Proteomic Analysis Of Heat Shock Proteins Related To Thermal Tolerance In The Silkworm, Bombyx Mori L

Due to economic importance, silkworm was the most important insect during 20th century and it was also the prominent genetic insect model after Drosophila melanogaster. Nearly every system studied in insects has demonstrated sensitivity to heat. Probably silkworm as a domesticated insect is the most sensitive insect to heat. Therefore understanding more about thermal tolerance and heat shock proteins (HSPs) in the silkworm provides valuable information in both agricultural and scientific aspects.Four breeds of silkworm, Bombyx mori L., were selected including Jingsong, a Chinese bivoltine breed, Haoyue, a Japanese bivoltine breed (both as heat-susceptible breeds), Nistari, a non-diapause polyvoltine as heat-tolerant breeds and P50, a diapause polyvoltine breed. There were four groups for each breed (two treatments and two sexes) which exposed to heat shock at 45℃for 30 minutes and 41℃for 1 hour (fifth instar, day 4) in a controlled growth chamber. Then the silkworms were returned to the standard rearing temperature (24℃) for a recovery period.2 and 4 hours after heat shock treatment fat body free of muscle were sectioned.2DGE was employed using Ettan IPGphor IEF unit and Ettan DALTsix multiple-gel electrophoresis unit for first and second step and image analysis using ImageMaster 2D software. MS and MS/MS spectra were obtained using the ABI 4700 Proteomics Analyzer MALDI-TOF/TOF mass spectrometer.The main results are presented according to the experimental layout: 2DGE proteome profile:From repeated experiments, we detected 25 proteins in the proteome of 4 silkworm breeds that were differentially expressed after heat shock exposure, that nine protein spots were commonly expressed in all experimented breeds including 5 proteins spots (no.1,2,3,4,9) from low molecular weight area of SDS-PAGE (2nd step of 2DGE) and 4 proteins spots (no.5 to no.8) from high molecular weight area. There were 16 specific response spots belonged to low molecular weight area of SDS-PAGE of each breed including 9,4 and 3 in Nistari, Jingsong and Haoyue respectively. P50 didn't express any specific response spots after heat exposure. The common response spots of silkworm to heat shock exposure was similar among different breeds. However, specific response spots were different among breeds HSPs identification:Ten out of 25 detected proteins in the proteome profiles were identified as HSPs (6 sHSP and 4 HSP70) by MALDI-TOF/TOF mass spectrometer in which 8 HSPs (4 sHSP and 4 HSP70) from commonly expressed proteins and 2 (sHSP) from specifically expressed proteins. While HSP19.9, HSP20.4, HSP20.8 and HSP23.7 were commonly expressed among all breeds but HSP20.1 and HSP21.4 were in Jingsong and Nistari respectively. Four HSP70 with different PI and MW from HSP70 family are inducible heat shock proteins in all of the experimented silkworm breeds in this study. Spot 2 from commonly expressed proteins obviously is a sHSP but PMF and MS/MS analysis could not match it with any sHSP in all the reapted experiments. It showed limitations of mass spectrometers to identify all differentially expressed proteins.Quantification:We quantified inducible HSPs expression in the fat body of two silkworm breeds after two hours of heat shock using qRT-PCR and image analysis of 2DGE. Statistical analysis of normalized volumes (%) of 8 protein spots including 4 sHSP and 4 HSP70 in different treatments, breeds, and sexes showed there are differences in the volume percentage of some spots related to identified HSPs between bivoltine Jingsong and polyvoltine Nistari breeds in both heat treatments. The expression of sHSPs in the Nistari breed was lower than Jingsong breed after the 45℃heat treatment, while there was no significant difference in the intensity of protein expression between breeds after the 41℃heat treatment. In other words, at mild and longer time heat exposure, silkworm breeds did not differ significantly in their response, while, at higher temperature and shorter time, the thermotolerant breed expressed significantly lower sHSPs (P<0.01). Protein expression intensity of HSP70 didn't show differences significantly (P<0.01) between the two heat exposure treatments and the silkworm breeds. Comparison of the spot volume percentage between the two sexes indicated that there are some differences in protein expression, although it was not significant among both sHSP and HSP70 proteins. These differences were approved by quantification of mRNA expression level of two genes including a small heat shock protein (HSP19.9) and HSP70 via qRT-PCR. For example while the expression of HSPs (HSP19.9 and HSP70) in Nistari breed was less than Jingsong for both mild and severe heat shock, the lowest expression was detected for HSP19.9 of Nistari breed when exposed to severe heat exposure. Data showed RNA transcription level in thermo-sensitive is more than thermo- tolerant breed,41℃more than 45℃treatment, HSP19.9 more than HSP70 and male higher than female when they were compared to grand mean of whole data. High variation was seen among sHSPs expression in the Japanese breed Haoyue:HSP19.9 and HSP23.7 had low expression, meanwhile HSP20.4 was highly expressed and the expression of spot 2 (non-identified protein) was inferior. Up-regulating and down-regulating were found among the expression of three HSPs including two small heat shock proteins (HSP19.9 and HSP20.4) and HSP70 in the ovary and testis of investigated breeds. ANOVA on normalized volumes of sHSP and HSP70 proteins after four hours were difference from that of two hour recovery duration so that differences between two breeds and two thermal treatments were not significant (P> 0.05).Totally, at least 7 small heat shock proteins are involved in protein homeostasis and induced thermotolerance of larva across silkworm breeds. Besides, four HSP70 with different PI and MW of HSP70 family were inducible HSPs in the silkworm across silkworm breeds. This suggests the sHSP and HSP70 confer thermotolerance in the silkworm larva. While 8 out of 9 common heat response proteins were identified as HSPs but from 16 specific heat response proteins only 6 proteins were identified. It showed limitations of current silkworm genome database to identify all differentially expressed proteins.Specific heat response proteins were among sHSP family. This implied the important role of sHSP in the induced thermotolerance of silkworm breeds. Also more variation was observed in the sHSP family than HSP70. Therefore probably some sHSPs have different role in the prevention of protein aggregation and protection of cells against heat stress. HSP70 constantly expressed after heat shock in thermotolerant Nistari breed and had low variation in all situations such as different treatments and recovery period but sHSPs were expressed in a high variation in changed conditions. Variation in the expression of sHSPs and specific expression of these stress proteins across breeds revealed the significance of small HSP family in thermotolerance of silkworm larvae.One of the aims of this study was finding some protein markers related to heat tolerant in the silkworm. However expression of specific proteins in each breed may suggests some candidate for specific proteins markers related to thermotolerance or thermosensitive characters in the silkworm but actually in this step of investigation we can not introduce any markers explicitly. So it is necessary to follow this hypothesis by doing more experiments related to mechanism of thermotolerance in the silkworm.Low expression of HSPs transcripts and protein in thermo-tolerant breed (Nistari) may represent genetic differences in HSPs expression. It is also probably related to natural section against these kinds of proteins in thermo-tolerant breed rather than adaptations to local thermal conditions. In other hand, High expression of HSPs mRNA in thermo-sensitive breed (Jingsong) shows importance of HSPs in survival and longevity of heat exposed silkworm larvae of this breed. Moreover since this breed is so sensitive to heat and doesn't have non-plastic tolerant, HSPs were extremely expressed in this breed. Therefore molecular mechanisms underlying up-regulation of heat-shock proteins differ between breeds with different genetic background.