【作者】 王文欢；

【导师】 张永安；

【作者基本信息】 中国林业科学研究院 ， 森林保护， 2009， 硕士

【摘要】 林业昆虫病毒作为一种高效无害专一性强的杀虫剂,现在已经在森林保护中起到了很大的作用。但其在林业上的使用率还不够广泛,主要是因为病毒防治害虫时潜伏期较化学杀虫剂长,没有后者那么立竿见影的效果,使得使用者对病毒治虫有质疑。现今对昆虫病毒防治效果的研究还仅仅停留在对病毒的形态观测阶段,本实验旨在建立几种以昆虫病毒核酸为基础的检测技术,从而填补昆虫病毒检测在分子生物学方面的空白,同时应用这些技术检测在昆虫体内低水平存在的病毒,为昆虫病毒的垂直传播及防治效果监测提供微观的、确凿的分子生物学证据,进而论证应用昆虫病毒防治林业害虫的可行性,确保森林生态保持稳定。根据HcNPV pe38基因设计两对引物,利用PCR法检测HcNPV基因组DNA,分别扩增出长为994和614 bp的片段,经测序确定为HcNPV pe38基因片段。应用两对引物分别检测了美国白蛾病虫的总DNA和获得的病毒多角体,其检测最低量分别为1 fg病虫总DNA和3~4 OBs/mL。用不同浓度的病毒感染试虫后,不同时间取其血淋巴用PCR方法检测。结果表明,接毒量为3.53×109 OBs/mL时,36 h后可检出病毒DNA;接毒量为3~4 OBs/mL时,120 h后可检出。病毒可检出时间随接毒浓度的递减而延后。从秦皇岛地区施用过HcNPV的林地和未施用病毒防虫的林地采集美国白蛾野外幼虫,取其血淋巴做模板进行PCR扩增,结果十分明显,说明之前建立的方法完全可以应用于实践中,同时说明HcNPV对该地区的美国白蛾种群具有持续控制作用。根据ClanGV egt基因设计两对引物,利用PCR方法检测ClanGV基因组DNA,分别扩增出401和491bp的片段,经测序确定为ClanGV egt基因片段。应用两对引物检测ClanGV病毒悬液,亦可成功扩增出目的基因片段,说明这两对特异性较高,可以用其检测ClanGV的存在,从而为昆虫病毒的野外检测奠定基础。更多还原

【Abstract】 Forest insect virus as a highly efficient sound-specific insecticide, is played a significant role in the forest protection now. But it is not be used popularly, mainly because virus has a long latent period, its reaction are slower than chemical pesticides, and these questioned the users. This experiment aimed at establishing several insect virus nucleic acid-based detection technologies, which fill the blank of the insect virus detection in the molecular biology. Applied the technologies to detect low concentration of OB suspension can provide some evidence for vertical transmission and reaction effect of insect virus, it also can demonstrate the feasibility of controlling pest with insect virus.Two pairs of specific primers were used for the detection of the Hyphantria cunea nucleopolyhedrovirus (HcNPV) by PCR. The two pairs of primers were both designed based on gene pe38 of HcNPV genome. The sizes of amplified fragments were 994 and 614 bp, respectively. The two pairs of primers were further used to detect the amount of viral DNA in diseased larvae or polyhedron of HcNPV. The minimum amount of detection could be as less as 1 fg of total DNA or 3?4 OBs/mL of polyhedron. The amount of viral DNA in hemolymph sampled from the larvae infected by different concentrations of polyhedron at different time points was also investigated, and it was found that the lower the concentration of polyhedron used, the later the viral DNA will be detected.Larvae of Hyphantria cunea were collected from Qinhuangdao. Their hemolymph is detected by PCR, the effect is also obviously. The result shows that the PCR-based method can be applied to practice, and HcNPV has a continuous control to Hyphantria cunea.Two pairs of specific primers were used for the detection of the Clostera anachoreta Granulovirus (ClanGV) by PCR. The two pairs of primers were both designed based on gene egt of ClanGV genome. The sizes of amplified fragments were 401 and 491 bp, respectively. The two pairs of primers were used to detect the genome DNA and OBs of ClanGV successfully.更多还原