刚毛柽柳腺苷甲硫氨酸脱羧酶(<em>ThSAMDC</em>)基因的克隆与胁迫...
摘要: S-腺苷甲硫氨酸脱羧酶(S-adenosyl-L-methionine decarboxylase,SAMDC)是一种通过参与多胺的代谢途径来调节植物生理生化过程的限速酶。通过分析刚毛柽柳(Tamarix hispida)的转录组数据,获得并克隆了SAMDC基因的cDNA序列,将其命名为ThSAMDC。该cDNA序列全长2085bp,包含tiny ORF(tORF)、upstream ORF(uORF)和main ORF(mORF)3个植物SAMDC基因特征ORF。主开放读码框(mORF)长1107bp,编码369个
氨基酸多肽,相对分子质量为40.34kD,理论等电点(PI)为4.72。ThSAMDC编码蛋白具有多个较强的亲水性区域,无明显跨膜区。通过与其他多个物种的氨基酸多序列比对结果表明,ThSAMDC具有两个典型的高度保守的结构域:酶原剪切位点(LSESSLF)与蛋白快速降解有关的PEST(TIHVTPEDGFSYAS)结构域。系统发育树结果表明ThSAMDC与菠菜(SoSAMDC)氨基酸序列一致性最高,为77%。实时荧光定量RT-PCR分析显示,ThSAMDC在NaCl、PEG、ABA、CdCl2诱导表达均上调,预示着ThSAMDC可能在刚毛柽柳非生物胁迫应答过程中发挥重要作用。 Abstract: S-adenosyl-L-methionine decarboxylase(SAMDC) is a rate-limiting enzyme that regulates the physiological and biochemical processes of plants by participating in the metabolic pathway of polyamines. The cDNA of S-Adenosine Methionine Decarboxylase gene(named ThSAMDC) was isolated and cloned by analyzing the transcriptome data of Tamarix hispida. The full-length of ThSAMDC is 2 085 bp, with three open reading frames, tiny ORF(tORF), upstream ORF(uORF) and main ORF(mORF). The mORF was 1 107 bp encoding 369 amino acids. The relative molecular weight and isoelectric points(PI) of the putative protein were 40.34 kD and 4.72. ThSAMDC-encoded protein has a number of strong hydrophilic regions, no obvious transmembrane region. Compared to the amino acid multiple sequence alignment of several other species, ThSAMDC includes two highly conserved domains: the proenzyme cleavage site(LSESSLF) and the PEST domain(TIHVTPEDGFSYAS) associated with the rapid degradation of the protein. Phylogenetic tree analysis shows that ThSAMDC has higher sequence similarity of 77% identities to the Spinach(SoSAMDC). Quantitative real-time PCR assay revealed that the mRNA level of ThSAMDC was significantly up-regulated under NaCl, PEG, ABA and CdCl2 treatments in T.hispida, suggesting that ThSAMDC may play an important role in drought resistance, salt tolerance and other stresses. Key words: Tamarix hispida, S-adenosylmethionine decarboxylase, stress responses, gene expression 张玉, 张悦, 张春蕊, 王艳敏, 王玉成, 王超. 刚毛柽柳腺苷甲硫氨酸脱羧酶(ThSAMDC)基因的克隆与胁迫下的表达分析[J]. 植物研究, 2018, 38(1): 132-140. ZHANG Yu, ZHANG Yue, ZHANG Chun-Rui, WANG Yan-Min, WANG Yu-Cheng, WANG Chao. Cloning and Expression Analysis of S-Adenosine Methionine Decarboxylase(ThSAMDC) Gene from Tamarix ramosissima[J]. Bulletin of Botanical Research, 2018, 38(1): 132-140. 1. Groppa M D,Benavides M P. Polamines and abiotic stress:Recent advances[J]. Amino Acids,2008,34(1):35-45.2. Kusano T,Berberich T,Tateda C,et al. Polyamines:essential factors for growth and survival[J]. Planta,2008,228(3):367-381.3. Fos M,Proa o K,Alabad D,et al. Polyamine metabolism is altered in unpollinated parthenocarpicpat-2 tomato ovaries[J]. Plant Physiology,2003,131(1):359-366.4. Boucherou A,Aziz A,Larher F,et al. Polyamines and environmental challenges:recent development[J]. Plant Science,1999,140(2):103-125.5. Shen W Y,Nada K,Tachibana S. Involvement of polyamines in the chilling tolerance of cucumber cultivars[J]. Plant Physiology,2000,124(1):431-440.6. Witzell J,Kuusela T,Sarjala T. Polyamine profiles of healthy and parasite-infected Vaccinium myrtillus plants under nitrogen enrichment[J]. Journal of Chemical Ecology,2005,31(3):561-575.7. Sharma S S,Dietz K J. The significance of amino acids and amino acid-derived molecules in plant responses and adaptation to heavy metal stress[J]. Journal of Experimental Botany,2006,57(4):711-726.8. 汪沛洪. 植物多胺代谢的酶类与胁迫反应[J]. 植物生理学通讯,1990(1):1-7.Wang P H. Enzymes of polyamine metabolism and stress response in plants[J]. Plant Physiology Communications,1990(1):1-7.9. Kasukabe Y,He L,Nada K,et al. Overexpression of spermidine synthase enhances tolerance to multiple environmental stresses and up-regulates the expression of various stress-regulated genes in transgenic Arabidopsis thaliana[J]. Plant Cell Physiology,45(6):712-722.10. Galston A W,Sawhney R K. Polyamines in plant physiology[J]. Plant Physiology,1990,94(2):406-410.11. Bhavna W,Manchikatla V R. Effect of increased polyamine biosynthesis on stress responses in transgenic tobacco by introduction of human S-adenosylmethionine gene [J]. Plant Sci,2003,164(5):727-734.12. Momtaz O A,Hussein E M,Fahmy E M,et al. Expression of S-adenosyl methionine decarboxylase gene for polyamine accumulation in Egyptian cotton Giza 88 and Giza 90[J]. GM Crops,2010,1(4):257-266.13. Malabika R,Ray W. Overexpression of S-adenosylmethionine decarboxylase gene in rice increases polyamine level and enhances sodium chloride-stress tolerance [J]. Plant Science,2002,163(5):987-992.14. Kusano T,Yamaguchi K,Berberich T,et al. The polyamine spermine rescues Arabidopsis from salinity and drought stresses[J]. Plant Signaling Behavior,2007,2(4):251-252.15. Bolle C,Hermann R G,Oelm ller R. A spinach cDNA with homology to S adenosylmethionine decarboxylase[J]. Plant Physiology,1995,107(4):1461-1462.16. Hao Y J,Zhang Z,Kitashiba H,et al. Molecular cloning and functional characterization of two apple S-adenosylmethionine decarboxylase genes and their different expression in fruit development,cell growth andstress responses[J]. Gene,2005,350(1):41-50.17. Tassoni A,Franceschetti M,Tasco G,et al. Cloning,functional identification and structural modelling of Vitis vinifera S-adenosylmethionine decarboxylase[J]. Journal of Plant Physiology,2007,164(9):1208-1219.18. 刘桂丰,侯英杰,王玉成,等. 干旱胁迫下刚毛柽柳消减
文库的构建及分析[J]. 植物研究,2005,25(1):69-73.Liu G F,Hou Y J,Wang Y C,et al. Construction and analysis of Tamarix hispida suppression subtractive hybridization library under drought stress[J]. Bulletin of Botanical Research,2005,25(1):69-73.19. Vandesompele J,De Preter K,Pattyn F,et al. Accurate normalization of real-time quantitative RT-PCR data by geometric averaging of multiple internal control genes[J]. Genome Biology,2002,3(7):RESEARCH0034.20. Livak K J,Schmittgen T D,Livak K J,et al. Analysis of relative gene expression data using real-time quantitative PCR and the 2- CT method[J]. Methods,2001,25(4):402-408.21. 张佳景,丁淑丽,邹宜静,等. 植物腺苷甲硫氨酸脱羧酶研究进展[J]. 细胞生物学杂志,2008,30(5):622-628.Zhang J J,Ding S L,Zou Y J,et al. Research advance on s-adenosylmethionine decarboxylase in plants[J]. Chinese Journal of Cell Biology,2008,30(5):622-628.22. Franceschetti M,Hanfrey C,Scaramagli S,et al. Characterization of monocot and diont plant S-adenesyl-l-methionine decarboxylase gene families including identification in the mRNA of a highly conserved pair of upstream overlapping open reading frames[J]. Biochemical Journal,2001,353(2):403-409.23. Hu W W,Gong H B,Pua E C. Molecular cloning and characterization of S-adenosyl-methionine decarboxylase genes from mustard(Brassica juncea)[J]. Physiologia Plantarum,2005,124(1):25-40.24. Hanfrey C,Franceschetti M,Mayer M J,et al. Abrogation of upstream open reading frame-mediated translational control of a plant S-adenosylmethionine decarboxylase results in polyamine disruption and growth perturbations[J]. Journal of Biological Chemistry,2002,277(46):44131-44139.25. 刘建新,胡浩斌,王鑫. 外源NO对盐胁迫下黑麦草幼苗活性氧代谢、多胺含量和光合作用的影响[J]. 植物研究,2009,29(3):313-319.Liu J X,Hu H B,Wang X. Effects of exogenous nitric oxide on active oxygen metabolism,polyamine content and photosynthesis of ryegrass(Lolium perenne L.) seedlings under salt stress[J]. Bulletin of Botanical Research,2009,29(3):313-319.26. Li Z Y,Chen S Y. Differential accumulation of the S-adenosylmethionine decarboxylase transcript in rice seedlings in response to salt and drought stresses[J]. Theoretical and Applied Genetics,2000,100(5):782-788.27. Roy M,Wu R. Overexpression of S-adenosylmethionine decarboxylase gene in rice increases polyamine level and enhances sodium chloride-stress tolerance[J]. Plant Science,2002,163(5):987-992.28. Wi S J,Kim W T,Papk K Y. Overexpression of carnation S-adenosylmethionine decarboxylase gene generates a broadspectrum tolerance to abiotic stresses in transgenic tobacco plants[J]. Plant Cell Reports,2006,25(10):1111-1121. 樊松乐, 王纪坤, 谢贵水, 王萌, 王立丰. R2R3型MYB转录因子HbMYB88结构和功能分析[J]. 植物研究, 2020, 40(1): 106-116. 孙丰坤, 李思达, 李吉祥, 陈晓慧, 曾凡锁. 外源NO对转基因白桦外源基因表达及DNA甲基化的影响[J]. 植物研究, 2017, 37(6): 870-875. [11] 王培龙, 刘中原, 张腾倩, 唐绯绯, 曲冠证, 高彩球. 刚毛柽柳ThPP2C基因的
克隆和表达分析[J]. 植物研究, 2017, 37(3): 395-401. [12] 冯德明, 温佩颖, 赵畅, 杨远彪, 杨桂燕, 于丽丽, 高彩球. 刚毛柽柳ThDREB基因在酵母中的表达及抗逆能力分析[J]. 植物研究, 2017, 37(1): 63-68. [13] 代丽娟, 郑唐春, 刘彩霞, 刘轶, 由香玲, 曲冠证. 烟草化学诱导表达系统的建立[J]. 植物研究, 2016, 36(6): 917-924. [14] 杨凯, 孙迎坤, 王奎玲, 刘庆华, 高捍东. 耐冬山茶抗寒基因CjCor1的全长克隆与表达分析[J]. 植物研究, 2016, 36(5): 753-759. [15] 贾园园, 张春蕊, 王玉成, 杨传平, 王超. 刚毛柽柳Na+/H+逆向转运蛋白基因的克隆与表达分析[J]. 植物研究, 2016, 36(3): 380-387.