| Lineage-specific genes?LSGs?are defined as a class of specific taxonomically restricted protein-encoding genes that share no significant sequence similarity with any other protein or peptide in the database.LSGs are also referred to as orphan genes or ORFans.As more and more genomes of some species were sequenced,LSGs have been studied in viruses,microbes,lower marine animals,plants,insects and primates.At the same time,it was confirmed that LSGs are a significantly abundant component of all genomes sequenced to date.Apart from being abundant,LSGs also play an important role in lineage-specific traits and adaptability.In addition,there is a correlation between the generation of new genes and the adaptability of lineage-specificity.The early work focused on the evolution of a novel protein from DNA sequence thought to be non-coding,and the evolution of lineage-specific transcription was largely ignored.Previous studies of several individual insect species-specific genes have also demonstrated these views.Lepidoptera is one of the most widespread insects orders in the world.They have huge family members and are distributed on all continents except Antarctica,this is inseparable from their adaptive evolution.However,there has been no systematic study on the specific genes for lepidopteran insects.In this study,the lepidoptera-specific proteins in silkworm were identified firstly by bioinformatics methods,and their basic characteristics,chromosome distribution,expression patterns and evolutionary relationships were analyzed.Then,the 4,5-dopa dioxygenase?DODA?,which was not reported in animal kingdom previously,was selected for further investigation using the silkworm,Bombyx mori,an important model organism of Lepidoptera,as a model.We cloned the orthologous DODA gene from silkworm,and investigated its sequence and expression patterns as well as the function of DODA in silkworm by prokaryotic expression.The main results obtained are as follows:1)Identification and characterization of lepidoptera-specific proteinsBased on five sequenced Lepidopteran species,we used the genome-wide comparison method to blast the proteins of five Lepidoptera species and non-Lepidoptera species?Chordata;Arthropoda:Diptera,Coleoptera,Hymenoptera,Hemiptera and Nematoptera;Crustacea;Arachnidea and Nematode?step by step,and candidate set of lepidoptera-specific proteins were screened.In order to ensure accuracy,the non-redundant databases of NCBI w used to further eliminate the protein data of lepidopteran insects.Thus,the lepidoptera-specific proteins in silkworm were identified.The result showed that 229 lepidoptera-specific proteins were identified in the silkworm.Comparing the basic characteristics of 14623 genes?or proteins?at the whole genome level of silkworm and 229 lepidoptera-specific genes?or proteins?in silkworm,it was found that the average length of lepidoptera-specific proteins in silkworm was significantly lower than the average length of proteins at the whole genome of silkworm.The average exon numbers of lepidoptera-specific genes in silkworm was also significantly lower than the average number of exons of silkworm genes at the whole genome level.Similarly,the average GC content of lepidoptera-specific genes in silkworm was significantly lower than the average of the GC content of the silkworm genes at the whole genome level.The chromosomal localization of lepidoptera-specific genes in silkworm showed that 205 genes were mapped to the silkworm chromosome,and the location of twenty-four genes was unknown.Lepidoptera-specific genes were distributed on the chromosomes of twenty-eight pairs of silkworm.2)Functional annotation and expression pattern of lepidoptera-specific proteins?or genes?in silkwormFunctional annotation of lepidoptera-specific proteins in silkworm was carried out,and forty-four proteins were annotated,of which twenty-eight proteins had Gene Ontology?GO?numbers.Ommochrome binding protein?OBP?accounts for a large proportion of annotated genes,and they are likely to be a large family.Secondly,three kinds of antibacterial peptide proteins were annotated:Moricin,Gloverin,and Lebocin.Some of these functional annotated proteins have the same function,they may be duplicate genes,or they may come from a gene family.We detected the expression patterns of 229 lepidoptera-specific genes by using twenty-one developmental stage microarray data.A total of seven typical expression patterns were summarized.Seven genes highly expressed in the egg stage,twenty-six genes highly expressed in the larval stage,six genes highly expressed in the molting stage,eighteen genes highly expressed in the metamorphosis stage,seven genes highly expressed in the pupal stage,thirty-three genes highly expressed in male moths and seventeen genes highly expressed in female moths.Functional annotation of highly expressed genes at various stages indicates that these genes are closely related to the specific developmental processes and physiological characteristics of the corresponding period.3)Identification and cloning of BmDODA1Phylogenetic analysis of antimicrobial peptides,ommochrome binding protein?OBP?and 4,5-dopa dioxygenase?DODA?revealed that most of the related genes of them were multiple copies,and some copies were ancestral copies shared by Lepidoptera,and some copies might be produced after differentiation of individual species.DODA was most likely to be derived from horizontal gene transfer of fungal donors.Therefore,we selected 4,5-dopa dioxygenase,which has no related research in lepidopteran insects and even animals,to carry out further basic characteristics and functional analysis in silkworm.Synteny analysis of DODA indicated that BmDODA1was an ancient copy shared by lepidoptera in two copies of BmDODA,so we used BmDODA1 as the representative of lepidoptera DODA.Primers were designed based on the predicted ORF sequence and the full-length sequence of BmDODA1?1079 bp?was obtained by RACE.4)Exogenous expression and activity detection of BmDODA1We exogenously expressed BmDODA1 protein using the prokaryotic expression system?Escherichia coli?.BmDODA1 recombinant protein was purified by histidine affinity chromatography and the purified recombinant protein of BmDODA1 was consistent with expected protein was identified by the Maldi–TOF–TOF.Subsequently purified BmDODA1 recombinant proteins were used as antigens to immunize mice for antibody production.The temporal expression profiles of BmDODA1 was examined:transcription level of BmDODA1 gradually increased from the fifth instar larvae 1th Day to 7th Day until the wandering stage began to decrease and an obvious high expression in the female of 7th Day of pupal stage.For spatial expression analysis of Day 3 of the fifth instar larvae:BmDODA1 showed high transcriptional level in midgut and head.This result was also consistent with translational levels detected by Western blotting.We examined the relative expressions of BmDODA1 following 3rd Day of the fifth instar larvae by infected by Bacillus bombyseptieus and Escherichia coli,the fat body of the infected domestic silkworm was dissected and used for templates preparation.At different time points?3 h,6 h,12 h?after infection,our results showed that expression of BmDODA1 significantly increased at transcription level after pathogen infection.This result suggested that BmDODA1 may be involved in basal resistance or immunization of the domestic silkworm.A typical 4,5-DOPA dioxygenase activity was detected with the soluble form of BmDODA1.The kinetic parameters of the recombinant protein BmDODA1 were calculated to obtain Vmax=0.1678+0.003nmol/mg/min and the Michaelis constant Km=1.06+0.05 mM.We also evaluated the effect of pH and temperature on enzyme activity of recombinant BmDODA1.The optimum pH for BmDODA1 at 8 and temperature at 30°C.It can be seen from the above results that the Lepidoptera-specific genes were distributed on each chromosome of the silkworm,and their sequence characteristics were significantly different from those of the whole gene.These lepidoptera-specific genes were likely to play an important role in the physiological processes of a specific period of silkworm growth and development.The annotated lepidoptera-specific protein functions were mainly associated with antimicrobial peptides and ommochrome-binding.These functions are particularly important for lepidoptera to adapt to the environment,which further indicates that lepidoptera-specific proteins contribute significantly to the adaptive evolution of lepidopteran insects.Functional analysis of 4,5-dopa dioxygenase from horizontal gene transfer indicated that the function of BmDODA1 in silkworm may not only involve the metabolism of dopa,but also play a role in the resistance of silkworm to pathogenic microorganisms.Our results provide an example of a functional horizontal gene transfer study that is rare between fungi and animals.It also confirmed the important role of lepidoptera-specific proteins in the evolution of lepidopteran insects. |
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