长白落叶松过氧化氢酶LoCAT1基因克隆及表达分析

doi:10.7525/j.issn.1673-5102.2019.04.008

摘要/Abstract

摘要： 为了解长白落叶松过氧化氢酶（CAT）基因的相关信息，探究该基因在长白落叶松不同组织中及不同逆境胁迫下的表达特性，本研究根据长白落叶松转录组数据库中获得的CAT1基因全长序列设计引物，克隆得到长白落叶松CAT1基因，命名为LoCAT1。该基因完整的开放阅读框（ORF）长度为954 bp，共编码317个氨基酸。系统进化树分析结果显示，LoCAT1基因与北美云杉、银杏等CAT基因亲缘关系较近。利用实时定量RT-PCR技术分析了LoCAT1基因在长白落叶松中的组织表达特异性和应对非生物胁迫的表达模式。结果表明：LoCAT1基因在长白落叶松的根、茎、叶中均有表达，其中在茎部表达量最低，在叶中相对表达量最高。在非生物胁迫下，LoCAT1基因在长白落叶松根、茎、叶中的表达均发生了变化，但表达模式不同。在NaCl处理后，根和茎中LoCAT1基因均表现为下调表达，在12 h时表达量最低，而叶中LoCAT1基因表达在24 h明显受抑制，随后被上调表达，胁迫96 h时表达量最高。PEG₆₀₀₀处理后，根和茎中LoCAT1基因的表达在胁迫早期被明显抑制，随后被上调表达。而叶中LoCAT1基因的表达在所有时间点均表现为上调表达。本研究推测长白落叶松LoCAT1基因可能参与了植物响应逆境胁迫的应答。

关键词: 长白落叶松, LoCAT1, 克隆, 生物信息学分析, 表达分析

Abstract: In order to know about genes of Larix olgensis catalase and determine the expression characteristics in different organs of L. olgensis under different stresses, a CAT1 gene was cloned based on specific primers designed according to the CAT1 gene sequences from transcriptome database of L. olgensis, named LoCAT1. The complete length of open read frame(ORF) is 954 bp, encoding 317 amino acids. The results of phylogenetic tree analysis indicated that there was a closer relationship between LoCAT1 and Picea sitchensisand Ginkgo biloba. By real-time quantitative polymerase chain reaction (qPCR) analysis, LoCAT1 is highly expressed in leaf, while less expressed in stems. The expressions of LoCAT1 were obviously difference in roots and stems and leaves of L. olgensis under abiotic stress. The expression patterns of LoCAT1 were not exactly similarity among these treatments. The expression levels of LoCAT1 in roots and stems of L. olgensis after the treatment of NaCl were down-regulated, with the lowest expression at 12 h. While expression level in leaves was significantly inhibited at 24 h, and then up-regulated, with the highest expression at 96 h. The expression levels of LoCAT1 in roots and stems of L. olgensis after the treatment of PEG₆₀₀₀ were significantly inhibited in the early stage and then up-regulated. While LoCAT1 expression level in leaves was up-regulated. It was suggested that LoCAT1 may play an important role in response to stresses in plant.

Key words: Larix olgensis, LoCAT1, cloning, bioinformatics analysis, expression analysis

中图分类号:

Q781
S791.22

白晓明, 董实伟, 杨宇宁, 宋跃, 张含国, 李淑娟. 长白落叶松过氧化氢酶LoCAT1基因克隆及表达分析[J]. 植物研究, 2019, 39(4): 539-546.

BAI Xiao-Ming, DONG Shi-Wei, YANG Yu-Ning, SONG Yue, ZHANG Han-Guo, LI Shu-Juan. Cloning and Expression Analysis of Catalase Gene(LoCAT1) from Larix olgensis[J]. Bulletin of Botanical Research, 2019, 39(4): 539-546.

参考文献

1. Gupta D K,Pena L B,Romero-Puertas M C,et al. NADPH oxidases differentially regulate ROS metabolism and nutrient uptake under cadmium toxicity[J]. Plant,Cell & Environment,2017,40(4):509-526.
2. Thapa G,Sadhukhan A,Panda S K,et al. Molecular mechanistic model of plant heavy metal tolerance[J]. Biometals,2012,25(3):489-505.
3. Loew O. A new enzyme of general occurrence in organismis[J]. Science,1900,11(279):701-702.
4. 刘灵芝,钟广蓉,熊莲,等. 过氧化氢酶的研究与应用新进展[J]. 化学与生物工程,2009,26(3):15-18.
Liu L Z,Zhong G R,Xiong L,et al. Research and application progress of catalase[J]. Chemistry & Bioengineering,2009,26(3):15-18.
5. Purev M,Kim Y J,Kim M K,et al. Isolation of a novel catalase(Cat1) gene from Panax ginseng and analysis of the response of this gene to various stresses[J]. Plant Physiology and Biochemistry,2010,48(6):451-460.
6. 王凤德,衣艳君,王海庆,等. 豌豆过氧化氢酶在烟草叶绿体中的过量表达提高了植物的抗逆性[J]. 生态学报,2011,31(4):1058-1063.
Wang F D,Yi Y J,Wang H Q,et al. Enhanced drought and photooxidation tolerance of transgenic tobacco plants overexpressingpea catalase in chloroplasts[J]. Acta Ecologica Sinica,2011,31(4):1058-1063.
7. 于德玲,王昌留. 过氧化氢酶的研究进展[J]. 中国组织化学与细胞化学杂志,2016,25(2):189-194.
Yu D L,Wang C L. Progresses in the research of catalase[J]. Chinese Journal of Histochemistry and Cytochemistry,2016,25(2):189-194.
8. 赵杨,魏颖娟,邹应斌. 低温胁迫下早稻幼苗叶片和根系的生理指标变化及其品种间差异[J]. 核农学报,2015,29(4):792-798.
Zhao Y,Wei Y J,Zhou Y B. Physiological index variation and its cultivar differences of seedling leaves and roots of early cropping rice under lower temperature stress[J]. Journal of Nuclear Agricultural Sciences,2015,29(4):792-798.
9. 刘俊祥,魏树强,翟飞飞,等. Cd²⁺胁迫下多年生黑麦草的生长与生理响应[J]. 核农学报,2015,29(3):587-594.
Liu J X,Wei S Q,Zhai F F,et al. Growth and physiology response of perennial ryegrass to Cd²⁺ stress[J]. Journal of Nuclear Agricultural Sciences,2015,29(3):587-594.
10. 蔡永智,祝建波,郝晓云,等. 转过氧化氢酶基因KatG棉花的抗旱性[J]. 西北农业学报,2013,22(12):56-61.
Cai Y Z,Zhu J B,Hao X Y,et al. Drought resistance of KatG transgenic cotton[J]. Acta Agriculturae Boreali-occidentalis Sinica,2013,22(12):56-61.
11. 彭丹,张霞,张富春. 盐穗木过氧化氢酶基因的克隆与功能分析[J]. 西北植物学报,2013,33(10):1933-1939.
Peng D,Zhang X,Zhang F C. Cloning and functional identification of catalase gene in Halostachys caspica[J]. Acta Agriculturae Boreali-occidentalis Sinica,2013,33(10):1933-1939.
12. 刘大丽,马龙彪,鲁振强. 过量表达OsCATb提高大肠杆菌对不同重金属逆境胁迫的耐受性[J]. 中国农学通报,2018,34(34):36-41.
Liu D L,Ma L B,Lu Z Q. Over-expression of OsCATb Enhanced E. coli stress tolerance to heavy metals[J]. Chinese Agricultural Science Bulletin,2018,34(34):36-41.
13. 王升平,杨金广,战徊旭,等. 烟草过氧化氢酶基因CAT1的克隆及表达特征分析[J]. 中国烟草学报,2014,20(5):103-109.
Wang S P,Yang J G,Zhan H X,et al. Cloning of catalase gene(CAT1) and its expression patterns in Nicotiana tabacum L.[J]. Acta Tabacaria Sinica,2014,20(5):103-109.
14. Frugoli J A,Zhong H H,Nuccio M L,et al. Catalase is encoded by a multigene family in Arabidopsis thaliana(L.) Heynh[J]. Plant Physiology,1996,112(1):327-336.
15. 林星谷,孔德仓,庞晓明,等. 冬枣过氧化氢酶基因的克隆及表达分析[J]. 西北植物学报,2012,32(6):1086-1092.
Lin X G,Kong D C,Pang X M,et al. Cloning and tissue expression analysis of catalase gene in Dongzao(Ziziphus jujuba)[J]. Acta Botanica Boreali-Occidentalia Sinica,2012,32(6):1086-1092.
16. 程华,李琳玲,许峰,等. 银杏过氧化氢酶基因CAT1的克隆及表达分析[J]. 林业科学研究,2010,23(4):493-499.
Cheng H,Li L L,Xu F,et al. Molecular cloning,characterization and expression of CAT1 gene from Ginkgo biloba[J]. Forest Research,2010,23(4):493-499.
17. 温庆放,刘建汀,朱海生,等. 丝瓜过氧化氢酶基因CAT1的克隆及表达分析[J]. 园艺学报,2016,43(10):2039-2048.
Wen Q F,Liu J T,Zhu H S,et al. Cloning and expression analysis of catalase CAT1 gene from Luffa cylindrical[J]. Acta Horticulturae Sinica,2016,43(10):2039-2048.
18. 胡晓晴. 一氧化氮处理后长白落叶松转录组测序及LoERF017基因功能研究[D]. 哈尔滨:东北林业大学,2016.
Hu X Q. Transcriptome sequencing of Larix olgensis Henry induced by nitric oxide and functional characterization of LoERF017 gene[D]. Harbin:Northeast Forestry University,2016.
19. 宋跃,李淑娟,白晓明,等. 响应面优化长白落叶松体胚形成早期培养条件的研究[J]. 植物研究,2017,37(4):613-620.
Song Y,Li S J,Bai X M,et al. Response surface optimization of culture conditions for the formation of early embryos in Larix olgensis[J]. Bulletin of Botanical Research,2017,37(4):613-620.
20. Willekens H,Van Camp W,Van Montagu M,et al. Ozone,sulfur dioxide,and ultraviolet B have similar effects on mRNA accumulation of antioxidant genes in Nicotiana plumbaginifolia L.[J]. Plant Physiology,1994,106(3):1007-1014.
21. Prasad T K,Anderson M D,Martin C R. Evidence for chilling-induced oxidative stress in maize seedlings and a regulatory role for hydrogen peroxide[J]. The Plant Cell,1994,6(1):65-74.
22. Mcclung C R. Regulation of catalases in Arabidopsis[J]. Free Radical Biology and Medicine,1997,23(3):489-496.
23. Suzuki M,Ario T,Hattori T,et al. Isolation and characterization of two tightly linked catalase genes from castor bean that are differentially regulated[J]. Plant Molecular Biology,1994,25(3):507-516.
24. Yang W L,Liu J M,Chen F,et al. Identification of Festuca arundinacea schreb CAT1 catalase gene and analysis of its expression under abiotic stresses[J]. Journal of Integrative Plant Biology,2006,48(3):334-340.
25. 张仁和,郑友军,马国胜,等. 干旱胁迫对玉米苗期叶片光合作用和保护酶的影响[J]. 生态学报,2011,31(5):1303-1311.
Zhang R H,Zheng Y J,Ma G S,et al. Effects of drought stress on photosynthetic traits and protective enzyme activity in maize seeding[J]. Acta Ecologica Sinica,2011,31(5):1303-1311.

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