摘要
家蚕可谓是二十世纪最为重要的经济昆虫,也是目前继果蝇以后最为重要的生物学研究模型昆虫之一。家蚕由于经过长期的室内饲养与经济性状的改良,逐渐的失去野性,变得对环境条件尤其是温度非常敏感。因此,研究家蚕的耐高温特性以及在分子水平上研究对高温冲击的反应如热激蛋白的合成等不仅具有重要的理论意义而且有很好的实践指导意义。特别是对于选育适应在热带和干旱地区及中国的夏秋蚕季节饲养的蚕品种,探明家蚕品种的耐热性能及机理尤为重要。自20世纪中叶第一次发现热激能诱导果蝇合成热激蛋白,进一步的研究发现从细菌到人类都具有合成热激蛋白的能力,因而认识到热激反应是一个普遍的生物学现象,热激蛋白研究也成为当前分子生物学研究的热点。本项目应用双向电泳技术,结合质谱技术和实时荧光定量PCR等分子生物学先进手段,选择遗传背景差异很大,耐高温性状差异特别明显的不同家蚕(Bombyx mori)品种为材料,在分子水平研究家蚕对热激反应特别是热激蛋的合成种类、品种间的差异及与耐高温性的关系。
本试验选取了四个不同家蚕品种分别为：菁松,中国系统二化多丝量品种,对高温比较敏感；皓月,日本系统二化多丝量品种,对高温很敏感；Nistari,热带无滞育多化性少丝量品种,对高温耐性好；P50,有滞育多化性少丝量品种,对高温耐性中等。家蚕各品种在标准饲养技术与条件下饲养至五龄第4天,分别用45℃热激30分钟或41℃热激1小时。然后在标准饲养温度(24℃)中恢复2小时或4小时,取样分别解剖血液、脂肪体、丝腺体、卵巢、睾丸等器官进行试验,得到如下研究结果。
1.双向电泳实验结果证明,本试验所用的所有品种经热激处理与对照比较,都有明显可见的热激蛋白产生。经过多次重复实验,都得到了重复性很好的试验结果,在4个不同品种、不同热激温度处理、雌雄性别及2次重复试验中都检测到了25个差异表达的热激蛋白,其中9个为普遍反应表达蛋白,这16个共有热激蛋白中,5个在凝胶的低分子区域,分别编号1、2、3、4、9号,4个在高分子区域,分别编号为5、6、7、和8号。
2.实验还在凝胶的低分子区域检测到了16个品种特异反应的热激蛋白表达,其中Nistari有9个,菁松有4个,皓月有3个,而P50一个也没有。因此不同家蚕品种有多个共有热激蛋白的合成,但特异反应则品种间有显著差异。
3.用MALDITOF/TOF质谱仪检测分析,热激处理后检测到的25个差异表达蛋白,有10个被鉴定为热激蛋白(HSPs),其中6个sHSP,4个为HSP70。这10个被鉴定为HSPs当中的8个为共有反应热激蛋白(4个sHSP,4个HSP70),2个为特异反应热激蛋白(都是sHSP)。被MS/MS鉴定出的蛋白质的氨基酸序列覆盖率为29%82%。本试验在所有品种中检测到四种可诱导的HSP70属于HSP70家属,具有不同等电点和分子量的共有热激蛋白；六种sHSP包括19.9、20.1、20.4、20.8、21.4和23.7为家蚕可诱导的sHSP。其中HSP19.9、HSP20.4、HSP20.8和HSP23.7为共有表达蛋白,HSP20.1只在菁松中特异表达,HSP21.4只在Nistari中特异表达。
4.热激处理后在实验中检测到的差异表达蛋白点2号很明显是sHSP,但PMF和MS/MS分析都不能与数据库中的任何sHSP相匹配,多次反复试验结果相同,可能是因为目前家蚕有限的基因组数据库而未能鉴定,有待进一步分析研究。
5.针对不同对热激反应蛋白质表达共性和特异性的差异,选择菁松和Nistari两个品种,热激处理后恢复2小时的脂肪体中可诱导HSPs,应用2DGE分析进行定量研究并进行统计检验。结果发现,45℃热激处理,sHSPs的表达在多化性品种Nistari中的表达比二化性品种菁松中的表达低,但是在41℃热激处理却区则两者之间并无显著差异。也就是说,在比较温和的热激处理后,不同耐热性的蚕品种在sHSPs表达量上没有显著差异,而在较高的温度热激时,耐高温好的Nistari在sHSPs表达量显著的低(P<0.01)。相反,HSP70的表达量在不同品种和不同热激温度之间都没有显著差异。
6.热激处理后恢复时间经过2小时和4小时之间在不同品种和处理的热激蛋白表达种类上没有差异,但在不同热激蛋白的表达量上有显著差异,特别是耐高温很强的Nistari品种中sHSP的表达量显著增加,这可能暗示了sHSP的表达与家蚕品种的耐高温性能有着更加密切的关系。
7.不同性别比较发现,sHSP和HSP70表达在雌雄之间有些可见的差异,但统计分析都没有达到极显著差异水平(P<0.01)
8.应用实时荧光定量PCR技术对上述定量差异进一步进行验证,对两个热激蛋白HSP19.9和HSP70的编码基因进行mRNA表达水平定量研究,结果非常一致。RNA转录水平：品种间耐热性低的菁松高于耐热性强的Nistari;处理间41℃热激高于45℃热激处理；不同热激蛋白间HSP19.9高于HSP70；雌雄间雄性高于雌性。
9.在日系耐热性差的品种皓月中检测到sHSPs之间表达差异很大：HSP19.9和HSP23.7表达较低,而HSP20.4表达很高,未被鉴定出的2号蛋白点表达量很低。
10.在所有本试验所选的品种中,其卵巢和精巢中可检测到三种HSPs两种sHSPs (HSP19.9、HSP20.4)和HSP70的表达,既有上调也有下调。
11.本实验应用双向电泳技术和MALDI-TOF-TOF光谱分析鉴定,检测到了家蚕基因组中所有的sHSP,认为所有家蚕品种中至少有7个小分子热激蛋白和4个具有不同PI和分子量的HSP70是热激可诱导的,并与家蚕幼虫的耐热性有关。
12.在本实验中检测到的9个特异表达热激蛋白中只有两个被鉴定为sHSP,说明现有的家蚕基因组数据库的限制,有待进一步完善。特异表达热激蛋白都落在小分子区域,并且其表达变化幅度也比HSP70大,推测可能sHSP在家蚕热激反应和家蚕品种耐热性差异中起更重要的作用。
13.本研究目的之一试图找到与家蚕品种耐热性有关的标记蛋白质。虽然在不同家蚕品种中找到了不同的特异表达热激蛋白,但要说明与品种耐热性的关系还有待用选择更多有特色的家蚕品种进行实验方可作出比较可靠的判断。
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.
引文
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