Application Of Crispr/Cas9 In Gene Editing Of Bactrocera Dorsalis

The oriental fruit fly,Bactrocera dorsalis(Hendel),is one of the most destructive agricultural pests in many areas of the world.The application of insecticides to manage this pest has led to serious resistance problems;therefore,new ways to control B.dorsalis are required.The CRISPR/Cas9 system is a simple and efficient genome-editing technique that shows significant potential for the genetic editing and exploring gene function in non-model animals.The intensely studied white gene is widely used as a genetic marker in Drosophila melanogaster.Here,we cloned and characterized the white gene in an important pest of the fruit industry,Bactrocera dorsalis.We obtained Bd White knockout strains,based on the wild-type strain,using the CRISPR/Cas9 genome editing system.The white mutants lost pigmentation in the compound eye and their black head spots.The simplicity and high efficiency of CRISPR/Cas9 creates an opportunity to investigate gene function and design novel management approaches for B.dorsalis.Our results provide the first evidence,to our knowledge,that white may play a functional role in cuticle pigmentation by affecting the expression of yellow.The main findings were as follows:1.The results of spatiotemporal expression analysis showed that:(1)Bd White was widely expressed in all developmental stages except the eggs,and the highest expression level was found in mature males.(2)After testing Bd White in various tissues of adult B.dorsalis,it was found that Bd White had the highest expression in compound eyes and Malpighian tube,relatively high levels of expression were also detected in the testis,and the lowest expression levels in brain,gut,and ovary.2.The Cas9 protein with two sg RNA mixtures were injected into the B.dorsalis embryo respectively,and the genome was extracted for PCR detection.Bd White-g1 mutant and Bd White-g2 mutant had different types of mutations at the target site.Potential off-target sites were detected by PCR,and no off-target effect was detected.Compared with the wild type,the compound eyes of Bd White mutant emerged with a pigment deficiency,and their offspring had a full-white compound eyes.In addition,wild-type flies exhibited much darker head spots than Bdwhite mutants.3.To identify mechanisms underlying the differences in head spot coloration between wild type and Bdwhite mutants,we identified candidate genes for melanin pigmentation in B.dorsalis,comprising Bd-TH,Bd-DDC,Bd-ebony,Bd-tan,Bd-aa NAT,Bd-yellow1 and Bdyellow2.The expression differences of the identified genes between wild type and Bdwhite mutants were detected by q PCR.The results showed that the expression of Bd-yellow1 in the mutant head was significantly down-regulated,and there were no significant changes in other genes.Furthermore,the expression of Bd-yellow1 in the wild-type head was significantly higher than that of the abdomen.These results indicate that head spot color may be regulated by Bd-yellow1 and white may play a functional role in cuticle pigmentation by affecting the expression of yellow.