Cloning, Expression And Functional Analysis Of The BmYki, A Key Member Of The Silkworm Hippo Pathway

The Hippo pathway is an important signaling pathway that found in recent years. It controls organ size through the regulation of cell growth, proliferation and apoptosis process.This pathway is very conservative in Drosophila and mammals. Transcriptional coactivator Yorkie(Yki) is a core member of the Hippo pathway, which regulates the function of the downstream target genes so as to control the size of organs. The silkworm is known as Lepidoptera insects and economic model insects. Studying the key genes and signaling pathways of regulating organ size in silkworm has important theoretical and practical significance for revealling the biological traits of Lepidoptera and the use of performance targets for the transformation of silkworm gene.In this study, the transcriptional coactivator Bm Yki downstream of the silkworm Hippo pathway was cloned and its gene structure, subcellular localization, temporal and spatial expression patterns and biological function was analyzed. The main results are as follows:1. Cloning and sequence analysis of the BmYki geneThe BmYki gene was identified from the silkworm and its CDS sequence was cloned. Three spliced forms(BmYki-S1, BmYki-S2 and BmYki-S3) and four spliced forms(BmYki-S1, BmYki-S2, BmYki-S3 and BmYki-S4) were cloned from the Bivoltine DZ strains and multivoltine yellow blood strains, respectively. This is the first time to report that the transcriptional coactivator Yki has several alternative splice forms.2. Subcellular localization of the BmYki geneTwo spliced forms of BmYki, BmYki-S1 and BmYki-S2, were fused with EGFP to construct the expression vectors used for subcellular localization. The constructs were transfected into BmNs cells followed by DAPI staining and fluorescence observation. The results showed that BmYki-S1 and BmYki-S2 were localized in the cytoplasm of BmNs, which is similar to the results of the Drosophila and mammalian Yki gene.3. Transcriptional expression analysis of the BmYki gene in tissues and periodsThe transcriptional expression of the BmYki gene in major tissues of day-3 fifth-instar larvae and developmental periods from day-1 first-instar larvae to day-1 moth was analyzed using qRT-PCR technology. The results showed that expression level of the BmYki gene in tissues and developmental periods is low. Comparatively, the expression level of the BmYki is most high in the posterior and middle silk glands, and a little high in the testis, ovary, trachea and head, but very low in the fat body, blood, malpighian tube and anterior silk glands. The BmYki gene has a expression peak in day-3 fifth-instar larvae and day-1 moth, but the former is the highest, and the expression level in other developmental periods is low. The BmYki gene expresses highly in the silk glands of the day-3 fifth-instar larvae, suggesting that it may be involved in the regulation of the silk glands or silk protein synthesis.4. Overexpression analysis of the BmYki gene in cultured cellsBased on the Gal4/UAS binary expression system, the BmYki gene was overexpressed in the silkworm embryonic cell line BmE, then expression of eleven downstream target genes of the Hippo pathway and eleven target genes associated with silk protein synthesis was analyzed using qRT-PCR technology. The results showed that the expression level of downstream target genes Myc, Ras, Diap1, Caspase1 and Kibra was significantly upregulated, Diap2 was increased slightly, while other target genes had no significant change. Besides, the expression level of silk protein synthesis-related genes FibH, P25, Ser1, Ser2, Ser3, SGF1, SGF2 and SGF3 was significantly downregulated, Sage was increased slightly, and other genes had no significant change. These results indicate that the BmYki(Hippo pathway) has the function of regulating biological processes such as cell proliferation and apoptosis, and might participates in regulating the expression of genes associated with silk protein synthesis.5. Knockout analysis of the BmYki gene in cultured cells By using the CRISPR/Cas9 system, the knockout of BmYki gene was performed in the silkworm embryonic cell line BmE, then expression of eleven downstream target genes of the Hippo pathway and eleven target genes associated with silk protein synthesis was analyzed using qRT-PCR technology. The results showed that the expression level of downstream target genes Myc, Ras, Diap1, Caspase9, Caspase1, Kibra and Wg was downregulated, which is opposite with the trends of BmYki overexpression. While the silk protein synthesis-related genes FibH, P25, Ser1, Ser2, Ser3, SGF1, SGF2 and SGF3 were also downregulated, which is similar with the trends of BmYki overexpression.