A Preliminary Study On Antp Gene Regulation In The Wing Of Bombyx Mori

Wing as a new trait during evolution, is of great importance for animals to evolve from water to land and from land to the air. Insects have been the first to own wings among invertebrates, which made insects quickly become the most various and widely spread species in the world.Silkworm is an important economic insect, and is also the model organism of Lepidoptera. Understanding the Silkworm wing formation mechanism can not only lay the foundation for understanding the regulation of wing development in other insect, but also provide a new theoretical support for the Lepidoptera pest control. Studies have shown that the emergence of insect wings is closely related to the expression of Hox genes. The wings of insects are usually limited to the second and third thoracic segments, it is because that Hox gene expression has suppressed wings occurrence in other segments. For example Scr suppressed the first thoracic segment wing primordia cell development, Ubx and abd-A inhibit the development of the abdominal wing. Ubx is only expressed in the hindwings, which lead to morphological differentiation between the forewing and hindwings. The gene expression of Antp does not seem necessary for wing development. The absence of Antp function in Drosophila and Tribolium showed that the wings were not affected. However, in the silkworm genebank of Southwest University, there are three wing mutants due to BmAntp gene mutation. They showed similar phenotypes that homozygote embryonic lethality, thoracic dysplasia, wing deformity in the adult of heterozygotes. It suggests that abnormal expression of the Antp gene could affect wing development in silkworm. We will start with these three materials, to investigate how BmAntp gene affect silkworm wing development, and to identify its function in wing regulation. The main results are as follows:1.Wing phenotype observation in the mutants and the wildtypeWe choose different periods of wing primordia of heterozygotes(Wes/+、Nc/+ and EM/+) and wild-type, and also pupal as well as moth wings, to observe and compare their phenotypic characteristics. It was found that, there was no significant difference in size and shape between the mutant and wild-type wing primordia before wandering. On the second day after wandering, we can observe that mutant wing discs cannot properly extend, displaying shrunken folded and curling. The pupal wings are incomplete short, and the adult wing display deformity and small size.2. BmAntp expression analysis during wing development in silkwormTo better understand BmAntp gene function in silkworm wing development, we investigated BmAntp gene expression patterns in the wing discs in forewings and hindwings of silkworm by quantitative PCR from the first day in fifth instar to moth. It was found that, BmAntp expressed consistently in both forewings and hindwings. The expression remained at a low level before wandering, and then went up and reached a peak before moth. After moth its expression became low again. It showed BmAntp expressed consistently in wings and had correspondence with wing development, suggesting that BmAntp may be closely related with wing regulation.3. The gene structure and expression analysis of BmAntp in the mutants and wildtypeBy analyzing the BmAntp gene sequences in the embryos of EM homozygote and the wildtype Dazao, we found out that there was sequences abnormalities with 3’UTR in type 1 transcript in EM homozygote. By RACE we obtained two new transcripts namely type 2 and type 3 of BmAntp in Dazao, while in mutant we obtained just type 3. We further analyzed BmAntp gene expression in the three heterozygotes and their wildtype at srveral times from fifth instar to moth. The result showed that BmAntp expressed higher in heterozygotes than wildtype. To further investigate such higher expression came from the normal transcripts or the mutant ones, we detected the Antp protein expression level. It showed that in heterozygote BmAntp is much lower.Thus we suspected the wing mutant resulted from loss-of-function of BmAntp.4. The preliminary study on BmAntp regulation in the wing development of silkwormWe selected Wes heterozygote which wing mutant was the most obvious among the three and the wildtype Dazao, and detected the expression level of wing related genes such as ap、vg and en. We found these genes expressed much lower in Wes mutants. Then we analysed the upstream of the genes and chose two critical genes ap and vg, to explore their interactions with BmAntp protein. EMSA showed that BmAntp could bind these two genes and thus involved in the wing development. We used the forewing and hindwing at 5 days after pupation in Wes and Dazao for microarray analysis. The result showed that there were 24 up-regulated genes and 14 down-regulated genes in forewing, and 78 up-regulated genes and 30 down-regulated genes in hindwings. We made annotations for these genes which lay a foundation for the next study.