| Sex is the basic characteristic of sexual reproduction organisms in nature,and there are huge differences between male and female individuals in physiology,morphology and behavior.Silkworm is a Lepidopteran insect,which is an important economical insect because it can secrete silk.There are huge differences between male and female individuals of the silkworm.Female individuals need to use a lot of energy to produce offspring,which leads to higher silk production efficiency and higher economic benefits for male individuals.On the other hand,the silkworm,as a Lepidopteran model insect,research on its sex determination can also provide us with new ideas for the prevention and control of Lepidopteran pests.BmSxl is a homologous gene of Drosophila BmSxl.Two alternative splicing forms have been reported in silkworm,BmSxl-L and BmSxl-S.Subsequent studies reported that BmSxl has a regulatory effect on the development of male sperm dimorphism.At the same time,our group previously had cloned two other alternative splicing forms of BmSxl,BmSxl-PA and BmSxl-PB.Among them,BmSxl-PB is a spliceosome form of testisspecific expression.In our existing research,it has been found that BmSxl is an RNAbinding protein that can bind to its own poly(U)sequence to achieve self-splicing regulation.And it was found that the BmSXL protein can bind to the poly(U)sequence in the 3'UTR region of nanos m RNA to regulate the expression of nanos.In this paper,we use RNAi to down-regulate the expression level of BmSxl and transgene up-regulate the expression level of BmSxl to further study the functions of BmSxl in the development of silkworm germ cells.At the same time,the effect and regulation of BmSxl were analyzed by EMSA,immunofluorescence,fluorescent quantitative PCR and cell flow cytometry.And obtained the following research results:1.Expression analysis of BmSxlFirst,we analyzed the expression pattern of BmSxl in various tissues and periods of silkworm by fluorescence quantitative PCR,RT-PCR and Western Blot.The results showed that BmSxl was highly expressed in the gonads of the silkworm,and was the highest in the male testis.And the spliced form of BmSxl-PB is expressed in the testis but not in the ovary.In silkworm gonads,the expression of BmSxl increased from the L5D7,and the increase in the testis was much greater than that in the ovary.Then we separated the outer membrane of the testis from the contents to analyze the expression of BmSxl.The results showed that BmSxl-PB was mainly expressed in the testis contents,while the BmSxl-PA was mainly expressed in the outer membrane.Therefore,we speculate that BmSxl may play a role in the development of silkworm germ cells.2.RNAi of BmSxl and phenotype of silkworm genital phenotype after interferenceIn order to further study the function of BmSxl in the development of silkworm germ cells,we used the common region of BmSxl-PA and BmSxl-PB to design interference double-stranded RNA primers,and then synthesized BmSxl double-stranded RNA for interference experiments.The results showed that in females with down-regulated expression level of BmSxl.No immature eggs are seen at the end of the fallopian tubes,and egg development is not continuous.The male external genitalia showed swelling of the excretory orifice.These results imply that BmSxl plays different physiological functions in males and females,and the decrease of BmSxl expression level will affect the development of eggs and the normal development of male external genitalia.3.Establishment and genital phenotype of BmSxl transgenic silkworm strainNext,we used transgenic methods to increase the expression of BmSxl on individuals.First,the silkworm c DNA template was used to clone the testis-specific BmSxl-PB form.And then connect it to the transgenic basic vector to obtain the piggy Bac[3×P3-Red-SV40,A4-BmSxl-PB-SV40] recombinant vector.Finally,the transgenic vector was microinjected into silkworm eggs.After screening and obtaining positive individuals,identification of transgene insertion sites and detection of target gene expression were performed to further confirm that BmSxl transgenic silkworms were obtained.Genital phenotypes of transgenic silkworm strains showed that the attracting glands of the external genitalia of females with increased expression of BmSxl-PB became irregular.No immature eggs are seen at the end of the fallopian tubes,and egg development is not continuous.Immunohistochemistry frozen section results showed that the outer membrane of the testis of the internal genitalia of male individuals became thicker.These results indicated that BmSxl-PB plays an important role in the development of the external reproductive organs and oogenesis of the silkworm.Based on the results of overexpression of transgenes and RNA interference experiments,BmSxl plays an important role in the development of silkworm reproductive organs and the regulation of germ cells.4.Regulation of BmSxl on cell cyclePreviously,it was reported in the literature that the germ cell development process of the silkworm is regulated by the cell cycle.Therefore,we analyzed the changes in the expression levels of cyclin A,cyclin D and CDK after the expression level of BmSxl was changed.The results showed that when the expression level of BmSxl changes,the expression level of cell cycle-related genes will also change,and the changing trends are inconsistent between male and female individuals.These results suggest that BmSxl can regulate the expression of cell cycle-related genes,and it plays different regulatory roles in males and females.Further,we up-regulated the expression level of BmSxl and down-regulated the expression level of BmSxl in BmNs cells,then collected the cells for PI staining and performed cell cycle flow cytometry analysis.The results of flow cytometry showed that after the expression level of BmSxl changed,the proportion of cells in different periods changed,that is,the cell division cycle changed.The results indicated that BmSxl has a regulatory effect on the division cycle of silkworm cells.We performed an immunofluorescence staining experiment to observe cell division.The Experimental results showed that the nucleus of the cells with up-regulated expression level of BmSxl-PA divided unevenly in the late stage of mitosis;the chromosomes of the cells with down-regulated expression level of BmSxl could not be arranged neatly on the equatorial plate in the nucleus.These results show that BmSxl affects the chromosomal behavior during cell division and the division of cell nuclei.4.Regulation of BmSxl on meiosis.5.The effect of BmSxl on meiosis-related genesThe previous literature reported that the Aly gene plays a regulatory role in meiosis.We tested the expression of Aly gene when the expression of BmSxl changed.The results showed that after the expression level of BmSxl changed,the expression level of Aly also changed correspondingly,and it changed in the same direction.The results indicated that BmSxl can positively regulate the expression of the meiotic gene Aly.6.Effect of BmSxl on Bmdsx,a key gene downstream of sex determinationBmdsx is a key gene downstream of the silkworm sex determination pathway.Our analysis found that there is a poly(U)sequence on the third intron of Bmdsx.We speculated that the BmSXL protein can bind to this poly(U)sequence,so we synthesized probes of this sequence for Gel Retardation(EMSA)binding experiments.The results showed that BmSXL protein can specifically bind to this poly(U)sequence.Next,we analyzed the changes of the male and female splicing forms of Bmdsx after the expression level of BmSxl changed.The results showed that after the expression level of BmSxl changed,the male and female splicing form of Bmdsx also changed,but we did not directly detect the change in the alternative splicing of Bmdsx.These results indicated that BmSxl has a certain influence on the alternative splicing of Bmdsx,but its influence is not indispensable.In summary,BmSxl plays an important role in the regulation of silkworm germ cells,and it may play a role in germ cells by affecting cell cycle,meiosis,and/or Bmdsx gene expression.Our results provide new data for in-depth understanding of silkworm sex determination and differentiation. |
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