Identification Of Pinta Homologous Genes In Silkworm,prokaryotic Expression And Binding Activity With Retinoids

The Drosophila PINTA protein has a CRAL_TRIO domain,which can bind to derivatives of similar retinol substances such as retinal and retinoic acid.Studies have shown that the function of PINTA protein is related to the formation of vision and plays a role in Drosophila retinal pigment cells.PINTA combined with all-trans retinol is a necessary pathway for the formation of rhodopsin.The loss of pinta gene can cause damage to Drosophila photoreceptor cells,thereby affecting Drosophila vision.The pinta homologous gene in the silkworm genome has not yet been reported in the literature.In this study,four genes that may have similar functions to Drosophila pinta homologous genes were selected from the silkworm genome through bioinformatics analysis and cloning and sequencing as candidate genes for silkworm pinta,and four of the pinta homologous genes were detected in spatiotemporal expression patterns and prokaryotic expression,and soluble proteins were obtained in vitro.The binding ability of PINTA homologous proteins and similar retinol substances was tested,which provides a basis for further clarifying the biological functions of pinta homologous genes in silkworms and the role of carotenoid metabolic pathways in silkworms.The main findings are as follows:1.Identification of pinta homologous genes in silkworm,Bombyx moriUsing the reported sequence of the Drosophila PINTA protein,a comparison search was performed in the silkworm genome data,and 25 silkworms with CRAL-TRIO domain homologous genes were identified.Based on their annotations and predictions in the NCBI,we selected them four genes predicted to have similar functions as Drosophila pinta homologous genes were used as candidate genes for silkworm pinta,and they were named pinta1,pinta2,pinta3,and pinta4,respectively.The 4 pinta homologous genes encode 6 proteins.Among these 6 proteins,the similarity between PINTA1 and PINTA2_X1 is as high as 93%,and the similarity between PINTA1 and PINTA2_X2 and PINTA1 and PINTA2_X3 are both 75%.The amino acid sequences of PINTA2 and PINTA3 are similar.The sex is also higher,at 47%.However,the amino acid sequence of PINTA4 and other PINTA proteins are quite different.PINTA2 and PINTA4 have 20% similarity in amino acid sequence.In addition,bioinformatics analysis found that all 6 proteins of PINTA homologous protein are extrinsic membrane proteins without transmembrane structure,and no signal peptide sequence is predicted for the 6 proteins,and all 6 proteins contain the CRAL_TRIO domain.2.Detection of temporal and spatial expression patterns of pinta2_x1,pinta3 and pinta4 homologous genes in silkworm,Bombyx moriRT-PCR and q PCR were used to detect the expression patterns of the four identified pinta homologous genes in different periods and different tissues of the silkworm.The results showed that:The four homologous genes of the silkworm pinta are expressed throughout the developmental period of the silkworm.The expression of pinta2_x1 in the pinta2 gene is the most abundant,and pinta2_x1 is highly expressed throughout the developmental period of the silkworm.The expression abundance of pinta3 and pinta4 is the second,pinta3 and pinta4 expressions were similar.They were all expressed at a high level during the developmental period of silkworm larvae,while the expression level was low at other stages;while pinta1_x2,pinta2_x2 and pinta2_x3 were weakly expressed throughout the developmental period of silkworm larvae.The pinta2_x1 has the highest expression abundance in different tissues of the 5th instar and 3days of the silkworm Dazao strain,with high expression in all tissues,and the highest expression in the ovary,followed by the midgut and silk glands.The pinta3 is highly expressed in body wall,midgut,head and fat.The pinta4 is highly expressed in the head and midgut.3.Prokaryotic expression,protein purification,antibody preparation and western blot detection of pinta2_x1,pinta3 and pinta4 genes in silkworm,Bombyx moriThe p ET-28a_pinta2_x1,p ET-28a_pinta3,and p ET-28a_pinta4 were successfully constructed by cloning the full-length CDS sequences of silkworm pinta2_x1,pinta3 and pinta4.The recombinant plasmids were introduced into the expression strain for prokaryotic expression.Under the conditions of 16? and 20 h,the protein expression was the highest,and the heteroprotein was relatively less.Three soluble proteins,PINTA2_X1,PINTA3 and PINTA4,were successfully induced in the supernatant.PINTA2_X1,PINTA3 and PINTA4 were purified from the supernatant solution containing soluble proteins by Ni-NTA column.Purification PINTA4 sent to the company for polyclonal antibody preparation,antibody preparation good silkworm after each organization and protein and antibody WB experiment,the result shows: PINTA4 proteins are expressed in different growth period of mulberry silkworm,higher expression level during the period of silkworm moth egg at home,larval stage gradually reduce,in mature larva stage expression quantity increased again.In tissues,the expression of PINTA4 was high in the head,body wall,midgut and fat,with the highest expression in the midgut;the expression of PINTA4 was weak in the ovary,blood and silk glands,and no expression in the testis.This result is basically consistent with the previous results of m RNA level detection.4.Immunohistochemical analysis of PINTA4 antibody in compound eyes of silkworm moth stageThe HE stained tissue sections of the compound eyes of the silkworm moth stage were made to observe the internal structure and composition.By comparing with the compound eye structure diagrams of other insects in the literature,the structure and composition of the compound eyes of the silkworm moth stage were understood.Subsequently,the PINTA4 antibody was used for immunohistochemical treatment.The results of the study showed that the distribution of PINTA4 was different from that of Drosophila.Compared with the control,the silkworm PINTA4 antibody did not react and precipitate in pigment cells,nor was it found in other retinas.5.Detection of the fluorescence competitive binding ability of silkworm PINTA2_X1,PINTA3 and PINTA4 and retinoid substances The PINTA2_X1,PINTA3 and PINTA4 purified by prokaryotic expression are combined with the fluorescent reagent 1,8-ANS to form a complex,and then retinoids(all-trans retinol,all-trans retinol all-trans retinaldehyde,all-trans retinoic acid,11-cis retinal,9-cis retinoic acid)were used for competitive binding experiments to explore the affinity of the three purified PINTA proteins with retinol and its derivatives.The experimental results showed that PINTA2_X1 protein has the strongest affinity with all-trans retinoic acid,followed by all-trans retinal affinity,and the weakest affinity with PINTA protein is all-trans retinol.The combination of PINTA3 and PINTA4 with retinoids is basically the same as PINTA2_X1.