当归延伸因子AsEF-1β基因的克隆及胁迫应答分析
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摘要
延伸因子1β(EF-1β)是蛋白质生物合成过程中肽链延长必需的调节因子之一。该研究采用同源克隆和RACE扩增技术克隆当归EF-1β基因序列,分析该基因序列特征、蛋白结构特点及UV-B辐射胁迫下的组织响应表达,以揭示当归栽培生境变迁过程中对UV-B胁迫适应的分子机制。结果显示:(1)成功克隆获得当归EF-1β基因全长序列(950 bp),编码225个氨基酸,命名为AsEF-1β(GenBank登录号:MG736314);AsEF-1β蛋白的分子量为24.5 kD,理论等电点为4.48,属亲水性氨基酸,在其C末端具有一个EF-1B超蛋白家族的典型结构域和保守区,鸟嘌呤核苷酸交换结构域;其氨基酸序列与同为伞形科的胡萝卜氨基酸序列相似性最高,达93%。(2)qRT-PCR分析结果显示,AsEF-1β基因在当归根部的表达量显著高于茎和叶(P<0.05);UV-B辐射胁迫下,茎及叶中的表达量均上调,分别是自然光照处理的2.43和3.76倍。研究表明,AsEF-1β基因可能参与当归对UV-B辐射胁迫的适应过程,为深入研究其在药用植物生长发育、逆境抗性形成及药效物质的生物合成代谢过程的生态调控奠定了基础。
Elongation factor 1β(EF-1β) is one of the essential regulatory factors of peptide chain prolongation in the process of protein biosynthesis. This study cloned the EF-1β gene sequence from Angelica sinensis using homologous cloning and RACE amplification techniques. We analyzed the sequence characterization, protein structure characteristics and expression profiling of EF-1β gene under UV-B radiation stress, to reveal the molecular mechanism of adaptation to UV-B radiation stress in the process of cultivation habitat change of A. sinensis. The results showed that:(1) the full-length sequence of EF-1β gene(950 bp), encodes 225 amino acids, which was named AsEF-1β gene(GenBank accession number: MG736314). The molecular weight of AsEF-1β is 24.5 kD, theoretical isoelectric point of 4.48. AsEF-1β belongs to hydrophilic amino acid, with the typical domain and conservative domain of family of EF-1 B super protein, and has a guanine nucleotide exchange domain at its C-terminal. The amino acid sequence of AsEF-1β gene showed the highest similarity with that of Daucus carota L. var. sativa Hoffm., which belongs to the same family of Umbrella.(2) the qRT-PCR analysis results indicated that AsEF-1β gene in root of A. sinensis was significantly higher than that in stem and leaf(P<0.05). Under UV-B radiation stress, the relative expression level of stem and leaf were up-regulated, which was 2.43 times and 3.76 times that of natural light condition(P<0.05), respectively. Studies showed that AsEF-1β gene may be involved in the adaptation process of A. sinensis to UV-B radiation stress, which will be laid for further exploration on its ecological regulations in the growth and development, formation of stress resistance and biosynthesis of pharmacodynamic substances of medicinal plants.
Elongation factor 1β(EF-1β) is one of the essential regulatory factors of peptide chain prolongation in the process of protein biosynthesis. This study cloned the EF-1β gene sequence from Angelica sinensis using homologous cloning and RACE amplification techniques. We analyzed the sequence characterization, protein structure characteristics and expression profiling of EF-1β gene under UV-B radiation stress, to reveal the molecular mechanism of adaptation to UV-B radiation stress in the process of cultivation habitat change of A. sinensis. The results showed that:(1) the full-length sequence of EF-1β gene(950 bp), encodes 225 amino acids, which was named AsEF-1β gene(GenBank accession number: MG736314). The molecular weight of AsEF-1β is 24.5 kD, theoretical isoelectric point of 4.48. AsEF-1β belongs to hydrophilic amino acid, with the typical domain and conservative domain of family of EF-1 B super protein, and has a guanine nucleotide exchange domain at its C-terminal. The amino acid sequence of AsEF-1β gene showed the highest similarity with that of Daucus carota L. var. sativa Hoffm., which belongs to the same family of Umbrella.(2) the qRT-PCR analysis results indicated that AsEF-1β gene in root of A. sinensis was significantly higher than that in stem and leaf(P<0.05). Under UV-B radiation stress, the relative expression level of stem and leaf were up-regulated, which was 2.43 times and 3.76 times that of natural light condition(P<0.05), respectively. Studies showed that AsEF-1β gene may be involved in the adaptation process of A. sinensis to UV-B radiation stress, which will be laid for further exploration on its ecological regulations in the growth and development, formation of stress resistance and biosynthesis of pharmacodynamic substances of medicinal plants.
引文
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[10] 程君,芮耀诚.真核延长因子eEF1A功能研究进展[J].药学实践杂志,2012,30(2):89-91,124.CHENG J,RUI Y C.Advance in function of the eukaryotic elongation factor eEF1A[J].Journal of Pharmaceutical Practice,2012,30(2):89-91,124.
[11] LE SOURD F,BOULBEN S,LE BOUFFANT R,et al.eEF1B:At the dawn of the 21st century[J].Biochimica et Biophysica Acta,2006,1 759(1-2):13-31.
[12] 彭亚兰,王友绍.红树植物桐花树EF1A基因的克隆与表达分析[J].生态科学,2014,33(4):704-712.PENG Y L,WANG Y S.Molecular characterization and expression analysis of elongation factor 1A from mangrove Aegiceras corniculatum[J].Ecological Science,2014,33(4):704-712.
[13] SASIKUMAR A N,PEREZ W B,KINZY T G.The many roles of the eukaryotic elongation factor 1 complex[J].Wiley Interdisciplinary Reviews,RNA,2012,3(4):543-555.
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[15] HWANG J,LEE S,LEE J H,et al.Plant translation elongation factor 1Bβ facilitates X (PVX) infection and interacts with PVX triple gene block protein 1[J].PLoS One,2015,10(5):e0128014.
[16] HWANG J,OH C S,KANG B C.Translation elongation factor 1B (eEF1B) is an essential host factor for tobacco mosaic virus infection in plants[J].Virology,2013,439(2):105-114.
[17] 陈炫,张天烨,羊健,等.小麦TaeEF1β基因的克隆、同源性及表达分析[J].浙江农业学报,2019,31(1):98-103.CHEN X,ZHANG T Y,YANG J,et al.Gene cloning,homology and expression analysis of TaeEF1β in Triticum aestivum L.[J].Acta Agriculturae Zhejiangensis,2019,31(1):98-103.
[18] 吴永娜,胡静,王引权,等.当归肌动蛋白基因片段的克隆及序列分析[J].中草药,2012,43(12):2 485-2 489.WU Y N,HU J,WANG Y Q,et al.Cloning and sequence analysis on actin gene fragment from Angelica sinensis[J].Chinese Traditional and Herbal Drugs,2012,43(12):2 485-2 489.
[19] SUN W,LI Y,ZHAO Y X,et al.Isolation and characterizing of a cDNA clone encoding an elongation factor EF-1α from halophyte Suaeda salsa[J].Acta Botanica Boreali-Occidentalia Sinica,2004,24(9):1 657-1 661.
[20] COSTA A,GIACOMO M D,MASSARELLI I,et al.Isolation,characterization and expression of an elongation factor 1α gene in potato (Solanum tuberosum) cell cultures[J].Giornale Botanico Italiano,2010,144(3):618-625.
[2] 王引权,王艳,陈红刚,等.海拔梯度对药用植物品质形成影响的研究进展[J].中国现代中药,2012,14(5):41-44.WANG Y Q,WANG Y,CHEN H G,et al.Advances in influences of altitudinal gradient on the distribution and quality formation of medicinal plants[J].Modern Chinese Medicine,2012,14(5):41-44.
[3] 潘红丽,李迈和,蔡小虎,等.海拔梯度上的植物生长与生理生态特性[J].生态环境学报,2009,18(2):722-730.PAN H L,LI M H,CAI X H,et al.Responses of growth and ecophsiology of plants to altitude[J].Ecology and Environmental Sciences,2009,18(2):722-730.
[4] WANG H,QIN F,CHENG P,et al.Effects of UV-B radiation intensity and timing on epidemiological components of wheat stripe rust[J].Journal of Integrative Agriculture,2018,17(12):2 704-2 713.
[5] YIN R,ULM R.How plants cope with UV-B:from perception to response[J].Current Opinion in Plant Biology,2017,37:42-48.
[6] ZHANG X X,TANG X X,ZHOU B,et al.Effect of enhanced UV-B radiation on photosynthetic characteristics of marine microalgae Dunaliella salina (Chlorophyta,Chlorophyceae)[J].Journal of Experimental Marine Biology and Ecology,2015,469:27-35.
[7] DWIVEDI R,SINGH V P,KUMAR J,et al.Differential physiological and biochemical responses of two Vigna species under enhanced UV-B radiation[J].Journal of Radiation Research and Applied Sciences,2015,8(2):173-181.
[8] 刁倩楠,蒋雪君,陈幼源,等.外源水杨酸预处理对低温胁迫下甜瓜幼苗生长及其抗逆生理特性的影响[J].西北植物学报,2018,38(11):2 072-2 080.DIAO Q N,JIANG X J,CHEN Y Y,et al.Effects of exogenous salicyic acid pretreatmenton on growth and resistance physiological indexes in melon seedling under chilling stress[J].Acta Botanica Boreali-Occidentalia Sinica,2018,38(11):2 072-2 080.
[9] 董天宇,朱旭东,张克坤,等.温度和盐胁迫对草莓开花进程和相关基因表达的影响[J].西北植物学报,2019,39(4):638-647.DONG T Y,ZHU X D,ZHANG K K,et al.Effect of temperature ang salt stress on the flowering process of strawberry[J].Acta Botanica Boreali-Occidentalia Sinica,2019,39(4):638-647.
[10] 程君,芮耀诚.真核延长因子eEF1A功能研究进展[J].药学实践杂志,2012,30(2):89-91,124.CHENG J,RUI Y C.Advance in function of the eukaryotic elongation factor eEF1A[J].Journal of Pharmaceutical Practice,2012,30(2):89-91,124.
[11] LE SOURD F,BOULBEN S,LE BOUFFANT R,et al.eEF1B:At the dawn of the 21st century[J].Biochimica et Biophysica Acta,2006,1 759(1-2):13-31.
[12] 彭亚兰,王友绍.红树植物桐花树EF1A基因的克隆与表达分析[J].生态科学,2014,33(4):704-712.PENG Y L,WANG Y S.Molecular characterization and expression analysis of elongation factor 1A from mangrove Aegiceras corniculatum[J].Ecological Science,2014,33(4):704-712.
[13] SASIKUMAR A N,PEREZ W B,KINZY T G.The many roles of the eukaryotic elongation factor 1 complex[J].Wiley Interdisciplinary Reviews,RNA,2012,3(4):543-555.
[14] FURUKAWA R,JINKS T M,TISHGARTEN T,et al.Elongation factor 1β is an actin-binding protein[J].Biochimica et Biophysica Acta (BBA) - General Subjects,2001,1 527(3):130-140.
[15] HWANG J,LEE S,LEE J H,et al.Plant translation elongation factor 1Bβ facilitates X (PVX) infection and interacts with PVX triple gene block protein 1[J].PLoS One,2015,10(5):e0128014.
[16] HWANG J,OH C S,KANG B C.Translation elongation factor 1B (eEF1B) is an essential host factor for tobacco mosaic virus infection in plants[J].Virology,2013,439(2):105-114.
[17] 陈炫,张天烨,羊健,等.小麦TaeEF1β基因的克隆、同源性及表达分析[J].浙江农业学报,2019,31(1):98-103.CHEN X,ZHANG T Y,YANG J,et al.Gene cloning,homology and expression analysis of TaeEF1β in Triticum aestivum L.[J].Acta Agriculturae Zhejiangensis,2019,31(1):98-103.
[18] 吴永娜,胡静,王引权,等.当归肌动蛋白基因片段的克隆及序列分析[J].中草药,2012,43(12):2 485-2 489.WU Y N,HU J,WANG Y Q,et al.Cloning and sequence analysis on actin gene fragment from Angelica sinensis[J].Chinese Traditional and Herbal Drugs,2012,43(12):2 485-2 489.
[19] SUN W,LI Y,ZHAO Y X,et al.Isolation and characterizing of a cDNA clone encoding an elongation factor EF-1α from halophyte Suaeda salsa[J].Acta Botanica Boreali-Occidentalia Sinica,2004,24(9):1 657-1 661.
[20] COSTA A,GIACOMO M D,MASSARELLI I,et al.Isolation,characterization and expression of an elongation factor 1α gene in potato (Solanum tuberosum) cell cultures[J].Giornale Botanico Italiano,2010,144(3):618-625.
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