Functional Properties Of Bombyx Mori BmMLs Protein Family And Its Association With Pheromones

As an important economic animal,silkworms are involved in lipid metabolism from chemical communication,odor recognition,and anabolic processes of ecdysone.The lipid-binding protein superfamily that recognizes lipid substances is widely distributed in organisms such as plants,animals,and fungi,and mediates different biological functions by interacting with specific lipids.ML family proteins are classified into three categories: Myeloid differentiation protein-2(MD-2)family,Nieman-pick type C2(NPC2)family and ML-related lipid recognition(ML)family.In vertebrates,MD-2 proteins mainly recognize lipid polysaccharides(LPS)assisted TLR4-induced downstream immune response;NPC2 proteins are mainly involved in cholesterol transport and metabolism.There are eight Bm ML protein sequences in the silkworm genome.They are named Bm NPC2 a,Bm NPC2 b,Bm Esr16,Bm ML-1,Bm ML-2,Bm ML-3,Bm ML-4 and Bm ML-5 in the silkworm genome database.In order to investigate the biological functions of BmMLs family members,this study preliminarily explored the tissue-specific expression characteristics,the function of binding to lipids and participation in antennal pheromone recognition of BmMLs in silkworm through bioinformatics analysis,fluorescence quantitative RT-qPCR,ELISA and RNAi experiments.(1)Bioinformatics analysis of BmMLs based on silkworm genome database revealed that Bm Esr16 and Bm ML-1 were closely related to NPC2 in mammals;structural prediction revealed that all eight Bm ML proteins had 7-8 beta-folds,and that the beta-fold layer directly formed hydrophobic binding chambers,predicting that they had the ability to bind lipids.(2)The tissue-specific expression of BmMLs in silkworm was detected by RT-qPCR.It was found that Bm NPC2 a was highly expressed in all tissues.Bm NPC2 b,Bm Esr16,Bm ML-1 and Bm ML-2 expressed differently in different tissues,while Bm ML-3,Bm ML-4 and Bm ML-5 was lowly expressed in all tissues.The transcriptional activity of eight members of the BmMLs gene family was higher in the antennae of adult silkworm,and the transcriptional expression of Bm ML-3 was different in females.(3)The full length of BmMLs gene was amplified by PCR and cloned into eukaryotic expression vector.The gene was transfected into S2 cells for eukaryotic expression and purified by Ni-NTA affinity chromatography.The purified proteins and lipids were tested by ELISA.Finding that Bm NPC2 a,Bm NPC2 b,Bm Esr16 and Bm ML-1 proteins had certain binding capacity with LPS,20 E and Cholesterol.Bm NPC2 a showed high binding capacity.(4)three-day-old 5th-instar silkworm larvae was injected with Cholesterol,20 E and LPS and were detected the transcriptional expression of BmMLs by RT-qPCR.It was found that all BmMLs were up-regulated by Cholesterol,while Bm ML-1 was not up-regulated by 20 E and LPS,and other genes were up-regulated.(5)After interfering with Bm NPC2 a,Bm NPC2 b,Bm ML-1 and Bm ML-3 genes of male adult silkworm by RNAi technology,abnormal mating phenomena appeared in Bm NPC2 a and Bm ML-3 interference groups,suggesting that Bm NPC2 a and Bm ML-3 may participate in the recognition of pheromones during mating of adult silkworm.The results showed that eight BmMLs expressed in silkworm tissues specifically.According to their binding with lipids,it was inferred that they played different biological functions in silkworm.RNAi experiments suggest that Bm NPC2 a and Bm ML-3 may be involved in the binding of pheromones in adult worms.The specific molecular biological mechanism needs further study to elucidate.