Functional Study Of Reproductive Development-related Genes Based On CRISPR/Cas9 Against Bactrocera Dorsalis

Bactrocera dorsalis is a dangerous fruit and vegetable pest that has brought huge economic losses to agricultural production.The rapid development of CRISPR/Cas9gene editing technology has brought new development opportunities for the scientific prevention and control of agricultural pests.In this study,three target genes(boule,rhomboid,sex peptide receptor)related to reproductive development of B.dorsalis were used as objects.Using CRISPR/Cas9 gene editing technology to study its function and explore the feasibility of CRISPR/Cas9 in the study of B.dorsalis.To compare the effects of editing these genes on the reproductive function of B.dorsalis.The screening will establish the genetic control of B.dorsalis in the future,especially the green control technology based on CRISPR/Cas9 gene editing to provide target genes.The research results are as follows:1.Bdbol gene was cloned and its expression in testis was the highest,which was significantly higher than that in ovary.Two highly active sgRNAs(B-sg2,B-sg5)were screened for CRISPR/Cas9 knockout.In the mutant line G0,64.70%of the individuals had gene mutations,and the average mosaic rate of the mutant individuals reached 63.42%.The embryonic hatching rate of the mutant Bdbol~-male backcross G1 showed a significant decrease(Student's t-test,P<0.05),even no hatching at all.The sperm viability test showed that the sperm viability of Bdbol~-G1 and G2males decreased significantly,the number of live sperm decreased significantly,the number of sperm death increased significantly(Student's t-test,P<0.05),and the total number of sperm did not change significantly.The ratio of live sperm in Bdbol~-G1 and Bdbol~-G2 was 55.9%and 41.6%,respectively.The reproductive development system of mutant males was abnormal.Both Bdbol~-G1 and G2 generations,semitransparent and albino testicular tissues were observed,and testicular size and development were abnormal.2.The Bdrho gene was cloned and found to be significantly higher in males than in females,and testis is highly expressed relative to other tissues.Screened two sgRNAs(R-sg1,R-sg2)for CRISPR/Cas9 knockout.In the mutant line G0,41.67%of the individuals showed gene mutations,and the average mosaic rate of the mutant individuals was 48.45%.The embryonic hatching rate of the mutant Bdrho~-male backcross G1 showed a significant decrease(Student's t-test,P<0.05).The total number of sperm in Bdrho~-G1 and Bdrho~-G2 is significantly reduced,and the proportion of live sperm is significantly reduced(Student's t-test,P<0.05).3.Bdspr gene was cloned and found to be highly expressed in females compared with males.The high expression of Bdspr gene in tissues was mainly concentrated in the central nervous system(CNS)distribution-head,thorax and gut.Two sgRNAs(S-sg1,S-sg5)were screened for CRISPR/Cas9 knockout.In 55.87%of the individuals in the mutant line G0,gene mutations occurred,and the average mosaic rate of the mutant individuals was 33.92%.The total number of single female oviposition of mutant Bdspr~-females decreased significantly.The average number of eggs production by Bdspr~-females was 193.8,which was significantly different from that of wild type(504 eggs per head)(Student's t-test,P<0.001).In the ovarian tissue of the mutant Bdspr~-G1 female,the egg was yellow coated and the egg color was abnormal.The ovarian morphology was abnormal,but the spermatheca was normal.Bdspr~-G2embryos also have abnormal yellow eggs.This indicates that Bdspr gene plays a role in regulating ovarian development and egg granule status.4.Among the three candidate genes,we found that the individual mutations proportion(number of mutated individuals/total survival)of Bdbol gene was 64.70%.higher than(Bdrho gene,41.67%)and(Bdspr gene,55.87%).The average mosaic rate of the mutant individuals of Bdbol gene(number of mutant cells in mutant individuals/total cells)reached 63.42%,much higher than(Bdrho gene,48.45%)and(Bdspr gene,33.92%).Moreover,after knockout,the effect of Bdbol~-mutation on offspring is more pronounced.Therefore,among the three target genes,Bdbol gene has the best compatibility with CRISPR/Cas9 system.In addition,considering the successful application of sterile insect technique(SIT)in pest control,selection of genes targeting male sterility has better operability.The average hatching rate of Bdbol~-male offspring was 23.17%,which was also lower than that of Bdrho~-male offspring(28.33%).In summary,Bdbol gene has higher application potential as a target gene for green control and genetic modification of B.dorsalis.