The Mechanism Research Of The Silkworm Olfactory Receptor Gene BmOR56 In Feeding Habits Regulation

Feeding is a complex behavior of insects,in which the olfactory system plays an important role.Bombyx mori is an oligovorous insect,that is greatly attracted by mulberry leaves,and that is closely related to the volatile cis-jasmine.BmOR56 is the receptor of cis-jasmine.In order to study the role of BmOR56 in domestic silkworm feeding,the BmOR56 gene was knocked out using CRISPR/Cas9 technology and two homozygous mutant lines were obtained through multigeneration screening,and the feeding behavior of silkworm was analyzed by feeding artificial diet and other foods.At the same time,the differentially expressed genes related to silkworm feeding habits and their functions were screened out based on the results of transcriptome analysis.The results are as follows:1.Two target gRNA were designed,and the BmOR56 gene was knocked out by CRISPR/Cas9 system.Through multi-generation screening identification,two BmOR56homozygous mutant lines（mutant 1 and mutant 2）were obtained.Mutant 1 showed 2 bases were exchanged by 113 bases at site 1,while no changes were observed at site 2.Mutant 2showed 4 bases were deleted at site 1,and 25 bases were deleted at site 2.Nonsense mutations in BmOR56 occurred in both mutant lines,resulting in complete loss of function.2.The expression profile of BmOR56 gene was analyzed.BmOR56 gene was mainly expressed in testis,head and epidermis,a small amount in ovarian tissue,and almost no expression in other tissues.From the developmental stage,BmOR56 gene was expressed at the highest level at the beginning of silkworm,and also at the end of the 4^th instar and the 3^rdday of the 5^th instar,but it was not expressed at the rest and mature stages.After BmOR56gene was knocked out,the expression of BmOR56 gene in testis,head and epidermis of mutant 1 and mutant 2 were significantly down-regulated,and the expression was also significantly down-regulated at the beginning of 5^th instar and the 3^rd day of 5^th instar.3.The transcription level of BmOR56 and olfactory receptor related genes were detected by q RT-PCR.Both BmOR56 and Bm Orco genes were up-regulated at 48 h and 96h after the two mutants emerged,and the expression of several ionotropic receptors was also significantly up-regulated,suggesting that the loss of BmOR56 may trigger other compensatory mechanisms.4.The feeding analysis of the BmOR56 homozygous mutant line of Bombyx mori.The results of the feeding behavior analysis showed that there was no significant difference between the wild-type silkworm and the mutant silkworm fed on mulberry leaves in terms of the setae dispersion rate,the newly exuviated larval rate and the body weight.The setae dispersion rate of the mutant silkworm fed on artificial diet were significantly lower than those of the wild type silkworm.In addition,mutant silkworms could feed on apples,pears,corn,lettuce and cabbage in varying degrees,which showed that compared with wild-type silkworms,the olfactory sensitivity of the mutant silkworms was weakened,and the feeding range was expanded.5.Screening and identification of differentially expressed genes in the antennae of the mutant 2 and the wild type silkworm Qiufeng on the 3^rd day of the 5^th instar.Transcriptome analysis showed a total of 1282 differentially expressed genes（DEGs）,of which 547 genes were down-regulated and 735 genes were up-regulated.Pathway analysis showed that these DEGs were mainly involved in binding activity,catalytic activity,structural molecular activity,transporter activity and metal ion binding.Further analysis of pathway genes showed that there were differences in the expression of a large number of genes related to feeding,protein synthesis and energy metabolism.In conclusion,we preliminarily speculate that the BmOR56 gene is closely related to the regulation of silkworm feeding,and there may be other compensatory mechanisms to maintain the silkworm’s preference for mulberry leaves.The weakening of mutant silkworm’s olfactory sensitivity leads to the expansion of its feeding range,indicating that BmOR56 can be used for silkworm breeding,especially the breeding of polyphagous feeding silkworm varieties.