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Identification, Cloning and Functional Analysis of BmMet, A Methoprene-tolerant Gene in the Silkworm, Bombyx Mori

Author: LiZhiQing
Tutor: ZhaoPing；XiaQingYou
School: Southwestern University
Course: Biochemistry and Molecular Biology
Keywords: Silkworm BmMet Clone Expression characteristics Functional Analysis
CLC: Q78
Type: Master's thesis
Year: 2009
Downloads: 14
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Abstract

Ecdysone insect growth, metamorphosis and reproductive development process (Molting hormone MH) and juvenile hormone (Juvenile hormone, JH) play an important role in the regulation. , MH regulation of insect growth and development of the molecular mechanisms have been clearer, receptors and other involved in the MH signaling cascade regulatory factors have been identified and the establishment of a MH cascade signaling network. And the the JH molecular mechanism study reported relatively few regulatory genes involved in the JH signaling the key has not yet been determined. Methoprene-tolerant (Met) gene was first in Drosophila is found that encodes a the bHLH-PAS-type transcription factor, has a typical DNA-binding domain. It has been reported, DmMet JH Ⅲ specific binding with high affinity mutant JH resistance, overexpression the DmMet will increase the sensitivity of the Drosophila exogenous JH and JH main types that, and can lead to Drosophila death in larval and pupal stage. In addition, by way of RNAi red castaneum TcMet gene transcriptional silencing, not only castaneum resistance to JH, but also causes precocious metamorphosis of the larvae. Show that the Met gene in JH the regulation process plays an important role, and thus speculated Met may be JH candidate receptor. Silkworm is an important economic insects and Lepidoptera model organisms. In recent years, researchers in the silkworm developmental hormone regulation Mechanism made many breakthrough. This study is based on the detailed map of the silkworm genome, the silkworm genome-wide chip expression data, RACE technology and RNAi technology Bombyx mori may the JH receptor BmMet gene length cDNA cloning and gene interference experiment, explore the BmMet gene function. The main findings are as follows: 1. The silkworm BmMet gene full-length cDNA clone bioinformatic analysis, we identified a detailed map of the silkworm genome predicted genes have a higher similarity with Drosophila DmMet gene gene, and named as BmMet. RACE-PCR cloning of full-length cDNA sequence of the gene. BmMet length cDNA of 1917 bp, which ORF of 1368bp, encoding 455 amino acids. 5'-UTR nucleotide is a 405 bp 3'-UTR nucleotide is 144bp and contains a 32 bp poly (A) tail. 2. The silkworm BmMet gene sequence structural chromosomal localization and genomic analysis found that the genes in the silkworm chromosome 8, only one of the whole gene exon and intron, which is in and other insects Met gene within The number of introns, such as the presence of an intron in the D. melanogaster, Aedes aegypti 9, the Anopheles gambiae 7, Tribolium castaneum 7. Domain predicted by the SMART program structure display, Bombyx mori BmMet, containing domains consistent with other insects Met that bHLH, PAS-A, PAS-B, and PAC speculated BmMet gene may have similar function and other insects Met gene. 3. The silkworm BmMet spatial and temporal expression of the gene characteristics using RT-PCR technology, we analyze the characteristics of the BmMet gene expression in the silkworm different developmental stages of the individual and the larvae in different tissues and organs in the fifth instar 3 days. The results showed that BmMet gene expression have continued throughout the developmental stages of the silkworm, also has its expression in various tissues and organs of the larvae of the fifth instar 3 days, and the silkworm JH titer rule change is no obvious correlation, suggesting BmMet gene expression has not been induced regulatory JH. 4. The silkworm BmMet gene function, RNAi analysis by dsRNA-mediated RNAi experiments, we conducted a Preliminary the silkworm BmMet gene function. 4 instar larvae of silkworm BmMet silencing of gene transcription, was found not to affect the normal development of the silkworm, Bombyx mori can normally slough off 4 age old epidermis and into the fifth instar and pupate normally of moth. Intends to the castaneum TcMet gene interference effect with red, while no obvious phenotype similar with Drosophila DmMet gene deletion, implying that the Met gene in silkworm and Drosophila Tribolium castaneum the mode of action of its different . In summary, this study identified and cloned the full-length cDNA sequences of the Met gene in the silkworm genome found that the silkworm BmMet genes with other insects Met gene sequence structure is very conservative, JH signaling pathway performance a difference. These results will be further studied BmMet gene function in silkworm lay the foundation.

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