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首页> 外文会议>International citrus congress >Amplification and Partial Sequencing of Ribosomal Intergenic Spacer (IGS) from the Citrus Fruit Rot Pathogens Penicillium digitatum, P. ulaiense and P. italicum
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Amplification and Partial Sequencing of Ribosomal Intergenic Spacer (IGS) from the Citrus Fruit Rot Pathogens Penicillium digitatum, P. ulaiense and P. italicum

机译：来自柑橘类腐败病原体上的核糖体Intercanic Spacer（Igs）的扩增和部分测序P. Ulaiense和P. Italicum

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摘要

Penicillium digitatum, P. italicum, and P. ulaiense are difficult to differentiate using morphological methods and the use of molecular techniques is challenged by the presence of a high number of phylogenetically related species. Moreover, these three species have almost identical Internal Transcribed Spacer (ITS) regions. The IGS regions have great potential to differentiate closely related species however their utilization has been limited mainly because of the difficulties related to their amplification and sequencing. In the present work, new universal primers were designed by comparing IGS flanking regions (28S and 18S genes) from a large number of fungi and utilised to amplify the complete IGS regions off. ulaiense, P. digitatum, and P. italicum. Amplicons were of approximately 4 000 bp for P. ulaiense and 3 500 bp for P. digitatum and P. italicum. Amplified regions were cloned with pCR?-XLTOPO? Kit and sequenced for approximately 1 000 nucleotide at both 3' and 5' sides for P. ulaiense and P. digitatum. As opposed, only a very short fragment was effectively sequenced for P. italicum. Alignment of IGS regions from P. digitatum and P. ulaiense with other GenBank available rDNA sequences and BLAST analyses evidenced very high levels of polymorphisms which are valuable for the development of specific molecular markers.
机译：青霉霉素，P.Tealicum和P.Ulaiense难以使用形态学方法来区分，并且通过存在大量的系统源相关物种的存在攻击分子技术的使用。此外，这三种物种具有几乎相同的内部转录间隔物（其）区域。 IGS区域具有很大的潜力，可以区分密切相关的物种，然而它们的利用主要是有限的，主要是因为与其扩增和测序有关的困难。在本作工作中，通过比较来自大量真菌的IGS侧翼区域（28s和18s基因）来设计新的通用引物，并利用用于放大完整的IGS区。 Ulaiense，P. Digitatum和P. Italicum。扩增子对于P.ulaiense和300bp的300bp，P. Digitatum和P.Tealicum的扩增子。用PCR克隆扩增区域 - XLTOPO？试剂盒并在3'和5'侧向P. Ulaiense和P. Digitatum的3'和5'侧测序约1 000个核苷酸。而不是，只有非常短的片段被有效地对P. iTalicum进行测序。来自P. Digitatum和P.Ulaiense与其他Genbank可用的RDNA序列和Blast分析的对准IGS区的对准证明了非常高水平的多态性，这对于特定分子标记的发展是有价值的。

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