Studies On The Pattern And Mechanism Of Sex Chromosome Dosage Compensation In Helicoverpa Armigera

The dosage compensation(DC)mechanism is used to balance the sex-linked gene products between male and female sex chromosomes,which is more common in the hermaphroditic eukaryotes.The mechanisms of dosage compensation found so far mainly include up-regulation of heterogametic chromosomes(XY or ZW)and down-regulation of homogametic chromosomes(XX or ZZ).The study on dosage compensation is helpful to elucidate the regulation of sex-linked genes and the sex regulatory networks of species.Helicoverpa armigera is an important agricultural pest of lepidoptera.The study on the sex chromosome dosage compensation in H.armigera can provide theoretical foundation for its environmentally-friendly control.In this study,we first analyzed the sexual differences in the expression levels of autosome and Z chromosome genes at both the transcriptome-wide level and individual gene level to identify the pattern of sex chromosome dosage compensation in H.armigera.Secondly,I identified the H.armigera homologues of the five Drosophila melanogaster male-specific lethal(MSL)complex genes by gene cloning and sequence phylogenetic analysis.I also compared spatiotemporal expression profiles of the five MSL genes in males and females of H.armigera,which implied MSL1 is the regulatory MSL gene for dosage compensation in this species.Finally,I injected siRNA of Hamsl1 into the 3~rdd instar larvae to verify if Hamsl1 acts as a regulatory gene for sex chromosome dosage compensation in H.armigera.The main results of this thesis can be summarized as follows:1.Determination of the type of sex chromosome dosage compensation in H.armigeraHomologous analysis of the chromosome sequences of the Bombyx mori genome and the transcriptome of H.armigera identified 9,199 H.armigera autosomal genes and 549 H.armigera Z-linked genes.After filtering out the genes with a low FPKM value(e.g.FPKM=0 in either sex)and/or sex-biased expression,the average expression and sexual differences of the remaining autosomal and Z chromosome genes of H.armigera were statistically analyzed to determine the pattern of sex chromosome dosage compensation in this species.It is clarified that H.armigera can achieve complete dosage compensation of Z chromosome by down-regulating the expression of two Z-chromosome genes in males and up-regulating the expression of one Z-chromosome genes in females,that is,Z≈ZZ<AA.Analysis of 15 Z-chromosome genes in different tissues of male and female H.armigera adults found between-gene variations in dosage compensation types,but the average expression of the 15 genes displayed complete dosage compensation.2.Identification of MSL complex genes in H.armigeraFive genes of H.armigera MSL complex were successfully obtained by homologous sequence alignment and gene cloning.Gene structure analysis revealed that Hamsl1 has two alternative transcripts,one of which belongs to the intron retention type.Domain prediction analysis revealed that the five H.armigera MSL proteins contain the dosage compensation-responsible domains of the five Drosophila melanogaster MSL complex proteins.Sequence comparison of the MSL complex genes in different orders of insect species showed that the sequence similarity between H.armigera and B.mori was the highest.Phylogenetic analysis the five MSL complex genes published in different insect species suggested that Hamsl1,Hamsl2,Hamsl3 and Hamof(males absent on the first)genes had high homology in insects.3.Screening of key MSL complex gene of H.armigera and its regulation of sex chromosome dosage compensationSpatiotemporal expression profiling showed that the HaMSL complex genes had spatiotemporal specificity and sex specificity at mRNA level.Combined with the identification of MSL complex,Hamsl1 gene was speculated as the key gene for dosage compensation in H.armigera.Injection of siRNA of Hamsl1 into the 3~rdd instar larvae of H.armigera yielded an interference efficiency of36.01%~64.27%and there was no specific death in the experimental insects.qPCR analysis of 15selected Z chromosome genes showed that 11 of them were down-regulated in male and female larvae with an overall down-regulation range of 30%~40%.Transcriptome sequencing found that knocking down Hamsl1 gene significantly down-regulated the expression of Z-linked genes and the overall down-regulation range was also 30%~40%.The RNA interference experiment showed that Hamsl1gene positively promoted the expression of male and female Z chromosome genes to regulate the Z chromosome dosage compensation of H.armigera.