【摘要】：白蠟蟲(chóng)(Ericerus pela Chavannes)二齡雄蟲(chóng)分泌的白蠟,是重要的工業(yè)原料,廣泛地應(yīng)用于化工、食品、醫(yī)藥、化妝品等行業(yè)。蠟酯合酶(wax synthase,WS)在白蠟生物合成途徑中催化長(zhǎng)鏈脂肪醇和脂酰輔酶A生成酯,是蠟酯生物合成的關(guān)鍵酶。本研究根據(jù)實(shí)驗(yàn)室前期獲得的ws全長(zhǎng)序列,擴(kuò)增其cDNA全長(zhǎng),用試劑盒體外轉(zhuǎn)錄了ws基因的dsRNA,注射于白蠟蟲(chóng)雌成蟲(chóng)體內(nèi),并用熒光定量PCR檢測(cè)ws基因的干涉效果,為驗(yàn)證ws的生理功能奠定了基礎(chǔ);同時(shí)利用Bac-to-Bac表達(dá)系統(tǒng)構(gòu)建了ws基因的pFastBac~(TM)HT B表達(dá)載體,轉(zhuǎn)化大腸桿菌DH10Bac~(TM),經(jīng)抗性和藍(lán)白斑篩選獲得重組桿粒rBacmid/EpelWS,轉(zhuǎn)染Sf9(Spodoptera frugiperda)細(xì)胞,在Sf9細(xì)胞中初步進(jìn)行表達(dá),免疫熒光觀測(cè)并用蛋白免疫印跡驗(yàn)證WS的表達(dá)。主要研究結(jié)果如下:(1)成功用試劑盒體外轉(zhuǎn)錄了ws基因的dsRNA,檢測(cè)結(jié)果表明,dsRNA已成功合成。(2)熒光定量PCR結(jié)果表明,注射ws基因的dsRNA一周后的試驗(yàn)組白蠟蟲(chóng)與經(jīng)gfp基因的dsRNA處理的對(duì)照組相比,試驗(yàn)組體內(nèi)ws mRNA轉(zhuǎn)錄量明顯下調(diào),為后續(xù)功能的研究奠定了基礎(chǔ)。(3)利用Bac-to-Bac表達(dá)系統(tǒng)成功構(gòu)建了WS表達(dá)載體,PCR鑒定及測(cè)序結(jié)果表明,目的片段已插入載體中。獲得了重組桿粒rBacmid/EpelWS,轉(zhuǎn)染Sf9細(xì)胞后收獲了P3病毒,蛋白免疫印跡表明:被P3病毒感染后的Sf9細(xì)胞總蛋白中,存在目的蛋白產(chǎn)生的雜交反應(yīng),WS被成功表達(dá)。本研究體外轉(zhuǎn)錄了白蠟蟲(chóng)ws基因的dsRNA,并在昆蟲(chóng)細(xì)胞中成功表達(dá)了WS,為白蠟蟲(chóng)ws的生理功能及WS的生化特性的進(jìn)一步研究奠定了重要基礎(chǔ)。
[Abstract]:White wax secreted by the second instar male worm of white wax worm (Ericerus pela Chavannes) is an important industrial raw material. It is widely used in chemical industry, food, medicine, cosmetics and other industries. Wax ester synthase (wax synthase,WS) catalyzes long chain fatty alcohol and fatty acyl coenzyme A to form esters in the biosynthetic pathway of white wax, which is the key enzyme in the biosynthesis of wax esters. In this study, according to the full-length ws sequence obtained earlier in the laboratory, the full-length cDNA was amplified. The dsRNA, of ws gene was transcribed in vitro and injected into the female adult of Wax Alba, and the interference effect of ws gene was detected by fluorescence quantitative PCR (FQ-PCR). It laid a foundation for verifying the physiological function of ws. At the same time, the pFastBac~ (TM) HT B expression vector of ws gene was constructed by Bac-to-Bac expression system and transformed into E. coli DH10Bac~ (TM),. The recombinant plasmid rBacmid/EpelWS, was transfected into Sf9 (Spodoptera frugiperda) cells by resistance and blue-white spot screening. The expression of WS was detected by immunofluorescence and Western blot. The expression of WS was detected by immunoblotting in Sf9 cells. The main results are as follows: (1) the dsRNA, detection results of ws gene were successfully transcribed with a kit in vitro. (2) the results of fluorescence quantitative PCR showed that dsRNA had been successfully synthesized. One week after injection of dsRNA with ws gene, the amount of ws mRNA transcript in the experimental group was significantly down-regulated compared with that in the control group treated with dsRNA of gfp gene. The results of PCR identification and sequencing showed that the target fragment had been inserted into the vector. (3) the WS expression vector was successfully constructed by Bac-to-Bac expression system, and the result of PCR identification and sequencing showed that the target fragment had been inserted into the vector. P3 virus was harvested after transfection of recombinant baculovirus rBacmid/EpelWS, into Sf9 cells. Western blot showed that there was hybridization reaction of target protein in the total protein of Sf9 cells infected with P3 virus and WS was expressed successfully. In this study, we transcribed the dsRNA, of white wax worm ws gene in vitro and successfully expressed WS, in insect cells, which laid an important foundation for the further study of the physiological function of ws and the biochemical characteristics of WS.
【學(xué)位授予單位】：中國(guó)林業(yè)科學(xué)研究院
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2016
【分類號(hào)】：S899.1

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