甜瓜自毒胁迫响应基因CmFe-SOD的克隆及表达分析Cloning and expression analysis of CmFe-SOD gene related to autotoxicity stress in Cucumis melo L.
张政达;王景荣;陈丽辉;樊佳茹;韩晓云;王革伏;张志忠;
摘要(Abstract):
【目的】克隆根系分泌物介导的甜瓜自毒胁迫响应基因Fe-SOD,分析基因序列特征和表达情况,探讨其功能。【方法】以甜瓜幼苗为材料,通过RT-PCR技术克隆Fe-SOD基因,命名为CmFe-SOD,进行生物信息学分析和亚细胞定位;通过qRT-PCR技术分析CmFe-SOD在自毒胁迫后甜瓜幼苗叶片和根系中的表达模式。【结果】CmFe-SOD基因组DNA全长1 255 bp,开放阅读框918 bp,编码305个氨基酸。生物信息学分析显示CmFe-SOD为酸性亲水蛋白,无信号肽和跨膜区域,二级结构中无规则卷曲比例最高,与三级结构预测结果一致,基因序列包含典型的Fe-SOD蛋白结构域。同源性分析表明,不同来源Fe-SOD在氨基酸序列上具有较高同源性,CmFe-SOD蛋白与黄瓜的关系较近。通过洋葱内表皮GFP荧光定位观察发现,CmFe-SOD定位于细胞质;qRT-PCR结果显示,根系分泌物处理后甜瓜幼苗叶片中CmFe-SOD基因表达水平整体呈增加趋势,在24 h时表达量最高;在根系中呈现先增加后下降趋势,6 h时表达量最高。【结论】CmFe-SOD基因在进化上较保守,参与了甜瓜根系分泌物介导的自毒胁迫响应,自毒胁迫后在叶片中表达量呈持续上升的趋势,在根部呈现先上升后下降的趋势。
关键词(KeyWords): 甜瓜;自毒胁迫;根系分泌物;Fe-SOD基因;亚细胞定位;qRT-PCR
基金项目(Foundation): 国家重大农技推广服务试点项目(KNJ-152046);; 福建农林大学创新专项基金(CXZX2016108,CXZX2017168);福建农林大学园艺学院青年学术骨干培养基金
作者(Author): 张政达;王景荣;陈丽辉;樊佳茹;韩晓云;王革伏;张志忠;
Email:
DOI: 10.13925/j.cnki.gsxb.20180132
参考文献(References):
- [1]徐小军,张桂兰,周亚峰,胡建斌,许彦宾.甜瓜设施栽培连作土壤的理化性质及生物活性[J].果树学报,2016,33(9):1131-1138.XU Xiaojun,ZHANG Guilan,ZHOU Yafeng,HU Jianbin,XU Yanbin. Studies on the physical-chemical and biological properties of soils cropped continuously with melon under protected cultivation condition[J]. Journal of Fruit Science,2016,33(9):1131-1138.
- [2] BERENDSEN R L,PIETERSE C M J,BAKKER P A H M. The rhizosphere microbiome and plant health[J]. Trends in Plant Science,2012,17(8):478-486.
- [3] TIAN Y Q,ZHANG X Y,WANG J G,GAO L H. Soil microbial communities associated with the rhizosphere of cucumber under different summer cover crops and residue management:A 4 year field experiment[J]. Scientia Horticulturae,2013,150:101-109.
- [4] JIA Z H,YI J H,SU Y R,SHEN H. Autotoxic substances in the root exudates from continuous tobacco cropping[J]. Allelopathy Journal,2011,27(1):87-96.
- [5] FANG C X,ZHUANG Y E,XU T C,LI Y Z,LI Y,LIN W X.Changes in rice allelopathy and rhizosphere microflora by inhibiting rice phenylalanine ammonia-lyase gene expression[J]. Journal of Chemical Ecology, 2013, 39(2):204-212.
- [6] SZAFRANSKA K,GLINSKA S,JANAS K M. Ameliorative effect of melatonin on meristematic cells of chilled and re-warmed Vigna radiata roots[J]. Biologia Plantarum,2013,57(1):91-96.
- [7] HERNANDEZ J A,FERRER M A,JIMENEZ A,BARCELO A R,SEVILLA F. Antioxidant Systems and O2.-/H2O2production in the apoplast of pea leaves:Its relation with salt-induced necrotic lesions in minor veins[J]. Plant Physiology,2001,127(3):817-831.
- [8] LEE D H,LEE C B. Chilling stress-induced changes of antioxidant enzymes in the leaves of cucumber:in gel enzyme activity assays[J]. Plant Science,2000,159(1):75-85.
- [9] THOMAS K,PAUL L F,SUSANNE B,STEFANIE P,SOPHIE D,OVE H G,WILLIAM L,SIMON K D. Transcriptomic characterization of the enzymatic antioxidants FeSOD,MnSOD,APX and KatG in the dinoflagellate genus Symbiodinium[J].BMC Evolutionary Biology,2015,15(1):1-20.
- [10] ALLEN R D,WEBB R P,SCHAKE S A. Use of transgenic plants to study antioxidant defenses[J]. Free Radical Biology Medicine,1997, 23(3):473-479.
- [11] ALSCHER R G,ERTURK N,HEATH L S. Role of superoxide dismutases(SODs)in controlling oxidative stress in plants[J].Journal of experimental botany, 2002,53(372):1331-1341.
- [12] KIM K Y,SANG Y L,CHO Y S,BANG I C,KIM K H,KIM D S,NAM Y K. Molecular characterization and m RNA expression during metal exposure and thermal stress of copper/zinc-and manganese-superoxide dismutases in disk abalone,Haliotis discus discus[J]. Fish&Shellfish Immunology,2007,23(5):1043-1059.
- [13]冯新,徐蕾,赖恭梯,谢晓清,林玉玲,赖钟雄.福州野生蕉FeSOD家族基因的克隆及在低温胁迫下的表达分析[J].西北植物学报,2015,35(5):853-864.FENG Xin,XU Lei,LAI Gongdi,XIE Xiaoqing,LIN Yuling,LAI Zhongxiong. Clone and expression analysis under cold stress of FeSOD family genes from the wild banana in Fuzhou[J]. Acta Botanica Boreali-Occidentalia Sinica,2015, 35(5):853-864.
- [14] LIN Y L,LA Z X. Superoxide dismutase multigene family in longan somatic embryos:a comparison of CuZn-SOD,Fe-SOD,and Mn-SOD gene structure,splicing,phylogeny,and expression[J]. Molecular Breeding, 2013,32(3):595-615.
- [15] RUIZ-LOZANO J M,COLLADOS C,BAREA J M,AZCON R. Cloning of cDNAs encoding SODs from lettuce plants which show differential regulation by arbuscular mycorrhizal symbiosis and by drought stress[J]. Journal of Experimental Botany,2001,52(364):2241-2242.
- [16]李钰,郑文寅,冯春,王富荣,李娟.非生物逆境胁迫下普通小麦烟农19幼苗FeSOD基因表达分析[J].作物杂志,2016(4):75-79.LI Yu,ZHENG Wenyin,FENG Chun,WANG Furong,LI Juan.Analysis of FeSOD gene expression in normal wheat Yannong19 seedlings under abiotic stress[J]. Crops,2016(4):75-79.
- [17] BADAWI G H,YAMAUCHI Y,SNIMADA E,SASAKI R,KAWANO N,TANAKA K. Enhanced tolerance to salt stress and water deficit by overexpressing superoxide dismutase in tobacco(Nicotiana tabacum)chloroplasts[J]. Plant Science,2004,166(4):919-928.
- [18] BARNOVA N E,KURENINA L V,SMIRNOV A N,BELOSHAPKINA O O,GULEVICH A A. Formation of the hypersensitivity response due to the expression of FeSOD1 gene in tomato when it is inoculated with Phytophthora infestans[J]. Russian Agricultural Sciences,2017,43(1):15-21.
- [19]奚玉培.甜瓜自毒作用的根系生理响应和自毒差异基因表达谱分析[D].福州:福建农林大学,2015.XI Yupei. The physiological response to autotoxicity in melon root and analysis of gene expression profiles associated with autotoxicity[D]. Fuzhou:Fujian Agricultural and Forest University,2015.
- [20] KLIEBENSTEIN D J,MONDE R A,LAST R L. Superoxide dismutase in Arabidopsis:an eclectic enzyme family with disparate regulation and protein localization[J]. Plant Physiology,1998, 118(2):637-650.
- [21] MOLINA-RUEDA J J,TSAI C J,KIRBY E G. The Populus superoxide dismutase gene family and its responses to drought stress in transgenic poplar overexpressing a pine cytosolic glutamine synthetase(GS1a)[J]. PLoS One,2013,8(2):e56421.
- [22] IVANA M,DANIJEL P,MASOUD H,JELENA S,DRAGAN V. Improved procedure for detection of superoxide dismutase isoforms in potato,Solanum tuberosum L.[J]. Acta Physiologiae Plantarum,2014,36(8):2059-2066.
- [23]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.
- [24] ZHANG Y,LI Z,PENG Y,WANG X J,PENG D D,LI Y P,HE X S,ZHANG X Q,MA X,HUANG L K,YAN Y H,MCPHEE D J. Clones of FeSOD,MDHAR,DHAR genes from white clover and gene expression analysis of ROS-scavenging enzymes during abiotic stress and hormone treatments[J]. Molecules,2015,20(11):20939-20954.
- [25]李琳玲,程华,许锋,王燕,常杰,程水源.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.LI Linling,CHENG Hua,XU Feng,WANG Yan,CHANG Jie,CHENG Shuiyuan. Molecular cloning,characterization and expression of iron superoxide dismutase gene from Ginkgo biloba[J]. Journal of Fruit Science,2009,26(6):840-846.
- [26] YU J Q,YE S F,ZHANG M F,HU W H. Effects of root exudates and aqueous root extracts of cucumber(Cucumis sativus)and allelochemicals, on photosynthesis and antioxidant enzymes in cucumber[J]. Biochemical Systematics and Ecology,2003,31(2):129-139.
- [27] LIU H C,SONG S W,CHEN R Y,SUN G W. Study on autotoxicity of aqueous extracts from different parts of flowering chinese cabbage[C]//Advanced Materials Research. Switzerland:Trans Tech Publications,2013:962-966.
- [28] ZHU X Z,GUO J,SHAO H,YANG G Q. Effects of allelochemicals from Ageratina adenophora(Spreng.)on its own autotoxicity[J]. Allelopathy Journal,2014,34(2):253-264.
- [29] YANG M,CHUAN Y C,GUO C W,LIAO J J,XU Y G,MEI X Y,LIU Y X,HUANG H C,HE X H,ZHU S S. Panax notoginseng root cell death caused by the autotoxic ginsenoside Rg1 is due to over-accumulation of ROS,as revealed by transcriptomic and cellular approaches[J]. Frontiers in Plant Science,2018,264, doi.org/10.3389/fpls.2018.00264.
- [30] DMITROVI?S,SIMONOVI?A,MITI?N,SAVI?J,CINGEL A,FILIPOVI?B,NINKOVI?S. Hairy root exudates of allelopathic weed Chenopodium murale L. induce oxidative stress and down-regulate core cell cycle genes in Arabidopsis and wheat seedlings[J]. Plant Growth Regulation,2015,75(1):365-382.
- [31]曾秀存,刘自刚,史鹏辉,许耀照,孙佳,方彦,杨刚,武军艳,孔德晶,孙万仓.白菜型冬油菜铜锌超氧化物歧化酶(Cu/ZnSOD)基因的克隆及其在低温条件下的表达[J].作物学报,2014,40(4):636-643.ZENG Xiucun,LIU Zigang,SHI Penghui,XU Yaozhao,SUN Jia,FANG Yan,YANG Gang,WU Junyan,KONG Dejing,SUN Wancang. Cloning and expression analysis of copper and zinc superoxide dismutase(Cu/Zn-SOD)GENE from Brassica campestris L.[J]. Acta Agronomica Sinica,2014,40(4):636-643.
- [32] SUN L H,SHEN L T,YE S. Simultaneous overexpression of both CuZn superoxide dismutase and ascorbate peroxidase in transgenic tall fescue plants confers increased tolerance to a wide range of abiotic stresses[J]. Journal of Plant Physiology,2007,164(12):1626-1638.