Cloning,Expression And Functional Analysis Of Clock Gene In Mythimna Separata (Walker)

The oriental armyworm,Mythimna separata(Lepidoptera: Noctuidae),a typical seasonal long-distance migratory insect,distributing in many countries and regions in Asia and Australia,can migrate between the north and south of China every year and threaten the safety of grain production of China.Due to the change of farmland ecosystem,the frequency of the M.separata outbreak was increased.It severely broke out in 2012 and 2013 causing serious impact on agricultural production.Circadian clock genes are a sort of regulatory genes which regulate biological rhythm in different levels including cells,tissues,organs and behaviors.The flying,mating and ovipositing of M.separata all happens in the dark period,which shows obvious circadian rhythms.However,the molecular regulation mechanisms of behavior rhythm in M.separata have not been reported.The ORF sequences of clock genes in M.separata were cloned in this study for the first time.Then the expression patterns of clock genes were analyzed then by real time quantitative PCR(RT-qPCR)method.In addition the function of clock genes were studied on the regulation of rhythm.The main results are as follows:1.Based on the transcriptome data,four clock genes of M.separata were cloned firstly,which were named as Msper(KY446805),Mstim(KY446806),Msclk(KY446807)and Mscyc(KY825136),with the open reading frames of 3579 bp,3132 bp,1851 bp,2094 bp,encoding 1192,1043,616,697 amino acids residues,respectively.The predicted molecular weight were approximately 132 kDa,117 kDa,69 kDa,75 kDa,and theoretical isoelectric point were 6.13,5.14,5.63,6.74 respectively.Amino acid sequences alignment results indicated that MsPER shared a high amino acid identity above 83% with PER of Mythimna unipuncta,Spodoptera exigua and Anagrapha falcifera.MsTIM shared a high amino acid identity above 80% with TIM of Helicoverpa armigera,Spodoptera exigua and Sesamia nonagrioides.MsCLK shared a high amino acid identity above 90% with CLK of Spodoptera exigua and Helicoverpa armigera.MsCYC shared a high amino acid identity above 68% with CYC of Sesamia nonagrioides,Bombyx mori,plutella xylostella,Antheraea pernyi and so on.The phylogenetic analysis confirmed the results of the sequences alignmen.2.qRT-PCR was performed to analyze the expression levels of Msper,Mstim,Msclk and Mscyc in different developmental stages,tissues,and zeitgebers.(1)The 4 clock genes differently expressed in different development stages.The relative mRNA expression levels at adult were significantly higher than other stages.The expression of Mscyc was significantly higher in 1 st larval stage than egg stage whereas the other three genes expressed lowest in larval stages.(2)The expressions of this 4 clock genes were different in various tissues.The Msper,Mstim and Mscyc expressed highest in antenna,while Msclk expressed higher in head than antenna.There were no obvious expression regularities of clock genes in flight muscle,legs and gonads.(3)The expression levels of this 4 clock genes possessed apparent circadian rhythm.Msper,Mstim and Msclk showed low expressions in scotophase and high expressions in photophase in male and female,while the expression of Mscyc was higher in photophase and lower in scotophase.3.The injection method was used to konckdown Msper and then detected the change of activity rhythm.The results showed that the expression of Msper decreased 60% at 24 h after interference.The peak of locomotor activities disappeared at night while the peak locomotor activities kept at the end of scotophase in control group after injection.Furthermore,the dsper moths did not mate with 48 h after injection,whereas mating behavior was recovered after interference function lost.In this study,the ORF sequences of 4 clock genes from M.Separata were cloned for the first time,and the expression levels of Msper,Mstim,Mscyc and Msclk genes were different at different development stages,tissues and time.Furthermore,it has been proved preliminarily that Msper was involved in activity rhythm regulation.The results may be beneficial for understanding the regulation mechanism of activity rhythm by clock genes in M.separata and lay the foundation for further explore the function of clock genes,aiming at establishing a genetic control system of M.Separata which targets insect behavior rhythm and realizing the goal of the sustainable pest management for M.separata.