采用同源克隆的方法,根据相近物种转铁蛋白(Transferrin, TF)基因的序列设计简并引物,在经感染真鲷的cDNA上进行PCR扩增,获得了真鲷TF的全基因编码序列(GeneBank Accession No.AY335444).该序列由2444bp核苷酸组成,含31 bp 5′非编码区...采用同源克隆的方法,根据相近物种转铁蛋白(Transferrin, TF)基因的序列设计简并引物,在经感染真鲷的cDNA上进行PCR扩增,获得了真鲷TF的全基因编码序列(GeneBank Accession No.AY335444).该序列由2444bp核苷酸组成,含31 bp 5′非编码区和340 bp 3′非编码区以及2073 bp的开放阅读框,可编码691个氨基酸,预测分子量约为74.3kD,等电点为5.63.同源性分析表明,真鲷TF与鱼类TF的相似性很高,约为65%～75%;与哺乳类转铁蛋白和乳铁蛋白的相似性约40%～50%;与脊索动物海鞘和无脊椎动物昆虫也有一定的相似性.进化分析表明,该基因是转铁蛋白而不是乳铁蛋白.真鲷TF与其它动物TF结构相似,是由两叶相似的结构域构成的单一肽链;该基因有一信号肽序列,缺乏糖基化位点,在它的两个结构域上,分别有三个TF家族的标签序列及一个MHC分子标签序列;与哺乳类相比,参与蛋白二硫键形成的半胱氨酸以及铁离子结合位点高度保守.RT-PCR结果显示,真鲷TF在肝脏成组成型表达,但在其它组织中的表达可受病原调控,表现为经病原刺激后转铁蛋白基因的组织分布显著增多.展开更多
赤眼蜂是我国乃至世界范围内应用最广的卵寄生蜂,其中玉米螟赤眼蜂Trichogramma ostriniae Pang et Chen是我国玉米种植区重要的天敌昆虫,但其不同的地理种群在生物防治效果上存在较大差异。为了有效利用玉米螟赤眼蜂的优势种群,有必要...赤眼蜂是我国乃至世界范围内应用最广的卵寄生蜂,其中玉米螟赤眼蜂Trichogramma ostriniae Pang et Chen是我国玉米种植区重要的天敌昆虫,但其不同的地理种群在生物防治效果上存在较大差异。为了有效利用玉米螟赤眼蜂的优势种群,有必要对其进行准确的鉴别。然而,采用传统形态学方法对同一种赤眼蜂的不同种群进行鉴别是非常困难的。本研究首先采用随机扩增多态性DNA(RAPD)标记对采自我国北京农科院、山东日照和山西太原的3个玉米螟赤眼蜂种群,进行了遗传差异分析,然后筛选了种群特异性位点,并通过克隆测序和引物设计构建了种群特异性分子标记,即序列特征性扩增区域(SCAR)标记。最后,利用SCAR标记对混合蜂群竞争试验结果进行了分子检测。该研究方法有助于玉米螟赤眼蜂优势种群的筛选,对于赤眼蜂的有效利用具有重要的意义。展开更多
Several species of the fungal genus Trichoderma establish biological interactions with various micro- and macro-organisms. Some of these interactions are relevant in ecological terms and in biotechnological applicatio...Several species of the fungal genus Trichoderma establish biological interactions with various micro- and macro-organisms. Some of these interactions are relevant in ecological terms and in biotechnological applications, such as biocontrol, where Trichoderma could be considered as an invasive species that colonizes a recipient community. The success of this invasion depends on multiple factors, which can be assayed using experimental communities as study models. Therefore, the aim of this work is to develop a species-specific sequence-characterized amplified region （SCAR） marker to monitor the colonization and growth of T. cf. harzianum when it invades experi- mental communities. For this study, 16 randomly amplified polymorphic DNA （RAPD） primers of 10-mer were used to generate polymorphic patterns, one of which generated a band present only in strains of T. cf. harzianum. This band was cloned, sequenced, and five primers of 20-23 mer were designed. Primer pairs 2F2/2R2 and 2F2/2R3 successfully and specifically amplified fragments of 278 and 448 bp from the T. cf. harzianum BpT10a strain DNA, respectively. Both primer pairs were also tested against the DNA from 14 strains of T. cf. harzianum and several strains of different fungal genera as specificity controls. Only the DNA from the strains of T. cf. hat-zianum was successfully amplified. Moreover, primer pair 2F2/2R2 was assessed by quantitative real-time polymerase chain reaction （PCR） using fungal DNA mixtures and DNA extracted from fungal experimental communities as templates. T. cf. harzianum was detectable even when as few as 100 copies of the SCAR marker were available or even when its population represented only 0.1% of the whole community.展开更多
文摘采用同源克隆的方法,根据相近物种转铁蛋白(Transferrin, TF)基因的序列设计简并引物,在经感染真鲷的cDNA上进行PCR扩增,获得了真鲷TF的全基因编码序列(GeneBank Accession No.AY335444).该序列由2444bp核苷酸组成,含31 bp 5′非编码区和340 bp 3′非编码区以及2073 bp的开放阅读框,可编码691个氨基酸,预测分子量约为74.3kD,等电点为5.63.同源性分析表明,真鲷TF与鱼类TF的相似性很高,约为65%～75%;与哺乳类转铁蛋白和乳铁蛋白的相似性约40%～50%;与脊索动物海鞘和无脊椎动物昆虫也有一定的相似性.进化分析表明,该基因是转铁蛋白而不是乳铁蛋白.真鲷TF与其它动物TF结构相似,是由两叶相似的结构域构成的单一肽链;该基因有一信号肽序列,缺乏糖基化位点,在它的两个结构域上,分别有三个TF家族的标签序列及一个MHC分子标签序列;与哺乳类相比,参与蛋白二硫键形成的半胱氨酸以及铁离子结合位点高度保守.RT-PCR结果显示,真鲷TF在肝脏成组成型表达,但在其它组织中的表达可受病原调控,表现为经病原刺激后转铁蛋白基因的组织分布显著增多.
文摘赤眼蜂是我国乃至世界范围内应用最广的卵寄生蜂,其中玉米螟赤眼蜂Trichogramma ostriniae Pang et Chen是我国玉米种植区重要的天敌昆虫,但其不同的地理种群在生物防治效果上存在较大差异。为了有效利用玉米螟赤眼蜂的优势种群,有必要对其进行准确的鉴别。然而,采用传统形态学方法对同一种赤眼蜂的不同种群进行鉴别是非常困难的。本研究首先采用随机扩增多态性DNA(RAPD)标记对采自我国北京农科院、山东日照和山西太原的3个玉米螟赤眼蜂种群,进行了遗传差异分析,然后筛选了种群特异性位点,并通过克隆测序和引物设计构建了种群特异性分子标记,即序列特征性扩增区域(SCAR)标记。最后,利用SCAR标记对混合蜂群竞争试验结果进行了分子检测。该研究方法有助于玉米螟赤眼蜂优势种群的筛选,对于赤眼蜂的有效利用具有重要的意义。
基金Project supported by the Chilean National Fund for Scientific and Technological Development(FONDECYT)(No.1120508)
文摘Several species of the fungal genus Trichoderma establish biological interactions with various micro- and macro-organisms. Some of these interactions are relevant in ecological terms and in biotechnological applications, such as biocontrol, where Trichoderma could be considered as an invasive species that colonizes a recipient community. The success of this invasion depends on multiple factors, which can be assayed using experimental communities as study models. Therefore, the aim of this work is to develop a species-specific sequence-characterized amplified region （SCAR） marker to monitor the colonization and growth of T. cf. harzianum when it invades experi- mental communities. For this study, 16 randomly amplified polymorphic DNA （RAPD） primers of 10-mer were used to generate polymorphic patterns, one of which generated a band present only in strains of T. cf. harzianum. This band was cloned, sequenced, and five primers of 20-23 mer were designed. Primer pairs 2F2/2R2 and 2F2/2R3 successfully and specifically amplified fragments of 278 and 448 bp from the T. cf. harzianum BpT10a strain DNA, respectively. Both primer pairs were also tested against the DNA from 14 strains of T. cf. harzianum and several strains of different fungal genera as specificity controls. Only the DNA from the strains of T. cf. hat-zianum was successfully amplified. Moreover, primer pair 2F2/2R2 was assessed by quantitative real-time polymerase chain reaction （PCR） using fungal DNA mixtures and DNA extracted from fungal experimental communities as templates. T. cf. harzianum was detectable even when as few as 100 copies of the SCAR marker were available or even when its population represented only 0.1% of the whole community.
