本文選題：桔小實蠅 + 多殺菌素�。� 參考：《海南大學》2015年碩士論文

【摘要】：桔小實蠅Bactrocera dorsalis (Hendel)是我國華南地區(qū)的重要果蔬害蟲,可為害柑桔、芒果、番石榴、楊桃等250多種果實,被許多國家和地區(qū)列為檢疫性害蟲。多殺菌素是在刺糖多胞菌(Saccharopolyspora spinosa)發(fā)酵液中提取的一種大環(huán)內(nèi)酯類無公害高效生物殺蟲劑,既作用于煙堿型乙酰膽堿受體(nicotinic acetylcholine receptor, nAChR),也作用于γ-氨基丁酸受體(GABAR),能有效控制鱗翅目、雙翅目和纓翅目等多種害蟲。目前,多殺菌素是生產(chǎn)上有效防治桔小實蠅的主要殺蟲劑之 但已有報道表明桔小實蠅對多殺菌素產(chǎn)生了一定的抗藥性風險。因此,為了合理使用多殺菌素防治桔小實蠅發(fā)生與危害,延長多殺菌素的使用壽命,本研究將昆蟲毒理學、生物信息學與蛋白質(zhì)組學相結(jié)合,初步開展了基于多殺菌素脅迫的桔小實蠅蛋白質(zhì)組學研究,建立了桔小實蠅幼蟲總蛋白的雙向電泳條件,分離和鑒定了多殺菌素脅迫前后的桔小實蠅差異表達蛋白,為深入探討多殺菌素的抗性形成機制研究奠定了基礎(chǔ)。主要結(jié)果如下： 1.建立并優(yōu)化了桔小實蠅幼蟲總蛋白的雙向電泳條件,確定使用24cm pH4-7的IPG膠條對BPP法提取的桔小實蠅幼蟲總蛋白進行雙向電泳,采用考馬斯亮藍法對雙向電泳凝膠進行染色為最適桔小實蠅蛋白質(zhì)組學研究的方法,為開展基于多殺菌素脅迫的桔小實蠅蛋白質(zhì)組學初步研究工作奠定了基礎(chǔ)。 比較了BPP法和3種TCA-丙酮法提取桔小實蠅幼蟲總蛋白的效果,結(jié)果表明：BPP法效果最好,可以獲得1187±42個蛋白點,且分離得到的蛋白點分辨率更高。比較了13、24cm pH4-7的IPG膠條分離蛋白質(zhì)的效果,發(fā)現(xiàn)使用24cm膠條分離得到1129±46個蛋白點,較13cm膠條增加了483個蛋白點。比較考馬斯亮藍染色與硝酸銀染色的效果發(fā)現(xiàn)二者染色效果相當,但考馬斯亮藍染色背景較硝酸銀染色干凈清晰。質(zhì)譜鑒定的BPP膠中10個特異的蛋白點以Metazoa (Animals)為檢索物種可成功檢索9個,以Drosophila (fruit flies)為檢索物種可成功檢索6個,且以Metazoa (Animals)為檢索物種可獲得更全面的信息。 2.分離和鑒定了5mg/mL多殺菌素短時間脅迫前后的桔小實蠅差異表達蛋白,發(fā)現(xiàn)所鑒定的蛋白主要參與物質(zhì)能量代謝、免疫反應(yīng)、解毒功能、細胞骨架生成等,為深入探討桔小實蠅對低濃度多殺菌素脅迫的應(yīng)答機制提供了基礎(chǔ)信息支撐。 采用浸漬法處理桔小實蠅3齡老熟幼蟲,浸泡時間10s,浸泡后置于土壤中。丙酮處理作為對照組(CK),置于土壤5min后取樣,5mg/mL多殺菌素處理作為處理組,分別置于土壤5min、2h后取樣。獲得豐度變化在2倍以上的58個差異蛋白點并進行了質(zhì)譜鑒定,共鑒定出44個,其中23個上調(diào)表達,21個下調(diào)表達。通過生物信息學分析,發(fā)現(xiàn)所鑒定的蛋白主要參與物質(zhì)能量代謝、免疫反應(yīng)、解毒功能、細胞骨架生成等,其中物質(zhì)能量代謝相關(guān)蛋白所占比例最多,達到43%。 3.分離和鑒定了10mg/mL多殺菌素長時間脅迫前后的桔小實蠅差異表達蛋白,發(fā)現(xiàn)所鑒定的蛋白包括物質(zhì)能量代謝相關(guān)蛋白、防御或免疫相關(guān)蛋白、神經(jīng)系統(tǒng)發(fā)展相關(guān)蛋白、細胞骨架蛋白及其它,為進一步探討桔小實蠅對高濃度多殺菌素脅迫的應(yīng)答機制提供了基礎(chǔ)信息支撐。 采用浸漬法處理桔小實蠅3齡老熟幼蟲,浸泡時間10s,浸泡后置于土壤中。丙酮處理作為對照組(CK),置于土壤6h后取樣,10mg/mL多殺菌素處理作為處理組,分別置于土壤6h、12h后取樣。獲得了54個表達量變化2倍以上的差異蛋白點,成功鑒定了42個蛋白點,其中上調(diào)蛋白點17個,下調(diào)蛋白點25個。鑒定的蛋白經(jīng)生物信息學分析后按功能分為5類：(1)物質(zhì)能量代謝相關(guān)蛋白,占67%；(2)防御或免疫相關(guān)蛋白,占2%；(3)神經(jīng)系統(tǒng)發(fā)展相關(guān)蛋白,占7%；(4)細胞骨架蛋白,占2%；(5)其它,占22%。
[Abstract]:Bactrocera ( Hendel ) , an important fruit and vegetable pest in South China , has been classified as quarantine pests in many countries and regions .

In order to prevent and control the occurrence and harm of Bactrocera citrinum and prolong the service life of polybactericidal agent , it has been reported that the two - dimensional electrophoresis condition of the total protein of Bactrocera Bactrocera is established , the two - dimensional electrophoresis condition of the total protein of Bactrocera Bactrocera is established , and the basis of the study is laid for further exploring the mechanism of the resistance formation mechanism of polybactericidal agent . The main results are as follows :

1 . Two - dimensional electrophoresis conditions were established and optimized for the total protein of Bactrocera citrinata larvae . Two - dimensional electrophoresis was carried out on the total protein extracted from BPP by IPG strips of 24 cm pH 4 - 7 .

The effects of BPP method and three TCA - acetone methods on the total protein of fly larvae were compared . The results showed that the results showed that the best results of BPP were 1187 鹵 42 protein spots and higher resolution .

2 . Isolation and identification of the differentially expressed proteins of Bactrocera citrinae before and after a short time stress of 5mg / mL , found that the identified proteins mainly involved in substance energy metabolism , immune response , detoxification function , cytoskeletal formation and so on .

Three - instar larvae of orange fly were treated with immersion method for 10 seconds , soaked and placed in soil . After 5 min of soil acetone treatment was taken as control group ( CK ) , the samples were sampled after 5 min of soil , and then sampled at 5 mg / mL for 2 hours .

3 . Isolate and identify the differentially expressed proteins of Bactrocera citrinae before and after long - term stress of 10 mg / mL , and found that the identified proteins include substance energy metabolism - related proteins , defense or immune - related proteins , nervous system development - related proteins , cytoskeletal proteins , and other proteins .

The 3 - year old larvae of orange fly were treated by impregnation . The soaking time was 10 seconds , soaked and placed in the soil . After 6 hours of soil , the samples were sampled after 6 hours of soil and 10 mg / mL multi - bactericide was taken as the treatment group .
( 2 ) defense or immune related protein , accounting for 2 % ;
( 3 ) nervous system development related protein , accounting for 7 % ;
( 4 ) cytoskeletal proteins , accounting for 2 % ;
( 5 ) Other , accounting for 22 % .
【學位授予單位】：海南大學
【學位級別】：碩士
【學位授予年份】：2015
【分類號】：S433

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