Function And Regulatory Mechanism Of The BmYki/BmSd In The Development Of Posterior Silk Glands In Bombyx Mori

How the organs of organisms are controlled during growth and development is one of the important frontier issues in the field of life sciences,which is of great significance to contribute to revealing the pathogenesis of major human diseases such as body developmental defects and malignant tumors.For economic animals and plants,organ development is closely related to biological traits such as yield and quality,analyzing its regulatory mechanism can provide theoretical guidance for the improvement of animal and plant varieties.The silkworm,Bombyx mori,known for its high efficiency in silk production,is the only silk-secreting animal that has been fully domesticated by humans and has formed a huge breeding industry（silk industry）.The silk gland is the exclusive place for silkworm to synthesize silk protein,which according to its shape and function,it can be divided into anterior silk gland（ASG）,middle silk gland（MSG）and posterior silk gland（PSG）,in which ASG is responsible for liquid silk fibrosis,MSG synthesizes and secretes silk sericin protein,and PSG synthesizes and secretes silk fibroin protein.Silk fibroin and sericin are the main components of silk,accounting for about 75%and 25%respectively.In view of the important industrial value of silk glands,the academic community has conducted long-term research on them,and has made abundant progress in the molecular regulation mechanisms of silk gland organogenesis,development,degeneration,and silk protein synthesis.However,the molecular mechanism of silk glands and their efficient synthesis of silk protein is too complicated,and existing researches have only revealed the tip of the iceberg.As a major basic scientific issue related to silk production and quality in the field of sericulture,research in this area still needs to be strengthened.This dissertation mainly focuses on the function and mechanism of silk gland development regulators.In previous studies,we identified an important signaling pathway Hippo and its members that regulate the development of organ size in silkworms.Interestingly,it was found that the only transcriptional co-activator downstream of Hippo pathway（named BmYki,through which the Hippo cascade pathway mainly outputs regulatory signals）was found to be highly expressed in the silkworm PSG,suggesting that it may be an important factor in regulating PSG development,but further research is needed to confirm.Therefore,this paper has carried out in-depth research around BmYki,and obtained the following main results.1.Identification and characterization of BmYki and its spliced isoformsTo analyze the function and role of BmYki in PSG,we firstly cloned BmYki gene and analyzed its basic characteristics using the silk gland as the material.The results showed that:1)There were at least four spliced isoforms of BmYki,named BmY1329,BmY1314,BmY1188 and BmY1173 according to the length of their coding sequence;2)All four spliced isoforms contained WW-type conserved domains,including two WW domains for BmY1329 and BmY1314,and one WW domain for BmY1188 and BmY1173;3)BmY1329,BmY1314,and BmY1173 were distributed in the nucleus and cytoplasm,while BmY1188 was mainly located in the cytoplasm and nuclear membrane,suggesting that it may play different functions from the other three isoforms;4)All four spliced isoforms were expressed in the main tissues of silkworm,and the expression level in PSG was relatively highest,suggesting that BmYki might play a regulatory role in PSG development.2.BmYki is a key factor regulating the development of the posterior silk gland inBombyx moriTo explore the role of BmYki in regulating PSG development,we carried out functional validation at the cellular and individual levels.First,overexpression and RNAi experiments were performed based on silkworm BmE and BmN cells.The results showed that overexpression or knockdown of the four spliced isoforms of BmYki not only significantly affected the expression of known target genes of the Hippo pathway,but also significantly affected the expression of silk fibroin genes（fib H,fib L,P25）.Subsequently,we took BmY1314 as a representative（BmYki mentioned below refers to BmY1314）,and made transgenic lines that specifically overexpressed and knocked out BmYki in PSG,named Yki^OE and Yki^KO,respectively.Investigating the silk gland phenotypes at different developmental stages found that:on the sixth day of fifth instar（L5D6）,the PSG organs of the Yki^OE strain were severely degenerated,significantly shortened in length,and significantly reduced in weight;at the cellular level,PSG cells were of different sizes,with uneven surface,wider cell layer,disordered cytoskeleton arrangement and increased intracellular DNA density.The PSG mutant phenotype of the Yki^KO line was in the opposite trend to that of the Yki^OE.Surprisingly,the mutant phenotype of PSG of Yki^OE line at the first day of first instar（L1D1）was opposite to that of L5D6,which showed significantly enlarged PSG,larger nuclei and increased intracellular DNA content,but no significant change in cell number,suggesting that the PSG increase in L1D1 may be caused by increased intranuclear replication.Further investigation found that the PSG nuclei of the Yki^KO strain showed rod-like nuclei on the first day of fourth instar（L4D1）,while the Yki^OE strain began to show increased branching of dendritic nuclei at L4D1.In addition,Cyc E,CDK2,E2F1,Myc,Cyc A,Fzr,MCM3,MCM6,MCM8,MCM9,MCM10 and other genes related to endoreplication were significantly increased,indicating that overexpression of BmYki can promote the endoreplication of PSG cells.In conclusion,overexpression or knockout of BmYki can induce severe PSG developmental mutations,indicating that BmYki is a key factor regulating PSG development in silkworm.3.BmSd is an important co-factor of BmYki regulating the development ofposterior silk gland in Bombyx moriRelevant studies in Drosophila and mammals have shown that Yki/YAP cannot directly bind DNA and can only perform regulatory functions with the assistance of co-factors（binding target gene DNA）,among which Sd/TEAD is the main co-factor of Yki/YAP.Through sequence analysis,we found that the N-terminal region of BmYki has a conserved domain that binds to Sd,suggesting that Sd may be a co-factor of BmYki.Subsequently,we cloned BmSd from silk gland in Bombyx mori and verified the interaction by Co-IP method.The results showed that BmSd binds to BmYki in a protein-protein form.Meanwhile,BmSd can also bind to Drosophila Yki or mammalian YAP,and BmYki can bind to Drosophila Sd or mammalian TEAD.Further,we analyzed the synergistic effect of BmSd and BmYki at the individual level.First,transgenic lines Sd^OE and Sd^KO that overexpressed and knocked out BmSd in PSG were made,and the PSG phenotype of L5D6 was investigated:It was found that compared with wild-type silk gland,the PSG of the Sd^OE line was almost completely degenerated,and only a few organ traces were seen;the PSG of Sd^KO mutant was significantly shorter,and the anterior region was enlarged and the posterior region was narrowed.This shows that BmSd is essential for the normal growth and development of PSG.Subsequently,we made the mutant Yki^OE/Sd^OE co-expressing BmYki and BmSd,and the mutant Yki^OE/Sd^KOoverexpressing BmYki and knocking out BmSd,to investigate the PSG phenotype in the early larval stage（L1D1）and found that the PSG of Yki^OE/Sd^OE was abnormally increased,and the increase was more significant than that of Yki^OE,and the expression of endoreplication-related genes in PSG was also significantly elevated.However,in the Yki^OE/Sd^KO mutant,the PSG phenotype originally induced by BmYki was suppressed by the knockout of BmSd and returned to the same size as the wild-type PSG.Taken together,these results indicate that BmSd is indeed a co-factor of BmYki in PSG,and is indispensable for BmYki to regulate the normal development of PSG.4.BmYki/BmSd regulate PSG degeneration in Bombyx moriAs mentioned above,the PSG of the Yki^OE/Sd^OE mutant showed excessive enlargement in the early larval stage（L1D1）,but continuous investigation found that the growth and development of the PSG began to slow down at L1D3,and the molting of first instar（L1M）showed a clear trend of degradation,to L5D6 almost completely disappeared.Studies have shown that ecdysone（20E）signaling regulates the degradation and degeneration of silk glands during metamorphosis（larvae-pupa）by inducing the expression of autophagy and other related genes.Therefore,is the premature degradation of PSG in the above Yki^OE/Sd^OE mutant due to the abnormal expression of 20E signaling-related factors induced by BmYki/BmSd?To this end,we detected the PSG of mutants in two key stages,L1D3 and L1M,and the results showed that 20E response factors Hr3,Hr4,Ftz-f1,E93 and E74A,as well as autophagy genes ATG3,ATG5,ATG6,ATG7 and ATG12 were markedly increased,indicating that BmYki/BmSd indeed induced the abnormal expression of 20E signal-related factors.Furthermore,we predicted a highly conserved DNA sequence（SBS for short）with the Drosophila Sd-binding motif in the promoter of Ftz-f1.Ch IP and EMSA validation results confirmed that BmYki/BmSd can bind to the SBS motif.In addition,we found that BmYki/BmSd can also interact with Hr3 in a protein-protein form.Taken together,the results indicated that BmYki/BmSd closely interacted with 20E signaling,and played an important role in regulating the degradation inhibition of PSG in larval stage.5.BmYki/BmSd regulate silk fibroin synthesisThe change of silk protein synthesis is an important reflection of whether the silk gland develops normally or not.From the above analysis,we confirmed that the PSG of Yki^OE,Sd^OE or Yki^OE/Sd^OE mutant was severely atrophied and degenerated.Further investigation of its cocooning characteristics found that the cocoon layer of Yki^OE,Sd^OE,and Yki^OE/Sd^OE was significantly thinner（showing a sericin cocoon phenotype）,while the cocoon layers of Yki^KO showed thick cocoon phenotype,which can be concluded that the dysregulated expression of BmYki/BmSd seriously affects the synthesis of silk fibroin.How does BmYki/BmSd regulate silk fibroin synthesis?We investigated this and found that:1)Potential SBS motifs are found in the promoters of the silk fibroin genes,and the double luciferase and EMSA experiments confirmed that BmYki/BmSd could bind to the SBS site in the fib H and P25 promoter regions and regulate their transcriptional activity.2)Dimm and FMBP-1 have been reported as two important transcription factors that directly regulate the expression of silk fibroin gene.In this study,it was found that the phenotype of the mutant PSG overexpressing Dimm was similar to that of Yki^KO,and the phenotype of the mutant PSG overexpressing FMBP-1 was consistent with that of Sd^OE.Interaction verification results showed that BmYki/BmSd can bind to the SBS sites in the promoter regions of Dimm and FMBP-1 to regulate their transcriptional expression.These results suggest that BmYki/BmSd can regulate silk fibroin synthesis in both direct and indirect manners.In conclusion,this thesis comprehensively used transgenic,gene knockout and other experimental methods to research the function and mechanism of silk gland regulatory factors BmYki and BmSd,confirming that BmYki/BmSd promoted the excessive growth of posterior silk gland through cell cycle and endoreplication related genes in early larval stage.It also significantly affected the expression of autophagy related genes and 20E response factors,and led to autophagy and apoptosis of posterior silk gland cells,thereby inducing abnormal degeneration of posterior silk gland.Furthermore,it was revealed that BmYki/BmSd can regulate silk fibroin synthesis in both direct and indirect ways.The results of this paper provide new insights for the research on silk gland in Bombyx mori,and the target genes of silk gland and mutant materials of silk gland have been identified,which can also contribute to the genetic modification of silk and the exploitation and utilization of silk protein.