导　　师： 黄自然

学科专业： I0402

授予学位： 博士

作　　者： ;

机构地区： 华南农业大学

摘　　要： 利用甜菜夜蛾(spodopteraexigua)幼虫虫体继代的一株野生型甜菜夜蛾核型多角本病毒(spodopteraexiguanucleopolyhedrovirus-senpv)感染人工饲料无菌饲育的甜菜夜蛾3龄幼虫,3日后发现幼虫染病死亡,镜检病虫尸体发现典型的病毒我角体.取染病幼虫的体液经过滤后感染甜菜夜蛾细胞株se301,72h发现大部分细胞核内形成病毒多角体,说明该病毒对se301细胞具有强烈的感染性. The 3~/(rd/) instar larvae of beet armyworm, Spodoptera exigua feeding withartificial diet were infected by a wide strain of Spodoptera exiguanucleopolyhedrovirus -SeNPV, which was obtained from beet regeneration inarmyworm larval body. The worms died three days later. Typical viruspolyhedra were observed through microscopic examination on the dead bodies.After filtering, the body fluid from infected worms was used to infect the beetarmyworm cell strain Se301. Virus polyhedra were found in 72h in most of thenuclei. This proved that this virus had strong infecting ability to Se301 cell. Se301 cells were infected by the culture solution containing wild-typeSeNPV virions. Through isolation and purification of plaque assay, fifteenclone strains were derived and designated as SeNPV G1 to SeNPV G15respectively. These 15 clones and the SeNPV strain isolated from Zika Countyof Japan /(S1/) were multiplied. The virus DNA was extracted and cleaved bythree restriction enzymes such as EcoR I, Pst I and Xho I. The electrophoresisresult showed 3 kinds of different DNA electropherograms variants wereobserved which were named SeNPV G1, SeNPV G3, SeNPV G4 ,respectively.The DNA electropherograms of them were different to that of S1, and thosereported from USA and China.. The size of DNA were G1 130.67kb, G3118.6kb, G4 129.3kb, S1 119.03kb respectively. The result showed thewild-type virus has spontaneous mutation ability. It further proved theviewpoint that the same wild-type virus might be a mixed population ofvarious variant strains. The three variant strains and the Japanes strain S1 were applied to infectSe301 cells and other six non-host insect cells to detect their multiplicationability. The results showed that four of them were highly infectious to Se301cells. They could form large quantity of budded virions and polyhedra,however, their infecting ability were different. The amount of BV wasG3>S1>G4>G1. All the four variant strains could infect Sf9 of S. Frugiperdaand Splm1229 of Spilosoma imparilis, and form polyhedra. They could formBV in the Sf9 cells, but the amount of polyhedra and BV produced in Sf9 cells were less than that in the infected Se301 cells. They could make the SpIm1229 cells to collapse and caused a apoptotic-like cytopathology. Althoughthey could not form BV and polyhedra in cell strains such BmN4 of Bombyxmori, Ld652y of Lymantria dispar, they could cause them show dead symptomsimilar to that in the infected SpIm 1229 cells. The dead symptom of Ld652ycells caused by G3 and S1 were more serious than that made by G1 and G4.These four variant strains showed no lesions and symptoms after infecting theSpLi221 of S. Litura and CLS-79 of S. Littoralis. They could carry onnormal proliferation.The virus DNA Slot Blot analysis of insect cells infected by the fourSeNPV variant strains showed that the viruses could not form virions afterinfection, but could replicate virus DNA. The four variants could replicatevirus DNA not only in Se301, SpIm and Sf9 cells, but also in BmN4 andSpLi221 cells, in which virions could not be formed. The DNA replicationamount was far less than that in infected Se301, SpIm and Sf9 cells. In theinfected CLS-79 and Ld652y cells, virus DNA replications could not bedetected.The Western Blot analysis of infected cells indicated that virus polyhedrincould be detected in Se301, SpIm, and Sf9 cells, which were infected bySeNPV variant strains. The amount of polyhedrin in the infected Se301 cellswas far more than that in SpIm and Sf9 cells. The amount of polyhedrosisproduced in infected Se301 cell was S1>G3,G4>G1. The protein amount of S1was about 2 times of G1, and 1.5 times of G3 or G4.In order to explore the way to acquire the host range–enlargedrecombinant virus, using the feature that G3 and BmNPV can both form viruspolyhedrin in SpIm cells, the two kinds of virus were used to co-infect SpImcells. After plaque culture, a new virus strain was derived. After restrictionenzyme cleaved, its DNA electropherogram showed similar to that of BmNPVDNA. After infection, it could form polyhedrin in Se301 and BmN4 cells inthe following 4 generations. In the cells of the fifth generation however, theformation of polyhedra could not be observed. This showed the virus was nonstable genetically. It could be inferred that this virus was not a geneticallystable recombinant. The result gave some useful hints to further research inthis field.

关 键 词： 甜菜夜蛾 变异株 昆虫细胞 增殖 生物学特性

分 类 号： [S435.663]

领　　域： [农业科学] [农业科学]