家蚕核型多角体病毒的PCR检测及其部分序列分析

为研究应用PCR技术进行家蚕核型多角体病毒广东株的敏感性检验以及探讨不同地理株系的基因水平的相互关系，本文通过对家蚕核型多角体病毒BmNPV广东株的人工繁殖与纯化，引用了一对根据多角体蛋白基因设计的引物phy35／phy36，对BmNPV的基因组模板DNA进行了PCR扩增，并对其产物进行测序分析。结果显示，PCR技术均可扩增检测出3×10^8个／mL至3×10^2个／mL不同浓度的BmNPV模板DNA，特异目标片段大小约为680bp，且扩增带的亮度随着病毒液浓度的降低而减弱，说明应用引物phy35／Dhy36进行PCR方法可以有效地应用于检测BmNPV病毒感染的家蚕。同时，测序获得了BmNPV广东株多角体蛋白pof佩edrin基因674bp大小的片段，GC含量为46．4％。经过BLAST比对分析，与BmNPV泰国株的相似性为99％，暗示家蚕BmNPV广东株与泰国株的BmNPV（登录号AY779044）亲缘关系非常相近，两者可能属于BmNPV的不同地理株系。通过系统发育树的进一步分析发现，家蚕核型多角体病毒广东株polyhedrin基因部分序列与家蚕NPV分离株s9多角体蛋白基因（DQ231336）关系很近。

PCR Detection and Partial Sequencing Analysis of Bombyx mori Nuclear Polyhedrosis Virus

In order to study the sensitivity of applying the PCR technique for detecting the BmNPV Guangdong strain （BmNPV GD） and to explore the relationship of the gene level between different geographic strains. In the study, BmNPV GD was artificially propagated in silkworm, and purified. Based on the BmNPV polyhedrin genes, one set of primer ofphy35/phy36 was quoted and synthesized for PCR identification and sequencing BmNPV. The results showed that the different template DNA, corresponding from BmNPV polyhedron concentrations of 3×10^8/mL to 3 ×10^2/mL was amplified and their specific bands were consistent with the size of target fragments （approximately 680 bp）. The brightness of amplified bands became weak gradually following the decreasing of virus concentration, it indicated that the primers phy35/phy36 could be applied to detect silkworm infecting BmNPV ef- fectively. Meanwhile, the above fragment was sequenced by two directions, BmNPV GD polyhedrin gene fragment was 674 bp in length and the contents of GC was 46.4% via Bioedit software analyses. After comparison analysis of BLAST, this fragment was 99% identical to the counterparts of the BmNPV from Thailand polyhedrin gene that had published in GenBank, they might be two different geographic strains of BmNPV. A phylogenetic tree was also constructed, the tree showed that there were very close relation between the BmNPV GD and BmNPV isolate S9 polyhedrin gene （DQ231336）.