Positional Cloning Of Silkworm Melanism(Mln) And Stony (St) Mutants And Functional Analysis Of Their Candidate Gene

Silkworm is an important agricultural economic insect, its moderate body scale, relative short life cycle, high rate of reproduction coupled with complete genomic data, molecular linkage map, hereditary variation map and abundant mutant materials accumulated over the century make it a model organism for Lepidoptera research, various mutation types exist in silkworm, including body color, appendages, body shape, metamorphosis, amino acid metabolism, uric acid metabolism, egg shape, egg color, lethality, etc. More than450Mendelian mutations were documented, among them, about300were mapped on silkworm linkage groups. Such ample resources play an ever increasing role in sericulture, fundamental biological research of silkworm as a model, using silkworm as a bioreactor and basic research of biology. Two kinds of mutations occupy larger proportions in silkworm mutants, body color mutation and body shape mutation. In lepidoptera insects, body color and body shape hold significance to their survival and reproduction. Study the mechanism of body color and body shape of silkworm can not only help researchers better understand metabolism and regulatory pathways of Lepidoptera pigmentation and development, but also provide clues for silkworm functional gene usage (such as breeding of practical strain on silkworm and Lepidoptera pest control). So, we selected melanism (mln) and stony (st) mutants as our study material, they are the representative mutation for melanism and larval body shape, respectively. By positional cloning, we found the candidate genes for these two mutations and carried out corresponding functional verifications. Our main findings are as follows: 1. Molecular mapping of mln and st locus and candidate gene screeningUsing SSR markers, we carried out preliminary mapping for mln and st loci, and constructed molecular marker linkage maps for mln and st. Marker S1807was tightly linked to mln locus. Although the nearest marker, S0809, had a distance of49.6cM from the st locus, the relative positions and distances of other marker on the linkage map were consistent with their physical positions and distances on the genome, so the result can be used for further analysis of st locus. According to the distance between the mutation locus and its nearest molecular marker and the correspondence between the molecular linkage map and the physical map, we calculated the physical position of the candidate genes, and screened the genes within the regions by bioinformatics analysis. For mln, we focused on the gene BGIBMGA008538. Ortholog to this gene in the fruitfly AANAT1can catalyze monoamines like dopamine. Dopamine is one of the precursors of melanin. Tissue microarray analysis showed that this gene was highly expressed in larva head, and head is the region where the mln phenotype is most obvious. Temporal microarray data showed that this gene was highly expressed during late stages of pupation and moth stage, and mln mutants were conspicuously melanized at these stages. If the function of the product of gene BGIBMGA008538was lost, it could cause dopamine to over accumulate, which in turn lead to melanization. Moreover, genotyping of marker P3C (polymorphic marker based on the candidate gene's sequence) among mapping group showed that no recombination existed between the candidate gene and the mln locus. Sequencing of P3C showed that in the mln mutant, a deletion was found on the third exon of BGIBMGA0085'38. Based on the evidences above, we conclude that BGIBMGA008538is the gene responsible for the mln mutation. For the st mutant, by combining genetic distance with physical distance, we calculated that the nearest marker--S0809had a distance of14.88M from the st locus. According the relative positions of the markers on the molecular linkage map and the length of scaffold and gap on the physical map of8th chromosome, we speculate that the st locus is on nscaf2827. By further analysis of st phenotype, we propose that the st mutation is caused by cuticle protein abnormality. Microarray analysis of three predicted cuticle genes on nscaf2827in association with genotyping and RT-PCR analysis showed that the gene G2expressed specifically during larval stage, and no recombination existed between this gene and st locus. Obvious differences were observed between cDNA amplifications of wild-type and st mutant. From the findings above, we propose G2is the gene responsible for the st mutation.2. Cloning of mln and st candidate gene and sequence analysis between wild-type and mutantWe cloned full-length cDNA of mln and st candidate gene and genomic sequences that exhibit polymorphism. Sequence comparison between wild-type and mutant showed that in the wild-type Dazao, a1460bp fragment was amplified, while in mln two fragments were amplified, measured1407bp and1253bp, respectively. Combine these three transcripts and the sequence of marker P3C mentioned previously, we found that in the mln mutant a deletion of96bp existed (67bp on exon), also was found was a29bp insertion in one of the introns. Bm-iAANAT in Dazao has5exons and encode a product of261amino acids, which possesses intact acetyl-transferase domain. While in the mutant, Dazao-mln, apart from the first2bp, all of the4th exon is lost in the type-1transcript, the resulting frameshift lead to a premature stop codon TAG. In the second abnormal transcript type-2, the last67bp of the4th exon are lost, and an insertion exists in one of the introns. The termination codon of type-2transcript is located at the3'UTR of the of the normal transcript. So, comparing with the product in wild-type, both type-1and type-2transcript encode potentially abnormal proteins, and both lost the acetyl-transferase domain. The candidate gene we select for st Bm-st is547bp long, consists of4exons. Its5'UTR is38bp, and3'UTR is77bp. Bm-st encodes143amino acids, has a typical chitin binding domain. In the st mutant, a58bp deletion and6nucleotide substitutions was found in Bm-st, resulting in a frameshift. The chitin binding domain of the mutated gene is completely disrupted, potentially loses its function. 3. Expression profile analysis of mln candidate gene and investigation of its differential expression between wild-type and mutantTemporal expression profile analysis of Bm-iAANAT was carried out from day3of4th instar to ecdysis. RT-PCR analysis showed that the expression of Bm-iAANAT gene fluctuated during this period. Bm-iAANAT expressed normally at day3of4th instar, and stopped to express at the beginning of4th molt(0-16hours). After molting (24hours later), Bm-iAANAT resumed to express. Bm-iAANAT was highly expressed at the beginning of5th instar, while its expression was not detected at the onset of wandering stage. At the end of wandering stage(2.5day of wandering), expression of Bm-iAANAT is highly elevated(32), while negatively correlated with20E titer. Highest expression of Bm-iAANAT was observed7days after pupation (P7) and8days after pupation (P8). Spacial expression profile showed that Bm-iAANAT was highly expressed in larva head, silkgland and cuticle, with head at the highest level. Expression of Bm-iAANAT was hardly detected in other tissues. Expression analysis of Bm-iAANAT at differently sclerotized regions showed that it expressed higher in highly sclerotized regions (head, thoracic legs and anal plate) than in less sclerotized region (cuticle). That's to say Bm-iAANATs expression is correlated with melanized regions of mln mutant but not its unmelanized region. Semiquantitative analysis of Bm-iAANAT in Dazao and Dazao-mln at day4of5th instar, day2of pupation and ecdysis showed that the two types of transcripts in mln were expressed lower than their normal counterpart in Dazao; qRT-PCR showed that the transcript level of normal Bm-iAANAT in Dazao was10-20times higher than the two abnormal transcripts in mln combined. Moreover, at the regions where mln mutants manifested the most evident phenotypic differences from Dazao, Bm-iAANAT expressed higher in Dazao than in mln.4. Expression profile analysis of st candidate gene, its differential expression in wild-type and mutant and influence exerted by20EThe candidate gene for st mutation Bm-st was highly expressed during mulberry feeding period at4th and5th instar; but was undetectable at4th molt and was evidently down-regulated from P2. Its expression was restricted to larval and early pupation stage. Spatial expression profile showed that Bm-st was highly expressed in larva head, cuticle, fat body and trachea, and was hard to detect in other tissues. The expression pattern of Bm-st corresponds well with st's phenotype. In the20E induction experiment, we found that expression level of Bm-st was lower in induced group than in control3hours after commence of experimentation(according to results of semiquantitative and quantitative analyses). After24hours, the induced group already developed into prematured larve, and their Bm-st expression was evidently lower than that of the control's (according to results of semiquantitative and quantitative analyses). This demonstrates that20E has a repression effect on Bm-st.5. Analyses of key melanin metabolism genes in wild-type and mutant and research on the function of Bm-iAANATWe analyzed the differential expression of key melanin metabolism genes in pigmented regions of wild-type and mln mutant, including:head, thoracic legs and anal plate. Results showed that, expression levels of Pale, Ddc and Yellow were higher in Dazao than in Dazao-mln. However, apart from in head, expression of ebony was lower in Dazao than in Dazao-mln. According to the expression profile of Ddc in mln, we speculate that the over-accumulated dopamine resulted from the abnormal Bm-iAANAT repressed the expression of Ddc, and this was verified by quantitation of dopamine in Dazao and Dazao-mln. In the RNAi experiment, knock down of Bm-iAANAT led to20%of the injected group to melanize, and their Bm-iAANAT expression was evidently lower than in the control group, showing that Bm-iAANAT did participate in consumption of dopamine and pigmentation of silkworm. This is the first report of the important role AANAT gene plays in insect body color pattern determination.6. Influences of the mutual regulation of catecholamines and melanin metabolism genes on color patterns and physical properties of mln sclerotized regionsWe analyzed differences in melanin gene expression level and catecholamine's content between Dazao and mln at various developmental stages. Result showed that mutual regulation of dopamine, NBAD, Ddc, ebony, black and tan coupled with AANATs function loss made mln mutant to color at its distinctive pattern at different developmental stages. After injection of β-alanine, we successful changed the pigment metabolism pathway in mln, and rescued its melanism phenotype. We also performed pigment precursor and related gene investigation. Scanning electron microscope analysis of cross-section of adult dorsal plate from Dazao and mln showed that stratification existed in mln's dorsal plate, and this was likely caused by lack of cross-linking agent NBAD in mln. Analysis of mechanical properties of adult wings from Dazao and mln showed that, the elastic modulus of mln was higher than Dazao, while damping of mln was lower than Dazao. Because we used near-isogenic systems to carry out the experiments, so the physical property differences were due to the catecholamine metabolism differences in wild-type and mln mutant.7. Quantitation of chitin between the wild-type and st mutant, RNAi and prokaryotic expression of Bm-stAnalysis of chitin (Glucosamine) content in cuticle of wild-type and st mutant at mulberry feeding stage showed that content of Glucosamine in st mutant was385.95±55.04119μg/mg, and in the wild-type was525.28±38.25767μg/mg. The content of Glucosamine in st mutant was lower than that in wild-type, this might be caused by dysfunction of Bm-st. Based on the expression pattern of Bm-st, we carried out RNAi on4th instar at decline-feeding period, phenotype documentation was performed right after4th molt. Result showed that about41%individuals in the injected group were unable to complete molting, and the cuticles of these silkworms were tight, and felt hard by hand-touching. These resembled the st phenotype. Some extreme individuals had abnormal cuticle and body shape, and might even bleed and melanize. Molecular investigation showed that the Bm-st level in the individuals that showed st-like phenotype was evidently lower than in the control group. To find out the chitin binding abilities of the Bm-st products in wild-type and st mutant, two prokaryotic vectors:pET32a-WT and pET32a-st were constructed, containing the CDS of the wild-type Bm-st and mutant Bm-st, respectively. After induced expression, the recombination protein of wild-type and mutant were obtained, provided experimental foundations for follow-up binding research.