The Function And Molecular Mechanism Of Oser1 In Regulating Lifespan

Health and longevity has always been the long-cherished wish of human beings since ancient times.Researchers have been trying to figure out a important scientific problem:How to delay aging and extend healthy lifespan?It is very necessary to explore the ways and methods to delay aging and extend healthy lifespan,especially with increasingly serious population aging at present.Lifespan is affected by many molecular signal pathways,among which insulin/insulin-like growth factor-1 signal（IIS）pathway is one of the most important lifespan regulation pathways.In different species,its function and molecular regulation mechanism are relatively conservative.Fox O is an important downstream transcription factor of IIS pathway.Studies have shown that Fox O can play a key role in biological processes such as growth,development and metabolism by regulating the transcription of target genes.In previous studies,our team found that Bm Fox O gene could regulate the longevity of silkworm and the target genes of Bm Fox O were screened and identified.It has been proved that there is a specific binding between Bm Fox O protein and transcriptional regulatory region of Bmoser1,and overexpression of Bmoser1prolongs the lifespan of silkworm.So we speculated that Bmoser1 may be involved in the process of Fox O regulating longevity.Therefore,this study mainly used CRISPR/Cas9 system to construct knockout strain of Bmoser1 gene to demonstrate its function of the protection on lifespan.Meanwhile,in order to further understand the conserved mechanism of oser1 regulatting lifespan,RNA-Seq is used to screen genes that responding to oser1,and Co-IP/MS is used to identify proteins that may interact with OSER1.The main results are as follows:（1）Establishment and statistical analysis of Bmoser1 gene knockout strainAccording to the principle of CRISPR/Cas9 system,gene editing was carried out in two ways,injection of knockout plasmid and mixed samples of g RNA and Cas9protein,to mutant fuction of Bmoser1.A Bmoser1 homozygous knockout strain with a total deletion of 71 bp in open reading frame from the 114th base to the 185th base was screened,and the deletion of 71 bp led to the early termination on translation of Bm OSER1 protein.The statistical results showed that the lifespan of female and male of the knockout strain was significantly shortened by 8.17%and 5.68%compared with the control group.The survival time of Bmoser1 knockout strain was significantly shortened by 28.13%under high temperature environment of 37°C.The knockout strain showed better tolerance under the starvation condition.After feeding H₂O₂,the maximum survival time of female individuals in Bmoser1 knockout group was significantly shortened by 13.32%.By detecting the expression level of antioxidant enzyme genes,it was found that the relative expression levels of Bmcat,Bmsod and Bmgpx genes in Bmoser1 knockout strain were significantly down-regulated compare with controls.The loss of function of Bmoser1 gene reduced the resistance of silkworm to heat stress and oxidative stress.（2）Screening and identification of the genes responding to changes in expression level of Bmoser1Transcriptomic sequencing was performed on Bmoser1 overexpressed individuals to screen genes responding to Bmoser1 expression.Compared with the control group,the Bmoser1 overexpression group showed 1,628 differentially expressed genes,of which1,313 genes were up-regulated and 315 genes were down-regulated.Combined with the log₂（FC）,P-value and FDR data of RNA-seq,Bmhsf-d,Bmgrxc4 and Bmwashc3were preliminarily identified as candidate genes for follow-up research.The expression of Bmhsf-d,Bmgrxc4 and Bmwashc3 was up-regulated with the overexpression of Bmoser1,which was consistent with the result of transcriptomic sequencing.After down-regulating the expression of Bmoser1 in silkworm cells,it was found that the expression levels of Bmgrxc4 and Bmwashc3 genes were significantly reduced.glrx2and washc3 can also respond to the upregulation of OSER1 expression in human cells.The expression of Bmgrxc4 and Bmwashc3 was significantly increased in the oxidative damage model of Bm N-SWU1 cells,which was consistent with the expression of Bmoser1,suggesting that Bmgrxc4 and Bmwashc3 may be related to the response of Bm OSER1 to H₂O₂.A Bmgrxc4 homozygous knockout strain with deletion of 1,842 bp in the genome was obtained,and theeffect of Bmgrxc4 on longevity will be investigated at the individual level.（3）Screening of OSER1 interacting proteinsIn order to explore the conserved mechanism of oser1 regulating lifespan,the interaction proteins of human OSER1 were identified and analyzed.The overexpression vector of oser1 was constructed,and its expression was detected after cell transfection.The results showed that the transcription and translation level of oser1 was significantly up-regulated,and there were obvious bands of target protein at 60 k Da can be detected.The result of subcellular localization of OSER1 protein was detected by Immunofluorescence（IF）showed that OSER1 was mainly located in the nucleus.The prediction results of Bio GRID database show that there may be an interaction between XPO1 and OSER1,and Net NES results show that OSER1 contains leucine-rich nuclear output signal（NES）that can be recognized by XPO1.At the same time,using OSER1 as bait protein,other potential interaction proteins were screened by Co-IP/MS.Four candidate proteins,including ATP1A2,ATP1A3,HSPD1 and PKM2,that could interact with OSER1 were screened by mass spectrometry data based on molecular weight,MASS spectrometry score and peptide coverage.The construction of overexpression vectors for these genes has been completed,and the identification of interactions with OSER1 is being completed.The study focused on the function and molecular mechanism of oser1 regulating lifespan,and found that the dysfunction of Bmoser1 significantly shortened the lifespan of silkworm.Bmgrxc4/glrx2 and Bmwashc3/washc3 can respond to the expression changes of Bmoser1/oser1,and its expression is significantly up-regulated in the cellular oxidative damage model.It is speculated that glrx2 and washc3 may be involved in the process of oser1 regulating lifespan.