Analysis Of Expression Pattern Of Ecdysone Synthesis And Related Receptor Genes Of Mythimna Loreyi

Ecdysteroids is an importanthormone during insects development.Its synthesis and signal transduction are mainly regulated by Halloween genes and downstream ecdysone receptors.It's of great biological significance on the regulation of growth,development and oviposition to clarify the fluctuation of 20E titer and the expression patterns of related genes.However,the research of above aspects in Mythimna loreyi,a key pest species on maize,is still vacant.Therefore,five Halloween genes and three downstream receptor genes were cloned.Then,the expression patterns of Halloween genes,the titer of 20E,and their relationship were elucidated in 5th and 6th larval instar,pupal and adult stages.The relationship between 20E titer and reproduction were also discussed.The main results were shown as follow:1.Five Halloween genes?Cyp307a1,Cyp306a1,Cyp302a1,Cyp315a1,and Cyp314a1?,and three ecdysone receptors,?EcR,USP and HR3?,were cloned.The complete ORFs were obtained in Cyp306a1,Cyp302a1 and Cyp314a1,and partial ORFs were obtained in the Cyp307a1,Cyp315a1.The predicated amino acid sequences of the five Halloween geneshave conserved domains of P450 super family.Phylogenetic tree analyses showed that different Halloween genes clustered into five different groups.The complete ORFs of USP and HR3,and the partial ORF of EcR were obtained.The DBD domain and the LBD domain were found in all the three receptor genes?Because of lacking 3'terminal,the LBD domain of EcR showed fragmentary?.Analysis of phylogenetic tree indicated that EcR of M.loreyi was clustered to EcR of Agrotis ypsilon,while USP and HR3 were clustered into HR3 of Heliothis armigera.2.Analysis of the expression patterns of the Halloween genes and related receptor genes indicates that:?1?It manifested that the expression level of all genes changed periodically during 5^thh and 6^th instar larvae.Halloween genes expressedhigher in late time of 5th or 6th larval instar.Besides,the expression level of EcR and USP during 5^th instar were significanthigher than that of 6^th instar,while the expression of HR3 showed contrary trend.?2?Although Halloween genes expressed in all peroids in pupa,there were differences between expression levels and patterns.The expression patterns of all genes seemed similar,the trend of which decreased firstly and went up later in female.The expression level of Cyp307a1 in initial pupa and Cyp314a1 at 72h pupa were significanthigher than others,while Cyp306a1,Cyp302a1 and Cyp315a1 expressed at 216h pupa were significanthigher than those at 72h.The expression difference among various peroids of EcR and USP was significant.HR3had a low expression level at 0h pupa,and then catched to thehighest value at 216h.Except Cyp307a1,the expression levels of other four Halloween genes increased first and then decreased in male pupa.Among them,Cyp306a1,Cyp302a1 and Cyp315a1had thehighest expression at 168h of pupation,which was significantlyhigher than other periods.Although Cyp314a1had thehighest expression at 120h of pupation,it was not significantly differ from 72h and 168h.The expression patterns of EcR,USP and HR3 were similar,and the peak of which appeared at 168h after pupation,then dropped to a lower level at 216h,the difference between them was significant.?3?All genes expressed at any development period,and the difference between female and male adults showed significance.3.The expression level of Cyp307a1,Cyp306a1,Cyp302a1,and Cyp315a1 werehighest in prothoracic gland,whereas,Cyp314a1 expressedhighest in malpighian tubule.And similar as Cyp314a1,EcR,USP and HR3 showedhighest at malpighian tubule,too.Besides,the lowest expression level of which were shown in prothoracic gland.And the former was 9.05,79.20 and 13.05 timeshigher than the later,respectively.4.The detection of 20E titer showed that during later stage of 5^th and 6^th instar,the 20E titer washigher than that in the previous stage.But in pupa stage,it washighest at initial time and then declined to the lowest level at 120h,after that,it returned back tohigher level gradually.During adult stage,20E titer remained at a low level within 48h after emergence,then reached to a small peak at 72h,and the floor level appeared at 96h after eclosion.Following that,it rebounded to the level of 72h.5.There was a strong correlation between the trend of 20E titer and the gene expression level in larva,pupa and adults stages of M.loreyi.Except for Cyp314a1,the expression changes of other genes were similar to the trend of 20E titer,both of which are low in the early stage of each instar buthigh in the late stage.However,compared with 20E titer,the expression peak of each gene in 6^th instar larva was advanced,and this phenomenon was also found in pupa stage.20E titer kept in low level during early stage,but reached to the peak at120h after eclosion,which was consistant with expression level of several genes named Cyp307a1,Cyp302a1,Cyp315a1 and HR3.Unlike the 20E titer,which fluctuated greatly throughout the adult stage,Cyp314a1 did not change much,although it washigher in early stage than that in late stage.6.There were two peaks of 20E titer in female adults.The first peak appeared at 72h after emergence when the females initiated reproduction.Then the content of 20E dropped to the lowest with the peak of egg laying.Twenty-fourhours later,the level of 20E titer drastically increased to the second peak when the daily fecundity began to decline.The results indicated that 20E was involved in adult reproduction,and the productive progress maybe inhibited byhigh titer of 20E but promoted by lower titer.