甘蔗ScHAK9基因克隆及表达分析
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摘要
KUP/HAK/KT家族基因在介导细胞内钾的积累及维持植物的生长发育中起重要作用,本研究以新台糖22号(ROC22)甘蔗为研究材料,采用同源克隆技术获得钾转运ScHAK9基因的完整编码序列(CDs),并利用生物信息学软件对蛋白质结构和功能进行预测分析,同时利用qPCR技术分析基因的组织特异性表达以及在不同干旱条件下的表达情况。结果表明,ScHAK9基因的cDNA完整编码区长度为2352 bp,编码783个氨基酸,属于碱性疏水蛋白,结构中包含了12个跨膜结构域,蛋白主要定位在细胞质膜上;蛋白质二级结构主要由α螺旋结构、无规则卷曲结构和扩展长链组成;系统发育树分析显示ScHAK9与玉米Zm HAK9基因同源性较高。qPCR分析结果表明,ScHAK9基因在不同组织间的相对表达量具有明显差异,叶片中表达最高,其次是茎,根系中表达最低,具有组织特异性;干旱胁迫可以诱导ScHAK9基因表达,其表达量随着胁迫程度加重表现出升高趋势,且在复水后才有所下降。初步推测该基因可能在甘蔗发育和抵御干旱胁迫中起着重要作用,本研究为进一步阐明ScHAK9的功能及作用机制奠定了分子基础。
The KUP/HAK/KT family gene plays an important role in regulation of potassium accumulation. In this research,we isolated the complete coding sequence of ScHAK9 and investigated the transcriptional levels in ROC22. ScHAK9 gene was composed of 2352 nucleotides that encodes 783 amino acids with 12 predictable transmembrane domains. The deduced protein was predicted to be a basic dewatering protein and localized to the cytoplasmic membrane. The secondary structure consisted of alpha spiral structure,irregular curling structure and extended long chain. ScHAK9 gene showed highly sequence identity with the homologous genes as compared with ZmHAK9 gene( Zea mays L.). Real-Time PCR showed that the ScHAK9 gene highly expressed in the leaves,followed by the stem and the root at last. ScHAK9 gene expression was induced under drought stress,and the higher expression decreased after re-watering. Thus,our work provided first insight about ScHAK9,which might play a role in drought stress resistance.
The KUP/HAK/KT family gene plays an important role in regulation of potassium accumulation. In this research,we isolated the complete coding sequence of ScHAK9 and investigated the transcriptional levels in ROC22. ScHAK9 gene was composed of 2352 nucleotides that encodes 783 amino acids with 12 predictable transmembrane domains. The deduced protein was predicted to be a basic dewatering protein and localized to the cytoplasmic membrane. The secondary structure consisted of alpha spiral structure,irregular curling structure and extended long chain. ScHAK9 gene showed highly sequence identity with the homologous genes as compared with ZmHAK9 gene( Zea mays L.). Real-Time PCR showed that the ScHAK9 gene highly expressed in the leaves,followed by the stem and the root at last. ScHAK9 gene expression was induced under drought stress,and the higher expression decreased after re-watering. Thus,our work provided first insight about ScHAK9,which might play a role in drought stress resistance.
引文
[1]张华,陈如凯.提升我国甘蔗核心技术竞争力的研究.甘蔗,2003,10(3):49-54
[2]姚瑞亮,李杨瑞,陈如凯.广西旱坡地甘蔗高产栽培措施的探讨.广西农业生物科学,1999,18(Z1):29-31
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[10]Su H,Golldack D,Zhao C S,Bohnert H J.The expression of HAKtype K+transporters is regulated in response to salinity stress in common ice plant.Plant Physiol,2002,129(4):1482-1493
[11]He C,Cui K,Duan A,Zeng Y,Zhang J.Genome-wide and molecular evolution analysis of the Poplar KT/HAK/KUP potassium transporter gene family.Ecology&Evolution,2012,2(8):1996-2004
[12]Song Z Z,Cong Y,Han L,Wang L,Su Y H.In silico analyses of KUP proteins based on grape genomic data.Genomics and Applied Biology,2011,6(30):728-737
[13]鲁黎明.主要农作物钾吸收转运基因及其进化关系分析.中国农业科学,2009,42(7):2271-2279
[14]Sato Y,Nanatani K,Hamamoto S,Shimizu M,Takahashi M,Tabuchi-Kobayashi M,Mizutani A,Schroeder J I,Souma S,Uozumi N.Defining membrane spanning domains and crucial membranelocalized acidic amino acid residues for K+transport of a Kup/HAK/KT-type Escherichia coli potassium transporter.Journal of Biochemist,2014,155(5):315-323
[15]Ahn S J,Shin R,Schachtman D P.Expression of KT/KUP genes in Arabidopsis and the role of root hairs in K+uptake.Plant Physiology,2004,134(3):1135-1145
[16]Zou N,Li B,Dong G,Kronzucker H J,Shi W.Ammonium-induced loss of root gravitropism is related to auxin distribution and TRH1 function,and is uncoupled from the inhibition of root elongation in Arabidopsis.Journal of Experimental Botany,2012,63(10):3777-3788
[17]Osakabe Y,Arinaga N,Umezawa T,Katsura S,Nagamachi K,Tanaka H,Ohiraki H,Yamada K,Seo S U,Abo M,Yoshimura E,Shinozaki K,Yamaguchi-Shinozaki K.Osmotic stressresponses and plant growth controlled by potassium transporters in Arabidopsis.The Plant Cell,2013,25(2):609-624
[18]艾兴辉.小麦转运蛋白基因Ta BASS2和Ta HAK11的功能研究.济南:山东大学.2013:20-21
[19]Sirichandra C,Wasilewska A,Vlad F,Valon C,Leung J.The guard cell as a single-cell model towards understanding drought tolerance and abscisic acid action.Journal of Experimental Botany,2009,60(5):1439-1463
[20]罗海斌,曹辉庆,魏源文,邓智年,潘有强,郭元元,何海波,李杨瑞.干旱胁迫下甘蔗苗期根系全长c DNA文库构建及EST序列分析.南方农业学报,2012,43(6):733-737
[21]韦丽君.甘蔗干旱胁迫的形态结构及生理机制研究.长沙:湖南农业大学.2014:14-16
[22]苏炜华,刘峰,黄珑,苏亚春,黄宁,凌辉,吴期滨,张华,阙友雄.甘蔗Ca2+/H+反向运转体基因的克隆与表达分析.作物学报,2016,42(7):1074-1082
[23]鲁黎明,杨铁钊.烟草钾转运体基因Nt HAK1的克隆及表达模式分析.核农学报,2011,25(3):469-476
[24]朱晓玲,陈海峰,王程,郝青南,陈李森,郭丹丹,伍宝朵,陈水莲,沙爱华,周蓉,周新安.大豆钾转运体基因Gm KT12的克隆和信息学分析.作物学报,2013,39(9):1701-1709
[25]Elumalai R P,Nagpal P,Reed J W.A mutation in the Arabidopsis KT2/KUP2 potassium transporter gene affects shoot cell expansion.Plant Cell,2002,14(1):119-131
[26]Gierth M,Mser P,Schroeder J I.The potassium transporter At HAK5functions in K+deprivation-induced high-affinity K+uptake and AKT1 K+channel contribution to K+uptake kinetics in Arabidopsis roots.Plant Physiol,2005,137(3):1105-1114
[27]晁毛妮,张志勇,宋海娜,李成奇,张新,胡根海,张金宝,王清连.陆地棉Pht1家族成员的全基因组鉴定及表达分析.棉花学报,2017,29(1):59-69
[28]Loutfy N,El-Tayeb M A,Hassanen A M,Moustafa M F,Sakuma Y,Inouhe M..Changes in the water status and osmotic solute contents in response to drought and salicylic acid treatments in four different cultivars of wheat(Triticum aestivum).Journal of Plant Research,2012,125(1):173–184
[29]孙洪荣,秦利军,赵丹,谭颖,赵杰宏,赵德刚.超量表达NtHAK1基因烟草对不同光辐射的光合响应.贵州农业科学,2013(12):24-27
[30]黄莹,敖俊华,陈迪文,周文灵,卢颖林,黄振瑞,李奇伟,江永.钾对水分胁迫下甘蔗幼苗生理和光合特性的影响.中国农学通报,2016,32(6):49-54
[2]姚瑞亮,李杨瑞,陈如凯.广西旱坡地甘蔗高产栽培措施的探讨.广西农业生物科学,1999,18(Z1):29-31
[3]徐超华,李纯佳,苏火生,陆鑫,李旭娟,刘洪博,林秀琴,毛钧,字秋艳,刘新龙.甘蔗非生物胁迫抗性研究进展.植物遗传资源学报,2017,18(3):483-493
[4]周会,雷敬超,桂意云,贤武,梁强,杨荣仲,李杨瑞.甘蔗种质资源自然条件下耐寒性评价.植物遗传资源学报,2012,13(6):968-973
[5]马小娟,戚金亮,印莉萍,黄勤妮.植物钾离子转运相关蛋白及基因研究进展.首都师范大学学报:自然科学版,2004,25(2):40-45
[6]Ashley M K,Grant M,Grabov A.Plant responses to potassium deficiencies:a role for potassium transport proteins.Journal of Experimental Botany,2006,57(2):425-436
[7]Santa-Maria G E,Rubio F,Dubcovsky J,Rodriguea-Navarro A.The HAK1 gene of barley is a member of a large gene family and encodes a high-affinity potassium transporter.Plant Cell,1997,9(12):2281-2289
[8]Zhang Z,Zhang J,Chen Y,Li R,Wang H,Wei J.Genome-wide analysis and identification of HAK potassium transporter gene family in maize(Zea,mys L.).Moleculur Biology Reports,2012,39(8):8465-8473
[9]Yang Z,Gao Q,Sun C,Li W,Gu S,Xu C.Molecular evolution and functional divergence of HAK potassium transporter gene family in rice(Oryza sative L.).Journul of Genetics and Genomics,2009,36(3):161-172
[10]Su H,Golldack D,Zhao C S,Bohnert H J.The expression of HAKtype K+transporters is regulated in response to salinity stress in common ice plant.Plant Physiol,2002,129(4):1482-1493
[11]He C,Cui K,Duan A,Zeng Y,Zhang J.Genome-wide and molecular evolution analysis of the Poplar KT/HAK/KUP potassium transporter gene family.Ecology&Evolution,2012,2(8):1996-2004
[12]Song Z Z,Cong Y,Han L,Wang L,Su Y H.In silico analyses of KUP proteins based on grape genomic data.Genomics and Applied Biology,2011,6(30):728-737
[13]鲁黎明.主要农作物钾吸收转运基因及其进化关系分析.中国农业科学,2009,42(7):2271-2279
[14]Sato Y,Nanatani K,Hamamoto S,Shimizu M,Takahashi M,Tabuchi-Kobayashi M,Mizutani A,Schroeder J I,Souma S,Uozumi N.Defining membrane spanning domains and crucial membranelocalized acidic amino acid residues for K+transport of a Kup/HAK/KT-type Escherichia coli potassium transporter.Journal of Biochemist,2014,155(5):315-323
[15]Ahn S J,Shin R,Schachtman D P.Expression of KT/KUP genes in Arabidopsis and the role of root hairs in K+uptake.Plant Physiology,2004,134(3):1135-1145
[16]Zou N,Li B,Dong G,Kronzucker H J,Shi W.Ammonium-induced loss of root gravitropism is related to auxin distribution and TRH1 function,and is uncoupled from the inhibition of root elongation in Arabidopsis.Journal of Experimental Botany,2012,63(10):3777-3788
[17]Osakabe Y,Arinaga N,Umezawa T,Katsura S,Nagamachi K,Tanaka H,Ohiraki H,Yamada K,Seo S U,Abo M,Yoshimura E,Shinozaki K,Yamaguchi-Shinozaki K.Osmotic stressresponses and plant growth controlled by potassium transporters in Arabidopsis.The Plant Cell,2013,25(2):609-624
[18]艾兴辉.小麦转运蛋白基因Ta BASS2和Ta HAK11的功能研究.济南:山东大学.2013:20-21
[19]Sirichandra C,Wasilewska A,Vlad F,Valon C,Leung J.The guard cell as a single-cell model towards understanding drought tolerance and abscisic acid action.Journal of Experimental Botany,2009,60(5):1439-1463
[20]罗海斌,曹辉庆,魏源文,邓智年,潘有强,郭元元,何海波,李杨瑞.干旱胁迫下甘蔗苗期根系全长c DNA文库构建及EST序列分析.南方农业学报,2012,43(6):733-737
[21]韦丽君.甘蔗干旱胁迫的形态结构及生理机制研究.长沙:湖南农业大学.2014:14-16
[22]苏炜华,刘峰,黄珑,苏亚春,黄宁,凌辉,吴期滨,张华,阙友雄.甘蔗Ca2+/H+反向运转体基因的克隆与表达分析.作物学报,2016,42(7):1074-1082
[23]鲁黎明,杨铁钊.烟草钾转运体基因Nt HAK1的克隆及表达模式分析.核农学报,2011,25(3):469-476
[24]朱晓玲,陈海峰,王程,郝青南,陈李森,郭丹丹,伍宝朵,陈水莲,沙爱华,周蓉,周新安.大豆钾转运体基因Gm KT12的克隆和信息学分析.作物学报,2013,39(9):1701-1709
[25]Elumalai R P,Nagpal P,Reed J W.A mutation in the Arabidopsis KT2/KUP2 potassium transporter gene affects shoot cell expansion.Plant Cell,2002,14(1):119-131
[26]Gierth M,Mser P,Schroeder J I.The potassium transporter At HAK5functions in K+deprivation-induced high-affinity K+uptake and AKT1 K+channel contribution to K+uptake kinetics in Arabidopsis roots.Plant Physiol,2005,137(3):1105-1114
[27]晁毛妮,张志勇,宋海娜,李成奇,张新,胡根海,张金宝,王清连.陆地棉Pht1家族成员的全基因组鉴定及表达分析.棉花学报,2017,29(1):59-69
[28]Loutfy N,El-Tayeb M A,Hassanen A M,Moustafa M F,Sakuma Y,Inouhe M..Changes in the water status and osmotic solute contents in response to drought and salicylic acid treatments in four different cultivars of wheat(Triticum aestivum).Journal of Plant Research,2012,125(1):173–184
[29]孙洪荣,秦利军,赵丹,谭颖,赵杰宏,赵德刚.超量表达NtHAK1基因烟草对不同光辐射的光合响应.贵州农业科学,2013(12):24-27
[30]黄莹,敖俊华,陈迪文,周文灵,卢颖林,黄振瑞,李奇伟,江永.钾对水分胁迫下甘蔗幼苗生理和光合特性的影响.中国农学通报,2016,32(6):49-54