本文選題：淡色庫蚊 + 溴氰菊酯�。� 參考：《南京醫(yī)科大學》2007年博士論文

【摘要】： 媒介昆蟲嚴重危害人類健康，據(jù)估計傳染病中2／3由媒介昆蟲傳播。世界史上許多危害嚴重的蟲媒傳染病如鼠疫、斑疹傷寒、黃熱病、瘧疾等都曾造成廣泛的流行并奪去成千上萬人的生命。當今世界隨著人類交往的日益頻繁，發(fā)現(xiàn)許多新的和重新出現(xiàn)的蟲媒傳染病。化學防治一直是媒介綜合治理的主要方法。殺蟲劑的大量、連續(xù)使用導致了媒介抗藥性的發(fā)生和發(fā)展。目前，世界范圍內(nèi)至少已有504種昆蟲產(chǎn)生了抗藥性，包括主要的媒介昆蟲，其中109種(亞種)蚊對一種或多種殺蟲劑產(chǎn)生了抗性。殺蟲劑抗性直接影響著蟲媒傳染病的流行和重新流行，因此，研究媒介抗藥性產(chǎn)生的機制并進行適宜治理實乃當務(wù)之急。本研究擬從克隆抗藥性相關(guān)基因入手，并對其進行功能鑒定，繼而研究其下游蛋白質(zhì)組學，為探討最終治理媒介抗藥性奠定基礎(chǔ)。為此，本文進行了以下幾個方面的研究。 一、淡色庫蚊核糖體蛋白L39基因的分子克隆、序列分析及表達差異鑒定 為克隆淡色庫蚊核糖體蛋白L39基因并對其進行表達差異的鑒定，本研究采用RT-PCR技術(shù)和cDNA末端快速擴增(RACE)策略，從淡色庫蚊(Culex pipiens pallens)對溴氰菊酯抗性品系4齡幼蟲中克隆核糖體蛋白L39基因(RPL39)，用相應的軟件進行生物信息學分析，并用熒光定量PCR和半定量RT-PCR分析敏感、抗性品系蚊的RPL39表達水平及對蚊各期RPL39進行表達差異鑒定。結(jié)果表明，成功克隆出RPL39基因。RPL39基因全長508bp，開放閱讀框為156bp，編碼51個氨基酸(GenBank／NCBI DQ080075)。序列分析顯示，該基因在各物種中保守性很高，與已報告的岡比亞按蚊(Anopheles gambiae)核糖體蛋白L39基因(GenBank／NCBI XP 320713)有99％同源性，并具有一個經(jīng)典的核糖體蛋白L39結(jié)構(gòu)域，沒有跨膜區(qū)域，無信號肽序列，屬于胞漿蛋白。熒光定量PCR結(jié)果表明，RPL39在淡色庫蚊對溴氰菊酯抗性品系中表達水平高于敏感品系，提示RPL39基因可能與對殺蟲劑的抗藥性相關(guān)。RPL39mRNA在淡色庫蚊抗性／敏感品系各個發(fā)育階段也有不同的表達，以雌性成蚊表達差異最大。 二、淡色庫蚊核糖體蛋白L39基因的初步功能研究 為鑒定核糖體蛋白L39基因與淡色庫蚊溴氰菊酯抗性的關(guān)系，，本研究擴增RPL39基因編碼區(qū)，構(gòu)建蚊細胞表達載體pIB／V5-His／RPL39，轉(zhuǎn)染蚊C6／36細胞。稻瘟菌素穩(wěn)定篩選和單克隆化后，細胞擴大培養(yǎng)，然后用RT-PCR和Western blot鑒定穩(wěn)定轉(zhuǎn)基因細胞的轉(zhuǎn)錄表達。不同濃度的溴氰菊酯處理穩(wěn)定轉(zhuǎn)基因細胞及對照未轉(zhuǎn)基因細胞和轉(zhuǎn)無關(guān)基因細胞后，用~3H-TdR測定細胞生存率法、細胞生長曲線測定及顯微鏡下觀察細胞存活率、細胞增殖速度及細胞生長情況等。結(jié)果顯示，成功構(gòu)建昆蟲細胞表達載體并建立穩(wěn)定轉(zhuǎn)染細胞系，RT-PCR和Western blot檢測重組表達載體(pIB／V5-His／RPL39)在穩(wěn)定轉(zhuǎn)染的蚊細胞內(nèi)高表達。不同濃度的溴氰菊酯處理轉(zhuǎn)染細胞后，在藥物壓力下，轉(zhuǎn)染核糖體蛋白L39目的基因細胞組的EC50顯著高于轉(zhuǎn)染無關(guān)基因CAT細胞組和未轉(zhuǎn)染空細胞組(p＜0.05)。細胞的增值速度也快于對照細胞；轉(zhuǎn)染RPL39基因細胞的形態(tài)、密度未見明顯變化，而對照細胞顆粒變粗、密度降低。初步研究結(jié)果提示，RPL39基因與溴氰菊酯抗性相關(guān)，可能為溴氰菊酯抗性相關(guān)基因。 三、淡色庫蚊核糖體蛋白L39下游蛋白質(zhì)組學的初步研究 為探討核糖體蛋白L39的抗藥性作用機制及與其他蛋白質(zhì)的相互作用，采用對轉(zhuǎn)染核糖體蛋白L39基因細胞進行二維電泳和質(zhì)譜鑒定的方法，對表達有差異的蛋白質(zhì)點進行鑒定。實驗結(jié)果提示，轉(zhuǎn)染核糖體蛋白L39基因的細胞高表達一系列與其他物種的各種酶高度同源的蛋白，例如酚氧化酶前體、絲氨酸／蘇氨酸蛋白激酶、絲氨酸蛋白酶、絲氨酸蛋白磷酸酶、精氨酸琥珀酸鹽移解酶、酪氨酸磷酯酶、以及具有降解多種藥物功能的多藥耐藥蛋白等；轉(zhuǎn)染細胞還高表達一系列和其他物種轉(zhuǎn)錄調(diào)控相關(guān)的蛋白高度同源的蛋白，例如ATP結(jié)合轉(zhuǎn)錄亞家族B、跨膜GTP酶、DNA復制準許因子、轉(zhuǎn)錄子的穩(wěn)定期調(diào)節(jié)蛋白、反轉(zhuǎn)錄轉(zhuǎn)座子gag蛋白等，以及高表達和其他物種同源的轉(zhuǎn)鐵蛋白和鐵反應元件結(jié)合蛋白，硒化物磷酸合成酶以及糖原合成酶等，因此推測轉(zhuǎn)染核糖體蛋白L39的細胞可能是通過解毒酶合成增加、保證細胞轉(zhuǎn)錄和翻譯蛋白的準確性以及增強細胞生存力等方面發(fā)揮抗藥性作用，從而為闡明核糖體蛋白L39的抗藥性作用機理提供理論基礎(chǔ)。
[Abstract]:In the present world , there are at least 504 kinds of insects in the world , including plague , typhus , yellow fever , malaria and so on .






Molecular Cloning , Sequence Analysis and Differential Expression of the L39 Gene of the Ribosomal Protein of 1 ,






The RPL39 gene ( RPL39 ) was cloned by RT - PCR and Rapid Amplification of cDNA Ends ( RACE ) . The results showed that the RPL39 gene was successfully cloned . The total length of RPL39 gene was 508bp , the open reading frame was 156 bp , and 51 amino acids were encoded . Sequence analysis showed that the gene has high conservation in each species , and has 99 % homology with the reported L39 gene of the ribosomal protein L39 gene of the Gambia , and has a classical ribosomal protein L39 domain . There is no transmembrane region and no signal peptide sequence . The results indicate that the RPL39 gene may be related to the drug resistance of the insecticide . The RPL39 mRNA has different expression in the development stages of the resistance / sensitive strain of the anti - mosquito resistance / sensitive strain , and the expression of RPL39 mRNA is the largest in the female adult .






Preliminary Study on the L39 Gene of the Ribosomal Protein of the Second , Light - color Cutians






The expression vector pIB / V5 - His / RPL39 was amplified by RT - PCR and Western blot .
The morphology and density of transfected RPL39 gene were not changed obviously , and the density of control cells was decreased . The results suggested that the resistance of RPL39 gene to fenvalerate was related to the resistance - related genes .






A Preliminary Study on the Proteomics of the downstream Proteomics of the Ribosomal Protein L39 in the Light - color Culicidae






In order to investigate the drug resistance mechanism of ribosomal protein L39 and its interaction with other proteins , the protein spots with different expression were identified by two - dimensional electrophoresis and mass spectrometry . The results suggested that the cells transfected with ribosomal protein L39 gene were highly homologous to various enzymes of other species , such as phenol oxidase precursor , serine / threonine protein kinase , serine protease , serine protein phosphatase , arginine succinate migration enzyme , tyrosine phosphoesterase , and multidrug resistance protein with multiple drug functions .
The transfected cells also highly express a series of proteins that are highly homologous to proteins associated with transcriptional regulation of other species , such as ATP - binding transcriptional sub - family B , transmembrane GTP enzyme , DNA replication grant factors , stabilized regulatory proteins of transcripts , anti - transcription transposable gag proteins , and the like , as well as high - expression and other species - homologous transferrin and iron - reactive element binding proteins , selenides phosphate synthase , glycogen synthase , and the like , thus suggesting that the cells transfected with ribosomal protein L39 may play a drug - resistant role through detoxification enzyme synthesis , ensuring cell transcription and translation protein accuracy , and enhancing cell viability , thereby providing a theoretical basis for the mechanism of resistance to ribosomal protein L39 .
【學位授予單位】：南京醫(yī)科大學
【學位級別】：博士
【學位授予年份】：2007
【分類號】：R346

【引證文獻】

相關(guān)碩士學位論文 前2條

1 段曉雷;PR-XP1基因的克隆及在C6/36耐藥細胞株中抗性的初步研究[D];西北農(nóng)林科技大學;2011年

2 溫雪梅;淡色庫蚊溴氰菊酯抗性基因XN-P450的克隆、分析及CYP6F1的表達[D];西北農(nóng)林科技大學;2012年

本文編號：1935987