The SAGE Construction Before And After Heat Treated In The5-instar Larvae Of The Silkworm, Bombyxmori, And The Research On Differently Expressed HSP Genes

The domesticated silkworm, Bombyx mori serves as a model for Lepidoptera. It isnot only an important economic insect, but also an ideal material for scientific andapplied researches. Nowadays, as the sericulture industry tends to be high efficient withless labor, improvement in strain resistance is urgently appreciated. On the other hand, abrand-new insect and basic biological field is being formed through insect researches.Systematic studies on expression disparity of certain larval genes against heat stress areway of practical and important to demonstrate the mechanism of heat-resistance insilkworm, even in insects, as well as culturing mild Bombyx strains possessed withheat-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 and female silkworms were used in both groups. Mature larvae and the5th instarlarvae fed after24h and72h were dissected to obtain fat body, silk gland and midgut.Total RNA was extracted from multiple tissues in all periods. Mixed tissues in strain7532females at high temperature, strain7532males at high temperature, strain7532females at normal temperature and strain7532males at normal temperature wereobtained for investigation. The technology of serial analysis of gene expression (SAGE)was used for the first time to construct female and male SAGE libraries under hightemperature and room temperature respectively, combining with the method ofbio-informatics method analyzed SAGE libraries. The cloning technology and RT-PCRtechnique was employed to clone a significantly different gene and analyze its tissueexpression pattern. 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. The2female 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 and specialized secondary database.3.58million and3.56million original labels (tags) wererespectively detected in control group and experimental group respectively.1062significantly different genes were obtained between the two libraries, of which298genes were up-regulated,764genes down-regulated. Five genes are randomly selectedto 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.2. GO analysis and KEGG path analysis of the two libraries before and after hightemperature treatment of the female silkworm Gene Ontology (GO) analysisrevealed that the distribution of genes in the two libraries are very similar, indicatingthat these genes have similar biological functions and participate in similarphysiological processes in different temperatures.732genes are distributed in176KEGG pathways by KEGG analysis, among which40pathways are tremendouslyenriched by differentially expressed genes (P <0.05). Half of the pathways are related tometabolism, bio-synthesis and signal transportation, which would help to identify thegenes resisting to high temperature and to explore the mechanism of gene regulatorynetworks.3. Cloning and analysis of the Cloning and Analysis BmsHSP27.4gene Manytags are annotated to the HSP in the female library, so four SAGE libraries are used toscreen and compare the genes commenting to the heat shock protein to comprehensivelyunderstand the HSP expression levels in both genders and temperature conditions.Finally, a total of27genes annotated to heat shock protein are obtained. The gene ofHSPgene10, which is expressed significant differences in both genders and differenttemperatures in this study, is selected for further study. Registry Number, its SilkDBBGIBMGA005823-TA, Zi Wen Li et al name the gene BmsHSP27.4in the paper and itsaccession number is BGIBMGA005823-TA in SilkDB database. The full sequence waselongated to944bp within which the longest ORF is741bp.5’ end of the tag is addedwith48bp while the3’ end added with187bp. The sequence is unanimous with thesequence from Japanese scholars Mita K (Accession No. AK382044). BmsHSP27.4encoded247amino acids and is localized to chromosome5and has no intron.Phylogenetic analysis showed that BmsHSP27.4is nearest to sHSP33.6ofChoristoneura fumigerana and belonging to different evolutionary branch comparing with most of the heat shock proteins. EST expression profile and the chip electronicexpression profiling all show that the BmsHSP27.4gene has higher expression levels inthe gonads and head.4.Space-time expression of BmsHSP27.4gene RT-PCR are used to investigatethe expression of BmsHSP27.4in three tissues (midgut, silk gland and fat body) fromfemale and male individuals with strain7532and strain Haoyue before and after thetreatment. The result shows that the gene appears a rapid upward trend in the threeorganizations of male and female in two different varieties after high–temperaturetreatment, indicating that BmsHSP27.4was sensitive to high temperature regardless ofstrains. The BmsHSP27.4gene displays higher expression level in male silkworm thanfemale one under high temperature environment with investing the expression situationof this gene in fat body and midgut from the different sex of strain7532after heattreatment.The SAGE library is of great scientific value, not only providing valuableinformation for the identification of high-temperature perceptual genes, but also helpfulto identify novel genes that are closely related to the high temperature. Meanwhile, it isbecoming an important database studying the molecular mechanism of resistance tohigh temperature in the near future.