Screening And Identification Of Y-specific Genes And Functional Study Of Bdyof Gene In Bactrocera Dorsalis

Bactrocera dorsalis(Hendel)is an important agricultural insect pest in China,which seriously affects agricultural economic benefits and food security.Spraying chemical insecticides has always been the main method for pest control of B.dorsalis.Although the sterile insect technology in Tephritidae based on radiation has been used in some field,it is hard to be widely used in agricultural production due to its high cost and low efficiency caused by technical defects.People urgently need to develop an environment-friendly and efficient green insect pests control technology.Y chromosome is vital for sex determination and male reproductive development.However,due to the incomplete information of genome of Y chromosome and the difficulty of sequencing,there are few studies on Y-specific genes of most insect species.In this study,we identified a novel Y-specific gene in B.dorsalis,which encodes a small protein that regulates the development of testes and sperm.Its deletion can cause male Oligozoospermia of B.dorsalis.We named it Bactrocera dorsalis Y-specific Oligozoospermia factor(Bdyof)gene.CRISPR/Cas9 gene editing technology and RNA interference(RNAi)technology were used to elucidate the important effect of Bdyof gene on male testis development and spermatogenesis of B.dorsalis.Our research provides a scientific theoretical basis and target for the development of radiation independent sterility technology of B.dorsalis.The main results are as follows,1.The transcriptome database of B.dorsalis was constructed with 327139 transcripts by de novo assembling the mixed transcriptome data of male and female B.dorsalis in egg,larva,pupa and adult stages.90 Y-specific candidate transcripts were screened by chromosome quotient(CQ).These Y-specific candidate transcripts were mainly clustered into energy metabolism and cell growth,and relatively conserved in Ceratitis capitata,Rhagoletis zephyria,Bactrocera latifrons,Bactrocera dorsalis,Bactrocera oleae,Rhagoletis pomonella and Bactrocera tryoni.A total of 14 Y-specific candidate genes which were most likely to affect the reproductive development of B.dorsalis were further screened out by annotation analysis,coding region prediction analysis,IGV analysis and other bioinformatics methods.In addition to the reported gyf,Mo Y and spermless gene,the remaining 11 genes were new Y-specific candidate genes,including 0245,0820,1522,3851,4657,5089,5661,9632,9681(Bdyof gene),2703 and 5354 gene.The 11 candidate genes were confirmed to be Y-specific by PCR amplification using the female and male genomic DNA as templates,respectively.The accuracy of these 11 genes de novo assembly prediction was confirmed by sequencing.2.The Y-specific gene Bdyof was cloned and analyzed.It has no intron and no homologous sequence in NCBI.Bdyof gene can encode a small protein(97 amino acid).Protein prediction results showed that Bdyof gene might be related to protein transport.The expression pattern of Bdyof gene by Quantitative Real-time PCR(q PCR)suggested that Bdyof gene was highly expressed in testis tissues from 1 d to 15 d after eclosion,but Bdyof gene was not expressed in ovaries.At different developmental stages of egg,the Bdyof gene was highly expressed at 6-9 h,and substantially decreased after 9 h.In adulthood,the Bdyof gene was highly expressed in male,but not expressed in female.Bdyof gene was marginally expressed in pupa and larva developmental stage.RNAi of Bdyof gene resulted in smaller testis of male B.dorsalis compared with control(ds EGFP injected)male.The expression of dsx-M gene was significantly lower than that of control male after interference of Bdyof gene,but the expression of tra-M gene was unchanged.Bdyof gene was knocked out by CRISPR/Cas9,and a homozygous mutant line of Bdyof gene was established(only for male).The egg hatching rate of homozygous mutant line was 11.0%,which was 52.6% lower than that of wildtype.The testes of homozygous mutant were abnormal(smaller,deformed,translucent).The death rate of sperm increased significantly and the number of sperm were significantly decreased.The expression of dsx-M gene in homozygous mutant was significantly lower than that in wildtype male but the expression of tra-M gene was unchanged.In addition,some of the mutant individuals had obvious yellow spots on the left and right sides of the abdominal segments 4-5,which was the same as the phenotype of dsx gene knockout in B.dorsalis.Knockout of Bdyof gene did not affect the sex ratio of offspring from mutant male,and no sex reversal or intersex individual was observed.These results suggest that Bdyof gene plays an important role in the development of testis and spermatogenesis of B.dorsalis,and Bdyof gene may interact with dsx-M gene as a downstream gene of sex determination pathway.