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Sequencing, Expression, and Functional Analyses of Four Genes Related to Fatty Acid Biosynthesis During the Diapause Process in the Female Ladybird, Coccinella septempunctata L.

ABSTRACT: The ladybird Coccinella septempunctata L., a predatory insect, serves as an excellent biological control agent against common agricultural pests. It undergoes a diapause phenomenon, during which a large amount of fat accumulates in the abdomen. A comprehensive analysis of this lipid accumulation can reveal the molecular mechanisms underlying diapause regulation, which can be exploited to improve the shipping and transport of the insect for agricultural applications. In this study, we compared the transcriptome of C. septempunctata during non-diapause, diapause, and post-diapause and screened four key genes related to lipid metabolism. The cDNA of these four relevant enzymes, acetyl-CoA carboxylase (ACC), long-chain fatty acid-CoA ligase (ACSL), elongase of very-long-chain fatty acids (ELO), and very-long-chain 3-oxoacyl-CoA reductase (KAR), were cloned using reverse transcription-polymerase chain reaction and rapid amplification of cDNA ends. Their expression profiles were analyzed during the preparation and maintenance phases of diapause and the post-diapause phase. The functions of these four key enzymes in diapause were further verified using RNA interference. All four genes were most closely related to the homeotic gene from Tribolium castaneum. The expression profiles of these four genes were significantly affected under diapause-inducing conditions; their expression level was the highest in the diapause preparation phase, and it gradually decreased with the diapause induction time. RNA interference showed that the target genes play important roles in fat storage during early diapause, and the decrease in their expression leads to a decrease in lipid content in C. septempunctata. These results indicate an important role of ACC, ACSL, ELO, and KAR in lipid accumulation. Our findings could help elucidate the production and accumulation of lipids by insects during the preparation for diapause and improve biological control.

SUBMITTER: Xiang M

PROVIDER: S-EPMC8417001 | biostudies-literature |

REPOSITORIES: biostudies-literature

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Project description:The most common ladybird beetle, Coccinella septempunctata L., is an excellent predator of crop pests such as aphids and white flies, and it shows a wide range of adaptability, a large appetite and a high reproductive ability. Diapause research plays an important role in the artificial propagation and shelf-life extension of insect products. Although this lady beetle's regulatory, physiological and biochemical characteristics in the diapause period are well understood, the molecular mechanism of diapause remains unknown. Therefore, we collected female adults in three different states, i.e., non-diapause, diapause and diapause termination, for transcriptome sequencing.After transcriptome sequencing using the Illumina HiSeq 2500 platform with pretreatment, a total of 417.6 million clean reads from nine samples were filtered using the program FASTX (version 0.0). Additionally, 106,262 contigs were assembled into 82,820 unigenes with an average length of 921 bp and an N50 of 1,241 bp. All of the unigenes were annotated through BLASTX alignment against the Nr or UniProt database, and 37,872 unigenes were matched. We performed further analysis of these unigenes using the Clusters of Orthologous Groups of proteins (COG), Gene Ontology (GO), and the Kyoto Encyclopedia of Genes and Genomes (KEGG) databases. Through pairwise comparisons of the non-diapause (ND), diapause (D), and diapause-terminated (DT) groups, 3,501 and 1,427 differentially expressed genes (DEGs) were identified between D and ND and between DT and D, respectively. Moreover, 443 of the DEGs were specifically expressed during the diapause period (i.e., DEGs that were expressed at the highest or lowest levels during diapause compared with the other stages). GO function and KEGG pathway enrichment were performed on all DEGs and showed that RNA-directed DNA polymerase activity and fatty acid metabolism were significantly affected. Furthermore, eight specific expressed genes were selected for validation using qRT-PCR. Among these eight genes, seven genes were up-regulated, and one gene was down-regulated; the change trends of the eight genes were the same between the qRT-PCR and RNA-seq analysis results.In this study, a new method for collecting and identifying diapause insects was described. We generated a vast quantity of transcriptome data from C. septempunctata L., providing a resource for gene function research. The diapause-associated genes that we identified establish a foundation for future studies on the molecular mechanisms of diapause.

Dataset Information

Sequencing, Expression, and Functional Analyses of Four Genes Related to Fatty Acid Biosynthesis During the Diapause Process in the Female Ladybird, Coccinella septempunctata L.

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