测的新技术。并发展了一种基于寡核苷酸微阵列芯片的MAPH技术。该方法根据所检测的DNA序列,制备若干具有通用引物的FCR产物作为可扩增探针组,与固定在尼龙膜上待测的基因组DNA杂交。用磁珠回收特异性杂交的探针,经生物素标记的通用引物扩增后,与相应的寡核苷酸微阵列芯片杂交。该特异性的寡核苷酸微阵列芯片包括10个抗肌营养不良基因的外显子探针和阴性、阳性探针。杂交清冼后,链霉亲和素-Cy3染色用芯片扫描仪得到杂交的荧光图像。分析荧光信号的强度差异给出特定基因片段拷贝数的变化。该方法用微阵列技术代替MAPH中的电泳检测技术,可大幅度增加检测的通量。选择了一个正常男性、一个正常女性和一个肌营养不良症患者的基因组DNA来进行验证。结果表明,该方法能够同时给出抗肌营养不良基因多个外显子中的基因片段拷贝数差异信息。     

伊犁鲈微卫星位点的筛选及近缘物种通用性

为开发伊犁鲈(Perca schrenkii)分子标记用于鲈属鱼类种质资源保护,以伊犁鲈为材料,应用磁珠富集法进行了微卫星标记的筛选.从伊犁鲈尾鳍提取总DNA,进行酶切、接头连接、PCR扩增,再采用生物素标记(CA)15探针及生物素标记(TG)15探针对扩增产物进行杂交富集,经再次PCR扩增及T-A克隆,成功构建了伊犁鲈基因组微卫星富集文库.采用重复序列引物筛选获得阳性克隆,随机选取48个阳性克隆进行测序,测得序列46个,其中38个克隆含有微卫星序列,41个位点的微卫星重复数在8次以上.根据测得序列设计17对微卫星引物,均能在伊犁鲈群体中扩增获得目的条带.采用该17对引物对河鲈(P.fluviatilis)及黄金鲈(P.flavescens)群体样本进行扩增,10对引物具有通用性,其中6对在河鲈中具有高度多态性(PIC>0.5),5对在黄金鲈中具有高度多态性.     

磁珠富集法分离小黄李基因组微卫星DNA片段

将微卫星探针5′端生物素化后与链亲和素磁珠特异结合,用磁珠和探针的结合物与两端连接已知序列人工接头的中国李品种小黄李(Prunus salicinacv.Xiaohuangli)基因组DNA酶切片段杂交,以此杂交片段为模板用人工接头序列为引物进行PCR扩增,根据PCR产物测序结果设计引物作为微卫星DNA的标记引物.结果在随机挑选的36个克隆进行菌落PCR检测时,从31个阳性克隆中挑选18个克隆进行测序后获得了12条特异序列,设计的8对SSR引物均在5个中国李受试品种上获得了预期的扩增产物,其中4对引物在受试品种上表现出多态性.     

东北虎中SOX基因的检测

应用特异于HMG bOX区域的兼并引物 ,扩增了东北虎的SOX基因。在扩增产物中发现五条大小分别为 2 2 0 ,2 70 ,3 5 0 ,4 3 0和 5 60bp的扩增带。经过与地高辛标记的人SRY基因进行Southern杂交表明这五条扩增带均呈现阳性 ,说明它们均为东北虎的SOX基因片段 ,这些基因保守区的长度在基因组DNA水平上存在着明显的差异。     

磁珠富集法分离刀鲚微卫星标记

利用磁珠富集法分离微卫星序列,以开发长江刀鲚微卫星分子标记。将长江刀鲚基因组DNA经限制内切酶Mse I酶切,回收400-1 000 bp片段,安装接头,构建长江刀鲚全基因组PCR文库。用生物素标记的微卫星探针(CA)12与其杂交,磁珠富集含有微卫星序列的DNA片段。将洗脱所得片段进行PCR扩增,然后进行克隆。经过菌落PCR检验后挑选出118个阳性克隆进行测序,其中97条含有微卫星序列。用设计合成59对微卫星引物对30尾养殖长江刀鲚进行引物的多态性筛选,得到9对多态性引物。     

雄牛特异的SRY同源序列的扩增和分析

利用人、兔、鼠SRY序列设计引物,应用PCR扩增牛的SRY序列,获得200bp的雄牛特异的扩增片段。克隆该扩增片段,获得重组质粒pCH21,进行序列分析,并与人、兔和鼠SRY的对应区域比较,具有高度同源性。用pCH21 DNA作探针与牛的基因组DNA酶切图谱杂交,显示了雄牛特异的I．7kb的杂交带。分析200bp的PCR扩增片段是牛的SRY基因片段。用同一对引物扩增人和山羊的DNA样品,也获得雄性特异的200bp的扩增片段。     

Use of a PCR method for controlling pure-breeding of honeybees Apis mellifera mellifera L. in the southern Urals

A key problem of honeybee (Apis mellifera mellifera) breeding in the Southern Urals is its cross-breeding with the Caucasian honeybee Apis mellifera caucasica. Mitochondrial DNA (mtDNA) in these subspecies differ in the length of a fragment localized between genes CO-I and CO-II, which can be used as a marker. A pair of 20-mer primers for PCR was chosen by means of computer design in order to determine the fragment size in both of the subspecies. The amplified fragment was shown to have a length of 350 bp in A. m. caucasica and 600 bp in A. m. mellifera. The difference in length results from the different ratio between two main elements P and Q, which comprise a major part of this sequence in these subspecies: a copy of P element and two copies of Q element in A. m. mellifera, and a copy of Q element only in A. m. caucasica. This sharply defined distinction allows us to use PCR for differentiating the subspecies, estimating the heterogeneity in the colonies, and rejecting queens in the selection process because of the maternal inheritance of the studied character. The nucleotide sequence of the amplified mtDNA fragment of A. m. mellifera was determined.     

蜜蜂属六蜂种间MRJP2基因C-端重复序列的比较分析

构建了中华蜜蜂（Apis cerana cerana,中蜂）8日龄工蜂头部cDNA文库,获得了中蜂王浆主蛋白2（major royal jelly protein 2,MRJP2）的全长cDNA序列,该序列长1 605bp,包含一个编码468个氨基酸的开放阅读框（open reading frame,ORF）。在中蜂MRJP2的cDNA序列的C-端,首次发现存在串联重复片段长度多态性（variable numbers of tandem repeat,VNTR）。克隆并测定了蜜蜂属Apis内其他5个种的MRJP2基因的C-端重复序列,结果表明: 在蜜蜂属的其他5个种中,C-端重复片段的核心序列是以碱基高度突变方式而表现出个体之间的多态性,而重复片段长度基本一致。中蜂与西方蜜蜂A. mellifera,大蜜蜂A. dorsata与黑大蜜蜂A. laboriosa,以及小蜜蜂A. florea与黑小蜜蜂A. andreniformis分别形成3个进化枝。中蜂和西方蜜蜂与大蜜蜂和黑大蜜蜂之间的亲缘关系较近,而与小蜜蜂和黑小蜜蜂的亲缘关系较远。     

王浆主蛋白MRJP7基因在意大利蜜蜂体内的表达

王浆蛋白是蜂王浆生物功能的物质基础,是由王浆蛋白基因家族(mrjps)编码合成的。但部分家族成员如MRJP7在王浆中的含量极少甚至检测不到。基因功能与其在生物体内的时空表达特性相关,为探究mrjp7的生物学功能,本研究利用荧光定量PCR技术对mrjp7在不同发育时期的工蜂和成年工蜂、雄蜂和蜂王的不同组织部位的表达进行定量检测。结果显示mrjp7在成年雄蜂体内的表达水平最低,成年蜂王次之,且在它们的各不同组织部位之间的表达量差异较小。该基因在工蜂幼虫和蛹期的表达同样较低,但在羽化后9日龄前后的哺育蜂王浆腺和头部特异性高表达,这与哺育蜂分泌蜂王浆哺育幼虫和蜂王的功能是相适应的,该结果在转录水平上证实了mrjp7的营养功能,为进一步的研究和应用打下了理论基础。     

A PCR-based method that permits specific detection of Paenibacillus larvae subsp. larvae, the cause of American Foulbrood of honey bees, at the subspecies level

AIMS: A reliable procedure for the identification of Paenibacillus larvae subsp. larvae, the causal agent of American Foulbrood disease of honey bees (Apis mellifera L.) based on the polymerase chain reaction (PCR) and subspecies - specific primers is described. METHODS AND RESULTS: By using ERIC-PCR, an amplicon of ca 970 bp was found among P. l. larvae strains but not in other closely related species. Based on the nucleotide sequence data of this amplicon, we designed the pair of oligonucleotides KAT 1 and KAT 2, which were assayed as primers in a PCR reaction. A PCR amplicon of the expected size ca 550 bp was only found in P. l. larvae strains. CONCLUSIONS: This PCR assay provides a specific detection for P. l. larvae. SIGNIFICANCE AND IMPACT OF THE STUDY: The developed PCR assay is highly specific because can differentiate Paenibacillus larvae subsp. larvae from the closely related Paenibacillus larvae subsp. pulvifaciens. The technique can be directly used to detect presence or absence of P. l. larvae spores in honey bee brood samples and contaminated honeys.     

Development of molecular RAPD marker for the identification of Pediococcus acidilactici strains

A RAPD analysis performed using a single primer targeted to the pediocin AcH/PA-1 gene was carried out on several P. acidilactici strains and on some related species of lactic acid bacteria. The high degree of genetic variability detected in P. acidilactici strains did not allow the selection of a common RAPD fragment that could be chosen as a potential species-specific DNA marker. Nevertheless a 700 bp fragment, that was found to be peculiar of all potential pediocin producer strains analyzed, was cloned and sequenced with the aim to develop a species specific PCR marker. Sequence analysis of the cloned 700 bp fragment showed one putative small open reading frame (ORF1), with no significant homology with known genes, and a partial putative second coding region (ORF2) with a high degree of similarity with several methionyl tRNA synthesis (metS) genes. The two coding regions were separated by a short spacer region. Primers targeted to ORF2 plus part of the spacer region and primers designed for the amplification of the entire cloned RAPD fragment were found to be species-specific for the detection of P. acidilactici strains. Furthermore primers designed on the ORF1 sequence allowed the amplification of a 439 bp fragment only in some P. acidilactici strains, including pediocin producing strains.     

A PCR detection method for rapid identification of Paenibacillus larvae

American foulbrood is a disease of larval honeybees (Apis mellifera) caused by the bacterium Paenibacillus larvae. Over the years attempts have been made to develop a selective medium for the detection of P. larvae spores from honey samples. The most successful of these is a semiselective medium containing nalidixic acid and pipermedic acid. Although this medium allows the growth of P. larvae and prevents the growth of most other bacterial species, the false-positive colonies that grow on it prevent the rapid confirmation of the presence of P. larvae. Here we describe a PCR detection method which can be used on the colonies that grow on this semiselective medium and thereby allows the rapid confirmation of the presence of P. larvae. The PCR primers were designed on the basis of the 16S rRNA gene of P. larvae and selectively amplify a 973-bp amplicon. The PCR amplicon was confirmed as originating from P. larvae by sequencing in both directions. Detection was specific for P. larvae, and the primers did not hybridize with DNA from closely related bacterial species.     

意大利蜜蜂甘油醛-3-磷酸脱氢酶基因amGAPDH2的cDNA克隆与序列分析

应用RT-PCR技术克隆了意大利蜜蜂(Apis mellifera)甘油醛-3-磷酸脱氢酶基因amGAPDH2,利用MEGA5.1、DNAMAN、PredictProtein和I-TASSER等软件对该基因进化关系以及其所对应的蛋白的同源性、理化性质和结构进行了预测和分析。从意大利蜜蜂cDNA文库中克隆得到了长1188 bp的amGAPDH2序列,GenBank登录号为MH152402。该序列具有完整的开放阅读框(ORF,114~1115 bp),其编码333个氨基酸。该基因编码的氨基酸序列与其它昆虫的GAPDH有较高的序列相似性(80%以上),系统进化分析表明amGAPDH2与中华蜜蜂、小蜜蜂的序列相似性最高。利用ProtParam等软件对amGAPDH2编码的蛋白质分析结果显示,amGAPDH2属于不稳定、亲水性蛋白;二级结构属于混合型,Helix占25.53%、Strand占24.62%、Loop占49.85%;am GAPDH2具有两个保守结构域:一个是N端的NAD(P)结合结构域,另一个是C端的催化结构域Gp_dh_C。该研究为后期amGAPDH2基因的生理功能研究提供了理论依据。     

西方蜜蜂不同级型王浆主蛋白MRJP8基因的表达差异

王浆主蛋白在蜜蜂的级型分化中具有重要的功能。为探究mrjp8在西方蜜蜂Apis mellifera不同级型的表达模式及功能差异。【方法】 利用荧光定量PCR技术对西方蜜蜂工蜂、 雄蜂和蜂王不同发育时期和不同组织的mrjp8表达水平进行检测。【结果】 工蜂体内mrjp8在9日龄前后的毒腺组织内特异性高表达, 为参照基因表达量的上万倍, 在其他发育时期和组织的表达量则明显较低, 其表达具有明显的时空特异性; 在雄蜂体内其表达量与对照相当; 在蜂王体内表达量可达参照的近1 000倍, 没有组织特异性。【结论】 mrjp8的这种表达模式提示其在工蜂防御及维系蜂王长寿命方面有积极作用, 这为进一步研究该基因乃至整个王浆蛋白基因家族的进化和功能分化提供了依据。     

Apis mellifera cytoplasmic elongation factor 1 alpha (EF-1 alpha) is closely related to Drosophila melanogaster EF-1 alpha

Using low stringency hybridisation with a Drosophila melanogaster EF-1 alpha gene fragment we have isolated a genomic DNA clone encoding elongation factor 1 alpha (EF-1 alpha) from Apis mellifera. The hybridising Apis mellifera sequence could be delineated to two small EcoRI fragments that were also revealed by genomic Southern hybridisation. By comparison with the corresponding Drosophila melanogaster data the complete translational reading frame has been deduced. It is interrupted by two intervening sequences of 220 and about 790 nucleotides. Comparison with known eucaryotic EF-1 alpha sequences further confirms that certain amino acid sequences seem to be invariable within the EF-1 alpha protein family.     

The complete mitochondrial genome of the Asiatic cavity-nesting honeybee Apis cerana (Hymenoptera: Apidae)

In the present study, we determined the complete mitochondrial DNA (mtDNA) sequence of Apis cerana, the Asiatic cavity-nesting honeybee. We present here an analysis of features of its gene content and genome organization in comparison with Apis mellifera to assess the variation within the genus Apis and among main groups of Hymenoptera. The size of the entire mt genome of A. cerana is 15,895 bp, containing 2 ribosomal RNA genes, 13 protein-coding genes, 22 transfer RNA (tRNA) genes and one control region. These genes are transcribed from both strands and have a nucleotide composition high in A and T. The contents of A+T of the complete genomes are 83.96% for A. cerana. The AT bias had a significant effect on both the codon usage pattern and amino acid composition of proteins. There are a total of 3672 codons in all 13 protein-coding genes, excluding termination codons. The most frequently used amino acid is Leu (15.52%), followed by Ile (12.85%), Phe (10.10%), Ser (9.15%) and Met (8.96%). Intergenic regions in the mt genome of A. cerana are 705 bp in total. The order and orientation of the gene arrangement pattern is identical to that of A. mellifera, except for the position of the tRNA-Ser(AGN) gene. Phylogenetic analyses using concatenated amino acid sequences of 13 protein-coding genes, with three different computational algorithms (NJ, MP and ML), all revealed two distinct groups with high statistical support, indicating that A. cerana and A. mellifera are two separate species, consistent with results of previous morphological and molecular studies. The complete mtDNA sequence of A. cerana provides additional genetic markers for studying population genetics, systematics and phylogeographics of honeybees.