Study On Male Sterility And Regulation Mechanism Of Bombyx Mori ?Ser2 And Plutella Xylostella ?Hanabi

There are many different speices of insects,lepidoptera is the second largest item insecta,there about seventy percent severer insect pests,such as the Plutella xylostella,Spodoptera frugiperda,Spodoptera litura,etc.These pests have brought huge losses to our country even to the world,however,the silkworm as model species of lepidoptera insect has significant economic benefits in our country.In this study,Bombyx mori,a model insect of lepidoptera,and Plutella xylostella,an agricultural pest,were used as the research objects to elucidate the molecular mechanism of insect sterility by exploring the functions of reproductive genes.In this paper,two male sterility genes were selected as the target genes,?Ser2 sterility mutants were constructed in silkworm,?Hanabi sterility mutants were constructed in diamondback moth by using CRISPR/Cas9 technique respectively,by analyzing the difference between the sterile mutants and wild-type,the mechanism and gene function leading to sterility were further explored to provide molecular and theoretical basis for Sterile Insect Technique.The main contents and results of this study are as follows:1.Study on the ?Ser2 male sterility mechanism of Bombyx moriSer2 is a member of the serine protease family that belongs to the accessory gland proteins mediates silkworm sperm functions.In this study,CRISPR/Cas9 technique was used to construct Ser2 deletion mutants in Bombyx mori,and reproductive mating experiments were conducted to verify that Ser2 deletion resulted in male sterility.Spermatozoa dissection and immunofluorescence staining were carried out to verify that Ser2 deletion did not affect the spermatozoa occurrence and development of silkworm,the sperm morphology of mutant was normal at all stages,we also use the STEM verified that the inner structure is normal.Micro-CT in situ non-destructive 3D images showed that the mutant semen protein could be successfully transfered into the female bursa copulatrix.These results suggest that Ser2 deficiency in Bombyx mori does not affect male ejaculation behavior,but may affect sperm-egg bonding.2.Study on the ?Hanabi male sterility mechanism of Plutella xylostellaThe CRISPR/Cas9 technique was used to knockout Hanabi gene in Plutella xylostella,and it was confirmed by mating experiments that Hanabi deficiency would cause sterility in Plutella xylostella.The eupyrene and apyrene sperm of Plutella xylostella were observed by immunofluorescence staining,the eupyrene sperm of the mutant was confirmed to have scattered nuclei,the apyrene sperm is normal.This study suggests that Hanabi plays an important role in the reproduction of diamondback moth,and the deletion of Hanabi may affect the development of eupyrene sperm and lead to sterility in Plutella xylostella.In conclusion,CRISPR/Cas9 technology was used to edit reproductive-related genes in two representative lepidopteran insects respectively,knockout Ser2 in Bombyx mori and Hanabi in Plutella xylostella.The sterility phenotypes were further verified by reproductive mating experiments.Moreover,the sterility mechanism was explored from the perspectives of sperm development morphology and ejaculation behavior separately,so as to elaborate gene functions.The results showed that both Ser2 and Hanabi are important genes for reproductive development and influence lepidopteran male normal reproduction.These genes can be used as effective target genes for the application of Sterile Insect Technique,it provides a molecular theoretical background for the study of pest control methods from the perspective of population regulation strategy,and provides a new strategy for pest control.