| The Zeugodacus cucurbitae is an important quarantine pest with worldwide distributions,it has strong reproductive ability and damages a wide range of areas.The adults lay eggs inside the fruit,and the larvae hatch and feed in fruit,causing flower and fruit drops.As an important part of the male reproductive system of Z.cucurbitae,the testis is directly and closely related to the reproduction and development of its offspring.The excavation of new technologies and targets for reproduction-related pests control has always been the focus and hotspot of the field of entomology.Testisspecific serine/threonine kinase gene is a family of critical genes that have been well studied in mammals but limited in insects.Accordingly,in this dissertation,transcriptome data of male tissues of Z.cucurbitae was sequenced.Two TSSK family genes(ZcTSSK1 and ZcTSSK3)highly expressed in the testis were screened and identified.Their open reading frame sequences were obtained through cloning,and the temporal and spatial expression pattern of ZcTSSK1 and ZcTSSK3 was analyed;in situ hybridization was used to localize analyze the expression sites of ZcTSSK1 and ZcTSSK3 in the testis of Z.cucurbitae adults.The physiological functions of ZcTSSK1 and ZcTSSK3 in the male reproduction process of Z.cucurbitae were further explored by RNAi technology.The research results will help understand TSSKs’ roles in the fruit fly and provide targets and foundations for developing environmentally friendly insecticides and sterility technologies for pest control.This will help develop green and ecological pest control strategies.The main results of this dissertation are as follows:1 Transcriptomic sequencing of tissues from male adult of Z.cucurbitaeThe transcriptomic sequencing of 16 tissue samples of male Z.cucurbitae was conducted.The clean data for each sample was > 6.16 Gb,and the percentage of Q30 bases was > 90.91%.The clean reads of each sample were aligned with the designated reference genome.The results showed that midgut sample has a lower mapping rate,which is 59.59%,the mapping rate of the other tissues were all more than 90%.Based on the quantitative results of expression levels,a total of 5343 differential genes between the testis and the three tissues of the malpighian tubules,fat body,and midgut of Z.cucurbitae were analyzed and screened.Among them,3130 genes were highly expressed in the testis.After increasing the screening fold,801 genes were highly expressed in testis,and these genes were functionally annotated.Eight genes were screened out from the testis as highly expressed genes,and quantitative data verification was performed with RT-q PCR,and the results showed that the q PCR data and transcriptome sequencing data have the same trends.The gene expression level in testis was significantly different from other tissues.Two TSSK family genes were screened and identified in the testis as highly expressed genes,i.e.,ZcTSSK1 and ZcTSSK3.2 ZcTSSK1 and ZcTSSK3 identificationThe full-lengths of the open reading frames of ZcTSSK1 and ZcTSSK3 were 900 bp and 1059 bp,encoding 299 and 352 amino acid residues.The proteins encoded by both genes have no signal peptides and transmembrane domain,and the molecular weights are 33.77 k Da and 39.66 k Da,respectively.Based on multiple sequence comparisons,the amino acid sequences of ZcTSSK1 and ZcTSSK3 have high homology with TSSKs of other species,and they have a conserved S-TKc domain,which contains a large number of identical amino acid residues.Most of the residues associated with ATP or substrates are well preserved.The phylogenetic analysis showed that ZcTSSK1 and ZcTSSK3 clustered with TSSK1 and TSSK3 of Bactrocera dorsalis.3 Analysis of expression patterns of ZcTSSK1 and ZcTSSK3Using RT-q PCR technology,the expression patterns of ZcTSSK1 and ZcTSSK3 in Z.cucurbitae various developmental stages,different ages of adults and different tissues were analyzed.The results showed that ZcTSSK1 and ZcTSSK3 were expressed at the pupal and male adult stages but not expressed at other stages.The expression patterns of ZcTSSK1 and ZcTSSK3 tended to be stably expressed in male adults of Z.cucurbitae,and the expression patterns in different tissues indicated that ZcTSSK1 and ZcTSSK3 were specifically and highly expressed in the testis.In situ hybridization results showed that both genes were expressed in the transformation zone of the testis,and the signals were mainly enriched in mature sperm cells.The expression region is connected to the vas deferens of the testis,which is the transformation and formation region of mature sperm(sperm cells transformed into flagellated sperm).4 Analysis of the involvement of ZcTSSK1 and ZcTSSK3 in regulating the male reproductive functionUsing RNAi technology,gene-specific ds RNA was synthesized in vitro.The target gene was silenced by microinjection,and the data showed that the ds RNA synthesized in vitro can effectively interfere the expression of target genes.Subsequently,continuous feeding was used to explore its physiological functions.After the newly emerged male adults of Z.cucurbitae were fed with ds RNA for nine days,the expression levels of ZcTSSK1 and ZcTSSK3 were down-regulated by 68% and 76%,these show that the feeding method can effectively silence the target gene.After 9 days,the treated males were mated with normal-fed females of the same age,and the number of egg-laying and the hatching rate of eggs within five consecutive days were counted.The results showed that the number of eggs produced by females mated with RNAimales was not significantly different from that of the control group,but the egg hatching rate was significantly reduced.Further analysis of the sperm counts showed that the sperm counts of males treated with ds ZcTSSK1 and ds ZcTSSK3 were significantly reduced by 61.23% and 47.32%.The results suggested that inhibition of ZcTSSK1 and ZcTSSK3 can significantly affect the spermatogenesis of Z.cucurbitae,reduce the number of sperm,and affect the fecundity of male adults. |
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