Transcriptome Sequencing And Positional Cloning Of I-lem Mutant In The Silkworm Bombyx Mori

The highly diverse color patterns in insects are of great evolutionary interests because of their association with natural selection.Among Lepidoptera,the silkworm Bombyx mori is the most suitable insect to identify genes responsible for colour patterns,because there are numerous pigmentation pattern mutants and its genome sequence and several high-density linkage maps are available.Body color mutants in B.mori are useful resources for understanding color pattern formation.In this study,we selected i-lem mutant,which is a representative body color mutant of silkworm,as the study object.Using the transcriptome sequencing technique,we compared and analyzed the differences in functional genes at transcription level and physiological metabolic pathways between i-lem and lem.By positional cloning method,we constructed a fine linkage map to screen the candidate gene for i-lem mutant.Through these two technologies,we look forward to outlining the molecular mechanism of i-lem and exploring the related functions in pigment synthesis and regulation.Main results obtain in this research are as follows.The aspect of transcriptome sequencing,1.We constructed i-lem and lem two transcriptome sequencing library successfully,Clean reads accounts for 98.7% of Raw reads,Q30 values are higher than 96.2% in all libraries.Total Mapped reads accounts for about 85.5% Clean reads,which uniquely mapped reads accounts for about 82.0% of Clean reads.Error rate and GC content control in a reasonable scope,which shows that the quality of the libraries are qualified.2.Between two libraries were found 86 differentially expressed genes,including35 up-regulate expression genes and 51 down-regulate expression genes.GO classification found 58 GO enrichmental term,KEGG analysis found 14 significant enrichmental pathway,and all the differentially expressed genes involved in GO term and KEGG pathway may affect multiple physiological and metabolic pathways in silkworm.3.All differentially expressed genes involved in GO classification and KEGG pathways are divided into two categories: the genes coding skin protein and the genes participate in insect congenital immune response system.The main difference between i-lem and lem are 28 genes coding cuticular protein and 9 immune response system related genes in insect such as PAH and Serpin-4,and the metabolic pathwaysof them.The aspect of positional cloning,1.For positional cloning of the i-lem locus,F1 heterozygous males were obtained from a single-pair cross between a lem train ah09 male and an i-lem strain l82 female and each was backcrossed with an l82 female.A total of 358 BC1 larvae were used for analysis.Using larva body color differences during the fourth dormany period to identify 159 i-lem mutant(light yellow)individuals and 199 lem mutant(dark yellow)individuals.By using 11 available markers and 358 BC1 individuals,we constructed i-lem linkage map.The map contains 72 exchanged individuals.The i-lem linked region was narrow to about 170 kb range on chromosome 2.The range contains 11 predicted genes.2.Using the Gene Ontology annotation,Smart website,Microarray Expression data information of silkworm genes and RT-PCR analysis results show that the predicted BMgn009799 genes encoding proteins belong to the aldo-keto reductase(AKR)superfamily,the gene between i-lem mutant and wild have different transcripts.In +i-lem/+i-lem transcript out a kind of ORF,which contains 7 exons and 1026 bp,encoding 341 aa protein;but in i-lem/i-lem the ORF contains only two exons: the first one and the last one.The length of it is 270 bp,encoding 89 aa protein.In summary,BMgn009799 exons 2-6 are deficient in i-lem mutant.3.Previous studies have shown that aldo-keto reductase(AKR)or carbonyl reductase(CR)can replace sepiapterin reductase(SPR)to take participate in the synthesis metabolic pathways of tetrahydrobiopterin(BH4)when SPR is deficient.It is worth noting that irregular body coloration of lem mutation phenotype is due to insufficient of BmSPR function.Therefore,we hypothesized that BMgn009799 encods a member of AKR,which is likely to be involved in pigment synthesis pathways in i-lem mutant.In conclusion,we serve BMgn009799 as a mutational candidate gene for i-lem.BMgn009799 encods a higher active AKR protein which plays a role in selective synthesis of BH4 in i-lem mutat(i-lem/i-lem;lem/lem),and promotes more pigment intermediates to generate BH4,thus reduced sepiapterin(SP)accumulation in larva surface body of lem relatively.This is the main performance of the inhibition i-lem to lem.In addition,according to the analysis of transcriptome sequencing,we found that the melanin synthesis pathway,which also affects the larval body wall hardening and shading,has been weaken in lem mutatant,Therefore,compared with lem mutant,i-lem mutant larva body color became lighter.