The Function And Mechanism Of Circadian Clock Gene Bmperiod On Diapause Of Bombyx Mori

Diapause is an important survival strategy formed by insects to adapt to environmental changes.It is regulated by biological clock system and endocrine system.Once the organism enters diapause state,it must go through a certain stages or physiological changes before it can end the diapause state.Even if the environment returns to normal conditions,it can not start to develop.Diapause process in Bombyx mori happens when the mother receives the stimulation from different external environmental signals at a specific stage of development.The stimuli will induces the occurrence of a series of metabolic pathways through the changes of hormones in the body.B mori is known as an important economic insect and as one of Lepidoptera model insect which undergo a typical embryonic diapause.It has always been regarded as an important model organism for egg diapause research.Owing to the importance of silkworm as an economic traits,research on diapause of silkworm is crucial for silkworm development and adaptation process.Moreover,it is important for better understanding of how silkworm deals with the changes of periodic environment..It is of great to provide an insight of diapause studies as a reference for the other species whilst contribute some information of pest control management.Diapause of silkworm is regulated by circadian clock gene.At present,the biological clock genes that have been found and identified including clock protein gene(Clock,Clk),cycle protein gene(Cycle,Cyc),cryptochrome gene(Cryptochrome,),cycle protein gene(Period,per),timeless protein gene(Timeless,Tim),rotary protein gene(Vrile,Vri)and double time protein gene(Double-Time,Dbt).The circadian rhythm of organisms is hereditary and controlled by genetic genes.The expression of these molecular clock genes is controlled by the regulatory feedback loop.The coordination and interaction of a series of biological clock related gene may leads to the rhythmic changes of various physiology metabolism and behavior in organisms.In this study,a silkworm mutant with circadian clock gene Bmperiod knocked out was constructed,which greatly changed the classical diapause pathway.Combined with the differential analysis of gene expression and the detection of double luciferase reporting system,the process of Period gene participating in diapause induction of silkworm was explored.The main results of this study are as follows:1.Analysis of Bmperiod gene expression patternDiapause of B.mori was affected by maternal effect.Embryos that incubated under25 ℃ with continuous light emission induced the release diapause hormone(DH)from hypopharyngeal ganglion which caused diapause eggs production by the adult moth.Under the condition of continuous dark incubation at 15 ℃,the adults produced non diapause eggs.Real time fluorescence quantitative PCR(qRT-PCR)was used to determine the expression of Bmperiod in silkworm eggs whcih were incubated at 15 ℃ and 25 ℃.The eggs were dissected and embryos were sampled at their environmentally sensitive stages(i.e.ST20,st21 a,st21b,st21 c,st22 and st23).The results showed that the expression of Bmperiod at25 ℃ was higher than at 15 ℃.Under the condition of 25 ℃,the expression of Bmperiod gene was up-regulated,indicating that silkworm embryos feel different cyanation temperature will induce the differential expression of Bmperiod gene.It is speculated that this gene may play a role in the process of receiving environmental signals(temperature and light)to induce diapause in silkworm embryos.2.Knockout of Bmperiod gene In order to explore the role of Bmperiod in the diapause determination of silkworm embryos,Bmperiod gene was knocked out using CRISPR / cas9 mediated gene editing technology in the dualistic silkworm strain Dazao.Two specific sgRNAs of Bmperiod gene were transcribed and synthesized in vitro and mixed with cas9 protein,and then microinjected into the newly oviposited non diapause silkworm eggs of Dazao incubated under low temperature(15 ℃).After injection,the G0 generation silkworm eggs were incubated at25 ℃ and the larvae were fed until the moth stage.The silkworms were self spawned,continued feeding,and the Bmperiod hybrid was obtained in the G1 generation.Interestingly,non diapause eggs were produced from the diapause destined moths.Sequence analysis of Bmperiod gene from the heterozygous individuals showed sequence variation located nearby the sgRNA target site of the gene sequence.The deletion of 2bp located at the second exon of the gene resulted in the frameshift mutation of Bmperiod gene and caused the loss of its function.Hence it shown that the Bmperiod gene was successfully knocked out.The G1 generation silkworm eggs were reared under the condition of 25 ℃,and the G1 generation hybrid moths mated with each other.Non diapause eggs were produced in 8 of the 18 cycles of silkworm generations.The non diapause silkworm eggs were reared under the condition of 25 ℃,and the Bmperiod homozygous individuals were screened from the G2 generation moths.The G2 generation homozygous moths were crossed link mating with each individual,and non diapause eggs were produced in 4 of the 10 eggs.In order to study whether Bmperiod gene will affect the expression of other carcadian rhythm genes,the expression of Bmperiod gene and carcadian rhythm genes(BmClock,BmCycle,BmTimeless,BmCry2)were analyzed in Bmperiod knockout homozygous individuals by fluorescence quantitative PCR(qRT-PCR).The results showed the expression of BmClock and BmTimeless genes in Bmperiod knockout homozygous individuals were up-regulated,the expression of Bmclock and Bmcry2 were down regulated compared to the wild-type Dazao at 72 hours after spawning.The results showed that Bmperiod gene function was important to regulate the diapause of silkworm embryos.3.BmiaaNAT gene responds to circadian rhythm gene to regulate diapause of silkwormIn the study of pupal diapause of Antheraea pernyi,it was found that iaaNAT gene may participate in the regulation of pupal diapause in response to circadian rhythm system.In order to further explore how the Bmperiod rhythm gene is involved in the regulation of embryonic diapause induction in silkworm,the expression of B gene in homozygous individuals with Bmperiod gene knockout was detected.It was found that the expression of BmiaaNAT gene was significantly up-regulated after Bmperiod gene knockout.According to previous studies,Clk/Cyc dimer can regulate the expression of iaaNAT,and period gene can inhibit this regulation.It is speculated that BmiaaNAT gene will respond to circadian rhythm signals to regulate diapause of silkworm.Prediction of the promoter region was made on the BmiaaNAT gene to determine the binding site of Bmclk/Bmcyc dimer.The result showed,BmiaaNAT gene contains E-box element that can bind to the dimer.HENCE,double luciferase assay was conducted to verify the promoter and the results showed that Bmclk/Bmcyc dimer promoted the expression of BmiaaNAT gene.Moreover,the expression level of BmiaaNAT gene and in embryos of environmentally sensitive period(i.e.ST20,st21 a,st21b,st21 c,st22 and st23)at different temperatures under diapause induction and non diapause induction conditions were evaluated using qRT-PCR in order to determine the whether BmiaaNAT gene was regulated by circadian rhythm gene and affect diapause of silkworm.The results showed that in wild-type Dazao,the expression of BmiaaNAT at 25 ℃was higher than that at 15 ℃.It is speculated that BmiaaNAT gene may play a role in receiving environmental signals to induce diapause.Further verification of BmiaaNAT gene role in diapause induction,BmiaaNAT gene interference was conducted in wild-type Dazao using RNAi technology.Two interference sites of BmiaaNAT gene were constructed through biological analysis software.The dsRNA of BmiaaNAT gene were transcribed and synthesized in vitro,and microinjected it into the silkworm eggs that newly oviposited by Dazao at low temperature(15℃).48 hours after injection,the expression of BmiaaNAT gene was detected by real-time fluorescence quantitative PCR(qRT-PCR)in 72 h and 96 h silkworm eggs.It was found that the expression of BmiaaNAT gene was significantly down regulated,indicating that the intervention was successful.The silkworm eggs injected with dsRNA were placed at 25℃ for rejuvenation.Non diapause silkworm eggs were found in the offspring eggs.The results showed that BmiaaNAT gene was regulated by rhythm genes(Bmclk and Bmcyc)and played a role in inducing diapause of silkworm.In short,this study represents the role of Bmperiod gene in the diapause induction process of silkworm.The preliminary study was conducted to explore its function through knockout of Bmperiod gene,double luciferase experiment and the interference of BmiaaNAT gene using RNAi technology.