黄粉虫脂肪酸合成关键基因的发育表达以及超长链脂肪酸延伸酶的功能研究

发布时间：2018-11-27 08:56

【摘要】：黄粉虫Tenebrio molitor Linneeus又名面包虫、黄粉甲等,现在主要人工饲养,由于其含有丰富的蛋白质和脂肪酸等营养成分,脂肪酸占其幼虫干重的30%以上,且不饱和脂肪酸含量又占黄粉虫脂肪酸比例的70%以上,不但可作为家养鸟类及宠物的饲料,也是一种食用昆虫,因而明确其脂肪酸合成途径及关键基因对于食品安全和食品成分的进一步加工非常重要。此外,在昆虫中脂肪酸的衍生物包括鞘脂类,甘油酯类、脂肪醇、蜡酯及烃类,对于信息素合成、表皮形成、能量提供、昆虫耐寒性等具有重要作用,通路上的关键酶有可能成为新农药靶标位点,用于害虫防治。基于上述原因,本研究以黄粉虫为研究对象,对其不同发育阶段转录组进行测序,分析了脂肪酸合成通路关键基因在发育阶段的表达。同时,基于转录组数据,扩增和克隆了超长链脂肪酸延伸酶(ELO),对黄粉虫卵和老熟幼虫进行RNA干扰,观察表型的变化;并对ELO进行异源表达,验证功能。主要研究结果如下:1.对黄粉虫7个发育阶段卵、1龄幼虫、2龄幼虫、老熟幼虫、蛹、雄成虫、雌成虫进行了转录组测序。获得104937个unigenes,获得有注释信息的unigenes 32806条。其中,脂肪酸合成通路关键基因乙酰辅酶A羧化酶基因(ACC)14个,脂肪酸合成酶基因(FAS)75个,超长链脂肪酸延伸酶基因(ELO)83个,脂酰辅酶A还原酶基因(FAR)101个,脂肪酸脱氢酶基因(FAD)75个,5种关键基因分别选择1个,对不同龄期表达量进行聚类分析和qRT-PCR验证,趋势重合达87.7%;ACC,FAS在雄成虫期高表达,其它阶段低,FAD基因在雌成虫期表达,其它阶段极低,FAR在各阶段表达。通过与KEGG数据库进行比对,共有44586条基因被分配到331条通路中,其中有113条contigs与脂肪酸合成途径相关,119条contigs与饱和脂肪酸合成途径相关,49条contigs与脂肪酸延伸途径相关,对这所有contigs进行聚类分析。另外,分析了黄粉虫老熟幼虫脂肪酸组成,不饱和脂肪酸含量高达73%。2.扩增得到3条TmELO cDNA(1005bp、972bp和936bp)全长,有HXXHH等ELO具有的特征模体,预测定位于内质网,具有跨膜结构。qRT-PCR显示,三个TmELO在卵、1龄幼虫、2龄幼虫、老熟幼虫、蛹、雌成虫、雄成虫阶段都有表达,TmELO1在卵期表达量低而TmELO2在卵期表达量高,TmELO3在卵期表达量高。3.通过注射dsRNA,干扰黄粉虫老熟幼虫和卵的转录水平表达,发现卵和老熟幼虫在一段时间内mRNA水平被大幅度抑制。卵的TmELO3基因被干扰后,其孵化率大幅度降低;老熟幼虫TmELO1和TmELO3基因被干扰后,死亡率上升。在INVSc1酿酒酵母菌株中表达了黄粉虫TmELO1、TmELO2和TmELO3,发现表达TmELO1能产生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表达后,酵母脂肪酸组分未变化。
[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.
【学位授予单位】：浙江农林大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：S899.9

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