| Silkworm (Bombyx mori) is not only an important economic insect, but also the important experimental animal for the genetic and molecular study. After thousands of years of human history in sericulture, through natural selection and artificial induction, we has accumulated plenty of stably inherited mutant resources of silkworm, involving the development of body and appendages, body color, markings, body shape, metamorphosis, etc. These resources are extremely valueable in sericulture and basic biological reasearch field.The E group of the silkworm is a complex locis of pseudoallelic genes group which is positioned at the21.1points in the linkage group6. The E group of the silkworm is good material for the genetics research. More than30mutations of the E group have been found so far, whose mutant phenotypes are related to excess or lack of markings or appendages, deform of segments and organs, in which some homozygous members dead at embryonic stage. In recent years, it has been confirmed that E group mutant Additional crescents (ECa) lose abd-A gene, while New additional crescents (EN) lose both Ubx and abd-A genes. Through the fine mapping, the candidate gene of Kp supernumerary legs (EKp) were locked to abd-A gene. In addition, in the Mu supernumerary legs (EMn),the expression site of Abd-B gene has changed. Therefore, E group mutation and Hox genes which play an important role in the process of the development of insect body pattern are closely linked.Triple crescents (ETc) mutant is one of the E group mutants of silkworm. Its phenotype is:the fourth, fifth, sixth segments have crescents, the eighth segment lose star pattern, homozygous type lose prolegs completely, semi-lethal at embryonic stage. The study of Triple crescents (ETc) mutant is helpful to understand the related genes’molecular regulation mechanism of E group mutations in Bombyx mori during development. This research is based on the classic genetic map and fine genome map of Bombyx mori. We used bioinformatics analysis, positional cloning, qRT-PCR, in situ hybridization technique and some other techniques to study the mechanism of Triple crescents (ETc). The main results are as follows:1.Morphology observation of Bombyx mori Triple crescents (ETc) mutationWith electron microscopy, We observed the silkworm Dazao and Triple crescents homozygous (ETc/ETc) embryos of the eighth day of development. The results showed that Triple crescents homozygous embryonic abdominal A3-A6lose appendages, even the slightest bulges was not found, this confirmed that ETc homozygous embryos lose prolegs completely. We observed the silkworm Dazao and Triple crescents heterozygous type (ETc/+) larvae in the third day of the fifth instar. The results showed that the Triple crescents has three crescents on the fourth, fifth, sixth segments, and lose star pattern on the eighth segment.2. Mapping of the Bombyx mori Triple crescents mutation E c geneTwo silkworm strain, obtained from the silkworm gene bank in Southwest University, ETc (06-720) and Dazao were used for mapping. For linkage analysis, BC]F(backcross female) was used, while another backcross BC1M (backcross male) was used for recombination analysis. We found polymorphism markers between two parents, from which13microsatellite (SSR) markers on the6th linkage in the silkworm SSR molecular linkage map and15self-designed markers. After verifying these polymorphic makers’linkage in22individuals genomic DNA of BC1F and analyzing192individuals genomic DNA of location combination BC1M of ETc, we got a molecular linkage map which genetic distance is7.5cM. The mark M-2and M-5are closely linked to ETc locus. These two markers are8.8Kb upstream of Bmabd-A gene, while other genes are very far away.Accordingly, Bmabd-A gene is identified as the candidate genes for the silkworm ETc.3.Bioinformatics analysis of Bmabd-AWe analysised mRNA splice variants of Bmabd-A based on the data from NCBI Database and Silkworm Genome Database. The results showed that:Bmabd-A-L type, the open reading frame (ORF) was1059bp which encodes352amino acids, predicted the protein pI/Mw:9.43/38.3KD; Bmabd-A-S type, the ORF is1044bp which encodes347amino acids, predicted the protein pI/Mw:9.43/38.3KD; Bmabd-A-3type, the ORF is1032bp which encodes343amino acids, predicted the protein pI/Mw:9.51/37.6KD. The analysis of protein domain online indicated that:all three isoforms of Bmabd-A have a Hox domain. The phylogenetic analysis showed that abd-A, Ubx and Abd-B gene formed a clade separately in insects. It Indicated that the abd-A, Ubx and Abd-B gene have high degree of homology in insect evolution, which suggested that their function play an important role in different insects. 4.Spatiotemporal expression pattern analysis of the Bmabd-A geneRT-PCR demonstrated that Bmabd-A expression began at the first day of embryonic period and reached its peak at the fifth day of embryonic period, then decreased. Bmabd-A was highly expressed in malpighian tubules, genital gland, fat body and midgut in the third day of the fifth instar. Therefore, we hypothesized that the Bmabd-A gene is involved in the regulation of growth and development of silkworm in early embryos, and it may be associated with the development of variety of organizations such as malpighian tubules, fat body, midgut.5. Sequence analysis of the Bmabd-A gene in wild-type and mutant and expression investigationWe cloned coding sequence cDNA of the Bmabd-A gene in mutant ETc by cloning technology. After sequence alignment and prediction of their spatial structure online, we found that no significant difference existed in the CDS and tertiary structure between the Dazao and ETc.However, qRT-PCR revealed that the expression level of Bmabd-A in ETc were significantly decreased, which is consistent with that abd-A gene have a role on the promotion and maintenance of the development of proleg in silkworm. We hypothesized that Bmabd-A gene expression was reduced in ETc, then affect genes and regulatory factors, which were related to the growth of prolegs in Bombyx mori, thus the ETc homozygote embryos lose peolegs. According to this, we speculate that Bmabd-A gene has dose-response effect.6. Analysis of the regulatory sequence in intron of Bmabd-AWe found that the first intron of Bmabd-A gene lose six different size of fragments, total of99bp, by cloning Bmabd-A gene in the ETc. We predicted transcription factor binding sites in the deletion region of the first intron in Bmabd-A gene online. The result showed that this deletion fragment contains6transcription factor bingding sites which are related to Hox gene transcription. We speculate that in the Triple crescents, the absence of the Hox transcription factor binding sites, which influence Bmabd-A gene transcription, leads to the down-regulation expression of Bmabd-A gene, then eventually leads to lose of prolegs completely in the Triple crescents homozygous embryos.7. In Situ hybridization of Bmabd-AIn order to investigate the expressions of Bmabd-A in the Triple crescents homozygous embryos, specific RNA probes were generated and performed in situ hybridization of silkworm embryos. The anti-sense chain of RNA was used as probe and sense chain as control.The results showed that there is light brown hybridization signals in the silkworm abdominal A1-A6segments. There was a faint hybridization signal in the posterior of the third thoracic segment (T3) and abdominal A7-A10segments.This result confirmed the previous literature of abd-A expression site in insect and showed directly the positions of embryos expression of Bmabd-A. |
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