| In this study,the multiple sequence alignment and phylogenetic analysis were performed for the cloning of 20 related genes of potato beetle,and the spatiotemporal expression of 20 melanin synthesis pathway genes were analyzed by qPCR technique.The response mechanism applied to juvenile hormone and ecdysone was explored,and the six genes of Ldtan,Ldebonyl,Ldebony2,Ldyellow-f1,Ldyellow-f2,Ldyellow-y were determined in the course of growth and development.The function of these six genes was determined by RNAi technique.The mechanism of interaction with hormones is described.The following results are obtained:1.Cloning of body color-related genes in LdecemlineataUseing the transcriptional and genomic data of L.decemlineata,Tribolium castaneum,Drosophila melanogaster NBAD hydrolase gene(Tan),the NBAD synthase gene family(Ebony),Yellow family gene.Using the program of BLAST,subgroup(cl,c2),d subgroup(d1,d2,d3),e subgroup(el,e2),f subgroup(f1,f2),g subgroup(g1,g2),h subgroup,x subgroup(x2,x3,x4,x6),y subgroup were cloned from Colorado potato beetle.The full-length cDNAs of the above 20 genes were cloned by extracting the first strand of total RNA of colorado potato beetles and a series of molecular biological techniques such as cDNA synthesis,PCR.and the sequences were submit to the NCBI and GenBank Accessesion number are as follows::KY221866?KY221864?KY221865?KY221856?KY221860?KY221853?KY221855?KY221854?KY221850?KY221857?KY221861?KY221862?KY221858?KY221849?KY221852?KY221851?KY221859?KY221848?KY221847?KY221863.2.Multiple alignment and phylogenetic analysis of color-related genes in LdecemlineataBy comparing the protein sequences of the corresponding genes such as colorado potato beetle,Drosophila melanogaster and Tribolium castaneum.By phylogenetic cluster analysis,the melanin biosynthesis pathway genes of colorado potato beetles were clustered separately with the Dendroctonus ponderosae and Tribolium castaneum.The most similar genes were named:Ld-tan?Ld-ebonyl?Ld-ebory2?Ldyellow-c1?Ldyellow-c2?Ldyellow-d1?Ldyellow-d2?Ldyellow-d3?Ldyellow-e?Ldyellow-e2?Ldyellow-f1?Ldyellow-f2?Ldyellow-g1?Ldyellow-g2?Ldyellow-h?Ldyellow-x2?Ldyellow-x3?Ldyellow-x4?Ldyellow-x6?Ldyellow-y.3.Tissue and temporal expression of body color-related genes in Ldecemlineata(1)Tissue expressionThe results showed that the 20 genes were expressed in all tissues,and there was no consistent expression pattern in each tissue,and there was no consistent expression pattern in all the tissues of the third instar larva of potato beetle and the gonad of adult beetle,the results showed that the 20 genes were expressed in all tissues,and none of them showed the same expression pattern.There are differences in expression between tissues,even if they are homologous genes,but there are differences between two genes.For example,the expression of Ld-ebony2 in the nervous system is more than 20 times higher than that of Ld-ebonyl.In the RNAi bioassay of Ld-ebony2;it was found that the larva had convulsive behavior,which may be related to neural regulation.The relative expression of Ld-tan gene was found to be relatively high in abdominal nerve,hindgut and epidermis,and in the anterior thymus,midgut,Markov tube,brain,foregut,ovary,fat body,blood cell.The expression of spermatozoa was relatively low,and the expression of spermatozoa was higher in female and male gonads than in male.Ldyellow-f1 and Ldyellow-f1,Ldyellow-y were highly expressed in blood cells and midgut:1.42±0.04,5.00±0.09?1±0.63,1.27±0.13?1±0.19,2.41±0.15.(2)Temporal expression By qPCR analysis,it was found that the expression of Ld-tan was relatively low in eggs,and increased gradually in the third and fourth instar larvae with the growth and development of the larvae,when the expression of Ld-tan was different in the different developmental stages of potato beetles,and the expression of Ld-tan was relatively low in the eggs,and then increased in the third and fourth instar larvae.It decreased gradually in the middle of 4th instar and reached the peak in pupal stage and adult stage.The expression of Ld-ebonyl increased with the growth and development of larva,reached its peak in the middle of 4th instar,and then decreased to a low level in prepupae stage.Ld-ebony2 gradually reached the first peak at the third instar,decreased at the beginning,end and pupa stage of the fourth instar,and reached the highest peak at the adult stage.With the growth and development of larva,there was a great fluctuation in the first to third instar of larva,but the Ldyellow-f1 and Ldyellow-y both reached the peak of expression in pupa stage and Ldyellow-f2 reached a small peak in prepupae stage.4.Cross-talk between Body Colour related genes and juvenile hormone and molting hormone in LdecemlineataBy RNAi,this study further reduces juvenile hormone synthesis gene LdJHAMT,juvenile hormone or hormone LdAS-c a juvenile hormone analogue LdMet,ecdysone synthesis related P450 gene Ldphm and Ldshd,the titer of ecdysone receptor and its signal transduction genes LdEcR and Ldhr3,After determining that these genes were down-regulated,the effect of knockout on the expression of chromation-related genes in the 20 individuals was examined.The results showed that Ld-tan may be regulated negatively by ecdysone and positive by juvenile hormone.Ld-ebonyl and Ld-ebony2 may be positively regulated by ecdysone.Ldyellow-f1 Ldyellow-f2 Ldyellow-y may be negatively regulated by ecdysone and positive by juvenile hormone.5.The effect on the growth and development of CPB by knockdown six melanin synthesis-related genesBy feeding the second instar larva dsRNA of Ld-tan,the expression level of Ld-tan decreased and the larva died to a certain extent,but no significant effect on the developmental duration,pupation rate and emergence rate was found.When the larvae developed to the end of the fourth instar,the body color of the larva was dark brown,and the R value of RGB was significantly different from that of control by Photoshop's colorimeter.On the other hand,the ebony gene led a significant decrease of survival rate compred with control treatment,so it was easy to form black pupae,which resulted in the abnormal emergence of the larva.There was no significant difference in mortality,pupation rate and emergence rate between Ldyellow-fl,Ldyellow-f2 and Ldyellow-y,but the body color of the elder larva showed golden color after interference,and the body color variation of Ldyellow-y was the most easily recognizable by the naked eye.6.Field Control by applying dsRNA-expressing bacteriaAccording to the results of RNAi,it was found that dsebony-1 and dsebony-2 had a certain lethality to larvae.After spraying 10-times dilution of dsRNA expression bacteria,it had certain control effects,and the duration could last more than 14 days. |
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