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研究生:饒筱芸
研究生(外文):Hsiao-Yun Jao
論文名稱:重組加州苜蓿夜蛾核多角體病毒提高對斜紋夜蛾幼蟲致病力
論文名稱(外文):Improvement in pathogenicity of Autographa californica nucleopolyhedrovirus to Spodoptera litura larvae by gene recombination
指導教授:石正人石正人引用關係
指導教授(外文):Cheng-Jen Shih
學位類別:碩士
校院名稱:國立臺灣大學
系所名稱:昆蟲學研究所
學門:生命科學學門
學類:生物學類
論文種類:學術論文
論文出版年:1999
畢業學年度:87
語文別:中文
論文頁數:66
中文關鍵詞:加州苜蓿夜蛾核多角體病毒斜紋夜蛾幼蟲基因重組寄主範圍
外文關鍵詞:Autographa californica nucleopolyhedrovirusSpodoptera litura larvaegene recombinationhost range
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本論文以斜紋夜蛾核多角體病毒為主要研究對象,嘗試選殖斜紋夜蛾核多角體病毒(Spodoptera litura nucleopolyhedrovirus , SpltNPV )對斜紋夜蛾幼蟲致病力高的基因,並利用基因重組的方法,將此影響寄主範圍的基因選殖到加州苜蓿夜蛾核多角體病毒(Autographa californica nucleopolyhedrovirus , AcMNPV)上,以提高重組病毒對斜紋夜蛾幼蟲的致病力,擴增AcMNPV之寄主範圍。首先利用EcoRI限制酵素切割SpltNPV DNA,建立基因庫,進而選殖基因片段於AcMNPV病毒傳送載體(pFastBacHTa)上,共篩選到13種含有不同DNA片段之傳送載體。利用Bac-to-Bac表現系統,將斜紋夜蛾核多角體病毒DNA EcoRI限制酵素片段,隨機選殖到AcMNPV上,產生含有SpltNPV DNA片段之AcMNPV重組病毒。本試驗共構築八種含有不同限制酵素片段之不同重組病毒,利用包埋前病毒(pre-occluded virus)形式,餵食斜紋夜蛾一齡幼蟲,進行生物檢定,發現這八種重組病毒對幼蟲致病力皆不超過30%,且八種重組病毒與野生型AcMNPV及無包涵體對一齡幼蟲之致病力,均無顯著差異。結果顯示能夠提高AcMNPV致病力之基因並未選殖至這八種重組病毒中。此外,本論文亦就去氧核糖核酸解旋酵素(DNA helicase)基因,探討其對桿狀病毒寄主範圍之關係。將AcMNPV之helicase基因選殖至傳送載體上(pAcSG2-LIC),命名為pALAcH,與SpltNPV DNA或線狀AcMNPV (BaculoGoldTM) DNA共轉染至SL7B細胞株中,測試helicase基因是否能夠幫助SpltNPV產生PIB。利用顯微鏡觀察,僅有與線狀AcMNPV共轉染的處理組細胞有明顯病徵出現,而與SpltNPV共轉染之處理組卻與未感染病毒之細胞十分相似,沒有PIB出現。但將轉染後繼代培養第一代及第三代之病毒液,萃取其DNA,利用 ScH primers (Kamita and Maeda, 1997)進行PCR複製,發現與線狀AcMNPV或與SpltNPV共轉染的處理組,或單獨轉染SpltNPV的對照組中,皆能增幅出同樣大小之核酸片段。結果顯示,若轉染SpltNPV至SL7B細胞株,SpltNPV能複製DNA,並且能產生有感染能力之子代。參照AcMNPV、 SpeiNPV及OpMNPV helicase 基因序列,分別設計之helicase gene primer,已能利用SpltNPV為模板增幅出核酸片段。將 ScH primers所增幅出之核酸片段經定序的結果與SpeiNPV、BmNPV、AcMNPV及LdMNPV之helicase基因核酸序列均有90%以上之相似度,以此DNA片段作成探針,進行南方氏轉漬雜合,結果在HindIII、BamHI、XbaI限制酵素片段約8-6 kb的位置有雜合反應。

This study was based on the Spodoptera litura nucleopolyhedrovirus (SpltNPV) to clone the gene which could increase pathogenicity to the larva of S. litura. Furthermore, the gene related to the host range was incorporated to Autographa californica nucleopolyhedrovirus (AcMNPV) by means of genetic engineering to generate recombinant virus. The recombinant virus was used to feed S. litura larvae for determining its the pathogenicity. First of all, the genomic library of SpltNPV was established by digesting DNA of SpltNPV with several restriction enzymes such as EcoRI. DNA fragments from established genomic library were cloned to the transfer vector pFastBacHTa of AcMNPV. Thirteen clones of recombinant transfer vector contained different EcoRI fragments of SpltNPV were obtained. These transfer vectors were used to generate recombinant AcMNPV by using Bac-to-Bac expression vector system. There were eight different recombinant viruses obtained. The pre-occluded form virus was used to bioassay 1st instar of S. litura. Results revealed that pathogenicity of 8 recombinant viruses were less than 30%, and there were no significant difference between recombinant viruses and wild type AcMNPV. This indicated that all recombinant viruses did not contain host range related gene of SpltNPV. In addition to construction of recombinant viruses containing EcoRI fragment of SpltNPV, the DNA helicase gene that is considered to be related expansion of to host range of AcMNPV was tested in this study too. DNA helicase gene from AcMNPV was cloned to transfer vector pAcSG2-LIC for generating recombinant transfer vector, pALAcH. The putative recombinant transfer vector was cotransfected into SL7B cell with DNA of SpltNPV and liner AcMNPV (BaculoGoldTM) respectively. Treatments after cotransfection was observed under phase inverted microscope to check the polyhedral inclusion bodies (PIB) formation. Only treatment with cotransfected liner AcMNPV appeared significant cytopathic effect, there was no PIB in treatment with cotransfeted SpltNPV. However, after 1-3 subcultures of cotransfection, the progeny virus DNA was extracted and amplified by PCR with AcMNPV helicase gene primer, all cotransfection treatments could obtain the same size of DNA fragments. This means that SpltNPV could replicate in SL7B cell in spite of PIB formation. In order to amplify the helicase gene of SpltNPV, PCR primer was designed by consulting the helicase gene sequence of AcMNPV, SpeiNPV and OpMNPV. Primer from OpNPV was used to amplify specific fragment to make a probe for Southern blot test of SpltNPV. A hybridized band was detected in XbaI fragment with ca. 3.5 kb. This fragment may contain DNA helicase gene of SpltNPV, but need further study.

目錄
中文摘要
英文摘要
圖次i
表次ii
壹、前言1
貳、前人研究 4
參、材料與方法 16
一、材料 16
二、方法 17
(一) 構築含SpltNPV DNA限制酵素片段之重組AcMNPV17
(二) AcMNPV DNA helicase gene與SpltNPV之共轉染試驗21
(三) 選殖SpltNPV DNA helicase gene24
(四) 病毒致病力之測試25
肆、結果與討論28
(一) 構築含SpltNPV DNA限制酵素片段之重組AcMNPV28
(二) AcMNPV DNA helicase gene與SpltNPV之共轉染試驗39
(三)選殖SpltNPV之helicase基因49
伍、結論 53
陸、參考文獻 55
柒、附錄 63
捌、誌謝 66

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