The SAGE Construction And Analysis Before And After Heat Treated In The5-instar Larvae Of The Male Silkworm, Bombyx Mori

Silkworm, Bombyx mori is not only an important economic insect, but also an idealmaterial for scientific. Systematic studies on expression disparity of certain larval genesagainst heat stress are way of practical and important to demonstrate the mechanism ofheat-resistance in silkworm, even in insects, as well as culturing mild Bombyx strainspossessed with heat-resistance.Strain7532was used as the material in this research. Heat treatment was employedin the experimental group while the control group was raised under room temperature.Male silkworms were used in both groups. Mature larvae and the5th instar larvae fedafter24h and72h were dissected to obtain fat body, silk gland and midgut. Total RNAwas extracted from multiple tissues in all periods. Mixed tissues in strain7532femalesat high temperature, strain7532males at high temperature, strain7532females atnormal temperature and strain7532males at normal temperature were obtained forinvestigation. The technology of serial analysis of gene expression (SAGE) was usedfor the first time to construct female and male SAGE libraries under high temperatureand room temperature respectively, combining with the method of bio-informaticsmethod analyzed SAGE libraries. The main results are as follows:1. Construction and analysis of the SAGE library The4SAGE libraries of the5th instar female and male larvae at normal and high temperature are constructed. The2male SAGE libraries are used to analyze library data and screen differential gene toexclude the interference of the gender differences. Besides, bioinformatics methods ofmulti-type analysis and multi-level data extraction were employed to provide a brief andspecialized secondary database.3.55million and3.58million original labels (tags) wererespectively detected in control group and experimental group respectively.917significantly different genes were obtained between the two libraries, of which590genes were up-regulated,327genes down-regulated. Three genes are randomly selected to verify whether the number of SAGE tags and the corresponding gene expressionabundance is consistent by the RT-PCR method, the results prove that the library data isreliable. Gene Ontology (GO) analysis revealed that the distribution of genes in the twolibraries are very similar, indicating that these genes have similar biological functionsand participate in similar physiological processes in different temperatures.917genesare distributed in142KEGG pathways by KEGG analysis, among which44pathwaysare tremendously enriched by differentially expressed genes (P<0.05). Half of thepathways are related to metabolism, bio-synthesis and signal transportation, whichwould help to identify the genes resisting to high temperature and to explore themechanism of gene regulatory networks. By combining both before and after hightemperature treatment5year-old female larvae SAGE library to comparative analysis,screening differences label1876, including high temperature label955, fall after riseafter high temperature label921, not gender factors in high temperature sensitivity318genes. After high temperature rises124genes,194genes. By analyzing GO availablethe difference of genes in catalysis, metabolism, binding the enrichment function is more.2. Disparate tags can be annotated by GLGI amplification Theoretically, longsequences (200bp-1000bp) can be obtained by GLGI amplification, but shortersequences would be obtained in practical experiments. This technology can be a highlyefficient for excavating unknown genes in the constructed SAGE libraries. In theexperiment,13elongated sequences of the18tags selected are detected, contributing toexcavating unknown genes in SAGE libraries.3. Space-time expressions of two different expression genesThere is significant difference on of two genes, heat shock protein, immune related proteins, its expression had tissues and development stage specificity. Under high temperature and normal temperature the two genes expression of space and time characteristics results show that immune related protein gene and heat shock protein of the transcription level in the bowel, silk gland, fat in the body is higher than normal temperature, Which expressed quantity is higher in fat body.