Functional Analysis Of The New Gene Tssor-2 In Plutella Xylostella

The diamondback moth(DBM),Plutella xylostella(L.),is a worldwide migratory pest that attacks cruciferous plants.The strong reproductive capacity of P.xylostella is one of the important internal causes of its population outbreak.New genes are speciesspecific genes that have no homology to other species.Most studies have shown that new genes can participate in the reproductive regulation of organisms and have an important role in the adaptive evolution of males.Therefore,we can identify some new genes that can regulate the reproduction of P.xylostella.In this study,we identified one new gene that encodes the seminal protein of P.xylostella,named Tssor-2.We analyzed the expression patterns of Tssor-2 including different development stages and different tissues.Besides,we used CRISPR/Cas9 to knock out Tssor-2 and statistically analyze the changes of the phenotype,which the main results are listed below:The full sequence length of Tssor-2 is 522 bp without intron.It encodes a protein with 173 amino acids with the first 20 amino acids are signal peptides.This protein does not contain transmembrane domains.The results of real-time quantitative PCR showed that the new gene Tssor-2 was highly expressed during the male adult stage.Tissue-specific expression profiles showed that Tssor-2 was highly expressed in the accessory gland which is an important tissue for activating sperms.This result was also confirmed by immunofluorescence result.CRISPR/Cas9-mediated Tssor-2 knockout created a homozygous mutant of Tssor-2 with two base pairs(2 bp)deletion.The results of immunofluorescence and LC/MS confirmed the deletions.It was found that the main reproductive organs of the mutant male had no obvious morphological or size change comparing with the wild type.However,when mated with wild-type females,the number of offspring eggs and hatchability of mutant males on the second day were significantly lower than those of wild-type males.Based on the multiple mating behavior of P.xylostella,further research found that sperm of wild-type male diamondback moth were significantly more competitive than sperm of mutant male diamondback moth that lacked Tssor-2.In order to understand the regulation mechanism of new gene Tssor-2 in males,we analyzed the metabolic pathways and regulatory networks involved in Tssor-2 based on RNA-seq.The results of KEGG and GO indicate that the new gene Tssor-2 is involved in the metabolic pathways related to the male P.xylostella spermatogenesis,including TCA cycle,glycolysis,lipid metabolism pathways and insulin metabolism pathways.We speculated that the protein encoded by Tssor-2 is an important auxiliary secretory protein that can affect male fertility and sperm competitiveness,which may regulate male reproduction through insulin metabolic pathway,fatty acid metabolism and sugar metabolism.In this study,we cloned a new gene Tssor-2 of P.xylostella and explained the role of the new gene in the reproduction of male P.xylostella.Our results provide a new clue for further studies of new genes in P.xylostella and other species.This study also provides new insights into the male adaptive evolution of diamondback moth,and provides the basis for the new specific targets for diamondback moth control.