Enolase Genes In Two Wild Silkmoths,Samia Cynthia Ricini And Antheraea Pernyi,and Mitochondrial Genome Of Loxostege Sticticalis

Enolase is a multifunctional protein that plays an important role in physiological pathology.At present,the understanding of enolase is mainly focused on human and pathogenic microbia;however,knowledgement on insect enolase is extremely limited.Identification of insect enolase started in our laboratory several years ago.In Antheraea pernyi,two enolase genes were first identified,and then named as enolase Ⅰ and enolase Ⅱ.respectively.The enolase Ⅱ appears to be Lepidoptera-specific and only present in the gonad,whereas enolase Ⅰ is present in all the insects.To address the evolution of enolase,it is necessary to investigate the sequence characteristics and tissue expression pattern of enolaseⅡ in more insect species,and to compare the enzymatic properties of enolase Ⅰ and Ⅱ.In this paper,the sequence characteristics and developmental expression pattern of the enolase Ⅰ andⅡ genes were investigated in Samia cynthia ricini,and named as Screno Ⅰ and Ⅱ,respectively.This paper also investigated the expression profile of the A.pernyi enolase Ⅰ(Apeno Ⅰ)and Ⅱ(Apeno Ⅱ)genes by qRT-PCR,the recombinant proteins rApeno Ⅰ and rApeno Ⅱ were expressed successfully,and then the high titer antibodies were obtained.Insect mitochondrial genome sequencing can provide basic sequence information for inderstanding of the structure of mitochondial genome and reconstructing the phylogenetic relationship of the target species.In this paper,the mitochondrial genome of Loxostege sticticalis previously completed in our laboratory was analyzed by bioinformatics,and the phylogenetic relationship among 24 Pyraloidea species(from 2 families of 9 subfamilies)was reconstructed.The results of this paper are as follows:1.The Screno Ⅰ and Ⅱ encode 433 and 431 amino acids,respectively.Screno Ⅰ and Ⅱonly exhibit 60%sequence identity,but Screno Ⅰ displays 91%-97%identities with enolase Ⅰfrom different species and Screno Ⅱ displays 84%-92%identities with enolase Ⅱ from different species.Evolutionary analysis further suggested that enolase Ⅱ and Ⅰ were evolved independently.The expression pattern of Screno Ⅰ and Ⅱ in the embryonic stage was detected by qRT-PCR,and the results showed that the expression level of Screno Ⅰ was higher,but Screno Ⅱ remained almost no expression.The expression level of Screno Ⅰ in the testis during pupal stage was lower and remained at this low level,while the expression of Screno Ⅱ was much higher than that of Screno Ⅰ and remained at a high level until eclosion.This result suggests that Screno Ⅱ is associated with the reproductive development.2.The expression level of Apeno Ⅰ was higher than that of Apeno Ⅱ that remained a very low level during both embryonic and larval stages;the expression level of Apeno Ⅰ in the testis during pupal stage was also higher than Apeno Ⅱ.The recombinant expression plasmids pET28b(+)-Apeno Ⅰ and Ⅱ were successfully constructed,and the recombinant proteins rApeno Ⅰ and rApeno II were expressed in the form of soluble or inclusion bodies,respectively.Western blot analysis showed that rApeno Ⅰ and rApeno Ⅱ were expressed correctly.The high titer antibodies were obtained by immunizing rabbits with the two recombinant proteins.Immunological test results show that Apeno Ⅰ and Apeno Ⅱ were expressed in blood,gonad and fat body of A.pernyi,which were inconsistent with RT-PCR results.Further tests revealed that the two antibodies had severe cross-reactivity.The success of the prokaryotic expression of the A.pernyi enolase Ⅰ and Ⅱ laid the foundation for the further study of the enzymatic properties.3.The AT content and preference of the mitogenome of L.sticticalis were similar to those of the mitogenome of the Pyraloidea previously published.The gene spacer is distributed in 15 regions,but only two spacers between tRNAGIn and ND2 and between tRANASer(UCN)and ND1 are present in all the Pyraloidea.In the A+T-rich region,three conserved sequences were identified.Based on the phylogenetic analysis of the mitogenome sequence,the Pyraloidea species used were divided into two families(the Pyralidae and the Crambidae).The Crambidae family was also divided into two branches,named as the PS and the non-PS branchs.The PS branch included Pyraustinae and Spiromelinae,and the non-PS branch included Crambinae,Acentropinae,Scopariinae and Schoenobiinae.This is the first time to use the mitogenome sequence to explore the phylogenetic relationship within the Pyraloidea.