小桐子ACO1基因的克隆与表达分析
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摘要
为探讨ACO1基因在小桐子抗逆中的作用,本研究基于小桐子最新注释的基因组数据库,首次克隆了小桐子ACO1基因的全长CDNA序列,命名为Jc ACO1,并对其功能结构域、系统进化、基因结构及器官和低温表达特性进行了分析。结果表明,克隆的Jc ACO1基因全长为1 022bp,编码319个氨基酸,预测分子量为36. 0kDa,等电点5. 5。序列比对表明,小桐子Jc ACO1在序列中部保守性较高,具有1个Pcbc superfamily保守结构域。进化树分析显示,小桐子Jc ACO1基因与毛果杨、橡胶树、木薯的同源性较高。qRT-PCR表达分析表明,小桐子幼苗Jc ACO1基因存在器官表达特异性,在叶与茎中表达量较高,而在根中表达量较低。另外,Jc ACO1基因在三种器官中都受低温诱导,低温胁迫24h时,基因在根与茎中表达量最大。说明Jc ACO1基因表达量升高,以响应小桐子的抗冷胁迫。
In order to study the role of JcACO1 gene in stress resistance of Jatropha curcas,the full-length coding frame sequence of ACO1( 1-aminocyclopropane-1-carboxylic acid oxidase) gene,named JcACO1,was cloned based on the genome database of J. curcas,and its functional domain,phylogenetic evolution,gene structure and low-temperature expression characteristics were analyzed. The results showed that the length of the cloned JcACO1 gene was 1 022 bp,encoded 319 amino acids with the molecular weight of 36. 04 kDa and the pI value of5. 50. Sequence alignment demonstrated that JcACO1 was highly conserved in the middle of the sequence and had a conserved domain of Pcbc superfamily. Phylogenetic tree analysis showed that the JcACO1 had high homology with Populus trichocarpa,Hevea brasiliensis and Manihot esculenta. qRT-PCR analysis revealed that Jatropha curcas seeding JcACO1 expressed in different organs,with abundant expression in leaves and stems,but scarcely in roots. In addition,JcACO1 gene was induced by hypothermia in all three organs,and the highest expression of JcACO1 gene was observed in roots and stems at 24 h of low temperature stress. The results showed that the expression of JcACO1 gene was increased in response to chilling stress.
In order to study the role of JcACO1 gene in stress resistance of Jatropha curcas,the full-length coding frame sequence of ACO1( 1-aminocyclopropane-1-carboxylic acid oxidase) gene,named JcACO1,was cloned based on the genome database of J. curcas,and its functional domain,phylogenetic evolution,gene structure and low-temperature expression characteristics were analyzed. The results showed that the length of the cloned JcACO1 gene was 1 022 bp,encoded 319 amino acids with the molecular weight of 36. 04 kDa and the pI value of5. 50. Sequence alignment demonstrated that JcACO1 was highly conserved in the middle of the sequence and had a conserved domain of Pcbc superfamily. Phylogenetic tree analysis showed that the JcACO1 had high homology with Populus trichocarpa,Hevea brasiliensis and Manihot esculenta. qRT-PCR analysis revealed that Jatropha curcas seeding JcACO1 expressed in different organs,with abundant expression in leaves and stems,but scarcely in roots. In addition,JcACO1 gene was induced by hypothermia in all three organs,and the highest expression of JcACO1 gene was observed in roots and stems at 24 h of low temperature stress. The results showed that the expression of JcACO1 gene was increased in response to chilling stress.
引文
[1] Johnson T S,Eswaran N,Sujatha M. Molecular approa-ches to improvement of Jatropha curcas Linn. as a sus-tainable energy crop[J]. Plant Cell Rep,2011,30(9):1 573-1 591.
[2]辛胡,颜岳辉,丁雪梅,等.小桐子MYB基因家族的鉴定及Jc MYB308基因的克隆与低温表达分析[J].生物技术通报,2018,1(1):110-118.
[3] Abeles F B. Ethylence in plant biology[M]. New Yorkand London:Academic Press,1973.
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[7]黄永红,易干军,周碧容,等.粉蕉ACC氧化酶c DNA的克隆及其序列分析[J].分子植物育种,2006,4(5):633-639.
[8]张丽,唐霞,马俊莲,等.富有柿果ACC氧化酶c DNA的克隆及其序列分析[J].华北农学报,2007,22(1):69-72.
[9]刘会超,李永春,巨关生,等.香石竹ACC氧化酶基因克隆及其反义表达载体构建[J].核农学报,2005,19(6):461-464,473.
[10] Chung M C,Chou S J,Kuang L Y,et al. Subcellularlocalization of 1-aminocyclopropane-1-carboxylicacid oxidase in apple fruit[J]. Plant Cell Physiol,2002,43(5):549-554.
[11]陈亮,赵丽萍,高其康.茶树新梢c DNA克隆测序和表达序列标签(ESTs)特性分析[J].农业生物技术学报,2005,13(1):21-25.
[12] Kende H. Ethylene biosynthesis[J]. Annu Rev PlantPhysio1 Plant Mo1 Bio1,1993,44:283-307.
[13] Varona J,Rodriguez E A,Jimenez E. Cloning andcharacterization of ripening-induced ACC oxidasegene from Carica papaya cv.‘Maradol roja’[OL]. ht-tp://www. ncbi. nlm. nih. gov/protein/AAL78058. 1.
[14]徐培华,李唯,杨德龙,等.桃果实ACC氧化酶基因的克隆及序列分析[J].华北农学报,2010,25(2):44-50.
[15] Tuskan G A,Difazio S,Jansson S. The genome of blackcottonwood,Populus trichocarpa(Tory.&Gray)[J].Science,2006,313(5 793):1 596-1 604.
[16] Calvo A,Lorenzo O,Nicolas C. Characterization andexpression of ACC synthase and ACC oxidase genes dur-ing the breaking of beechnuts dormancy[OL]. http://www. rcbi. nlm. nih. gov/protein/CAD21844. 1.
[17] Kongsawadworakul P,Chrestin H. Cloning and charac-terization of a c DNA encoding 1-aminocyclopropane-l-carboxy-late oxidase from Hevea bark:expression inresponse to various stresses[OL]. http://www. ncbi.nlm. nih. gov/protein/AAP41850. 1.
[18] Barry C S,Blume B,Bouzayen M. Differential expres-sion of the 1-aminocyclopropane-1-carboxylate ox-idase gene family of tomato[J]. Plant,1996,9:525-535.
[19] Rudus I,Sasiak M,Kepczynski J. Regulation of ethyl-ene biosynthesis at the level of 1-aminocyclopropane-l-carboxylate oxidase(ACO)gene[J]. Acta Physiolo-giae Plantarum. 2012,6:1-13.
[20] Cara B,Giovannoni J J. Molecular biology of ethyleneduring tomato fruit development and maturation[J].Plant Sci,2008,175; 106-113.
[21] Babula D,Misztal L H,Jakubowicz M. Genes involvedin biosynthesis and signalisation of ethylene in Brassicaoleracea and Arabidopsis thaliana; identification and ge-nome comparative mapping of specific gene homologues[J]. Theor Appl Genet,2006,112(3):410-420.
[22] Ouyang S,Zhu W,Hamilton J. The TIGR rice genomeannotation resource:improvements and new features[J]. Nucl Acid Res,2007,35(1):883-887.
[23] Wagstaff C,Channasut U,Harren F J M,et a1. Ethyl-ene and flower longevity in Alstroemeria:relationshipbetween tepal sene-scence,abscission and ethylene bi-osynthesis[J]. J Exp Bot,2005,56(413):1 007-1 016.
[24]王新力,彭学贤.香蕉果实成熟相关基因ACO启动子区的克隆及其功能初探[J].生物工程学报,2001,17(4).
[25]张晓海,雇廷茂,海燕,等.番茄ACC合成酶基因的反义RNA核酶嵌合基因植物表达载体的构建及对番茄的转化[J].内蒙古大学学报:自然科学版,2001,32(1):74-78.
[26] Kim Y S,Kim H S,Lee Y H,et a1. Elevated H2O2production via overexpression of a chloroplastic Cu/Zn-SOD gene of lily(Lilium oriental hybrid ‘Marco Polo’)triggers ethylene synthesis in transgenic potato[J]. PlantCell Rep,2008,27:973-983.
[27] Zhang M,Yuan B,Leng P. The role of ABA in trigge-ring ethylene biosynthesis and ripening of tomato fruit[J]. J Exp Bot,2009,60(6):1 579-1 588.
[28] Vriezen W H,De Graaf B,Mariani C,et al. Submer-gence induces expansin gene expression in flooding-tol-erant Rumex palustris and not in flooding-intolerant R.acetosa[J]. Planta,2000,210:956-963.
[2]辛胡,颜岳辉,丁雪梅,等.小桐子MYB基因家族的鉴定及Jc MYB308基因的克隆与低温表达分析[J].生物技术通报,2018,1(1):110-118.
[3] Abeles F B. Ethylence in plant biology[M]. New Yorkand London:Academic Press,1973.
[4] Ausubel F M,Kingston R E,Seidman J G. Short proto-cols in molecular biology[M]. Beijing:Science Press,2005. 442-446.
[5] Bradford M M. A rapid and sensitive method for thequantitation of microgram of protein utilizing the principleof protein in-dye binding[J]. Anal Biochem,1976,72:248-254.
[6] Holdsworth M J,Schuch W,Grierson D,Organisationand expression of a wound/ripening-related small multi-gene family from tomato[J]. Plant Mol Biol,1988,11(2):81-88.
[7]黄永红,易干军,周碧容,等.粉蕉ACC氧化酶c DNA的克隆及其序列分析[J].分子植物育种,2006,4(5):633-639.
[8]张丽,唐霞,马俊莲,等.富有柿果ACC氧化酶c DNA的克隆及其序列分析[J].华北农学报,2007,22(1):69-72.
[9]刘会超,李永春,巨关生,等.香石竹ACC氧化酶基因克隆及其反义表达载体构建[J].核农学报,2005,19(6):461-464,473.
[10] Chung M C,Chou S J,Kuang L Y,et al. Subcellularlocalization of 1-aminocyclopropane-1-carboxylicacid oxidase in apple fruit[J]. Plant Cell Physiol,2002,43(5):549-554.
[11]陈亮,赵丽萍,高其康.茶树新梢c DNA克隆测序和表达序列标签(ESTs)特性分析[J].农业生物技术学报,2005,13(1):21-25.
[12] Kende H. Ethylene biosynthesis[J]. Annu Rev PlantPhysio1 Plant Mo1 Bio1,1993,44:283-307.
[13] Varona J,Rodriguez E A,Jimenez E. Cloning andcharacterization of ripening-induced ACC oxidasegene from Carica papaya cv.‘Maradol roja’[OL]. ht-tp://www. ncbi. nlm. nih. gov/protein/AAL78058. 1.
[14]徐培华,李唯,杨德龙,等.桃果实ACC氧化酶基因的克隆及序列分析[J].华北农学报,2010,25(2):44-50.
[15] Tuskan G A,Difazio S,Jansson S. The genome of blackcottonwood,Populus trichocarpa(Tory.&Gray)[J].Science,2006,313(5 793):1 596-1 604.
[16] Calvo A,Lorenzo O,Nicolas C. Characterization andexpression of ACC synthase and ACC oxidase genes dur-ing the breaking of beechnuts dormancy[OL]. http://www. rcbi. nlm. nih. gov/protein/CAD21844. 1.
[17] Kongsawadworakul P,Chrestin H. Cloning and charac-terization of a c DNA encoding 1-aminocyclopropane-l-carboxy-late oxidase from Hevea bark:expression inresponse to various stresses[OL]. http://www. ncbi.nlm. nih. gov/protein/AAP41850. 1.
[18] Barry C S,Blume B,Bouzayen M. Differential expres-sion of the 1-aminocyclopropane-1-carboxylate ox-idase gene family of tomato[J]. Plant,1996,9:525-535.
[19] Rudus I,Sasiak M,Kepczynski J. Regulation of ethyl-ene biosynthesis at the level of 1-aminocyclopropane-l-carboxylate oxidase(ACO)gene[J]. Acta Physiolo-giae Plantarum. 2012,6:1-13.
[20] Cara B,Giovannoni J J. Molecular biology of ethyleneduring tomato fruit development and maturation[J].Plant Sci,2008,175; 106-113.
[21] Babula D,Misztal L H,Jakubowicz M. Genes involvedin biosynthesis and signalisation of ethylene in Brassicaoleracea and Arabidopsis thaliana; identification and ge-nome comparative mapping of specific gene homologues[J]. Theor Appl Genet,2006,112(3):410-420.
[22] Ouyang S,Zhu W,Hamilton J. The TIGR rice genomeannotation resource:improvements and new features[J]. Nucl Acid Res,2007,35(1):883-887.
[23] Wagstaff C,Channasut U,Harren F J M,et a1. Ethyl-ene and flower longevity in Alstroemeria:relationshipbetween tepal sene-scence,abscission and ethylene bi-osynthesis[J]. J Exp Bot,2005,56(413):1 007-1 016.
[24]王新力,彭学贤.香蕉果实成熟相关基因ACO启动子区的克隆及其功能初探[J].生物工程学报,2001,17(4).
[25]张晓海,雇廷茂,海燕,等.番茄ACC合成酶基因的反义RNA核酶嵌合基因植物表达载体的构建及对番茄的转化[J].内蒙古大学学报:自然科学版,2001,32(1):74-78.
[26] Kim Y S,Kim H S,Lee Y H,et a1. Elevated H2O2production via overexpression of a chloroplastic Cu/Zn-SOD gene of lily(Lilium oriental hybrid ‘Marco Polo’)triggers ethylene synthesis in transgenic potato[J]. PlantCell Rep,2008,27:973-983.
[27] Zhang M,Yuan B,Leng P. The role of ABA in trigge-ring ethylene biosynthesis and ripening of tomato fruit[J]. J Exp Bot,2009,60(6):1 579-1 588.
[28] Vriezen W H,De Graaf B,Mariani C,et al. Submer-gence induces expansin gene expression in flooding-tol-erant Rumex palustris and not in flooding-intolerant R.acetosa[J]. Planta,2000,210:956-963.