The Expression And Functional Analysis Of BmPP Gene In The Silkworm, Bombyx Mori

Silkworm (Bombyx mori) is a typical representative of Lepidoptera and a model organism of genetic study. In the processes of insect growth and development, the molting hormone (Molting hormone, MH) and juvenile hormone (Juvenile hormone, JH) play very important roles. The hormone regulation mechanism has been the concern of this research filed. The paralytic peptide of silkworm(Bombyx mori paralytic peptide, BmPP) has important functions in silkworm development. Hence, the study of its expression before and after the pupation, the location in thee brain tissue, and its function will provide not only the ideas and methods for preparation the permanent silkworm pupa, but also lay a foundation for the study of hormone regulation mechanism. In the present study, bioinformatics analysis of BmPP, its expression profile, in situ hybridization, and RNAi interference analysis were carried out. The main results are as follows:1. Bioinformatics analysis of BmPPThe sequence alignment result of BmPP with its homologies from other insects clearly indicated that all of the ENF family members have conserved Glu-Asn-Phe sequence and two bases standard cleavage site. In the typical structure of domain, gene cluster analysis was performed using the amino acid sequence of BmPP with those from the other six species. The phylogenetic tree showed that Pseudaletia separata (PsGBP), Pseudoplusia includens (PiPSP), Spodoptera litura (Sp1GBP) and Mamestia brassicae (MbGBP) first of four together, the first two moths together, and then together with the Bombyx mori(BmPP), Bombyx mori (BmPP) is close to that of Theretra japonica(TjENF) and Manduca sexta (MsPP); and far away from that of the other insects, which with the investigation between the traditional evolutionary line.2. Expression pattern of BmPPThe RNAs were extracted from the brains at the day-7 5th instar larvae and day 1 pupae, the prothoracic gland and central nervous system at the day-3 pupae as well as day-5 pupae. The qRT-PCR results revealed that BmPP gene was expressed in all the brain tissues at the above stages. However, even in the silkworm brain, BmPP gene was expressed at the day-7 5th instar larvae with the highest abundant. Besides, the distribution of BmPP gene in tissues at the same developmental stage was distinct. For example, at the pupa day-1, the BmPP gene was transcribed with the highest level in the central nervous system, with the lower level in prothoracic gland and brain tissue. These results indicated that the expression of BmPP gene is tissue-and stage-specific.3. In situ hybridization of BmPPThe RNA of BmPP was used to prepare the digoxigenin-labeled probe. The anti-sense and sense RNA probes were employed to hybridize the brain tissue at the day-7 5th instar larvae. The signals were visualized from anti-sense probes and not from sense probes. The result clearly revealed the spatial location of BmPP gene expression.4. RNAi analysis of BmPPBased on the expression profile of BmPP gene, the RNAi analysis of this gene was performed. The siRNA was injected to the day-6 5th instar larvae and day-1 pupa seperately. The results showed that there was no effect on development and eclosion when the pupae were treated by RNAi. On the other hand, BmPP siRNA-injection in larvae resulted in delayed development. The RNAi analysis indicated that down regulation of BmPP gene expression in specific stage generated observable phenomenon, suggesting that BmPP gene has an important role in the metamorphosis of silkworm.In summary, the present study provided for the first time the basic bioinformatics analysis of BmPP gene. The expression profile of this gene was investigated. The functional analysis of BmPP gene was carried out by RNAi treatment. All of these data will assist in the elucidation of the functions of BmPP gene.