| Male accessory glands(MAGs)play important roles in the mating process of insects,and their secretions enter into the female together with sperm,which in turn affect the reproductive behavior of female insects.With the rapid development of sequencing technology,the identification of male accessory gland secretion proteins has gradually become a hot spot in life science.Takeout is a secreted protein that can bind small lipid granules and exhibits a pronounced male bias in insect expression,and contains specifically expressed takeout in male accessory glands.Zeugodacus cucurbitae(Coquillett)is an important agricultural quarantine pest that seriously infests fruit and vegetable production.It belongs to the Tephritidae,Diptera.It has a wide host range and strong fecundity and is widely distributed in tropical,subtropical and temperate regions,mainly harming cucurbit and nightshade crops.The identification of the male accessory gland proteins(ACPs)of Z.cucurbitae and the functional study of the important protein takeout will play an important guiding role in developing the control strategy of Z.cucurbitae based on reproductive regulation.This dissertation took the Z.cucurbitae as the research object,used transcriptome and proteome sequencing technology to obtain the whole transcriptome and proteome data of male accessory glands,and then used bioinformatics analysis to identify the takeout family genes,comprehensively using quantitative real-time PCR(RT-q PCR),western blot(WB),immunohistochemistry,RNAi and CRISPR/Cas9,the physiological function of the takeout specifically expressed in the MAGs of the fruit fly was determined.The main results are as follows:1.Transcriptomic and proteomic sequencing of male accessory gland in Z.cucurbitae.Based on the whole transcriptome data of MAGs of Z.cucurbitae,a total of 12,958 transcripts were obtained.The sequence alignment rate of the clean reads of each sample and the melon fly genome reached more than 94%,and the correlation between samples was more than 95%.Based on signal peptide prediction,700 genes with signal peptides were screened.Besides,1,863 proteins were identified based on the proteome sequencing data of the MAGs of Z.cucurbitae,and 242 secreted proteins were screened out according to the prediction results of signal peptides.Based on combined transcriptome and proteome analysis,68 secreted proteins were identified,functionally annotated and classified,and the results showed that most of them were enzymes,followed by binding proteins.Among the 68 proteins secreted by MAGs,the highly expressed proteins content serine protease,lectin and takeout.The top 10 genes and proteins expressed in the transcriptome and proteome were selected to analyze expression patterns in different development stages and tissues.The results showed that most of them were expressed in male adults and were specifically expressed in MAGs.2.Identification,sequence analysis,and the spatio-temporal expression of takeout genes in Z.cucurbitae.A total of 32 takeout family genes were identified through transcriptome and genome data,including 26 takeout and 6 JHBP.The open reading frames were 300 – 800 bp,encoding 100 – 300 amino acids,and all have signal peptides.The multiple sequence alignment results showed that the insect takeout proteins had high homology,with two conserved cysteine residues,four highly conserved glycine residues,and contained a conserved domain JHBP.In addition,the RT-q PCR results showed that Zctakeout1 was only highly expressed in the male adult stage,and the expression level gradually increased as the age increased of the male adult.The expression level of Zctakeout1 became stable until the sexual maturity stage.The expression patterns of different tissues showed that Zctakeout1 is specifically expressed in MAGs.Immunohistochemical analysis confirmed that Zctakeout1 was expressed in the two mesoderm epidermal accessory glands,and the signal was mainly enriched around the binuclear cells.3.Function of Zctakeout1 based on CRISPR/Cas9-mediated genome editing in Z.cucurbitaeAfter continuous feeding ds RNA of Zctakeout1 for 6 d,the expression of Zctakeout1 decreased significantly.The silencing efficiency was 76.29%,and the mating rate of Z.cucurbitae also decreased significantly.The results indicated that the gene might be involved in the reproductive behavior of Z.cucurbitae.The physiological function of Zctakeout1 was furtherly studied by using CRISPR/Cas9,and a homozygous strain with a deletion of 8 bp was successfully obtained.With mating with wild-type males,Zctakeout1 protein was detected in homozygous and wide females by Western blot.The behavioral observation result showed that the mating rate of the homozygous was significantly lower than that of the wild type.The number of eggs laid by females after mating with homozygous males was continuously counted for 7 days.The results showed that the number of eggs laid by wild-type females mating with homozygous males was significantly lower than that of wild-type females mating with wild-type males,and the number of eggs laid by homozygous females mating with homozygous males was also significantly lower than that of control,both decreased by about 50%.However,there was no significant difference in the hatching rate of eggs.These results indicated that the male adult of Z.cucurbitae can transfer the Zctakeout1 protein into the female with sperm during mating,thereby affecting female fertility. |
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