| The cotton whitefly Bemisia tabaci(Gennadius)(Hemiptera: Aleyrodidae)is a phytophagous,piercing-sucking pest.It has a wide host range and feeds on more than 500 host plant species,including fruits,flowers,vegetables,and ornamental crops.The results of behavioral and molecular phylogenetic studies show that whiteflies are not a single species but a species complex containing at least 36 cryptic species.In China,the most damaging cryptic species of this complex are B.tabaci Mediterranean(MED)and B.tabaci Middle East-Asia Minor 1(MEAM1),which were formerly referred to as the Q and B’biotypes,’ respectively.Hepatocyte nuclear factor 4(HNF4),as a member of the nuclear receptor family,has highly conserved structural features and can regulate the expression of hepatocyte-specific genes and carbohydrate and lipid metabolism.In this study,based on the genome and transcriptome data of B.tabaci,a nuclear receptor gene was successfully cloned in B.tabaci and named Btab HNF4 according to its sequence characteristics,and its spatiotemporal expression and expression level under different humidity conditions were detected by real-time quantitative PCR.RNAi technology was used to explore the target gene’s biological function in insect environmental adaptability and female reproduction and further study the effects of nuclear receptor gene silencing on adult juvenile hormone content,egg production,hatching rate,ovarian development,and the relative gene of the juvenile hormone signal transduction pathway.1.Cloning and sequence analysis of Btab HNF4.The analysis results showed that the complete open reading frame(ORF)of the Btab HNF4 was 1302 bp,encoding 434 amino acids,and the predicted molecular weight and theoretical isoelectric point were 48.56 k Da and 4.79,respectively.The predicted amino acids are hydrophilic amino acids without a signal peptide or transmembrane domain,including one glycosylation site and multiple phosphorylation sites.The spatial structure prediction results showed that the predicted amino acid secondary and tertiary structures of Btab HNF4 were mainly composed ofα-helices and random coils.Amino acid sequence analysis showed a highly conserved N-terminal zinc finger DNA-binding domain(DBD)and a conserved C-terminal ligand-binding domain(LBD).The results of multiple sequence alignment showed that Btab HNF4 had a high degree of identity with the HNF4 of other insects.The results of phylogenetic tree analysis showed that the entire phylogenetic tree was divided into five major branches,among which Bemisia tabaci,Thrips palmi,Drosophila melanogaster,and Coccinella septempunctata gathered on the same branch,indicating that they are closely related and may have similar biological functions.2.The expression pattern of Btab HNF4 in different developmental stages,different tissues,and different humidity conditions was detected by using real-time quantitative PCR technology in B.tabaci.The results showed that the Btab HNF4 gene was widely expressed in different developmental stages of B.tabaci,but there were obvious differences,showing a fluctuating change rule.Among them,the expression level of Btab HNF4 was the highest in the female adult,followed by the egg stage,and the expression level was the lowest in the 1-2 and 3rd instar nymphs.In different tissues,the expression level of Btab HNF4 was highest in the abdomen,followed by the thorax,and the expression level in the head tissue was the lowest.In addition,under different humidity conditions,the expression level of the target gene increased with humidity.3.The biological functions of Btab HNF4 were verified by RNAi.The results showed that the expression level of Btab HNF4 was significantly lower than that in the control group,and the silencing efficiency was over 60%,indicating that the target gene was successfully silenced.The results of RNA interference in adults showed that after Btab HNF4 silencing,the mortality of adults under desiccation stress conditions was significantly increased in B.tabaci.However,in normal humidity conditions,there was no significant difference between the treatment and control groups,suggesting that Btab HNF4 plays an important role in the adaptation of adults to dry environments.In addition,the results of RNAi in females showed that Btab HNF4 silencing in females would significantly affect the female’s egg production,hatching rate,and ovary development,indicating that Btab HNF4 is involved in the female reproductive process.4.The effects of Btab HNF4 silencing on the content of juvenile hormone in females and genes related to the juvenile hormone signal transduction pathway in B.tabaci.The results showed that Btab HNF4 silencing would reduce the content of juvenile hormone in B.tabaci.It was speculated that Btab HNF4 silencing affected the biosynthetic pathway of juvenile hormone,thereby affecting the content of juvenile hormone.In addition,after silencing the target genes,the expression levels of Vg and Vg R genes were measured by a real-time quantitative method in B.tabaci.The results showed that the silencing of Btab HNF4 significantly affected the transcription level of the Vg in B.tabaci.The above results indicated that Btab HNF4 played an important role in the reproductive regulation of females.Therefore,we speculate that Btab HNF4 affects the reproductive process of females by regulating the content level of juvenile hormone in insects and the transcription level of key genes in the juvenile hormone signal transduction pathway.In summary,this study identified and analyzed the nuclear receptor based on the information in the B.tabaci genome and transcriptome database and clarified the expression level of the target gene in different developmental stages,different tissues,and different humidity conditions.The function of the target gene in adult desiccation tolerance and female fecundity was further explored by RNAi technology.These results can further enrich the functional research of nuclear receptor genes and lay a theoretical foundation for exploring the reproductive regulation mechanisms of insects. |
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