| The oriental fruit fly,Bactrocera dorsalis(Hendel),belonging to Tephritidae of Diptera,can severely infest many fruits and vegetables.The broad host range(～450 plant species),adaptability and rapid development of resistance to pesticides are among the reasons why B.dorsalis is a devastating pest to fruits and vegetables.ATP-binding cassette transporter(ABC transporter)is a kind of protein that can use ATP to promote the reverse concentration gradient transmembrane transport of substances.It can transport a large number of substrates,such as hormones,lipids,toxic metabolites and drugs,then affect the physiological and biochemical processes,growth and development,detoxification and metabolism of insects.In addition,there are many members of ABC transporters,and the functions of each subfamily are greatly differentiated,therefore it is necessary to explore their functions in depth and systematization.In this study,the ABC transporter G subfamily genes(BdABCGs)of B.dorsalis were identified,and 15 BdABCGs were obtained.Based on the analysis of their molecular characteristics,spatiotemporal expression patterns and their expression patterns in response to stress were comprehensively analyzed.Additionally,RNA interference(RNAi)and CRISPR/cas9 technology were used or established to explore the function of the target gene.The main results of this dissertation are as follows:1.Bioinformatics and spatiotemporal expression pattern analysis of ABCGs genes in B.dorsalisBioinformatics analysis was used to identify the molecular characteristics of 15 genes encoding the ABC transporter G subfamily BdABCGs(BdABCG1-BdABCG15).The amino acid sequences of the 15 BdABCGs have high degree of similarities with the corresponding genes of Bactrocera latifrons and Bactrocera oleae,reaching more than 90%.The similarities of ABCGs amino acid sequences between B.dorsalis and Drosophila melanogaster were among 49.2%-90.5%.All the 15 proteins have typical structural characteristic sequences of ABC transporter subfamily,including ABC transporter signature motif,Walker A/P-loop,ATP binding site,Q-loop/lid,Walker B,D-loop,H-loop/switch region and 5-7transmembrane helix.The 15 BdABCGs contain one TMD and one NBD,respectively,which are arranged in a reverse pattern of NBD-TMD.They are typical half transporters,which are need to formation homologous or heterodimer for performing their transport functions.Phylogenetic analysis showed that 15 BdABCGs belong to G subfamily,and have close evolutionary relationship with ABCA and ABCH subfamily members.The expression patterns of 15 BdABCGs in different developmental stages were determined by qPCR.It was found that BdABCGs were expressed throughout the development process,and most of the genes were highly expressed in white pupae and newly emerged adults.Except for BdABCG1 and BdABCG8,most of these ABCGs were highly expressed in eggs,sexually mature females and ovaries,and most of these genes in males were significantly higher expressed than those in females.In addition,the expression patterns of pigment transport related genes in different developmental stages of compound eyes were consistent with the development of compound eyes.BdABCGs were mainly expressed in the Malpighian tubules of adults or larvae,fat bodies of adults and midgut of larvae.In conclusion,all above results suggest that BdABCGs have multiple functions and may be involved in detoxification metabolism,growth and development,pigment transport and other processes.2.Expression of BdABCGs under different environmental stress in B.dorsalisIn this study,qPCR was used to analyze the m RNA expression of BdABCGs under insecticide treatment,high and low temperature treatment,starvation and desiccation treatment,as well as in warmand cold-acclimation strains.The expressions of these BdABCGs to the above environmental stresses were different in some extent.BdABCG1,BdABCG3 and BdABCG8 were significantly up-regulated after treatment with avermectin,and BdABCG14 was significantly up-regulated after treatment with malathion and avermectin,suggesting that these genes may be involved in the physiological process of coping with the above two insecticides stress.After starvation,desiccation and low temperature treatment,most of BdABCGs were significantly up-regulated.Only one gene was significantly up-regulated in warm-acclimation strain,but no gene was significantly up-regulated in high temperature treatment.These results indicated that the response of BdABCGs is more sensitive to low temperature than that to high temperature.It is worth mention that BdABCG6 was significantly up-regulated in short time of low temperature treatment,warm-acclimation strain,cold-acclimation strain,starvation and desiccation treatment,suggesting that BdABCG6 may play an important role in stress resistance.In conclusion,most of the BdABCGs(10)were significantly up-regulated after different stress,indicating their important roles in the process of resistance in B.dorsalis.3.Detection of RNAi efficiency and phenotypic observation of BdABCGs in B.dorsalisThe usage of RNAi technology is very limited in the gene functional studies of B.dorsalis,and which are mainly due to the inability or instability of silencing efficiency of most genes.In present study,ds RNA of BdABCGs were injected into 5-day-old adults,and the silencing efficiency of the target gene was detected after 24 hours.The results showed that the silencing efficiency and stability of 10 BdABCGs were different.The expression levels of BdABCG3,BdABCG4,BdABCG6,BdABCG10 and BdABCG11 were significantly decreased stably compared with the control group(ds GFP).Meanwhile,these genes can be also silenced in larvae,while other BdABCGs i.e.,BdABCG1,BdABCG2,BdABCG5,BdABCG8,BdABCG15 could not be effectively silenced or the silencing efficiency was not stable.Interestingly,we found that those BdABCGs highly expressed in fat body usually have more stable effective RNAi efficiency.At 3,5 and 7 days of age,ds RNA of BdABCG4,BdABCG6 and BdABCG11 were injected into adult females of B.dorsalis,and the egg laying amount decreased significantly,suggesting that these 3 genes were related to the reproduction of B.dorsalis.4.Construction of BdABCG2 homozygous mutant strain of B.dorsalis based on CRISPR/cas9 technologyA homozygous mutant strain with stable inheritance of BdABCG2 was constructed using CRISPR/Cas9 technology.The ratio of mutants from G0 generation to adult was 25.3%(22/87).Results of genotyping showed that there were 4 kinds of deletion fragment genotypes(-5 bp,-6 bp and two kinds of-10 bp).Homozygous mutants with-10 bp deletion were successfully obtained in G3 generation after mutation screening.The expression of BdABCG2 gene in homozygous mutants was significantly reduced at the transcriptional level and maintained at a low level,indicating that gene editing effectively reduced the expression of this gene.In view of the correlation between ABCG2 gene and compound eye coloring in D.melanogaster,we observed the color of B.dorsalis mutant strain by super depth of field microscope in this study,and found that the eye color of BdABCG2 homozygous mutant and heterozygous mutant strains did not have significant changes.There was no significant difference in the contents of drosopterins pigments and xanthommatin between the homozygous mutant and the wild type,It is speculated that the function of BdABCG2 involved in the transport of ocular pigment precursors has changed.Comparative transcriptome analysis of BdABCG2 homozygous mutants and non-mutants showed that there were 379 differentially expressed genes.The differentially expressed genes mainly focused on metabolism.It was speculated that BdABCG2 might be involved in other physiological functions related to metabolism. |
