| The silkworm(Bombyx mori)is a completely metamorphic insect.The pupa is a special stage in which the larva is transformed into an adult and is ready for reproduction.It is an important period for the development and maturity of the reproductive system.The silkworm does not feed in the pupal stage,and all the materials and energy needed for development come from the storage of the larva stage.Silkworm pupae are rich in lipids,accounting for about one-third of their dry weight,and fatty acids are their main component,accounting for about 80% of total lipids.Fatty acids have been found to affect their ability to reproduce in many organisms.Silkworm is not only an important model organism of Lepidoptera,but also an important economic insect.So far,there are few research reports on the relationship between fatty acids and reproduction.This topic explored the relationship between fatty acids in the pupal stage of the silkworm and female reproduction.The results are as follows:1.Division of the developmental stages of the ovaries of the silkworm pupaObservating the ovarian tissue of silkworm from 1st day to 9th day(the day before moth)after pupation through anatomy.It was found that the ovaries grew slowly on the first and second days after pupation,and began to rapidly grow on the3 rd day until they were full-body cavity before moth.There is no obvious accumulation of nutrients in the first 5 days.On the 6th day,the color of silkworm eggs at the far end of the ovariole(the earliest formed silkworm eggs)changes from white to yellow,the eggs got bigger,and the accumulation of nutrients is obvious.As they develop,mature eggs accumulated nutrients increase from far to near in the ovariole.Almost all silkworm eggs have accumulated nutrients before the moth.The analysis found that the ovaries were in previtellogenesis on the 3rd day of the pupae,and the ovaries were in the vitellogenesis on the 6th day by detecting the expression of the marker gene Bm EP80 of the vitellogenesis and the marker gene Bm A.L12 of the choriogenesis.2.Analysis of the changes in the content of fatty acids of silkworm pupaeGas chromatography(GC)was used to determine the relative content of fatty acids in the female pupae(without the ovaries)and ovaries on the 1st day,3rd day,6th day,and 9th day respectively.It was found that 5 major fatty acids(C16:0,C18:0,C18:1,C18:2,C18:3)and 7 trace fatty acids(C15:1,C16:1,C17:0,C17: 1.C20:0,C20:3,C14:0).Among them,C18:3 has the highest relative content,accounting for37.69%-48.64% of total fatty acids,C18:1 accounting for 19.26%-20.28%,C16:0accounting for 14.09%-15.72%,C18:0 accounting for 8.61%-14.12 %,C18:2 accounts for about 6.35%-8.50%.The content of the main fatty acids in the pupal body is stable during 1st to 6th days after pupation,but the C18:3 in the pupal was reduced from48.21% to 37.69%,and the C18:0 was increased from 8.61% To 14.19% since the 6th day to the 9th day after pupation.This may be because the energy required for life activities in the pupal stage is more inclined to be provided by C18:3,or the synthesis of C18:3 in the late pupal stage is reduced.The major fatty acids in the ovary vary greatly with the development of the pupae.Saturated fatty acids(SFA)of ovaries have a dominant position at the early stage of pupation,accounting for about 63% of the total fatty acids,but on the third day,the SFA significantly drops to 43%,while C18 :1increased from 12.94% to 20.30%,and C18:3 significantly increased from 10.76% to26.18%.On the 9th day,the relative content of C18:3 in eggs is higher than that in pupae,so it may also be caused by preferential selection of polyunsaturated fatty acids(PUFA)for lipid transport during egg maturation.The relative content of the three fatty acids that make up ginkgolic acid,C15:1,C17:0,and C17:1,increased on the 9th day of pupa,which may be related to its ability to inhibit the growth of pathogens.The content of C20:0 increases day by day during the pupal stage,and C20:3 increases significantly on the 9th day of pupa,which may be related to the synthesis of active lipids.The trace fatty acid in the ovary approaches the proportion of the pupal since the vitellogenesis,which may be caused by a large amount of lipid transport from the fat body.3.Transcriptome sequencing of silkworm ovariesTranscriptome sequencing was performed on the ovaries on the 3rd day and the6 th day,and a total of 912 differentially expressed genes(DEGs)were detected.Functional annotation and enrichment analysis of up-regulated genes found that DEGs are mainly involved in transmembrane transport,post-translational modification,cell membrane composition,lipid and carbohydrate metabolism,intracellular transport,and actin synthesis pathways.On the 6th day,highly expressed DEGs were consistent with the phenomenon that the rapid accumulation of nutrients in the ovary.Further analysis of GO enrichment of reproduction-related DEGs found that they involved CYP genes,signal coupling protein genes,enzyme genes,channel protein genes and membrane receptor genes,and so on,covering reproductive behavior,gonadal development,oogenesis,hormone regulation,and signal transmission,cell migration and many other aspects.Further analysis of the DEGs related to lipid transport and metabolism found that genes are mainly enriched in the PPAR signaling pathway,fat digestion and absorption,unsaturated fatty acid synthesis,fatty acid elongation and other pathways.The DEGs in the PPAR pathway are three SCD family genes,which may be related to the synthesis of C18:1.The genes enriched in the fatty acids elongation pathway are the HADH gene and two elongases of very-long-chain fatty acid genes,Bmelo10945(BGIBMGA010945)and Bmelo842(BGIBMGA000842).At the same time,Bmelo842 is also enriched in the unsaturated fatty acid synthesis pathway,indicating that except for the fatty acid extension function,it may also have the function of unsaturated fatty acids synthesis.Expression correlation analysis found that Bmelo842,Bmelo10945 and Bmcyp4g25 co-expressed with a variety of reproduction-related genes.It is speculated that their expression changes may affect the reproductive performance of female silkworms.4.The function of elongases of very-long-chain fatty acid synthase on the fatty acid content of silkworm pupaeThe changes in the expression levels of Bmelo842 and Bmelo10945 of the ovaries of silkworm pupae from 3rd day to 6th day were detected by RT-q PCR.It was found that the former had the highest expression on the 6th day of pupation,and the latter had a peak expression on the 5th day.The intervention was selected when the female pupae developed to the 4th day,and the injection was superimposed after 24 hours.The female pupae were dissected on the 6th and 9th day of their development,and the relative content of fatty acids in the pupae and ovaries was detected by GC.The results showed that the C18:1 content decreased significantly after interference with Bmelo10945,and the relative content of even carbon fatty acids in trace fatty acids decreased,indicating that Bmelo10945 may have two functions,the elongation of fatty acids and the synthesis of monounsaturated fatty acids(MUFA).After interfering with Bmelo842,it was found that the relative content of C18:1 and C18:3 decreased significantly,while their synthetic substrates C18:0 and C18:2 increased significantly,indicating that Bmelo842 may participate in the biosynthesis of C18:1 and C18:3.The substrate was accumulated after the gene being silenced.5.The function of elongases of very-long-chain fatty acid synthase on moths egg-layingAfter gene interference,the female pupae can develop normally,and the progress of ovarian development and egg maturation is not significantly different from that of the control group.The female moths in the experimental group and the normal male moths mated and investigated their eggs.15 moth circles are counted in each group.The control group laid an average of 354 eggs,the ds Bmelo10945 group laid an average of 254 eggs,which is a significant decrease.The ds Bmelo842 group laid an average of 336 eggs,and there is no significant change.Also,gene correlation analysis found that the hormone receptor 3(Bm HR3)involved in ecdysone signal transduction has a significant expression correlation with Bmelo842 and Bmelo10945.Therefore,ds Bm HR3 were injected and found that the expressions of two elongases of very-long-chain fatty acid synthase genes were significantly down-regulated,and the moth lays an average of 284 eggs after the intervention,and the number of eggs was significantly reduced.Similar results were obtained with the interference Bmelo10945.This indicates that Bm HR3 may act as an upstream gene to regulate the expression of Bmelo842 and Bmelo10945 during the vitellogenesis.6.The function of cytochrome P450 Bm Cyp4g25 on the development of silkwormThe expression of Bm Cyp4g25 was silenced by injection of ds RNA on the 4th day and the pre-pupa period.It was found that,compared with the control group,the female pupae developed during the pupal period had no change,and the ovarian development had no change.In the pre-pupa stage,it was observed that the silkworm stopped developing after injection of ds RNA,and remained in the pre-pupa stage after6 days.Its larval gastropod,thoracic foot and epidermis could not degenerate.The above results indicate that Bm Cyp4g25 is related to ecdysone synthesis and plays an important role in the larva-pupa transition process. |
