Mitochondrial Genome And Biosynthesis Regulating Genes Of Dastarcus Helophoroides And Its Population Genetic Structure Based On COXI Gene

Dastarcus helophoroides(=longulus)(Coleoptera: Bothrideridae)is an important natural enemy of long-horned beetles.Mitochondria are the main source of energy required for cell and living activity.Mitochondrial DNA has been widely used for phylogeography,population genetic structure,phylogenetic,molecular evolution,comparative evolutionary genomics analyses and investigations,because of its relatively small molecular weight,high rates of mutation,matrilineal inheritance and high copies in the cell.The mitochondrial DNA coded genes can't independently complete the process of oxidative phosphorylation to produce ATP,and nuclear DNA regulates the mitochondrial genes by a serious of transcription factor and coenzyme factor.In this study,we used long-PCR and primer-walking sequencing method to investigate the mitochondrial genome of D.helophoroides,the complete sequence was obtained and the molecular biology information was analyzed.Based on the whole sequence of the mitochondrial genome,the genetic structure and genetic difference of different D.helophoroides population was studied using COXI gene.According to the transcriptome information data of D.helophoroides,the mitochondria biosynthesis related genes including AMPK genes and PGC-1? signal pathway genes were identified,characterized and expression analyzed.The main results are as follows:1)The complete mitochondrial genome of Dastarcus helophoroides which consists of 13 PCGs,22 tRNA genes,two rRNA genes and a non-coding region(D-loop),is sequenced for its nucleotide sequence of 15,878 bp(GenBank: KF811054.1).Except for COI gene generally starts with non-canonical initial codon,all protein-coding genes start with ATN codon and terminate with the stop codon TA(A)or TAG.The secondary structure of rrnL and rrnS consists of 48 helices(contains four newly proposed helices)and 35 helices(contains two newly proposed helices)respectively.All 22 tRNAs in D.helophoroides are predicted to fold into typical cloverleaf secondary structure,except trnS1(AGN),in which the dihydrouracil arm(DHU arm)could not form stable stem-loop structure.Protein-coding genes(nucleotide dataset and nucleic acid dataset)have been used to infer the phylogenetic relationship;the results showed that D.helophoroidesis can be classified into Cucujoidea(Bothrideridae)and the result consisted with the current morphology-based hypothesis.The analyses will provide effective molecular information in exploring the population genetic structure,species identification and the evolution and phylogeny of D.helophoroidesis.2)The genetic sequence of COXI was used to investigate the genetics and relationships within and among D.helophoroides populations collected from five different geographic locations.A total of 26 haplotypes with 51 nucleotide polymorphic sites were defined,and low genetic diversity was found among the different populations,the genetic variations were observed mainly within populations.Low pairwise fixation index values and high gene flows show that there was high gene exchange between populations.The co-distributed haplotype DH01 was suggested to be the most ancestral haplotype,and other haplotypes thought to have evolved from it through several mutations.Low genetic diversity among different populations is related to a relatively high flight capacity,host movement,and human-aided dispersal of D.helophoroides.The high gene exchange among populations AG,MA and MR may suggest that cross naturally of these populations in the field.This analysis provided the theoretical foundation in genetic differences of D.helophoroides,and provided molecular basis for utilizing the nature enemy in biological control.3)The full-length cDNAs of three AMPK submits were indentified from D.helophoroides transcriptome and obtained using rapid amplification of cDNA ends(RACE).Phylogenetic analysis indicated that D.helophoroides AMPK submits had high identity with other insects,especially closely related to orthologs species Tribolium castaneum(>85%).The real-time RT-PCR was used to quantify dynamic changes of AMPK mRNA.AMPK genes could be detected in all treatments,and the levels showed significant expression specificity.The higher expressions of the three genes were observed in male reproductive systems,fatbody,first instar larva and pupa.Along with the survival time extension of D.helophoroides,the expressions of AMPK genes showed distinctly increased trend in older adults,this may be related to its response to environmental changes and ease aging.In different stressed conditions,the expressions of AMPK genes in female adults were higher than that in male adults.The genes showed increased firstly and then decreased patterns with the increase of processing time.The results show that AMPK genes plays an important role in insect metamorphosis,life extension and stress.4)The PGC1-?,NRF1,mtTFA,COXI and ATP6 genes were identified from D.helophoroides transcriptome and mitochondrial genome.In the phylogenetic relationships,different amino acid sequences of D.helophoroides genes showed highly homology with the corresponding sequences of other species.The real-time qPCR results of the five mitochondria biosynthesis genes showed that the expressions existed significant tissue specificity,which were higher in male reproductive systems rather than that in other tissues.The expressions of these genes were highest in 1 instar larva and lowest in pupa.Twenty months later of D.helophoroides adult eclosion,the genes expressions were increased along with the survival time extended,and reached the maximum value on 30 months.In the tissues,stages and adult ages with n DNA coding genes(PGC1-?,NRF1 and mtTFA)which highly expressed,the mt DNA coding genes(COXIand ATP6)also showed high expressions.The analyses provide the foundation further study of the energy synthesis of D.helophoroides in the life-span.