Functional Analysis Of MicroRNA Let-7 And MiR-2 Regulating Development In Bombyx Mori

After nearly fifty years of effort,the development of space life science has become the important content of space science development.At the same time,the investigators need to select suitable animal models for the research of space life science.As a representative of lower organisms and an important economic insect,the silkworm,Bombyx mori,will become an ideal model to unravel a variety of important questions in space environment.B.mori has of important role for space science experiments in ?SJ-10? recoverable satellite,and the change of genes in development initiated by space environment has been one of the experiment projects for space life science.However,the regulation net of gene expression is multidimensional and complex with many regulation factors.And the exact mechanism of the effect of space environment is still unclear.micro RNA(mi RNA)are a class of endogenous small RNA molecules transcribed and processed from genome non-coding region and playing important roles in gene expression regulation by leading to their target m RNA degradation or translational inhibition.Thus,this subject mainly studies the mi RNA regulation roles and functions in B.mori growth and development on the application of the most advanced mi RNA technologies and genetic manipulation methods.let-7 and mi R-2 are likely to be involved in development and metamorphosis of Bombyx mori according to the early selection work in our lab group.But both of them have not been functional annotated and verified.Thus,this study inhibited the function of mi RNA let-7 and investigated the effect of let-7 on the development of silkworm using the transgenic mi RNA sponge technology in the living organism of silkworm.Secondly,the transgenic mi RNA cluster overexpression technology has been used to make an ectopic functional enhancement of mi RNA family mi R-2 and to analyze the regulation of mi R-2 on the wing development of silkworm.The research above can provide new idears and a new layer of genomic manipulation to reveal the mechanism of gene express change and developmental mutation in space condition.At the same time,it verified possibility of the mi RNA transgenic methods in silkworm and provided fundamental base for the further research on the spatial and temporal-specific functional roles.The study and obtained results in this thesis are shown below.(1)Establishment of the mi RNA sponge technology in Bombyx mori and functional analysis of the mi RNA let-7 in Bombyx mori.This thesis focused on the evolutionarily highly conserved mi RNA let-7.The mi RNA sponge technology has been applied in analyzing the effect of mi RNA let-7 on the development of silkworm in vivo,combining with the GAL4/UAS transgenic system.And the target genes Bm FTZ-F1 and Bm E74 regulated by let-7 have been verified in vivo and in vitro.Firstly,using the transgenic mi RNA sponge technology the mi RNA loss-of-function line Bm Let-7Sp has been established successfully.This silkworm line could express the mi RNA sponge sequence which can base pairing imperfectly to the endogenous Bm Let-7 mature form.The transcription of this sponge sequence were happened ubiquitously after the activation driven by A3(Actin3)-GAL4.The reporter gene m Cherry(improved monomeric red fluorescent proteins)was put in the upstream of sponge sequence.let-7 sponge can decay the activity of let-7 by competitive decoy the endogenous let-7 mature form in vivo.Secondly,this thesis obtained individuals with abnormal development phenotype.Around 75%(n=150)of larvae could not access the third molting and died at the end of third instar.They had leaner body,different colour and little undigested food in the gut compared to UASLet-7Sp(control group)during L3D3 when they stop eating.10% larvae died during the fourth and fifth larval instar with phenotype similar to L3D3 larvae.And the remaining 15% of animals showed arrestment during pupation and died within several days after cocooning.These animals appeared to be bound in the apolysed cuticle,unlike the control.But more of the Bombyx pupal body was formed except for the head which was bound tightly.Thirdly,among the transgenic mutants,the expression level of FTZ-F1 and Eip74EF(E74)were up-regulated significantly as well as the down-regulation of Bm Let-7.FTZ-F1 is a member of the nuclear hormone receptor superfamily,while E74 is a steroid-triggered transcription factor.Both of them are the predictional target genes of Bm Let-7 and has been verified in cell culture experiment.And they are the key regulators in ecdysone signaling pathway.The expressions of them were inhibited by Bm Let-7.To date,this is the first report on such analyses in non-drosophilid insects where transgenic mi RNA sponge technology has been used.And the let-7 has been verified to be able to regulate the processs of molting and metamorphosis in Bombyx mori.When the regulation balance was destroyed,the larval-larval transformation and larval-pupal transformation were affected.The effect of let-7 on developmental timing in silkworm is similar to its function in drosophila and nematode.At the same time,let-7 was verified to be able to inhibit the expression of the key regulators in ecdysone signaling pathway,Bm FTZ-F1 and Bm E74.Both of them have the target sites of let-7 in 3'UTR(3'-untranslated region).(2)The analysis of mi R-2 regulation in wing developmentThe invertebrate-specific mi RNA mi R-2 family was focused in this part.The transgenic mi RNA cluster overexpression technology was used to analyz the regulation of mi R-2 family during wing development in Bombyx mori.And the target genes of mi R-2 family were verified in vivo and in vitro.At the same time,the CRISPR/Cas9 knock out technology has been applied innovatively to analyz and verify the target genes of mi R-2 family.Firstly,the transgenic silkworm line A3>mi R-2 was established by using the transgenic mi RNA cluster overexpression technology,which can ectopic express the pre-mi RNA sequence of mi R-2 cluster after activation driven by A3-GAL4 ubiquitously to enhance the function of mi R-2 family in vivo.Secondly,this thesis obtained individuals with deficient wings which occupied 80 percent ratio among A3>mi R-2.In the mutants,the mature form level of the mi R-2 family members(mi R-2a,mi R-2b,mi R-13 a and mi R-13b)were increased,while the relative expression level of abnormal wing disc(awd)and fringe(fng)were decreased significantly.Both of them are the important positive regulators in Notch signaling pathway.After software analysis,these two genes are known as the preditional target genes of mi R-2 family.In the cell cultural experiment,mi R-2 family has been verified to be able to target their 3'UTRs and inhibited their expression.Otherwise,the transcript level of Bm Wnt-1,a down stream gene in Notch signaling pathway,was decrease in the mutants.Moreover,somatic mutagenesis analysis of awd and fng using the CRISPR/Cas9 system and knock-out mutants also resulted in deformed wings similar to those observed in the mi R-2 cluster over-expression transgenic animals.These results indicated that mi R-2 family play an important role in wing development of Bombyx mori,which can inhibit the expression of Bmawd and Bmfng genes.In conclusion,the transgenic mi RNA research methods can be applied in Bombyx mori.let-7 can inhibit two key genes in ecdysone signaling pathway,Bm FTZ-F1 and Bm E74,and regulate the molting and metamorphosis in Bombyx mori.mi R-2 family can inhibit two key genes in Notch signaling pathway,Bmawd and Bmfng,and regulate the wing development in Bombyx mori.ds RNA-mediated RNAi by using directly injection of ds RNA was seldom effective in silkworm,especially during larval stage.An alternative approach is to apply Cas9 system in Bombyx mori.The Cas9 sg RNA-mediated mutagenesis of the mi R-2 family target genes in this study produced abnormal wing phenotypes similar to those in the A3>mi R-2 over-expression line.Bombyx mori with the easy rearing and easy handle characters was chosed as the model organism in this thesis.By transgenic methods,the mi RNAs function were decayed or enhanced in vivo in a long term and heritable manner to understand the biological function of let-7 and mi R-2 and their molecular mechanism.It helps us understand the function of mi RNA,but also be useful to the research of gene expression regulation and mutation mechanism in space condition.It is one of the center contents that mi RNA can be used as a new layer of manipulation for the regulation of gene expression.And the silkworm will become an ideal model organism for space research.