蛋白核小球藻Fesod基因的克隆及表达分析

doi:10.13560/j.cnki.biotech.bull.1985.2015.05.027

摘要/Abstract

摘要： 利用RACE技术克隆了蛋白核小球藻Fesod全基因序列，得到1 215 bp序列，其5'非翻译区长52 bp，3'非翻译区长452 bp，共编码236个氨基酸。生物信息学分析结果表明，该Fesod基因编码蛋白的分子量为26.42 kD，等电点为6.98；位于线粒体中的概率是73.9%；该蛋白没有信号肽序列；蛋白质二级结构预测结果表明FeSOD中α-螺旋和无规卷曲分别占53.39%和39.41%。再利用实时荧光定量PCR技术检测了不同盐度和水杨酸浓度对蛋白核小球藻Fesod基因表达的影响，结果表明Fesod表达量随着盐度升高而增加，45‰盐度表达量为15‰盐度的3.61倍；而植物激素水杨酸的添加一定程度抑制了45‰盐度培养藻Fesod基因的表达，并且随其浓度增加呈现先升后降的趋势。表明蛋白核小球藻Fesod受盐度诱导，而水杨酸对其影响不明显。

关键词: 蛋白核小球藻, 铁超氧化物歧化酶, 转录表达, 盐度, 水杨酸

Abstract: The Fesod gene of Chlorella pyrenoidosa was cloned by RACE method. Total 1 215 bp Fesod sequence was obtained, containing 52 bp 5' untranslated region, 452 bp 3' untranslated region, and an open reading frame of 711 bp. The bioinformatics analysis showed that the molecular weight of FeSOD was 26.42 kD and isoelectric point was 6.98; the FeSOD protein located in the mitochondria with a probability of 73.9%; the FeSOD protein had no signal peptide; and the secondary structure prediction results showed that a-helix and random coil in FeSOD accounted for 53.39% and 39.41%, respectively. Then, the transcriptional expression of Fesod at different concentrations of salinity and salicylic acid was detected by real-time fluorescent quantitative PCR. The results showed that Fesod expression increased with the salinity varying from 15‰ to 45‰, and the expression quantity was 3.61 times under 45‰ salinity culture than that under 15‰ salinity. Moreover, salicylic acid could inhibit the mRNA accumulation of Fesod to a certain extent under 45‰ salinity, and its accumulation firstly increased and then decreased with the salicylic acid concentration from 0.1 to 2.0 mmol/L. In conclusion, the expression of Fesod was induced by salinity, while salicylic acid showed no significant effect on its expression.

Key words: Chlorella pyrenoidosa, Fe-superoxide dismutase, transcriptional expression, salinity, salicylic acid

沈佳, 江灵芝, 孙雪. 蛋白核小球藻Fesod基因的克隆及表达分析[J]. 生物技术通报, 2015, 31(5): 173-178.

Shen Jia, Jiang Lingzhi, Sun Xue. Gene Cloning and Expression Analysis of Fesod Gene from Chlorella pyrenoidosa[J]. Biotechnology Bulletin, 2015, 31(5): 173-178.

参考文献

[1] 覃鹏, 刘飞虎, 梁雪妮. 超氧化物歧化酶与植物抗逆性[J]. 黑龙江农业科学, 2002, 24（1）：31-34.
[2] 赵咏梅. 植物SOD在抵抗干旱胁迫中的作用[J]. 生物学教学, 2011, 36（3）：4-5.
[3] McCord JM, Fridovich I. Superoxide dismutase：An enzymic func-tion for erythrocuprein（hemocuprein）[J]. J Biol Chem, 1969, 224（22）：6049-6055.
[4] Matés JM. Effects of antioxidant enzymes in the molecular control of reactive oxygen species toxicology[J]. Toxicology, 2000, 153（1-3）：83-104.
[5] 焉婷婷, 周亚维, 李朋富, 等. 广盐性硅藻披针舟形藻在高盐和低盐胁迫下的抗氧化响应[J]. 盐业与化工, 2010, 39（5）：15-19.
[6] Jahnke LS, White AL. Long-term hyposaline and hypersaline stresses produce distinct antioxidant responses in the marine alga Dunaliella tertiolecta[J]. Plant Physiol, 2003, 160（10）：1193-1202.
[7] 马旭俊, 朱大海. 植物超氧化物歧化酶（SOD）的研究进展[J]. 遗传, 2003, 25（2）：225-231.
[8] Hayat Q, Hayat S, Irfan M, et al. Effect of exogenous salicylic acid under changing environment：A review[J]. Environmental and Experimental Botany, 2010, 68（1）：14-25.
[9] Parashar A, Yusuf M, Fariduddin Q, et al. Salicylic acid enhances antioxidant system in Brassica juncea grown under different levels of manganese[J]. Int J Biol Macromol, 2014, 70：551-558.
[10] Li T, Hu Y, Du X, et al. Salicylic acid alleviates the adverse effects of salt stress in Torreya grandis cv. Merrillii seedlings by activating photosynthesis and enhancing antioxidant systems[J]. PLoS One, 2014, 9（10）：e109492.
[11] Mutlu S, Atici O. Alleviation of high salt toxicity-induced oxidative damage by salicylic acid pretreatment in two wheat cultivars[J]. Toxicol Ind Health, 2013, 29（1）：89-96.
[12] Al-Whaibi MH, Siddiqui MH, Basalah MO. Salicylic acid and calcium-induced protection of wheat against salinity[J]. Protoplasma, 2012, 249（3）：769-778.
[13] 李建宏, 浩云涛, 翁永萍. Cd^2＋胁迫条件下椭圆小球藻的生理应答[J]. 水生生物学报, 2004, 28（6）：659-663.
[14] 王淑智, 李利, 张道勇, 等. NaCl与Cd对小球藻光系统Ⅱ（PSⅡ）活性的影响[J]. 应用与环境生物学报, 2011, 17（6）：839-846.
[15] 许超, 李梅梅, 曾伟, 等. 高效氯氰菊酯对蛋白核小球藻的毒性影响[J]. 环境科学与技术, 2013, 36（12M）：6-10, 41.
[16] 邓意龙, 孙雪, 徐年军, 等. 蛋白核小球藻（Chlorella pyrenoi-dosa）Rubisco活化酶基因的克隆与表达分析[J]. 海洋与湖沼, 2012, 43（1）：41-46.
[17] Livak KJ, Schmittgen TD. Analysis of relative gene expression data using real-time quantitative PCR and the 2^-ΔΔCTmethod[J]. Method, 2001, 25（4）：402-408.
[18] Yang R, Liu W, Zhang XL, et al. Sequences ofMn-sodgene fromPyropia haitanensis（Bangiales, Rhodophyta）and its expression under heat shock[J]. Botanica Marina, 2013, 56（3）：249-259.
[19] Lu N, Zang X, Zhang X, et al. Gene cloning, expression and activity analysis of manganese superoxide dismutase from two strains of Gracilaria lemaneiformis（Gracilariaceae, Rhodophyta）under heat stress[J]. Molecules, 2012, 17（4）：4522-4532.
[20] 郭建军, 龚兴国, 鲁宇文, 等. 钝顶螺旋藻Fe-SOD基因的克隆及序列分析[J]. 浙江大学学报：理学版, 2004, 31（6）：674-678.
[21] 樊寅卯, 张蕾, 秦松, 等. 雨生红球藻铁型超氧化物酶基因的克隆与序列分析[J]. 海洋科学, 2012, 36（4）：857-865.
[22] McKim SM, Durnford DG. Translational regulation of light-harves-ting complex expression during photoacclimation to high-light in Chlamydomonas reinhardtii[J]. Plant Physiol Biochem, 2006, 44（11-12）：857-865.
[23] 李娟, 杨立新, 郑文寅, 等. 小麦FeSOD基因的克隆与序列分析[J]. 核农学报, 2013, 27（8）：1111-1117.
[24] Kanematsu S, Okayasu M, Ueno S. Atypical cytosol-localized Fe-superoxide dismutase in the moss Pogonatum inflexum[J]. Bull Minamikyushu Univ, 2013, 43 A：23-31.
[25] Salin ML. Toxic oxygen species and protective systems of the chloroplast[J]. Physiologia Plantarum, 1988, 72（3）：681-689.
[26] 卢雪景, 刘红涛, 柴玉荣, 等. 外源一氧化氮缓解盐胁迫下杜氏盐藻氧化损伤的初步研究[J]. 河南农业科学, 2009, 37（5）：47-52.
[27] 李琳玲, 程华, 许锋, 等. 银杏铁型超氧化物歧化酶基因（GbFeSOD）的克隆与表达[J]. 果树学报, 2009, 26（6）：840-846.
[28] Kurepa J, Herouart D, Van MM. Differential expression of CuZn-and Fe-superoxide dismutase genes of tobacco during development, oxidative stress, and hormonal treatments[J]. Plant Cell Physiol, 1997, 38（4）：463-470.