| Bactrocera dorsalis(Hendel)is a kind of detrimental pest for the fruit and vegetable.With the development of molecular biology technology,pest sterility control techniques(SIT)reducing the number of pest populations by modifing population at genetic level has gradually become an effective means for the control of B.dorsalis.Studying the mechanism of sex determination is the basis of SIT.In typical XY determination system,male differentiation factor(M factor)located on Y chromosome mainly controls male differentiation.However,the mechanism of sexual determination in B.dorsalis is far to be elucidated,and there is very little research on the Y chromosome and its functional genes.The Chromosome Quotient(CQ)calculation is a novel method for searching specific genes in Y chromosome and has been successfully applied in several Diptera pests.With the RNA interference and CRISPR/Cas9 gene editing system,the functional gene analysis in the B.dorsalis could be performed readily.In this thesis,we applied CQ to screen the Y-specific genes and found C73309,a Y chromosome specific gene in the dataset.Further,RNAi and CRISPR/Cas9 were applied to verify the function of C73309.It was found that C73309 plays an important role in the development of male reproductive system,especially in the process of testis development and spermatogenesis.We named it spermatogenesison-Y chromosome(Bdsy).Bdsy gene can be used to develop a molecular marker to distinguish the sex of early embryos of B.dorsalis,and using RNAi or CRISPR/Cas9 to target Bdsy gene to establish male sterile lines,develop sex-targeted green pest control,provide new ideas and scientific basis for the green control of B.dorsalis.The main results were presented as follow:1.We firstly performed transcriptome analysis of 0h-1h,2h-4h,5h-8h early stage embryo and sexually mature males.Mature male and female pest individuals were sampled to perform genome sequencing.The transcripts of 2h-4h and 5h-8h embryo were assembled and aligned to the male and female genomes respectively with CQ<0.3 and male reads>10.From the primary sequence dataset,1011 and 2583 candidate sequences were screened from the 2h-4h and 5h-8h embryo datasets respectively due to their higher expression levels in the male individuals.After the filtering,the candidate sequences were annotated and screened with Y chromosome specific property.Finally,12 of 108 contigs were annotated successfully in 2h-4h embryo;meanwhile there were 3 annotated contigs in the 69 candidate sequences from 5h-8h embryo.By gene fragments amplification and agarose electrophoresis,7 Y-specific sequences(34040?52579?67915?73309?74210?107270?152374)and 2-Y-biased sequences(116796?151939)were identified in 2h-4h embryo.Likewise,one Y-specific sequences(32995)and one Y-biased sequences(9898)were harvested from 5h-8h embryo data.Sequence alignment of Y chromosome specific fragments revealed that the 73309 and 74210 belonged to gene C73309.2.Further,a Y chromosome specific gene,Bdsy,which highly relative to spermatogenesis,was cloned and selected to investigate its spatial-temporal expression and function to the male reproduction organ.Bdsy contained 2 exons and 1 intron,encodes 214 amino acids.There is a homologous gene in Bactrocera tryoni(MK504335.1)which showed protein sequence similarity at 84.86% to Bdsy.Spatialtemporal expression analysis of Bdsy showed highest accumulation 15 d testis(increased during developmental stage).To validate the function of Bdsy,we interfered Bdsy in newly emerged males.The interfered individuals developed abnormal testis(shown as smaller and albinistic testis)and notably decreased number of sperms.Knockdown expression of Bdsy in 0h-1h embryo resulted in similar phenotypes to that which interfered with newly emerged males.Knockout Bdsy using CRISPR/Cas9 also led to abnormal testis and male sterility in G0.Totally,we found the Bdsy gene participated in testis development and spermatogenesis,and is a significant gene in the male development of B.dorsalis. |
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