Abstract: 【Aim】 The thermal sensitive transient receptor potential ankyrin 1 (TRPA1) is activated when the eggs of biovoltine Bombyx mori are stimulated under 25 ℃, which may then activate an unknown signaling pathway related to the release of diapause hormone (DH). The aim of this study is to investigate the molecular mechanism of BmTRPA1 regulating DH secretion and the relationship between BmTRPA1 and GABAergic signaling pathway in B. mori. 【Methods】 CRISPR/Cas9 was used to knockout BmTRPA1 in B. mori stimulated under 25 ℃. Transcriptome sequencing was used to screen the differentially expressed genes in the brain-suboesophageal ganglion (Br-SG) complex between B. mori wild type (WT) and BmTRPA1 mutant type BmTRPA1^-/- on the day-3 of the pupal stage. GO classification and KEGG functional enrichment analysis were performed for the differentially expressed genes and specifically expressed genes. The relative expression levels of six differentialy expressed genes, 10 key genes in GABAergic signaling pathway and neuropeptide gene K5 were detected by qRT-PCR. 【Results】 A total of 13 039 genes were expressed in the Br-SG complex of WT and 12 937 genes were expressed in the Br-SG complex of BmTRPA1^-/-, among them 12 484 genes were co-expressed in the Br-SG complex of WT and BmTRPA1^-/-, 555 genes were specifically expressed in the Br-SG complex of WT and 453 genes were specifically expressed in the Br-SG complex of BmTRPA1^-/-. Compared with Br-SG complex of WT, 32 genes in the Br-SG complex of BmTRPA1^-/- were up-regulated and 13 genes in the Br-SG complex of BmTRPA1^-/- were down-regulated. These differentially expressed genes were mainly enriched in key life processes such as positive feedback regulation, metabolism, response to stimulus, and cell regulation. The differentially expressed protein GAT, which is enriched in the process of protein transport and endocrine, belongs to the plasma membrane GABA transporter and is a key protein in regulating the release of DH from B. mori. qRT-PCR result showed that the relative expression levels of GAT and K5 in the Br-SG complex of BmTRPA1^-/- were significantly down-regulated as compared with those in the Br-SG complex of WT. 【Conclusion】 This omics level study showed that the knockout of TRPA1 can reduce the expression of GAT, thereby regulating GABAergic signaling pathway and reducing DH release. The results provide target proteins and data references for further exploration of molecular regulatory mechanisms of diapause induced by B. mori in response to ambient temperature.

Key words: Bombyx mori, diapause, transient receptor potential ankyrin, transcriptome, brain-suboesophageal ganglion complex