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Analysis of Structure, Expression and Function of Bombyx Mori Diapause Receptor Gene

Author: WangLiGang
Tutor: ShenXingJia
School: Jiangsu University of Science and Technology
Course: Biochemistry and Molecular Biology
Keywords: Silkworm Diapause hormone receptor Incubation temperature Gene Expression Analysis Real-time PCR Promoter Insect hormones
CLC: S881.26
Type: Master's thesis
Year: 2011
Downloads: 31
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Abstract

Silkworm (Bombyx mori) is an economically important insect model organisms, the silkworm diapause molecular mechanism has important theoretical and practical significance. The silkworm egg diapause in the long-term evolutionary process characteristics, the diapause is dominated by hereditary and environmental conditions. Silkworm diapause can be divided into univoltine, bivoltine and multi-resistance varieties. Silkworm bivoltine varieties Diapause by previous generations of the environmental impact, the egg stage is 25 ° C high temperature tomorrow is produced in incubating diapause eggs, while the low temperature of 15 ℃ dark incubation on the production of non-diapause eggs. Silkworm embryonic diapause pupal stage swallow ganglion (subpharyngeal ganglion, SG) hormone synthesis and secretion of diapause (diapause hormone, DH) is closely related to, the DH must by diapause hormone receptor (BmDHR) to play a role. To explore the incubation temperature regulation of the bivoltine silkworm diapause molecular mechanism, the the paper Bmdhr gene structure, the spectrum of expression and functional characteristics of a preliminary study. 1, Bmdhr gene structure analysis from NCBI downloaded Bmdhr mRNA-1 ~ Bmdhr mRNA-5 sequence (Login number: AB164386 ~ AB164390), the online gene group comparison in the silkworm genome database, ORF analysis using DNASTAR software package and the derivation of the corresponding The amino acid sequence of the protein transmembrane region; TMHMM service v. 2.0 program analysis. The results show that a single copy gene Bmdhr, whole length of about 33.79 kb and contains seven exons and six introns; 5 mRNA from the same mRNA transcript is present comes through different splicing, wherein Bmdhr mRNA- 1 and Bmdhr mRNA-2 encoding the amino acid sequence is the same for the same receptor; BmDHR-1 contains seven transmembrane regions, BmDHR-3, BmDHR-4 and BmDHR-5 respectively contain five transmembrane regions. Spatial and temporal expression of Bmdhr gene characteristics and incubation temperature on the expression silkworm bivoltine varieties Qiufeng silkworm eggs moth half points method is divided into 2 groups, respectively dark incubation at 15 ℃ and 25 ℃ Ming reminder Green, using real-time quantitative PCR technique to analyze the impact of incubation temperature on the silkworm at different developmental stages and tissues Bmdhr gene mRNA transcript levels. The results showed: Bmdhr mRNA in-1 is mainly expressed in the pupal stage ovarian 4 d after the most sensitive pupation of diapause hormone and its transcription level rapidly rose to the peak, and the high temperature incubation is higher than the low temperature incubation; Bmdhr mRNA -4 expression in various developmental stages of blood, high temperature under the Ming incubation of its in the pupa blood in the transcriptional level is 7.7 times that of the low-temperature incubation, BmDHR-4 may be decided whether the second generation of the silkworm bivoltine varieties one of the key factors in diapause; Bmdhr mRNA-5 in 2 ~ 3 d after pupation ovarian transcriptional level high and low temperature incubation transcriptional level is higher than the high-temperature incubation, 3 d transcription levels after pupation 4 ~ 5 d to pupate after the incubation treatment between the two kinds of transcription level no significant differences. 3, Bmdhr gene promoter characteristics cloned from the silkworm genome DNA analysis in bioinformatics analysis based on different regulatory sequences Bmdhr gene promoter reporter plasmid using pGL3.0 basic vector construction, different length start The promoter sequence activity analysis, comparing different concentrations of juvenile hormone analogue (JHA), ecdysone (MH) and diapause hormone (DH) and hormone the combination of Bmdhr gene promoter activity. The results show that the -941 ~ -1364 nt interval Bmdhr transcription start site upstream negative regulatory element may exist. MH concentration 4μg/mL, the promoter activity was significantly weakened, and the remaining concentration was significantly enhanced. JHA concentrations were 2,4,6,10 μg / mL, the promoter activity extremely significantly enhanced when 1μg/mL significantly weakened, 8μg/mL is very significantly weakened. MH and JHA concentration of 1,2,4,6 μg / mL, the promoter activity was significantly increased when 1μg/mL highest activity; while both concentration was 8,10 μg / mL under the conditions, a promoter activity not significant. DH concentration between 0 to 60 nm, the promoter activity was significantly increased, the highest activity, of which about 20 nM when the concentration is greater than 60 nM, promoter activity did not change significantly. Eukaryotic expression of BmDHR and functions to build the IE-1 promoter of-BmDHR expression plasmid pcDNA3.1 vector for the skeleton, and were detected by a variety of BmDHR promoter activity of silkworm trehalase gene (Bmtrehelase) impact. The results show that the four kinds of diapause hormone the receptor BmDHR-1, 3 BmDHR-, BmDHR-4 and BmDHR-5 individually abundantly expressed, can raised Preparation trehalase gene promoter activity; different ratios (1:3 , 1:1 and 3:1) BmDHR-1 / BmDHR-5 can be raised the trehalase promoter activity, and the two together strong expression BmDHR-5 has a certain inhibition; BmDHR-1/BmDHR-5 ratio of 1:3, a considerable raise multiples BmDHR-1 alone. This thesis Bmdhr gene structure and expression of the spectrum and features, and the accumulation of experimental data to elucidate the molecular mechanism of silkworm diapause.

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