Screening Of Candidate Genes For Sel Mutant And Analysis Of Alternative Splicing Of BmADAR In The Silkworm, Bombyx Mori

The silkworm,Bombyx mori,is a mode of Lepidoptera insect with very clear genetic background.So far,at least 600 mutations have been identified and more than 200 of them have been placed on linkage maps.The study of body color mutants in silkworm not only helps us to understand the related genes and regulatory networks of pigment metabolism in insect of Lepidoptera,but also provides new ways for the utilization of related functional genes.Sel is a body color mutant in which larvae is pale yellow and it has been mapped in the 24-0.0 loucus.According to the previous research,the activity of sepiapterin deaminase in Sel mutation is much higher and the accumulation of sepialumazine in Sel is more than normal silkworm.The preliminary mapping for Sel have put it in the position which is12 cM from the SSR marker S2404.The molecular substance of Sel has not been identified so far,and people have little understanding in Sel which involved in the regulation of insect pigment synthesis and the formation molecular mechanism in body color pattern.According to silkworm genetic linkage map analysis and the relationship between the function of candidate genes and the phenotype of Sel mutant,we made GTP Cyclohydrolase I(BmGTPCHI),Porphobilinogen deaminase(BmPBGD)and Adenosine deaminase acting on RNA(BmADAR)as three candidate genes for Sel.We had gene cloning,sequence alignment and expression pattern analysis in wild type and mutant,respectively.Besides,we analysied the alternative splicing of BmADAR,and identified different transcripts of expression characteristics and possible editing sites.The mainly results are as follows:(1)According to the results of gene cloning and secquence analyzing,there is no difference in coding secquences of BmGTPCHIa and BmGTPCHIb between p50 and U12(Sel/+).The expression profiles in different developmental stages of BmGTPCHIb between the two strains are basically consistent,and the differences in tissue expression profiles is Malpighian tubule.Whether the function of BmGTPCHIb which has no expression in Malpighian tubule of Sel mutant and significantly different between p50 affects the formation of phenotype of Sel mutation or not remains to be further research.(2)There are four different amino acid sites of BmPBGD between p50 and U12(Sel/+)according to gene cloning and secquence analyzing,but sequencing results in the F2 generation of separating individuals showed no linkage with Sel.The expression profiles in different developmental stages of BmPBGD between p50 andU12(Sel/+)basically consistent.However,the expression profiles in differnet tissues of fifth instar larvae have several obvious differences between the two strains.Whether the higher expression of BmPBGD in head of Sel mutant is the reason that deaminase activity in vivo of Sel mutant higher than normal silkworm or not yet to be in-depth study.(3)We comprehensively cloned and abtained a variety of BmADAR transcripts with RT-PCR and RACE for the first time.We abtained 2 transcripts with complete ORF and 4transcripts without termination codon,altogether six isoforms which encoding different amino ends or carboxyl ends.The common feature of the four transcripts without termination codon is that the termination codon TAG occures A-to-I RNA editing by itself.Gene structure analysis of the 2 complete ORF transcriptions shows that BmADAR mRNA alternative splicing has happened in the mature process.The results of spatial and temporal expression profiles showed that silkworm mainly express BmADARa isoform.(4)According to the results of BmADARa inquired in silkworm genome database,it consists of 19 exons,18 introns constitute and coding 717 aa.Homologous sequence alignment and conservative analysis showed that,like fruit flies and other insects ADAR,BmADARa belongs to the vertebrate ADAR2 homologue.It is reported that expressing and purificating ADAR using prokaryotic expression system is out of enzyme activity.To study the characteristics of enzyme activity of BmADARa,we carried out a research into the prokaryotic expression of the enzyme protein,which helps a large number of expression,purification and identification of this enzyme in vitro enzyme activity features in the future.In conclusion,BmADAR transcriptional expression pattern in Sel mutant needs further identification,we cann't rule out the correlation between the three genes(BmGTPCH,BmPBGD and BmADAR)and Sel.The mutation of the translation area,especially the change of the activity of the promoter region,may also cause abnormal gene function and lead to mutation type.In order to determine the genetic nature of the Sel,we can map it by positional cloning and lock the candidate gene through chromosome map in the future.The results obtained in this paper provide a necessary theory foundation for further analisis of A-to-I RNA editing mechanism of BmADAR and its interaction mechanism with dsRNA target gene of target genes.