【摘要】：黃粉蟲(chóng)Tenebrio molitor Linneeus又名面包蟲(chóng)、黃粉甲等,現(xiàn)在主要人工飼養(yǎng),由于其含有豐富的蛋白質(zhì)和脂肪酸等營(yíng)養(yǎng)成分,脂肪酸占其幼蟲(chóng)干重的30%以上,且不飽和脂肪酸含量又占黃粉蟲(chóng)脂肪酸比例的70%以上,不但可作為家養(yǎng)鳥(niǎo)類(lèi)及寵物的飼料,也是一種食用昆蟲(chóng),因而明確其脂肪酸合成途徑及關(guān)鍵基因?qū)τ谑称钒踩褪称烦煞值倪M(jìn)一步加工非常重要。此外,在昆蟲(chóng)中脂肪酸的衍生物包括鞘脂類(lèi),甘油酯類(lèi)、脂肪醇、蠟酯及烴類(lèi),對(duì)于信息素合成、表皮形成、能量提供、昆蟲(chóng)耐寒性等具有重要作用,通路上的關(guān)鍵酶有可能成為新農(nóng)藥靶標(biāo)位點(diǎn),用于害蟲(chóng)防治�；谏鲜鲈�,本研究以黃粉蟲(chóng)為研究對(duì)象,對(duì)其不同發(fā)育階段轉(zhuǎn)錄組進(jìn)行測(cè)序,分析了脂肪酸合成通路關(guān)鍵基因在發(fā)育階段的表達(dá)。同時(shí),基于轉(zhuǎn)錄組數(shù)據(jù),擴(kuò)增和克隆了超長(zhǎng)鏈脂肪酸延伸酶(ELO),對(duì)黃粉蟲(chóng)卵和老熟幼蟲(chóng)進(jìn)行RNA干擾,觀察表型的變化;并對(duì)ELO進(jìn)行異源表達(dá),驗(yàn)證功能。主要研究結(jié)果如下:1.對(duì)黃粉蟲(chóng)7個(gè)發(fā)育階段卵、1齡幼蟲(chóng)、2齡幼蟲(chóng)、老熟幼蟲(chóng)、蛹、雄成蟲(chóng)、雌成蟲(chóng)進(jìn)行了轉(zhuǎn)錄組測(cè)序。獲得104937個(gè)unigenes,獲得有注釋信息的unigenes 32806條。其中,脂肪酸合成通路關(guān)鍵基因乙酰輔酶A羧化酶基因(ACC)14個(gè),脂肪酸合成酶基因(FAS)75個(gè),超長(zhǎng)鏈脂肪酸延伸酶基因(ELO)83個(gè),脂酰輔酶A還原酶基因(FAR)101個(gè),脂肪酸脫氫酶基因(FAD)75個(gè),5種關(guān)鍵基因分別選擇1個(gè),對(duì)不同齡期表達(dá)量進(jìn)行聚類(lèi)分析和qRT-PCR驗(yàn)證,趨勢(shì)重合達(dá)87.7%;ACC,FAS在雄成蟲(chóng)期高表達(dá),其它階段低,FAD基因在雌成蟲(chóng)期表達(dá),其它階段極低,FAR在各階段表達(dá)。通過(guò)與KEGG數(shù)據(jù)庫(kù)進(jìn)行比對(duì),共有44586條基因被分配到331條通路中,其中有113條contigs與脂肪酸合成途徑相關(guān),119條contigs與飽和脂肪酸合成途徑相關(guān),49條contigs與脂肪酸延伸途徑相關(guān),對(duì)這所有contigs進(jìn)行聚類(lèi)分析。另外,分析了黃粉蟲(chóng)老熟幼蟲(chóng)脂肪酸組成,不飽和脂肪酸含量高達(dá)73%。2.擴(kuò)增得到3條TmELO cDNA(1005bp、972bp和936bp)全長(zhǎng),有HXXHH等ELO具有的特征模體,預(yù)測(cè)定位于內(nèi)質(zhì)網(wǎng),具有跨膜結(jié)構(gòu)。qRT-PCR顯示,三個(gè)TmELO在卵、1齡幼蟲(chóng)、2齡幼蟲(chóng)、老熟幼蟲(chóng)、蛹、雌成蟲(chóng)、雄成蟲(chóng)階段都有表達(dá),TmELO1在卵期表達(dá)量低而TmELO2在卵期表達(dá)量高,TmELO3在卵期表達(dá)量高。3.通過(guò)注射dsRNA,干擾黃粉蟲(chóng)老熟幼蟲(chóng)和卵的轉(zhuǎn)錄水平表達(dá),發(fā)現(xiàn)卵和老熟幼蟲(chóng)在一段時(shí)間內(nèi)mRNA水平被大幅度抑制。卵的TmELO3基因被干擾后,其孵化率大幅度降低;老熟幼蟲(chóng)TmELO1和TmELO3基因被干擾后,死亡率上升。在INVSc1釀酒酵母菌株中表達(dá)了黃粉蟲(chóng)TmELO1、TmELO2和TmELO3,發(fā)現(xiàn)表達(dá)TmELO1能產(chǎn)生C20:0脂肪酸并延伸至C24:0,TmELO2主要在酵母中使脂肪酸C16:0和C16:1占比增加;TmELO3表達(dá)后,酵母脂肪酸組分未變化。
[Abstract]:Powdery mildew (Tenebrio molitor Linneeus), also known as bread worm and yellow powder A, is now mainly raised in captivity. Because of its rich protein and fatty acids, fatty acids account for more than 30% of the dry weight of its larvae. The content of unsaturated fatty acids accounts for more than 70% of the fatty acids of powdery mildew, which can be used not only as a feed for domestic birds and pets, but also as an edible insect. Therefore, the identification of fatty acid synthesis pathway and key genes is very important for food safety and further processing of food ingredients. In addition, the derivatives of fatty acids in insects, including sphingolipids, glycerides, fatty alcohols, waxes and hydrocarbons, play an important role in pheromone synthesis, epidermis formation, energy supply, insect cold tolerance, etc. The key enzymes in the pathway may become new pesticide target sites for pest control. For the above reasons, the key genes of fatty acid synthesis pathway were analyzed by sequencing the transcriptome at different developmental stages. At the same time, based on the transcriptional data, the ultra-long chain fatty acid extension enzyme (ELO),) was amplified and cloned to interfere with the RNA of the egg and mature larvae of Molitor, and the phenotypic changes were observed, and the heterologous expression of ELO was carried out to verify the function. The main results are as follows: 1. The transcriptome sequence of 7 developmental stage eggs, 1st instar larva, 2nd instar larva, mature larva, pupa, male adult and female adult were sequenced. 32806 items of unigenes with annotated information obtained from 104937 unigenes,. Among them, there are 14 (ACC) genes, 75 (FAS) genes, 83 (ELO) genes and 101 (FAR) genes of fatty acid synthesis pathway key genes acetyl coenzyme A carboxylase, fatty acid synthase (FAS) 75, hyperlong chain fatty acid extension enzyme (ELO) 83 and lipoyl coenzyme A reductase (FAR) 101, respectively. Fatty acid dehydrogenase gene (FAD) 75, 5 key genes were selected respectively, the expression of fatty acid dehydrogenase at different age was analyzed by cluster analysis and qRT-PCR verification. The trend of overlap was 87.7%. The expression of ACC,FAS was high in male adult stage and low in other stages. FAD gene was expressed in female adult stage and very low in other stages. FAR was expressed in all stages. By comparing with KEGG database, 44586 genes were assigned to 331 pathways, 113 of which were related to fatty acid synthesis pathway, 119 to saturated fatty acid synthesis pathway and 49 to fatty acid extension pathway. All of the contigs were analyzed by cluster analysis. In addition, the fatty acid composition of the mature larvae was analyzed, and the unsaturated fatty acid content was as high as 73.2. Three full-length TmELO cDNA (1005bpc972 BP and 936bp were obtained, with characteristic motifs of ELO such as HXXHH, which were predicted to be located in endoplasmic reticulum and had transmembrane structure. QRT-PCR showed that three TmELO were in eggs, 1st instar larva, 2nd instar larva, older mature larva and pupa. The expression of TmELO1 was lower in the egg stage, higher in the egg stage, and the expression of TmELO3 was higher in the egg stage. By injecting dsRNA, to interfere the transcription level of mature larvae and eggs, it was found that mRNA levels in eggs and mature larvae were significantly inhibited for a period of time. The hatching rate of egg TmELO3 gene was significantly decreased, and the mortality of mature larva TmELO1 and TmELO3 genes was increased. TmELO1,TmELO2 and TmELO3, were expressed in INVSc1 Saccharomyces cerevisiae. It was found that the expression of TmELO1 could produce C20: 0 fatty acids and extend to C24: 0 TmELO 2. The proportion of fatty acids C16: 0 and C16: 1 increased mainly in yeast. After TmELO3 expression, the fatty acid composition of yeast did not change.
【學(xué)位授予單位】：浙江農(nóng)林大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2017
【分類(lèi)號(hào)】：S899.9

【參考文獻(xiàn)】

相關(guān)期刊論文 前10條

1 汪露;張鳳娟;李肇陽(yáng);陶波林;馬紀(jì);;中華齒刺甲和黃粉蟲(chóng)抗凍蛋白基因3'-UTR序列的克隆及其差異性比較[J];基因組學(xué)與應(yīng)用生物學(xué);2015年04期

2 易杰群;鄒志文;張古忍;;昆蟲(chóng)脂肪酸及其脫飽和酶與耐寒性的關(guān)系概述[J];環(huán)境昆蟲(chóng)學(xué)報(bào);2015年01期

3 朱小奇;趙肖;武玉飛;謝彩云;陳小娜;王俊剛;申紅;;大腸桿菌誘導(dǎo)黃粉蟲(chóng)產(chǎn)生抗菌肽的提取及抑菌活性的測(cè)定[J];石河子大學(xué)學(xué)報(bào)(自然科學(xué)版);2014年04期

4 石萌;劉小寧;馬紀(jì);;利用細(xì)菌表達(dá)dsRNA介導(dǎo)黃粉蟲(chóng)抗凍蛋白基因的RNA干擾[J];生物技術(shù)通報(bào);2014年08期

5 王南溪;楊偉;楊春平;楊樺;史亮;黃敏;龍玲;;黃粉蟲(chóng)油脂對(duì)小鼠體重及學(xué)習(xí)記憶能力的影響[J];食品工業(yè)科技;2015年01期

6 張棋麟;袁明龍;;基于新一代測(cè)序技術(shù)的昆蟲(chóng)轉(zhuǎn)錄組學(xué)研究進(jìn)展[J];昆蟲(chóng)學(xué)報(bào);2013年12期

7 張振華;陳介南;盧孟柱;章懷云;劉伯斌;;胡蘿卜與黃粉蟲(chóng)抗凍融合基因在擬南芥中的表達(dá)與抗凍性分析[J];中國(guó)農(nóng)學(xué)通報(bào);2012年31期

8 黃瓊;胡杰;周定剛;王勤;楊偉;朱天輝;;兩種色型黃粉蟲(chóng)抗凍蛋白cDNA克隆、序列分析與表達(dá)分析[J];昆蟲(chóng)學(xué)報(bào);2012年06期

9 馮云甲;徐洪富;王憲輝;;黃粉蟲(chóng)不同抗凍蛋白基因家族成員的低溫應(yīng)激表達(dá)[J];應(yīng)用昆蟲(chóng)學(xué)報(bào);2012年03期

10 唐馨;毛新芳;熱西力·克來(lái)木;劉忠淵;;黃粉蟲(chóng)抗菌肽TmAMP3在大腸桿菌中的高效表達(dá)及活性檢測(cè)[J];昆蟲(chóng)學(xué)報(bào);2011年10期

，

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