摘要
为研究DsCPV在松毛虫种群中的垂直传递及对松毛虫灾害的持续控制作用 ,通过反转录聚合酶链式反应 (RT PCR)建立了DsCPV的早期检测技术。依据家蚕质型多角体病毒 (BmCPV)质型多角体蛋白的核苷酸序列设计一对引物 ,从纯化的DsCPV、BmCPV和舞毒蛾质型多角体病毒 (LdCPV)的基因组dsRNA可成功地扩增出长 6 14bp的目的片段 ,从提取的健康松毛虫幼虫肠组织的DNA、舞毒蛾核型多角体病毒 (LdNPV)基因组核酸、以及棉铃虫质型多角体病毒 (HaCPV)基因组核酸 ,未能扩增出目的片段。DsCPV基因组dsRNA扩增片段的序列与BmCPV相应基因序列具有 87%的同源性 ,检测敏感度为 1pg的DsCPV基因组dsRNA。由于松毛虫与家蚕、舞毒蛾相互之间食性不同 ,而且BmCPV和LdCPV对松毛虫无感染性 ,即在松毛虫体内不会有Bm CPV病毒和LdCPV病毒的感染 ,因此该结果一方面从分子水平上证实了DsCPV与BmCPV、LdCPV存在基因同源性 ,同时也表明了依据BmCPV的基因序列设计引物对DsCPV基因组核酸建立的RT PCR扩增体系 ,可以作为松毛虫种群中DsCPV的一种敏感、特异、早期、快速的检测手段。
In order to understand vertical transmission of DsCPV and its role in sustained controlling pine caterpillars,this study was undertaken to develop the assay technique for detection of DsCPV in early stage of infection of pine caterpillars,based on the reverse transcription of RNA followed by the polymerase chain reaction amplification (RT-PCR).A pair of primers producing 614bp amplification fragment were designed based on the sequence of the C-polyhedrin gene of Bombyx mori CPV (BmCPV),and the expected amplification products were obatined when genomic dsRNAs isolated from purified BmCPV,DsCPV and Lymantria dispar CPV (LdCPV) were used as templates.Genomic dsRNAs isolated from Heliothis armigera CPV and DNAs from Lymantria dispar nuclear polyhedrosis virus (LdNPV) and DNAs from midgut tissue of healthy Dendrolimus spectabilis (Walker) larva did not yield any amplification products.The detection limit of purified DsCPV genomic dsRNAs was 1.0pg.The nucleotide sequence of the 614bp DNA amplification product from DsCPV was 87% homogenous with the corresponding part of C-polyhedrin gene of BmCPV.These results demonstrated that BmCPV,DsCPV and LdCPV were homologous as classfied in the same electropheres type I,and that this RT-PCR assay could be used for early,rapid,sensitive and specific detection of DsPCV infection in the natural population of the pine moth,due to the apparent different feeding habits of Bombyx mori,Dendrolimus ssp.and Lymantria dispar larva each other and the no infection of BmCPV and LdCPV to Dendrolimus ssp.
出处
《林业科学》
EI
CAS
CSCD
北大核心
2001年第3期78-82,共5页
Scientia Silvae Sinicae
基金
国家自然科学基金资助项目 (3 95 70 5 96)
