The Knockout Line Of Flotillin-2 Contructed By CRISPR-Cas9 In Laodelphax Striatellus(hemiptera: Delphacidae)

The small brown planthopper,Laodelphax striatellus,is one of most destructive pests in rice production in China.The insects not only cause the decreasing of rice production by directly feeding behavior,but act as insest vector of Rice stripe virus which is so-called“cancer”pathogen resulting rice stripe disease.RSV is a representative species of Tenuivirus and only transmitted by insect.RSV is a negative-sense ssRNA virus with four RNA strands.The concentration of CP protein encoded by RNA3 is positively correlated with the amount of virus,so its RNA3 and CP proteins can indicate the amount of virus.As persistent propagative virus,it can not only replicate in host plants,but also replicate in the small brown rice planthopper.Here are four invasive barriers when RSV transmit in the vector of vector with a persistent propagative manner:the first barrier is the midgut infection barrier.We envisage starting with the first barrier to reduce the entry of viruses into cells or reduce the spread of viruses between cells,thereby reducing the toxicity of the gray planthopper and the transmission efficiency of RSV.Most viruses enter the cell through endocytosis,and some viruses spread through the action of actin between cells.Flotillin-2 protein is involved in endocytosis and regulation of the actin cytoskeleton.It may be involved in the entry of RSV into the Laodelphax striatellus’s cells and the transmission between cells.We compared the high-virus Laodelphax striatellus with non-virus Laodelphax striatellus by qPCR.The expression of Flotillin-2 in high-virus planthopper is significantly higher than that of non-virus.Five days after feeding crude RSV,it was found that the expression of Flotillin-2 is significantly increased which is compared with the non-virus planthopper.It is concluded that the expression of Flotillin-2 is related to RSV.To further validate this phenomenon,this study transiently interfered with the Flotillin-2gene by microinjection of 3⁴ instar larvae of Laodelphax striatellus.Take appropriate amount of 3⁴ instar larvae with non-virus,and put them on rice which have been fed RSV for 10-15 days to seed.On the third day,the Flotillin-2 gene of the Laodelphax striatellus was interfered with dsRNA.After the dsRNA was injected,the small brown rice planthopper was placed on fresh rice seedlings for 3 days.By qPCR,compared with the interference GFP gene,the interference effect of the Flotillin-2 gene and the relative expression of the CP and RNA3of RSV were detected.The results showed that the relative expression of Flotillin-2 gene was significantly decreased after interference,and the relative expression of CP and RNA3 genes in RSV of Laodelphax striatellus was also significantly decreased.At the same time,it was confirmed by Western that the CP protein was almost not expressed after interfering with Flotillin-2,indicating that Flotillin-2 was positively correlated with the amount of virus.Because RNAi has the problem of off-target,temporary gene inactivation,and inability to completely knock out the gene,we used CRISPR/Cas9 gene editing technology to knock out Flotillin-2.First,we injected Cas9/sgRNA1 and Cas9/sgRNA2 into the eggs within 3 hours of hatching,respectively,and injected 150 eggs into each group to detect the mutation rate of the two sgRNAs to screen the sgRNA used in the formal experiment.Of the 21 eggs that survived the injection of Cas9/sgRNA1,12 were mutated with a mutation rate of 57.14%;among the16 eggs injected with Cas9/sgRNA2,12 eggs were also mutated with a mutation rate of 75%.Therefore,sgRNA2 was selected for subsequent experiments.Eggs of the Laodelphax striatellus were injected with Cas9/sgRNA2,and a total of 730 eggs were injected,27 eggs were hatched,and only 9 of them survived to the adult,and the survival rate was 1.2%.The mutated G0 generation is mated with the wild-type Laodelphax striatellus.Because the larvae are small in size,the detection of body tissues will affect their life activities.Therefore,the whole worm can be tested for genotype after mating and oviposition.After 4 generations of mating screening,two different genotypes of the Flotillin-2 knockout homozygous mutant line of Laodelphax striatellus were successfully constructed.After that,by performing qPCR,the mutant strain was compared with the wild type,the relative expression level of the homozygous mutant Flotillin-2 was significantly decreased when the primer of qPCR was set at the mutation site.After Western detection,it was found that the Flotillin-2 protein of the homozygous knockout strain was not expressed.The mutation was verified at the transcriptional level and protein level.After the construction of the Flotillin-2 knockout homozygous mutant line of the Laodelphax striatellus,the mechanism of interaction with the virus needs further verification.The deletion of the Flotillin-2 gene may contribute to the entry of RSV into the small brown rice planthopper’s cells and its proliferation,thereby reducing the efficiency of Laodelphax striatellus transmiting RSV.The mutants of Flotillin-2^-/-could heip to reveal the mechanism of replication and transmission of RSV in its insect vector.It is one important breakthrough in arbovirus studying area and the potential target to control RSV transmission.