以SAM (S-腺苷酰-L-甲硫氨酸)基因作为内参基因, 利用实时荧光定量PCR技术对稀杯盔形珊瑚(Galaxea astreata)共生虫黄藻(Symbiodinium spp.) Hsp70、Hsp90、psaA和psbA基因在升温至32℃持续胁迫及恢复至28℃后的表达情况进行了研究。结果表明, 升温至32℃时, Hsp70、Hsp90表达量无显著变化(p>0.05), 随着高温胁迫的持续, Hsp70与Hsp90的表达量显著增高(p<0.01), 到32℃ 7d时2个基因表达量达到最大值(分别为胁迫前的10.3倍和28.2倍); 在随后的降温过程中, Hsp70和Hsp90的表达量显著下降(p<0.05), 当温度恢复至28℃ 5d时2个基因的表达量均下降至较低水平(分别为胁迫前的0.6倍和2.0倍, p<0.01), 且趋于稳定。随着温度的升高, psaA的表达量略有升高(p>0.05), 在31℃ 24h时表达量达到最高(为胁迫前的1.2倍), 随后显著下降(p<0.01); 而psbA的表达量随着温度的升高显著增加(p<0.05); 在31℃ 24h时表达量达到最高(为胁迫前的1.9倍), 继续升温至32℃持续胁迫24h后才开始显著下降(p<0.01)。当温度降至28℃时, 2个基因的表达量有一定程度的增加, 但是仍显著低于胁迫前的表达水平(p<0.01), 28℃ 9d时分别为胁迫前的0.27倍和0.15倍, 表明热应激可能使虫黄藻的光合功能受到损伤。
The relative expressions of stress genes Hsp70, Hsp90 and choloplast psaA, psbA (SAM as reference gene) in Galaxea astreata endosymbiotic zooxanthella (Symbiodinium spp.) under heat stress were detected by using real-time PCR technique. All samples were exposed to elevated temperature at 32 ℃ over a 7-day period, and then the temperature was reduced to 28 ℃ gradually, with the temperature change from 28 ℃ to 32 ℃ at a rate of 1 ℃ per 24 hours. The results showed that the expression patterns of Hsp70 and Hsp90 were similar. The induced expression levels were upregulated but showed no significant difference compared to the control group (p>0.05) during the early stage of temperature climbing, subsequently reaching the maximize level after 168h tolerance period at 32 ℃. The lowest abundance of the gene products was detected when returned to the normal condition of 28 ℃ after 5-day recovery. Moreover, we examined the expression levels of chloroplast psaA and psbA genes stimulated by heat. Both were increased at the initial stage of thermal stress, and reached maximize at 31 ℃ for 24 h, with 1.2 and 1.9 folds compared to the level of the untreated group, respectively. Subsequently at 32 ℃, psbA transcripts maintained the high level until 24 h consecutive hours of stress, while psaA immediately decreased significantly (p<0.01). When temperature recovery reached 28 ℃, the transcripts of the two genes were significantly lower than the initial level (p<0.01), which indicates that the photosynthetic function of endosymbiotic zooxanthella may be injured (or inhibited).
1 佘大为, 张海波, 王月, 等, 2007. 坛紫菜叶绿体 psaA 基因片段的序列分析[J]. 水产科学, 26(5): 289-291. SHE DAWEI, ZHANG HAIBO, WANG YUE, et al, 2007. Sequence analyses of chloroplastic psaA gene fragment from Porphyra haitanensis [J]. Fisheries Science, 26(5): 289-291 (in Chinese).
2 赵美霞, 余克服, 张乔民, 2006. 珊瑚礁区的生物多样性及其生态功能[J]. 生态学报, 26(1): 186-194. ZHAO MEIXIA, YU KEFU, ZHANG QIAOMIN, 2006. Review on coral reefs biodiversity and ecological function[J]. Acta Ecologica Sinica, 26(1): 186-194 (in Chinese).
3 朱葆华, 2005. 几种腔肠动物共生藻的离体培养及其相关的生理学研究[D]. 青岛: 中国科学院海洋研究所. ZHU BAOHUA, 2005. Axenic cultivation of Symbiodinium sp. from several coelenterates and investigation for their physiology[D]. Qing Dao: The Institute of Oceanology, Chinese Academy of Sciences (in Chinese).
4 ARO E M, VIRGIN I, ANDERSSON B, 1993. Photoinhibition of Photosystem Ⅱ. Inactivation, protein damage and turnover[J]. Biochimica et Biophysica Acta, 1143(2): 113-134.
5 BAIRD A H, BHAGOOLI R, RALPH P, et al, 2009. Coral bleaching: the role of the host[J]. Trends in Ecology & Evolution, 24(1): 16-20.
6 BELLANTUONO A J, HOEGH-GULDBERG O, RODRIGUEZ- LANETTY M, 2012. Resistance to thermal stress in corals without changes in symbiont composition[J]. Proceedings of the Royal Society B: Biological Sciences, 279(1): 1100-1107.
7 BHARGAV D, SINGH M P, MURTHY R C, 2007. Toxic Potential of municipal solid waste leachates in transgenic Drosophila melanogaster ( Hsp70 laez): Hsp70 as a marker of cellular damage[J]. Ecotoxicology and Environmental Safety, 2(12): 105-109.
8 CLARK M S, PECK L S, 2009. Triggers of the Hsp70 stress response: environmental responses and laboratory manipulation in an Antarctic marine invertebrate (Nacella concinna)[J]. Cell Stress and Chaperones, 14: 649-660.
9 COLOMBO-PALLOTTA M F, RODRÍGUEZ-ROMÁN A, IGLESIAS-PRIETO R, et al, 2010. Calcification in bleached and unbleached Montastraea faveolata evaluating the role of oxygen and glycerol[J]. Coral Reefs, 29(4): 899-907.
10 CSERMELY P, SCHNAIDER T, SӦTI C, et al, 1998. The 90-kDa molecular chaperone family: structure, function and clinical applications. A comprehensive review[J]. Pharmacology & Therapeutics, 79(2): 129-168.
11 FITT W K, BROWN B E, WARNER M E, et al, 2001. Coral bleaching: interpretation of thermal tolerance limits and thermal thresholds in tropical corals[J]. Coral Reefs, 20(1): 51-65.
12 HENNIGE S J, MCGINLEY M P, GROTTOLI A G, et al, 2011. Photoinhibition of Symbiodinium sp. within the reef corals Montastraea faveolata and Porites astreoides : implications for coral bleaching[J]. Marine Biology, 158(11): 2515-2526.
13 IGLESIAS-PRIETO R, MATTA J L, ROBINS W A, et al, 1992. Photosynthetic response to elevated temperature in the symbiotic dinoflagellate Symbiodinium microadriaticum in culture[J]. Proceedings of the National Academy of Sciences, 89: 10302-10305.
14 JOKIEL P L, 2011. The reef coral two compartment proton flux model: a new approach relating tissue-level physiological processes to gross corallum morphology[J]. Journal of Experimental Marine Biology and Ecology, 409(1/2): 1-12.
15 KETTUNEN R, PURSIHEIMO S, RINTAMÄKI E, et al, 1997. Transcriptional and translational adjustments of psbA gene expression in mature chloroplasts during photoinhibition and subsequent repair of photosystem Ⅱ[J]. European Journal of Biochemistry, 247(1): 441-448 .
16 KUSNETSOV V V, MIKULOVICH T P, KUKINA I M, et al, 1993. Changes in the level of chloroplast transcripts in pumpkin cotyledons during heat shock[J]. FEBS Letters, 321(2/3): 189-193.
17 LESSER M P, 2006. Oxidative stress in marine environments: biochemistry and physiological ecology[J]. Annual Review of Physiology, 68: 253-278.
18 LUAN W, LI F H, ZHANG J Q, et al, 2010. Identification of a novel inducible cytosolic Hsp70 gene in Chinese shrimp Fenneropenaeus chinensis and comparison of its expression with the cognate Hsp70 under different stresses[J]. Cell Stress and Chaperones, 15(1): 83-93.
19 MCGINLEY M P, ASCHAFFENBURG M D, PETTAY D T, et al, 2012. Transcriptional response of two core photosystem genes in Symbiodinium spp. exposed to thermal stress[J]. PloS One, 7(12): e50439.
20 MIDDLEBROOK R, HOEGH-GULDBERG O, LEGGAT W, 2008. The effect of thermal history on the susceptibility of reef- building corals to thermal stress[J]. The Journal of Experimental Biology, 211(7): 1050-1056.
21 RAGNI M, HENNIGE S, SUGGETT D J, et al, 2010. PSⅡ photoinhibition and photorepair in Symbiodinium (Pyrrhophyta) differs between thermally tolerant and sensitive phylotypes[J]. Marine Ecology Progress Series, 406: 57-70.
22 ROSIC N N, PERNICE M, DOVE S, et al, 2011. Gene expression profiles of cytosolic heat shock proteins Hsp70 and Hsp90 from symbiotic dinoflagellates in response to thermal stress: possible implications for coral bleaching[J]. Cell Stress and Chaperones, 16(1): 69-80.
23 SAMMARCO P W, STRYCHAR K B, 2013. Responses to high seawater temperatures in Zooxanthellate Octocorals[J]. PloS One, 8(2): e54989.
24 SINGH A K, SINGHAL G S, 2001. Regulation of plastid gene expression by high temperature during light induced chloroplast development[J]. Photosynthetica, 39(3): 353-361.
25 STAT M, CARTER D, HOEGH-GULDBERG O, 2006. The evolutionary history of Symbiodinium and scleractinian hosts-symbiosis, diversity, and the effect of climate change[J]. Perspectives in Plant Ecology, Evolution and Systematics, 8(1): 23-43.
26 TAKAHASHI S, WHITNEY S M, ITOH S, et al, 2008. Heat stress causes inhibition of the de novo synthesis of antenna proteins and photobleaching in cultured Symbiodinium [J]. Proceedings of the National Academy of Sciences, 105(11): 4203-4208.
27 TAKAHASHI S, WHITNEY S M, BADGER M R, 2009. Different thermal sensitivity of the repair of photodamaged photosynthetic machinery in cultured Symbiodinium species[J]. Proceedings of the National Academy of Sciences, 106(9): 3237-3242.
28 WEIS V, 2008. Cellular mechanisms of Cnidarian bleaching: stress causes the collapse of symbiosis[J]. The Journal of Experimental Biology, 211: 3059-3066.
29 WANG W X, VINOCUR B, SHOSEYOV O, et al, 2004. Role of plant heat-shock proteins and molecular chaperones in the abiotic stress response[J]. Trends in Plant Science, 9(5): 244-252.
30 YELLOWLEES D, REES A, LEGGAT W, 2008. Metabolic interactions between algal symbionts and invertebrate hosts[J]. Plant Cell and Environment, 31(5): 679-694.
