| Plutella xylostella,a species of Lepidoptera,is a worldwide migratory pest that seriously harms cruciferous plants and causes huge economic losses to global agricultural production.Fecundity is an important factor in the rapid expansion and outbreak of P.xylostella populations.Therefore,it is of great significance to analyze the molecular mechanisms of its reproductive development to control the population growth.Egg shell formation plays a vital role in insect oogenesis and embryo development.Insect egg shells are mainly divided into inner and outer egg shells,which is the vitelline membrane layer,and the waxy layer with hardened egg shell.Compared with the outer egg shell,there are relatively few studies on the vitelline membrane,mainly in model insects such as Drosophila melanogaster and Bombyx mori.At present,the study on the formation of vitelline membrane in P.xylostella is still blank.To identify the role of vitelline membrane gene in egg formation and embryo development of P.xylostella can provide a potential molecular target for P.xylostella control.Therefore,we analyzed the molecular characteristics and evolutionary relationship of the vitelline membrane protein gene(VMP26),and clarified the expression pattern of PxVMP26 in the different developmental stages and different tissues based on the mRNA and protein levels.Based on this,the PxVMP26 gene was successfully knocked out using the CRISPR/Cas 9 technology,and the biological characteristics,such as egg formation and production,hatching rate,embryonic water retention,and vitelline membrane formation were analyzed.The main results are as follows:The PxVMP26 is a single exon gene contained an ORF of 852 base pairs(bp)encoding 283 amino acids.The theoretical molecular weight is 26 k Da.Phylogenetic tree analysis showed that the PxVMP26 is most closely related to Bm VMP30 of B.mori,but overall,it is less conservative.It is speculated that the evolution rate of VMPs is higher and its function may be difference among different species.The results of qPCR and Western blot analysis of PxVMP26 in different developmental stages showed that mRNA and protein of PxVMP26 were both specifically expressed in female adults.The expression began within 24 h of emergence,reached its peak at 48 h,and gradually decreased at 72 h.Tissue-specific expression profiles showed that PxVMP26 gene and protein were specifically expressed in the ovary of P.xylostella adult,indicating that PxVMP26 gene plays an important role in the reproductive development of P.xylostella females.Based on CRISPR/Cas9 technology,target sites were designed at 292-311 and320-339 bp regions of the PxVMP26 gene.The homozygous strains of 8 and 46 bp frameshift mutation were obtained.The knockout efficiency was verified by immunofluorescence and Western blot.The results showed that No PxVMP26 protein was detected in the mutant strains(Mut),while that was normal in the wild-type strain(WT),indicating that the PxVMP26 gene was successfully knocked out.At the same time,the PxVMP26 transcripts were effectively suppressed after VMP26 knockout.Biological analysis showed that PxVMP26 gene knockout had no significant effect on the number of laid eggs of P.xylostella,but it could significantly reduce the egg hatchability.In addition,PxVMP26 gene knockout caused abnormal vitelline membrane formation in P.xylostella.The egg length was significantly shortened,and the water retention of the embryo was reduced,indicating that the PxVMP26 gene is involved in the egg formation and embryonic development of P.xylostella.In this paper,the molecular characteristics and expression patterns of PxVMP26 gene were identified,and the role of PxVMP26 gene in egg formation and embryonic development of P.xylostella was first investigation by using CRISPR/Cas9.The results can provide basic data for the analysis of the reproductive regulation mechanism of P.xylostella and help to screen new genetic-control target for P.xylostella population. |
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