| Yellow mealworm(Tenebrio molitor Linneeus)is currently mainly used for small-scale animals as feed.It contains rich protein,fatty acids and other nutrients andfatty acids account for more than 30% of the dry weight of the larvaewhile unsaturated fatty acids account for more than 70% of its fatty acids,in addition,Tenebrio molitor is not only a kind of feed of domestic birds and pets,but also an edible insect.Based on the above reasons,it’s very important to find out the fatty acid synthesis pathway and key genes of yellow mealworm for food safety and futher processing of food composition.Besides,fatty acids are precursors of sphingolipids,glycerololipids,fatty alcohols,wax esters and hydrocarbons in insects,furthermorefatty acids and their derivatives are very important for pheromonebiosynthesis,insect epidermis formation,energy availability,and insect cold tolerance.In summary,the key enzymes of fatty acid synthesis pathway are likely to be the new pesticides target site.Therefore,T.molitor was closed as the objectin this research,T.molitor from seven developmental stages were used to perform transcriptome sequencing using Illumina HiSeqTM 2000 platform and a large amount of unigeneswereobtained.And then we analyzed the expression of key genes in fatty acid biosynthesis of yellow mealworms.Meanwhile,based on the transcriptome data,three ELO full-length cDNAs were amplification and cloned.The eggs and mature larvae were injected by dsRNA and observed,and ELO genes were expressed in Saccharomyces cerevisiae INVSc1 to certify the functions.The main results are as follows,1.We analyzed the transcriptome data of seven developmental stages of T.molitor including eggs,1st larvae,2nd larvae,mature larvae,pupa,female adults and male adults.A total of 104937 unigenes were obtained and 32806 unigenes were annotated by Uniprot database,among which,key genes of fatty acids biosynthesis,14 ACC genes,75 FAS genes,83 ELO genes,101 FAR genes and 75 FAD genes were included.Then we selected one gene from every different gene kind,perform cluster analysis and used qRT-PCR to verify.The results showed that the relative transcript levels of ACC and FAS were high in male adults and low in other developmental stages,FADwas high in female adults and very low in otherstages,and FAR was expressed in the seven developmental stages.Compared with KEGG database,44586 contigs were obtained and assigned to 331 pathways.Fatty acid biosynthesis pathway has 113 contigs,biosynthesis of unsaturated fatty acid pathway has 119 contigs and fatty acid elongation pathway has 49 contigs,and cluster analysis was performed for these contigs.In addition,total fatty acid compositions of T.molitor mature larvae were analyzed by gas chromatography and the results showed that unsaturated fatty acids account for as high as 73%.2.Three TmELOs full-length(1005bp,972 bpand 936bp)were amplified and cloned.There were motifs such as HXXHH in three TmELOs and the TmELOs were predicted to locate in the endoplasmic reticulum.A phylogenetic tree showed that they had high similarity with the ELO of Tribolium castaneum.All TmELO genes were expressed in eggs,1st and 2nd instar larvae,mature larvae,pupae,male and female adults.The egg stages had a lower expression level of TmELO1 and higer expression level of TmELO2.TmELO3 was high in eggs but low in other developmental stages.3.Injection of dsTmELO1 or dsTmELO2 or dsTmELO3RNA significantly decreased the transcript level of TmELO1 or TmELO2 or TmELO3respectively.The injection of mature with ds TmELO1 and dsTmELO3significantly increased mortality and the injection of egg with dsTmELO1 significantly decreased the hatchability.However,RNAi did not produce any obvious changes in the components of fatty acids.Heterologous expression of TmELO genes in yeast revealed that TmELO1 and TmELO2 have functions to elongate fatty acids expecially TmELO1 could produce C20:0 to C24:0 of fatty acid and TmELO2 mainly increased the levels of C16:0 and C16:1. |
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