Annotation And Functional Study Of Sex-determination Genes In Bemisia Tabaci

As a major worldwide agricultural pest,Bemisia tabaci has the characteristics of wide host range,high reproduction rate,good adaptability and high risk,which is difficult to be controlled.The long-term chemical control leads to the resistance of B.tabaci.It is urgent to propose new methods of control and develop related basic theoretical research.Insect sex determination is the result of long-term evolutionary selection,which affects the development and reproduction of insects.This research used the genome and transcriptome of B.tabaci Q,annotated and cloned all possible sex determination genes(SDGs)in B.tabaci,and analyzed the gene expression patterns and alternative splicing(AS),and on this basis,used RNAi technology for function verification.It can lay a theoretical foundation for screening potential targets and developing new ways for effective B.tabaci control.The main results are as follows:The combined genome-and transcriptome-wide analyses identified 26 genes putatively associated with sex-determination in B.tabaci Q.Among these genes,PSI,tra2 and dsx were documented previously and the other 23,including tra,fru,and Sxl,were first identified.Comparative genome analysis indicates that SDGs showed species-specific.For example,tra is absent in A.pisum,N.lugens,A.aegypti and B.mori.her only exists in Drosophila melanogaster,while sisC and sisA are specific for D.melanogaster and M.domestica.The temporal profiles of 26 SDGs throughout B.tabaci developmental stages displayed a consistent expression pattern: the highest transcript levels were detected in eggs(21 genes,80.8%)and the lowest in adults(24 genes,92.3%).In addition,15 genes(57.7%)showed significant differences between the two sexes.The expression patterns were further validated by RT-qPCR,in which 92.3% of these genes were consistent with RNA-seq.The preliminary study of AS events of 26 SDGs in B.tabaci were investigated using female/male transcriptome data,the results showed da,dsx,Imp,PSI,tra2 and mle were different in the female/male transcriptome.PCR validation showed da,dsx,PSI and mle have sex-specific isoforms in B.tabaci adults,while Imp,tra2 does not.Traditionally,AS can be classified into four categories: intron retention(IR),exon skipping(ES),alternative acceptor site(AA),and alternative donor site(AD).In B.tabaci,ES was validated for dsx,tra2,PSI and da,suggesting a common splicing mechanism as well.The BtPSI gene was cloned by using the transcriptome data and RACE technique.The ORF of this gene is 2208 bp encoding for 735 amino acids and exhibits structural features characteristic of known insect PSI.A total of 92 BtPSI splice variations were found in B.tabaci adults,of which 70 are female-specific,14 are male-specific.The RNAi experiments showed that BtPSI was involved in regulating the expression of Vg gene,but it was not related to the transcription regulation of dsx.In addition,silencing BtPSI did not affect the fecundity of females and the sexual characteristics of two sexes.The Btix gene was cloned by the annotation information.The ORF of this gene is 546 bp encoding for 181 amino acids and has a conservative structure region of typical IX protein: Med29.A total of 4 Btix splice variations were found in B.tabaci adults,of which only Btix1 and Btix2 have complete Med29 domain.RNAi experiments showed that: 1)silencing Btix did not change the AS and mRNA expression level of dsx;2)silencing Btdsx had no effect on the expression of Btix;3)silencing Btix led to a significant decrease in the oviposition of the RNAi females;4)silencing Btix occasionally appeared a female with an abnormal abdomen.The results indicated that Btix could be used as a potential target gene for B.tabaci control.Based on annotation and transcriptome data,we identified two transcripts of fru,fru-a and fru-b in B.tabaci adults.The fru-a contains a 1263 bp ORF that codes for 420 amino acids.The fru-b contains a 1143 bp ORF that codes for 380 amino acids.For all the insects in our analysis,Fru contain BTB domain and most zinc fingers,and the zinc finger of fru-a and fru-b belongs to ZnA and ZnG respectively.Both fru-a and fru-b showed high expression in the egg stage and showed significant differences in B.tabaci females and males.AS analysis showed that,fru-a and fru-b both have sex-specific splicing in B.tabaci adults.Compared the fru and dsx between B.tabaci and Nasonia vitripennis,they all have the putative TRA/TRA-2 binding sites,suggesting that fru-a and fru-b may also be regulated by tra and tra2.