糜子PmASR2基因克隆与表达分析
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摘要
为了解糜子PmASR2基因在逆境中(干旱、Na Cl、糖及各种植物激素胁迫)的作用,从河曲红糜子中克隆出PmASR2基因,其全长541 bp,保守域长336 bp,共编码112个蛋白,通过生物信息学分析可知,该基因是由缺水胁迫引起的植物ABA/WDS诱导蛋白质(ASR),且为亲水蛋白,糜子PmASR2基因分别在单子叶植物和双子叶植物之间进化保守(76.4%～86.4%),其中,与柳枝稷相似度最高(86.4%)。实时荧光定量PCR结果显示,PmASR2基因在PEG、甘露醇、ABA、过氧化氢、Na Cl、茉莉酸甲酯、生长素、水杨酸胁迫下基因相对表达量均发生改变,并且根>叶>茎,其中,在茎中相对表达量变化不明显。各种胁迫整体上相对表达量随时间增加呈现先升高后降低的变化趋势,赤霉素胁迫下该基因在糜子不同部位(根、茎、叶)中相对表达量未发生明显变化。
To understand the role of broomcorn millet(Panicum miliaceum L.)PmASR2 gene in stress(drought, Na Cl, sugar and various phytohormonal stress), this study cloned PmASR2 gene from Hequ red broomcorn millet, which was 541 bp in length and 336 bp in conserved domain. 112 proteins, via bioinformatics analysis showed that the gene was a plant ABA/WDS-inducible protein(ASR)caused by water stress and was a hydrophilic protein. The broomcorn millet PmASR2 gene was evolutionarily conserved between monocots and dicots(76.4%-86.4%), which had the highest similarity with switchgrass(86.4%). Real-time PCR showed that the relative expression of PmASR2 gene under PEG, Na Cl, mannitol, ABA, H2 O2, Me Ja, IAA and SA were changed, and root >leaf>stem, the relative expression of stems did not change significantly. The relative expression of various stresses increased firstly and then decreased with time. Under the GA stress, the relative expression of the gene in different parts of the broomcorn milletc(root, stem, leaf)did not change much. In this experiment, the cloning and expression analysis of the PmASR2 gene of broomcorn millet was used to further study the regulation mechanism of this gene on stress.
To understand the role of broomcorn millet(Panicum miliaceum L.)PmASR2 gene in stress(drought, Na Cl, sugar and various phytohormonal stress), this study cloned PmASR2 gene from Hequ red broomcorn millet, which was 541 bp in length and 336 bp in conserved domain. 112 proteins, via bioinformatics analysis showed that the gene was a plant ABA/WDS-inducible protein(ASR)caused by water stress and was a hydrophilic protein. The broomcorn millet PmASR2 gene was evolutionarily conserved between monocots and dicots(76.4%-86.4%), which had the highest similarity with switchgrass(86.4%). Real-time PCR showed that the relative expression of PmASR2 gene under PEG, Na Cl, mannitol, ABA, H2 O2, Me Ja, IAA and SA were changed, and root >leaf>stem, the relative expression of stems did not change significantly. The relative expression of various stresses increased firstly and then decreased with time. Under the GA stress, the relative expression of the gene in different parts of the broomcorn milletc(root, stem, leaf)did not change much. In this experiment, the cloning and expression analysis of the PmASR2 gene of broomcorn millet was used to further study the regulation mechanism of this gene on stress.
引文
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[9]谢静静,王笑,蔡剑,等.苗期外源脱落酸和茉莉酸缓减小麦花后干旱胁迫的效应及其生理机制[J].麦类作物学报,2018(2):221-229.
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[16]胡伟.小麦TaASR1基因克隆及抗逆功能研究[D].武汉:华中科技大学,2013.
[17] YANG C Y,WU C H,JAUH G Y,et al. The LLA23 protein translocates into nuclei shortly before desiccation in developing pollen grains and regulates gene expression in Arabidopsis[J]. Protoplasma,2008,233(3/4):241-254.
[18] LIU H Y,DAI J R,FENG D R,et al. Characterization of a novel plantain Asr gene,MpAsr,that is regulated in response to infection of Fusarium oxysporum f. sp. cubense and abiotic stresses[J]. Journal of Integrative Plant Biology,2010,52(3):315-323.
[19]LI X,LI L,ZUO S,et al. Differentially expressed ZmASR genes associated with chilling tolerance in maize(Zea mays)varieties[J].Functional Plant Biology,2018,45:1173-1180
[20] HU Y X,YANG X,LI X L,et al. The SlASR gene cloned from the extreme halophyte Suaeda liaotungensis,K. enhances abiotic stress tolerance in transgenic Arabidopsis thaliana[J]. Gene,2014,549(2):243-251.
[21] KIM S J,LEE S C,HONG S K,et al. Ectopic expression of a cold-responsive Os Asr1 c DNA gives enhanced cold tolerance in transgenic rice plants[J]. Molecules&Cells,2009,27(4):449-458.
[22]高阳,刘国宝,刘科,等.大豆ASR表达提高酵母和烟草细胞对Cu2+耐受力[J].深圳大学学报(理工版),2015(2):137-144.
[23]蒋晓梅,张新全,严海东,等.柳枝稷根组织实时定量PCR分析中内参基因的选择[J].农业生物技术学报,2014,22(1):55-63.
[24]梁潘霞,邢颖,廖青,等.甘蔗ASR基因克隆及水分胁迫下的表达分析[J].西南农业学报,2017,30(10):196-220.
[25] KIM I S,KIM Y S,YOON H S. Rice ASR1 protein with reactive oxygen species scavenging and chaperone-like activities enhances acquired tolerance to abiotic stresses in saccharomyces cerevisiae[J]. Molecules&Cells,2012,33(3):285-293.
[26]李冉辉.一种大豆固有无序蛋白-Gm ASR蛋白的多重保护功能研究[D].长春:东北师范大学,2013.
[27]YANG C Y,CHEN Y C,JAUH G Y,et al. A lily ASR protein involves abscisic acid signaling and confers drought and salt resistance in Arabidopsis[J]. Plant Physiology,2005,139(2):836-846.
[28]张会,郑洁旋,简曙光,等.厚藤ASR基因克隆及功能初步分析[J].植物科学学报,2018,36(3):402-410.
[29]许灿,王朝丽,冯健君,等.枇杷ASR基因分离及其在果实发育中的表达分析[J].中国南方果树,2017,46(3):20-22.
[30]朱攀攀.桑树ASR基因的克隆及表达特征分析[J].蚕业科学,2016(1):16-22.
[31]FENG Z J,XU Z S,SUN J,et al. Investigation of the ASR family in foxtail millet and the role of ASR1 in drought/oxidative stress tolerance[J]. Plant Cell Reports,2016,35(1):115-128.
[32]杨晓月.二穗短柄草BdASR基因家族的克隆、表达分析和遗传转化[D].武汉:华中科技大学,2015.
[2]邵艳军.高粱、玉米苗期抗旱生理与分子机制的比较研究[D].杨凌:西北农林科技大学,2005.
[3]陈秀晨,熊冬金.植物抗逆性研究进展[J].湖北农业科学,2010,49(9):2253-2256.
[4]王星玉.山西省黍稷(糜)品种资源研究[J].种子,1985(3):13-16.
[5]王星玉.黍稷种质资源描述规范和数据标准[M].北京:中国农业出版社,2006.
[6]林凡云,胡银岗,宋国琦,等.干旱复水条件下诱导的糜子特异表达基因的分离与分析[J].农业生物技术学报,2006,14(4):537-541.
[7]林凡云,胡银岗,宋国琦,等.糜子干旱后复水过程中基因表达谱的初步分析[J].西北农林科技大学学报(自然科学版),2007,35(3):81-86.
[8]王俊雅,胡兵辉.覆盖条件下不同生育期干旱胁迫对玉米植株生长的影响探讨[J].绿色科技,2017(9):113-116.
[9]谢静静,王笑,蔡剑,等.苗期外源脱落酸和茉莉酸缓减小麦花后干旱胁迫的效应及其生理机制[J].麦类作物学报,2018(2):221-229.
[10] MASLE J,GILMORE S R,FARQUHAR G D. The ERECTA gene regulates plant transpiration efficiency in Arabidopsis[J]. Nature,2005,436:866-870.
[11] IUSEM N D,BARTHOLOMEW D M,HITZ W D,et al. Tomato(Lycopersicon esculentum)transcript induced by water deficit and ripening[J]. Plant Physiology,1993,102(4):1353-1354.
[12]李建锐.谷子SiASR4基因参与植物响应干旱和盐胁迫的功能研究[D].北京:中国农业大学,2018.
[13] HITZ W D,SCOLNIK P A. Tomato(Lycopersicon esculentum)transcript induced by water deficit and ripening[J]. Plant Physiology,1993,102(4):1353-1354.
[14] VIRLOUVET L,JACQUEMOT M P,GERENTES D,et al. The Zm ASR1 protein influences branched-chain amino acid biosynthesis and maintains kernel yield in maize under water-limited conditions[J]. Plant Physiology,2011,157(2):917-936.
[15]RICARDI M M,GONZ魣LEZ R M,ZHONG S,et al. Genome-wide data(ChIP-seq)enabled identification of cell wall-related and aquaporin genes as targets of tomato ASR1,a drought stress-responsive transcription factor[J]. BMC Plant Biology,2014(1):29.
[16]胡伟.小麦TaASR1基因克隆及抗逆功能研究[D].武汉:华中科技大学,2013.
[17] YANG C Y,WU C H,JAUH G Y,et al. The LLA23 protein translocates into nuclei shortly before desiccation in developing pollen grains and regulates gene expression in Arabidopsis[J]. Protoplasma,2008,233(3/4):241-254.
[18] LIU H Y,DAI J R,FENG D R,et al. Characterization of a novel plantain Asr gene,MpAsr,that is regulated in response to infection of Fusarium oxysporum f. sp. cubense and abiotic stresses[J]. Journal of Integrative Plant Biology,2010,52(3):315-323.
[19]LI X,LI L,ZUO S,et al. Differentially expressed ZmASR genes associated with chilling tolerance in maize(Zea mays)varieties[J].Functional Plant Biology,2018,45:1173-1180
[20] HU Y X,YANG X,LI X L,et al. The SlASR gene cloned from the extreme halophyte Suaeda liaotungensis,K. enhances abiotic stress tolerance in transgenic Arabidopsis thaliana[J]. Gene,2014,549(2):243-251.
[21] KIM S J,LEE S C,HONG S K,et al. Ectopic expression of a cold-responsive Os Asr1 c DNA gives enhanced cold tolerance in transgenic rice plants[J]. Molecules&Cells,2009,27(4):449-458.
[22]高阳,刘国宝,刘科,等.大豆ASR表达提高酵母和烟草细胞对Cu2+耐受力[J].深圳大学学报(理工版),2015(2):137-144.
[23]蒋晓梅,张新全,严海东,等.柳枝稷根组织实时定量PCR分析中内参基因的选择[J].农业生物技术学报,2014,22(1):55-63.
[24]梁潘霞,邢颖,廖青,等.甘蔗ASR基因克隆及水分胁迫下的表达分析[J].西南农业学报,2017,30(10):196-220.
[25] KIM I S,KIM Y S,YOON H S. Rice ASR1 protein with reactive oxygen species scavenging and chaperone-like activities enhances acquired tolerance to abiotic stresses in saccharomyces cerevisiae[J]. Molecules&Cells,2012,33(3):285-293.
[26]李冉辉.一种大豆固有无序蛋白-Gm ASR蛋白的多重保护功能研究[D].长春:东北师范大学,2013.
[27]YANG C Y,CHEN Y C,JAUH G Y,et al. A lily ASR protein involves abscisic acid signaling and confers drought and salt resistance in Arabidopsis[J]. Plant Physiology,2005,139(2):836-846.
[28]张会,郑洁旋,简曙光,等.厚藤ASR基因克隆及功能初步分析[J].植物科学学报,2018,36(3):402-410.
[29]许灿,王朝丽,冯健君,等.枇杷ASR基因分离及其在果实发育中的表达分析[J].中国南方果树,2017,46(3):20-22.
[30]朱攀攀.桑树ASR基因的克隆及表达特征分析[J].蚕业科学,2016(1):16-22.
[31]FENG Z J,XU Z S,SUN J,et al. Investigation of the ASR family in foxtail millet and the role of ASR1 in drought/oxidative stress tolerance[J]. Plant Cell Reports,2016,35(1):115-128.
[32]杨晓月.二穗短柄草BdASR基因家族的克隆、表达分析和遗传转化[D].武汉:华中科技大学,2015.