Genome Identification And Analysis Of The Members Of The Silkworm Hippo Pathway

The silkworm Bombyx mori which belongs to Lepidoptera Bombycidae, is so far the unique economic insect that has been fully domesticated and used for mass rearing. The silkworm has lots of advantages such as short life cycle, clear genetic background, highly efficient biosynthesis and secretion of silk proteins, low-cost feeding and large-scale breeding etc., and has become one of the most important model insects after Drosophila. Since the determination of the whole genome sequence of the silkworm, it’s very necessary to identify at the genomic level and analyze functional genes associated with important economic traits and biological traits, which has great significance for promoting the value of the silkworm as a model of Lepidoptera, as well as the genetically modification of silkworm varieties and creation of novel materials.Organ size is an important biological characteristics of multicellular organisms. For some economic animals, organ size also determines their yield or quality traits. The Hippo pathway is a major signaling pathway that controls the organ size of animals. Since the first discovery of this pathway in Drosophila in 1995, there has been great progress in the study of the Hippo pathway, and it also shows complex and diverse regulatory mechanisms of this pathway.Based on the whole genome data and microarray expression data of the silkworm, this study aims to identify at the genomic level and analyze the members of the silkworm Hippo pathway, clone the core members BmHpo and analyze its expression profiles, the main results are as follows:1. Identification and analysis of the core members of the silkworm Hippo pathwayBy using the method of sequence homology comparison, we identified four coremembers of the silkworm Hippo pathway, BmHpo, BmMats, BmSav and BmWts. BmHpo and BmWts contains a serine/threonine protein kinase catalytic domain. BmSav containing four tandem WW domains. BmMats contains a Mob1 phocein domain. All the domains are similar to the core members of the Drosophila Hippo pathway, indicating that their function or modes of action might be similar. Microarray expression analysis showed that the BmHpo was highly expressed in ovary and martensite, BmMats was highly expressed in fat body, and BmWts and BmSav had similar expression pattern in the metamorphosis stage of male and female.2. Identification and analysis of the upstream members of the silkworm Hippo pathwayBy using the method of sequence homology comparison, we identified 49 upstream members of the silkworm Hippo pathway. Among the 47 members that has tissue expression information, BmMop, BmJub, BmCrb, BmCi, BmSmurf, BmLft and BmMer were expressed specifically in tissues; BmEx, BmZyx, Bm Fex L7, BmDpp, BmDlg, BmRassf, BmSTRIPAK,BmPP1, BmMop, Bmβ-cat, BmSlmb, Bm DREF, Bm CK2, Bm14-3-3 and BmCsk were highly expressed in ovary or testis, suggesting that these members might participate in the regulation of the spermatogenesis and oogenesis. Besides, these members were also highly expressed in the silk glands, which suggests that they may regulate the growth and development of the silk glands.3. Identification and analysis of the downstream members of the silkworm Hippo pathwayBy using the method of sequence homology comparison, we identified 10 downstream members of the silkworm Hippo pathway, including the transcription co-activator BmYki. The BmYki showed the relative high expression level in ovarian, anterior and posterior silk glands; BmCbt, BmBrm and BmWbp2 were highly expressed in silk glands, ovary and testis.BmMad was highly expressed in testes and fat body; BmMor was highly expressed in integument and fat body; BmTsh was highly expressed in anterior and posterior silk glands;BmHth was highly expressed in males and the posterior silk glands of females; BmSdBP was highly expressed anterior and posterior silk glands of females; BmSd was highly expressed in the fat body of males.BmYki contains two WW domains and highly conserved tryptophan residues, which can specifically recognizes and binds to the proline-rich motifs and phosphorylated ammonia acid/tryptophan-proline peptides. The proteins of another nine downstream members are also similar with the corresponding downstream members of the Drosophila Hippo pathway. Wespeculate that the downstream members of the silkworm Hippo pathway have similar functions or modes of action with the Drosophila. In other words, as the most crucial downstream members, the BmYki plays key roles in regulating the expression of downstream target genes, and the BmSd, BmTsh, BmHth, BmMad, BmCbt, BmBrm, BmMor, BmWbp2 and BmSdBP combinate with BmYki and promote it enters the nucleus to regulate downstream target genes.4. Cloning and expression analysis of the BmHpo, a core member of the silkworm Hippo pathwayThe core member of the silkworm Hippo pathway, Bm Hpo, was amplified and cloned from silkworm using RT-PCR technology. The length of CDS sequence of BmHpo is 1479 bp,which encodes 492 amino acids; BmHpo contains a domain of Ster I-20 family S commonly exists in Hpo family. Subcellular localization showed that BmHpo was localized in the cytoplasm, which was consistent with the localization of Hpo gene in Drosophila and mammals. Transcriptional expression analysis revealed that the BmHpo was expressed in a low level in different tissues, but showed a relative high level in testis and head. BmHpo was also expressed in a low level in different developmental stages, which is in agreement with the fact that the Hippo pathway is a cell growth inhibitory signal pathway.5. Construction and transcriptional expression of three CRISPR/Cas systemsAccording to corresponding sequences of three CRISPR/Cas system downloaded from Addgene plasmid database, we synthesized sequences of Sp Cas9, SaCas9 and AsCpf1 fused with nuclear signals and corresponding gRNA expression vector sequences after codon optimization. Then, we constructed three Cas expression vectors termed with pUC57[A4-SpCas9-Ser1pA], pUC57[A4-SaCas9-Ser1pA] and pUC57[A4-AsCpf1-Ser1pA]which was controlled by the promoter of silkworm actin4(A4). Cell transient expression detection assay showed that SpCas9, SaCas9 and AsCpf1 wrer highly expressed in BmE cells,indicating that all of them had the potential to be used for genome editing in silkworm. These results laid a preliminary foundation for the implementation of directional knockout of the silkworm Hippo pathway members at the individual level.