| The ladybird Coccinella septempunctata L.is an important predator,which has attracted considerable attention in biological control program.It is capable of entering a facultative adult diapause to survive winter or summer.Diapause provides a possibility that could be used for increasing shelf life,improving the control effect,and promoting the mass rearing and application of the ladybirds.C.septempunctata can accumulate large amounts of lipids during diapause.Lipid is an important nutrient and energy resources for the development of insect diapause.The study of lipid accumulation is crucial for revealing the molecular mechanism of diapause regulation.Fatty acid synthesis is vital for lipid accumulation,and the enzymes Fatty acid synthase(FAS)and acyl-CoA delta(11)desaturase(FADΔ11)are key regulators involving in the fatty acid synthesis of the ladybird.Fatty acid synthase is a multifunctional enzyme,and its catalytic product,palmitic acid,can store energy in the form of triglycerides.FAS also participates in the synthesis of phospholipids,cell membrane structure,etc.FADΔ11 is a kind of desaturase,which can catalyze saturated fatty acids as unsaturated fatty acids.In addition,FADΔ11 could also play a role in the synthesis of insect sex pheromone.However,the function of the two enzymes and its regulation mechanism during diapause are not clear.In this study,we cloned three genes,Fas1,Fas2 and FadΔ11 from C.septempunctata,and determined the relative mRNA levels of the target genes under different treatments by qRT-PCR.The function of CsFas and CsFadΔ11 during diapause of C.septempunctata also has been studied using RNAi.The main results are as follows:1.The primers were designed based on the sequences which encode fatty acid synthase and acylCoA Delta(11)desaturase according to the transcriptome database of C.septempunctata.We obtained the full-length cDNA of the two CsFas genes and a CsFadΔ11 gene,the lengths of which were 6563 bp,7642bp,and 1447 bp,respectively.The two CsFas genes own different conservative domains,which belong to different branches of phylogenetic trees.It is speculated that the CsFas involves in different metabolism pathways.Amino acid sequence analysis showed that CsFadΔ11 contains three histidine boxes and six transmembrane domains,which is a typical fatty acid desaturase.2.The qPCR assays indicated that CsFas1,CsFas2 and CsFadΔ11 were all expressed under different treatments(newly emerged adult,at the day of 10 or 20 after diapause induction,early diapause,late diapause,post diapause and normal development adult).However,the results displayed that the highest expression occur at the day of 20 after diapause induction and in the early diapause.The expression pattern of the target genes are consistent with the change of lipid content during diapause.3.The dsCsFas1,dsCsFas2 or dsCsFadΔ11 were guided into C.septempunctata by microinjection(dsGfp as control).The relative expression of CsFas1,CsFas2 or CsFadΔ11 was significantly lower than the control,and the level of total lipid and triglyceride decreased extremely.Whereas,the supercooling point increased distinctly.Diapause-destined individuals appear similar phenotype,such as normal development of digestive tract,with the nondiapause individuals after interference of CsFas1,CsFas2 or CsFadΔ11.4.Diapause induction condition as a signal stimulate the expression of CsFas in C.septempunctata,thus promoting the synthesis of palmitic acid.Palmitic acid is the precursor of other fatty acids.Besides,CsFadΔ11 was already confirmed to participate in the regulation of lipid metabolism during diapause of the C.septempunctata in this paper.It is speculated that the CsFadΔ11 gene is related to the contents and species of unsaturated fatty acids.And the ladybird will get higher chilling-tolercence. |
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