| Sericulture is an agricultural industry closely related to forestry resources,which has always played an important role in the development of rural agriculture and in increasing income of farmer.In the production of sericulture,the quantity of cocoon is directly related to the economic benefits and development prospects of the industry.Therefore,the primary goal of sericulture production is to pursue a strain with high cocoon yield.Silk gland is the only organ for silkworm to synthesize and secrete silk protein,which is one of the most important biological basis for the development of sericulture.For thousands of years,through cross breeding,the silk gland of silkworm has become larger and larger,and the cocoon silk production is getting higher an higher.However,it is difficult to improve cocoon yield by traditional cross breeding,and molecular breeding has been paid more and more attention.The key step of silkworm molecular breeding is to find the key genes that affect silk gland size.Transcription factor Senseless is a zinc finger protein,which homologues Gfi-1,Gfi-1B and pag-3 are involved in many processes such as tumorigenesis,apoptosis,proliferation,cell fate and differentiation.In Drosophila melanogaster,the size of the salivary gland of the mutant is only half of that of the wild type,indicating that senseless plays a key role in the development of salivary gland.The silk gland of Bombyx mori and salivary gland of Drosophila melanogaster are homologous organs.However there was no relevant report about how senseless regulates the development of silk gland in Bombyx mori and the effect on silk yield.In the present study,the expression pattern of senseless in the silk gland and its effect on the silk yield of Bombyx mori were discussed,which would provide an important guiding significance for improving the cocoon yield in production.In this study,the protein structure and evolutionary relationship of Bmsenseless(Bmsens)were cloned and analyzed,respectively.Further more,q PCR,immunofluorescence staining,luciferase reporter test and gel retardation test were used to explore the spatio-temporal expression pattern of Bmsens in silk gland and its regulatory relationship with transcription factor SGF1 and SAGE.Finally,RNAi technology was used to explore the effect of Bmsens on cocoon silk yield.The main results are as follows:(1)In order to explore the protein structure characteristics of BmSens,homologous protein sequences of Homo sapiens,Rates norvegicus,Gallus Gallus,Drosophila melanogaster and Caenorhabditis remanei were downloaded from NCBI database,and the amino acid sequences of the above proteins were compared by DNAMAN to analyze the motifology characteristics.The results showed that the amino acid consistency between BmSens protein and the homologous proteins of the above species was about 30% which was at a relatively low level.However,the zinc finger domain of Senseless proteins from different sources was more conserved.The homologous sequences of 13 species were selected from the NCBI data,and the results showed that BmSens were most closely related to Manduca sexta..(2)The expression level of Bmsens in silk glands from the first to the fifth instar at mulberry-feeding and dormant stages was detected by q PCR technique.The results showed that the expression level of Bmsens increased with the development of silk glands.Compared with each instar of mulberry feeding stage,the expression level of the corresponding dormant stage was lower.The expression level increased gradually at the 1st to 3rd instar feeding stage,which reached the highest at the 3rd instar feeding stage,and then decreased gradually at the 4th instar feeding stage on the 3rd day of the 5th instar feeding stage.The expression level reached the highest at the second instar of molting and then decreased gradually.PCR was used to detect the expression of Bmsens in silkworm silk glands,head,fat body,trachea,digestive tube and martensians tube on the third day of the fifth instar,and the results showed that Bmsens were expressed in all the above tissues.(3)Two sets of primers were designed according to the full-length CDS sequence of Bmsens found in NCBI database,and two segments of Bmsens were amplified by PCR using silk gland c DNA as template,which were named Bmsens-LC and Bmsens-SC,respectively.Two recombinant expression vectors p GEX-4T-3-Bmsens were constructed and transformed into BL21 prokaryotic competent cells to express the fusion protein.The purified protein was dissolved in normal saline and immunized mice.After immunization for four times,the blood was collected by eyeball remove and obtained Bmsens polyclonal antibody.The specificity of the antibody was verified by western blot using anterior,middle and posterior silk gland proteins as antigens.The results showed that the antibody obtained from p GEX-4T-3-Bmsens-SC could bind to the antigen specifically,and the target band size was correct.(4)Immunofluorescence staining of silkworm silk glands at mulberry feeding and resting stages from the first to the fifth instars with BmSens antibody and nucleic acid dye DAPI showed that Bmsens were located in the cytoplasm of silk glands from the first to the fifth instars and weakly located in the nucleus at the 3rd day of the 5th instars.Cytoplasmic nuclear proteins were extracted from the silk glad of 3rd day of 5th instar silkworm,western blot further confirmed that Bmsens were expressed in the cytoplasmic nucleus of the 5th instar and 3rd day of silk glands.(5)The luciferase reporting assay and gel migration assay were used to explore the regulatory relationship between Bmsens and FOX-type transcription factors SGF1 and BHLH-type transcription factors SAGE.The results showed that SGF1 and SAGE had positive regulation on Bmsens.When the binding sites of transcription factors SGF1 and SAGE on the Bmsens promoter were mutated,the regulatory effect disappeared.Gel migration experiments further demonstrated the regulation between Bmsens and SGF1 and SAGE.(6)The expression of Bmsens in the 3rd day of 4th instar of feeding period was interfered by RNAi.The results of q PCR showed that the expression of Bmsens was significantly decreased after the intervention,the silkworm body size was significantly reduced as well as the body weight.At the same time,the whole silk gland became smaller,especially the middle silk gland and the posterior silk gland became thinner and shorter compared with the control group.In addition,the cocoon became smaller and the cocoon layer became thinner,and the dry cocoon weight also decreased significantly.The results showed that Bmsens played an important role in silk gland development and cocoon yield. |
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