| With further aviation technology development,exploitation and utilization of the space frontier has been paid more and more attention.The lepidopteran insect,Bombyx mori,presents a lot of advantages when compared with the other animals models,which including small in body size,light weight,short life cycle,easy material availability and clear genetic background.Based on the comprehensive consideration of resource constraints of return satellite platforms,the influence of space environment on the embryonic development and further explore the mechanism of variation in silkworm was studied in here.In this study,we send the silkworm embryos into space by the China's SJ-10 recoverable satellite.The silkworms larval were analysis by high-throughput sequencing that hatched after returning to the ground surface,and also to detect larvae phenotypically.The conservative miRNA-14 and BmTCTP gene were selected,and using the CRISPR/Cas9 transgenic system to explore the molecular mechanism.The main research and the results in this thesis and are as the follows:(1)Ground simulation before launched.The simulation was performed under simulated space conditions(including confined environment and vibration).In order to ensuring the embryo could hatched,the optimum conditions(temperature,humidity,barometric pressure,oxygen supply,etc.)were explored.Given that the orbital cycle is 12 days for satellites,1-3 days for sample collection and ground preparation,the temperature control to 21-22 degrees on orbit because the silkworm eggs hatch for 10 days at 24-25 degrees.(2)Studied on the embryonic development of silkworm after flight.In accordance with the launching site requirements,the silkworm incubator has been validation the loading process,exercise of co-transport,matching test and launch site whole-site electrical test in the launching site.The silkworm incubator completed the sample loading on April 4,2016 and completed the silkworm sample loading and tower transfer delivery on April 5.Dur ing the flight phase,silkworm incubators completed in-orbit experiments for 12 days and 15 hours according to in-orbit experimental procedures,and achieved space-enclosed silkworm culture and cryogenic fixation of silkworm in 5 culture units at low temperature.As the result,the silkworm embryos process image was obtained the first time.The silkworm embryos cultured in space environment can hatch properly after returning to the ground,and the larval development time is shortened by 3 days,without any other developmental abnormalities.(3)Whole genome gene expression profile.After the silkworm embryos being returned to the ground,to exploit the molecular effects of space environment on embryo,the RNA extract from hatching larval for comparative transcriptional analysis by RNA-Seq analysis including the embryos cultured in space environment,the embryos cultured in the ground control group at 21? and the embryos hatched at 24? in the ground control group.The results showed that the differential expressed genes were mainly enriched the metabolic pathway,trypsin secretion,protein digestion and absorption pathways which space group compared with the control group at 21?.There were significant different genes are mainly assembled in trypsin secretion,protein digestion and absorption,galactose metabolic pathway between the space flight group and the ground control group 24 ?.In order to avoid temperature changes result in different genes expressed,we screened from these two differential expressed genes which only different in the space flight group at 21? in and the ground control group genes that were significantly different,and were mainly enr iched in the metabolic pathway.Besides,there are many cuticular protein were significant changed in this pool differential expressed genes.These results showed that the space environment can cause silkworm embryonic metabolism and cuticular protein changed that allowing them to better adapt to changing conditions in space environment.(4)Explored the genes function.The miRNA-14 functions.MicroRNAs(miRNA)regulate multiple physiological processes including development and metamorphosis in insects.miRNA-14(miR-14)is a non-coding RNA which conserved in invertebrate.Ubiquito us transgenic overexpression of miR-14 using the GAL4/UAS system resulted in delayed silkworm larval development and smaller body size of larva and pupa with decrease in ecdysteriod titers.On the contrary,miR-14 disruption using the transgenic CRISPR/Cas9 system led to a precocious wandering stage with increase in ecdysteriod titers which similar with the phenotype in space environment cultured.We identified that the E75 and EcR-B,which both serve as essential mediators in the ecdysone signaling pathway,as putative target genes of miR-14 by in silico target prediction.Dual-luciferase reporter assays confirmed the binding of miR-14 to the 3'UTRs of E75 and EcR-B in a mammalian HEK293 T cell line.Furthermore,transcription levels of E75 and EcR-B were significantly affected in both miR-14 overexpression and knockout transgenic animals.Taken together,our data suggested that the canonical invertebrate miR-14 is a general regulator in maintaining ecdysone homeostasis for normal development and metamorphosis in B.mori.We also speculate that the spatial environment may affect the silkworm ecdysone signing pathway.The TCTP functions The translationally controlled tumor protein(TCTP)is a highly conserved and multifunctional protein with activities ranging from cytoskeletal regulation to transcription regulation in numerous organisms.In insects,TCTP is essential for cell growth and proliferation.There are also many reported that TCTP related to radiation.Recently,TCTP has been reported to affect the innate intestinal immune pathway in the Bombyx mori silkworm,a lepidopteran model insect.However,the comprehensive physiological roles of TCTP in the silkworm remain poorly understood.Here,performed functional analysis of BmTCTP by using a binary transgenic CRISPR/Cas9(clustered regularly interspaced short palindromin repeats/RNA-guided Cas9 nucleases)system.Disruption of BmTCTP led to developmental arrestment and subsequent lethality in third instar larvae.Histological analysis revealed that growth impairment originated from decreased cell size,and the proliferation and differentiation of intestinal epithelial cells were also affected.RNA-seq analysis revealed that genes involved in carbohydrate metabolism,lipid metabolism and digestive system pathways were significantly affected by BmTCTP depletion.Besides,the relative expression of BmTCTP was significantly down-regulated in space environment cultured silkworms.Together,the results demonstrated that BmTCTP plays a key role in controlling larval growth and development.To date,this is the first report which using the satellites to achieve the embryo culture in the space environment,it is also the first time to successful acquired the silkworm embro images.We using the high-throughput technology for genes analysis.Studying the important genes in the space environment on the development of silkworm embryos will become an integral part of space exploration and also contribute to the development of space insect biology. |
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