Positional Cloning Of Albino (al) Mutant And Its Formation Mechanism In Silkworm, Bombyx Mori

Silkworm is an important economic insect, as well as an important model organism of Lepidoptera insects. It has the characteristics, such as moderate body size, short life cycle, and high reproduction rate etc, of experimental animal. Also, the genome frame, fine mapping, molecular linkage map, hereditary variation map of silkworm has been accomplished and the classical genetics research over the past100years has accumulated abundant genetic background. Silkworm has abundant mutant resource, of which, the types include body color, body shape, appendage development, metamorphosis, egg color, egg shape, amino acid metabolism, lethality, etc. It is an important materials for the study of Lepidoptera insects.The insect body color is closely related to its foraging, mating, thermoregulation, avoiding predators and adapting to the environment. Therefore the study of insect body color is significant for its survival and reproduction. Body color mutant occupy large proportion in silkworm mutants. Study on the formation mechanism of silkworm body color mutant can not only help researchers better understand related genes and regulatory networks of Lepidoptera pigmentation metabolism, but also provide clues and lay a foundation for silkworm functional gene usage. In this study, we selected al mutant, which is a representative of silkworm body color mutant, as the study object. By positional cloning, we successfully separated the candidate gene for al mutant, and then carried out corresponding functional research, as well the analysis on its formation mechanism. Our main research results are as follows:1. Molecular mapping of al locus and candidate gene screening Two silkworm strains, Dazao and al, reared in silkworm gene bank were used as the parents, and from which the BC,F (F,(+al/al)♀×01-070(+al/al)♂) for linkage analysis, and BC,M (01-070(+al/al)♀×F1(+al/al)♂) for recombination analysis were produced. Because the classical genetic research has located al mutation locus on37.9centimorgan of the fifth linkage group, we used18pairs of SSR markers on the fifteenth linkage group and60pairs of walking primers on the5chromosome to screen polymorphism, linkage test and molecular mapping, and eventually narrowed the al locus within400kb between the marker C24and D20on nscaf2674, tighly linked to marker C10.By bioinformatic analysis in this region, we found17predicted genes. And the functional analysis showed that only BGIBMGA003643gene could associated with the metabolism of melanin in the candidate region. Studies have shown that BGIBMGA003643gene encodes6-pyruvoyl-tetrahydropterin synthase(PTPS), which is a key enzyme participated in BH4metabolism. BH4is an indispensable coenzyme for partially aromatic hydroxylase (PAH、TH and TPH), while PAH and TH are the key enzyme genes in the pathway of melanin metabolism. So we suspected that the BH4could involve in melanin metabolism in the form of coenzyme, which coincides well with the phenotype characteristics of al mutant. Therefore, we proposed BGIBMGA003643gene responsible for the al mutant, and named it BmPTPS.2. Cloning and expression pattern analysis of al candidate geneWe cloned full-length cDNA and genomic sequences of al candidate gene in Dazao and al mutant. Sequence comparison showed that, the genome size of the two strains is1893bp and1881bp, respectively. But comparing with the wild-type, two deletions at the the upstream sequence of BmPTPS gene were found in al mutant, which the length is1192bp and314bp respectively. Informational analysis found that the two missing sequences are repetitive sequence. The full-length cDNA of BmPTPS gene in Dazao and al were measured, and the lengh is1014bp and1002bp, respectively. Compared with Dazao, a11bp deletion in the exon2(also an another1bp deletion in the5’UTR) were identified in al mutant, resulting in a frameshift and eventually formed a premature stop codon TAA, and for which the BmPTPS gene can not be translated into protein.The temporal and spatial expression pattern of BmPTPS gene were measured in our experiment, and the former showed that this gene has an expression during each period of the larvae (from newly hatched larvae to day7of5th instar), and the latter showed that the BmPTPS gene was expressed in all tissues from day3of5th instar. Among them, strong expression was detected feeding mulberry period of the first instar, at the beginning of second instar, molting period of second instar, beginning of5th instar and day2of5th instar. Spatial expression profile showed that BmPTPS was highly expressed in larva head, blood, nervous, trachea, genital gland and silkgland. Expression pattern analysis showed that the level of BmPTPS was changed significantly in the first instar and second instar, which is consistent with the emergence period of al mutant phenotype. Simultaneously, we compared the expression of BmPTPS between Dazao and al at the beginning of the second instar. By qRT-PCR, we found that the expression level of BmPTPS were significantly down-regulated in al compared with Dazao at the beginning of the second instar. These results further indicated that the BGIBMGA003643gene is a candidate gene of al mutant.3. The expression of key genes in BH4and melanin pathway, and the substance content detection in al mutantTo explore the effect of the BmPTPS mutation on the expression of other key genes in BH4pathway, we quantified the expression levels of these genes, including GTPCH, SPR and DHFR, by qRT-PCR. The results showed that the expression level of key genes in pathway all have a corresponding change due to the mutation of BmPTPS gene in al mutant. In the al mutant, level of GTPCH, a gene on the upstream of the candidate was lower than that in the wild-type, and the expression of SPR and DHFR, the genes on the downstream were significantly upregulated in al mutant.Because BH4could be used as an indispensable coenzyme of phenylalanine hydroxylase (PAH) and tyrosine hydroxylase (TH) involved in melanin synthesis, we compared the differences in melanin genes expression and catecholamine’s content between Dazao and al at the beginning of second instar. Result showed that in al mutant, levels of PAH and TH genes were considerably higher than that in Dazao. Simultaneously, the content of phenylalanine and tyrosine, substrate of these two genes, in al mutant were36times and4times higher than that in Dazao, while the content of dopa and dopamine, the products of the two genes were2.1times and5times lower, than that in the Dazao strain, respectively. These results indicated that the formation mechanism of al mutant:mutation of BmPTPS gene results in insufficient of tetrahydrofolate, then the melanin metabolic pathway was blocked, and finally cause the deficiency of melanin, producing the al mutant phenotype.4. After inhibitor DAHP treatment, emergence of al mutant similar phenotype and related genes expressionWe treated N4newborn larvae with GTPCH inhibitor, DAHP, and observed their phenotype at the beginning of the second instar, and the group treated with0.1mol/L NaOH were used as control. The result showed that DAHP-treated group obtained a extremely al mutant similar phenotype on day1of the second instar, while the control group has no significant changes. Simultaneously, we detected the expression of BH4-metabolizing genes and melanin related genes. This is an extremely exciting result that the level of each gene in DAHP-treated group was consistent with that of the al mutant. Concretely, GTPCH and PTPS, two genes involved in BH4pathway, were expressed at sharply lower levels in the DAHP-treated group, but the level of SPR, DHFR, PAH and TH increased significantly. Further we redesigned the following experiment:At the beginning of the second instar, the larvae selected from the DAHP-treated group were refed with BH4, and these only treated with DAHP were as control. We found that the BH4refeeding larvae recovered to the normal black cuticle, while the DAHP-treated group remained a similar al phenotype. These findings not only proved that inhibitory effect of DAHP was through BH4pathway, but also indirectly proved that the al mutant was due to loss function of BmPTPS gene.5. BH4feeding experimentsIn order to further demonstrate that the phenotype of al mutant is due to the deficiency of BH4, we fed30mM BH4on newly hatched al individuals (Theoretically each batch contains1/4albino individuals) and examined their phenotypes on day2of second instar, and these fed with H2O were as control. The BH4treatment group was evidently melanized, while the control group had no obvious change. Meanwhile, the expression of the key genes in the pathway of BH4biosynthesis and the melanin related genes were investigated and the result revealed that compared with the control, the expression level of each gene in BH4treatment group has a significant recovery trend to that in Dazao, falling in the level between wild-type Dazao and the al mutant. Specifically, compared to the control, the level of GTPCH and PTPS in BH4-treated group was increased significantly but still below the Dazao, while the expression level of SPR, DHFR, PAH and TH were significantly down-regulated but still higher than in Dazao.