【作者】 李永丹；

【导师】 高希武；

【作者基本信息】 中国农业大学 ， 农业昆虫与害虫防治， 2005， 博士

【摘要】 本文对意大利蝗痘病毒Calliptamus ualicus EPV（CiEPV）、两伯利亚蝗疸病毒Gomphocerussibiricus EPV（GsEPV）、红胫戟纹蝗痘病毒Dociostarus kraussi EPV（DkEPV）包涵体蛋白基因进行了克隆、测序、分析,对亚洲小车蝗痘病毒Oedaleus asiaticus EPV（OaEPV）的增效性进行了初步研究,并生物测定了OaEPV与化学杀虫剂混用的杀虫效果,这些工作有利于蝗虫痘病毒在生物防治上的开发利用。 CiEPV、GsEPV与DkEPV sph基因序列全长分别为4242 b p、4488 bp与 4242 bp,分别包含2922bp、2967bp与2922bp的开放阅读框架,编码的蛋白分子量为109.2、111.1及109.4 kDa。序列分析表明,CiEPV、GsEPV与DkEPV与直翅目昆虫痘病毒的包涵体蛋白,氨基酸水平上同源性都大于75%:而与鳞翅目EPV和鞘翅目EPV的包涵体蛋白,氨基酸水平上同源性都小于20%。CiEPV、GsEPV与DkEPV的包涵体蛋白分别包含19、21、19个半胱氨酸残基,并且聚集在蛋白的C-末端。基因的5′端非编码区包含了基因转录的启动区域,具有典型痘病毒的晚期基因启动标志序列TAAATG。在启动子ATG上游45个核苷酸的区域,可能为基因转录的启动区域,直翅目昆虫此序列的同源性达98%。基冈的3′非编码区,均发现另一个不完全的开放阅读框架（ORF）,与sph基因方向相反。此序列与MsEPV的潜在蛋白基因MSV072序列同源性80%以上。 接种最为1.67×10⁵OBs/头的OaEPV与1.67×10⁵孢子/头的绿僵菌混合感染黄胫小车蝗及东亚飞蝗,比用同量的绿僵菌孢子单独感染两种试虫,杀虫效果更为明显,增效率7d分别可达41.7%,28.9%。OaEPV的增效作用与OaEPV的用量有关,OaEPV的剂量越大,增设作用越强。OaEPV经2×SDS包涵体碱性裂解缓冲液裂解,离心,分别用上清液及沉淀与绿僵菌孢子混合感染黄胫小车蝗,上清液的增效活性比沉淀大2.5倍。用三种不同方法（2×SDS包涵体碱性裂解液法,0.02 mol/L NaOH裂解法,8 mol/L尿素裂解法）裂解OaEPV所得蛋白液与绿僵菌混用,生测结果表明,包涵体裂解液法制备所得蛋白增效活性最高,NaOH裂解法次之,尿素裂解法制备的蛋白几乎没有增效活性。将OaEPV用包涵体碱性裂解液裂解后,用葡聚糖G-200凝胶柱层析进行分离,出现两个洗脱峰,这两个峰的洗脱液浓缩后进行生物测定,初步表明增效作用主要为第二个峰的蛋白片段。经SDS-聚丙烯酰胺凝胶电泳,第二个峰的蛋白片段的分子量为40 kDa左右。 OaEPV与化学杀虫剂辛硫鳞、甲基对硫磷、溴氰菊酯、氰戊菊酯、卡死克、抑太保及西维因7种农药混合感染黄胫小车蝗的LC₅₀,均比单独使用农药均有所下降,卡死克、抑太保的下降率达49.0%与42.8%,效果最为明显。更多还原

【Abstract】 In this thesis, The complete nucleotide sequences of spheroidin genes of Calliptamus italicus entomopoxvirus (CiEPV), Gomphocerus sibiricus entomopoxvirus (GsEPV) and Dociostarus kraussi EPV (DkEPV) were determined. The enhancement of metarhizium inffection in grasshoppers by Oedaleus asialicus entomopoxvirus (OaEPV) was tested. The effect of 7 chemical insecticides mixing with OaEPV on Oedaleiis infernalis was studied. These works would impact the CiEPV. GsEPV. DkEPV and OaEPV to be used as biological control factor.The CiEPV, GsEPV and DkEPV spheroidin genes respectively harbored ORFs of 2922 bps, 2967 bps and 2922 bps. that were capable of coding polypeptides of 109.2, 111.1 and 109.2 kDa. Computer analysis indicated that CiEPV, GsEPV and DkEPV spheroidins shared less than 20% aa identities with lepidopteran AmEPV and coleopteran AcEPV spheroidins, but more than 80% aa identities with orthopteran OaEPV, MsEPV and AaEPV spheroidins. The CiEPV, GsEPV and DkEPV spheroidins respectively contained 19, 21 and 19 cysteine residues that were particularly abundant at the C-termini, as is the case with those of the other orthopteran EPV spheroidins. The promoter regions of the there spheroidin genes were highly conserved (99%) among the orthopteran EPVs and also contained the typical very A+T rich and TAAATG signal mediating transcription of poxvirus late genes. We also sequenced an incomplete ORF downstream of the pheroidin gene of CiEPV, GsEPV and DkEPV. The ORF was in the opposite direction to the spheroidin gene and was homologous to MSV072 putative protein of MsEPV.The mortalities of 3-instar nymphs of Oedaleiis infemalis and Locusta migraloria manilensis inoculated with the mixture of OaEPV (1.67 × 105OBs per larva)and metarhizium(1.67× 105 spores per larva)were higher than of those inoculated the metarhizium (1.67 × 105 spores per larva) alone. The rates of enhancement were 46.7% . 32.5% in the 7th day. In addition, the enhancing activity was relation to the does of OaEPV. The enhancing activity increased as the does of OaEPV increased. Oedaleiis asialicus entomopoxvirus contained a factor, which enhanced the infecton of Metarhizium flaroviride in the grasshopper, Oedaleus infemalis. When the spheroids of OaEPV dissolved with 2× SDS spheroid alkaline solution and centrifuged, the enhancing activity of the supernatant was 2.5 times greater than the pellet. The enhancing activity of spheroid proteins depended on the alkali used to dissolve the spheroids. The greatest activity occurred with spheroid alkaline solution, followed with 0.02 mol/L NaOH, and none with 8 mol/L urea. Spheroid proteins were fractionated on a sephadex G-200 column. Two fractions were obtained and the second fraction had the enhancing activity. SDS-PAGE indicated the molecular weight of the active fraction was 40 kDa.The LC50 of 7 chemical insecticides mixed with OaEPV on Oedaleiis infemalis are lower than that of the insecticides alone. The ratios of decrease of LC50 of Flufenoxuron and Chlorfluazuron are 49.0% and 42.8%.更多还原