黃粉蟲脂肪酸合成關(guān)鍵基因的發(fā)育表達(dá)以及超長鏈脂肪酸延伸酶的功能研究
發(fā)布時間：2018-11-27 08:56
【摘要】：黃粉蟲Tenebrio molitor Linneeus又名面包蟲、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)和脂肪酸等營養(yǎng)成分,脂肪酸占其幼蟲干重的30%以上,且不飽和脂肪酸含量又占黃粉蟲脂肪酸比例的70%以上,不但可作為家養(yǎng)鳥類及寵物的飼料,也是一種食用昆蟲,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,在昆蟲中脂肪酸的衍生物包括鞘脂類,甘油酯類、脂肪醇、蠟酯及烴類,對于信息素合成、表皮形成、能量提供、昆蟲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),用于害蟲防治�；谏鲜鲈�,本研究以黃粉蟲為研究對象,對其不同發(fā)育階段轉(zhuǎn)錄組進(jìn)行測序,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,基于轉(zhuǎn)錄組數(shù)據(jù),擴(kuò)增和克隆了超長鏈脂肪酸延伸酶(ELO),對黃粉蟲卵和老熟幼蟲進(jìn)行RNA干擾,觀察表型的變化;并對ELO進(jìn)行異源表達(dá),驗(yàn)證功能。主要研究結(jié)果如下:1.對黃粉蟲7個發(fā)育階段卵、1齡幼蟲、2齡幼蟲、老熟幼蟲、蛹、雄成蟲、雌成蟲進(jìn)行了轉(zhuǎn)錄組測序。獲得104937個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,脂肪酸合成酶基因(FAS)75個,超長鏈脂肪酸延伸酶基因(ELO)83個,脂酰輔酶A還原酶基因(FAR)101個,脂肪酸脫氫酶基因(FAD)75個,5種關(guān)鍵基因分別選擇1個,對不同齡期表達(dá)量進(jìn)行聚類分析和qRT-PCR驗(yàn)證,趨勢重合達(dá)87.7%;ACC,FAS在雄成蟲期高表達(dá),其它階段低,FAD基因在雌成蟲期表達(dá),其它階段極低,FAR在各階段表達(dá)。通過與KEGG數(shù)據(jù)庫進(jìn)行比對,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),對這所有contigs進(jìn)行聚類分析。另外,分析了黃粉蟲老熟幼蟲脂肪酸組成,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,有HXXHH等ELO具有的特征模體,預(yù)測定位于內(nèi)質(zhì)網(wǎng),具有跨膜結(jié)構(gòu)。qRT-PCR顯示,三個TmELO在卵、1齡幼蟲、2齡幼蟲、老熟幼蟲、蛹、雌成蟲、雄成蟲階段都有表達(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.通過注射dsRNA,干擾黃粉蟲老熟幼蟲和卵的轉(zhuǎn)錄水平表達(dá),發(fā)現(xiàn)卵和老熟幼蟲在一段時間內(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)位級別】：碩士
【學(xué)位授予年份】：2017
【分類號】：S899.9
本文編號：2360140
[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)位級別】：碩士
【學(xué)位授予年份】：2017
【分類號】：S899.9
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