甘蔗钾转运蛋白基因SsHAK2的克隆及表达特性分析
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摘要
甘蔗(Saccharum species hybrid)是最重要的糖料作物,由于其生育周期长,生物量大,对钾的需求量也大。南方是我国甘蔗的主要种植区域,由于土壤本身特性,有效钾含量低,限制了甘蔗的生产。因此,培育耐低钾甘蔗品种是提高甘蔗钾吸收效率的有效途径之一。本研究以甘蔗品种新台糖22号为材料进行低钾胁迫处理,利用RT-PCR技术从其根系中克隆得到钾转运蛋白基因,命名为SsHAK2(Gen Bank登录号:KM98738)。该基因全长为2 798 bp,包含一个完整的2 352 bp的ORF,编码784个氨基酸,相对分子量为87.602 k D,等电点为8.85,预测其为碱性蛋白。SsHAK2苷酸序列包含了12个跨膜结构域(S1~S12),有80%的概率定位在细胞质膜上。同时,该基因还包含3个保守结构域,分别为钾转运蛋白结构域和氨基酸转运蛋白结构域。SsHAK2基因与玉米(Zea mays)、大麦(Hordeum vulgare)、水稻(Oryza sativa)等其他作物中HAK基因具有高度的同源性,一致的核苷酸比例在52%~95%。q PCR分析结果表明,在低钾、干旱和盐胁迫下,SsHAK2的表达都发生改变。在低钾胁迫条件下处理96 h,该基因相对表达量最大,约达到对照的1.70倍;在盐胁迫条件下处理48 h,该基因的相对表达量出现显著上调,96 h相对表达量最高,约为对照的4.37倍。在干旱胁迫12 h,该基因的相对表达量达到最高,约为对照的4.07倍。干旱胁迫处理24 h,该基因表达量约为对照的1.69倍。但是,在干旱胁迫24到48 h过程中表达由下调转为上调。干旱胁迫48 h后,该基因的表达迅速下调,96 h时该基因表达量最低,约为对照表达量的1/4。q PCR分析结果表明该基因在低钾、干旱和盐胁迫下发挥重要的调控作用。本研究结果为进一步研究甘蔗钾吸收分子机制提供基础。
Sugarcane(Saccharum species hybrid) is the most important crop for the production of sugar. Due to its high biomass production and lengthy growing season, sugarcane needs to absorb a large amount of potassium(K) throughout its life cycle. In the southern China where sugarcane is cultivated, most of the soilhave low content of available K, which limits the production of sugarcane. To improve the productivity, one of the efficient means is to improve sugarcane cultivars that have efficient uptake capacity of K and hence are tolerant to low K. This study was conducted in which a major commercial cultivar, ROC22, was subject to low K stress. From the experiment, a potassium transporter gene, named SsHAK2(Gen Bank accession number:KM98738), was cloned by reverse transcription-polymerase chainreaction(RT-PCR) from total RNA of sugarcane roots. The full length of the SsHAK2 gene was 2 798 bp, and contained a complete open reading frame of 2 352 bp encoding a protein with 784 amino acids. The molecular weight of SsHAK2 was 87.602 k D and the isoelectric point of 8.85, as the alkaline protein. It contained twelve transmembrane segmentand(S1~S12) and the possibility of subcellular localization in plasma membrane was 80%. The deduced polypeptide of SsHAK2 had three transmembrane domains, including potassium transporter domain and amino acid transporter domain. The homology analysis indicated that SsHAK2 shared sequence homology with other members of HAK family in plant, such as Zea mays, Oryza sativa, Hordeum vulgare, with the level of sequence identity ranging from 52% to 95%. The changes in expression of SsHAK2 under low K, drought and salt stresses were detected by q PCR analysis. Under low K stress condition, the gene had the highest inducible expression level at 96 hours, which was 1.70-fold than that of control. Under salt stress, the expression level began to increase rapidly after 48 hours stress of salt and also reached the highest at 96 hours or 4.37-fold than that of control. Under drought stress, the gene had the highest level at 12 hours or 4.07-fold than that of control, and then dropped to 1.69-fold at 24 hours. Afterwards from 24 to 48 hours, it was up-regulated and after 48 hours became down-regulated rapidly, by 96 hours which had the lowest inducible expression level or only 1/4 of that in control. It suggests that SsHAK2 might take part in responses to various stress in addition to low K. This study has established a foundation for future research on understanding the molecular mechanism of K uptake in sugarcane.
Sugarcane(Saccharum species hybrid) is the most important crop for the production of sugar. Due to its high biomass production and lengthy growing season, sugarcane needs to absorb a large amount of potassium(K) throughout its life cycle. In the southern China where sugarcane is cultivated, most of the soilhave low content of available K, which limits the production of sugarcane. To improve the productivity, one of the efficient means is to improve sugarcane cultivars that have efficient uptake capacity of K and hence are tolerant to low K. This study was conducted in which a major commercial cultivar, ROC22, was subject to low K stress. From the experiment, a potassium transporter gene, named SsHAK2(Gen Bank accession number:KM98738), was cloned by reverse transcription-polymerase chainreaction(RT-PCR) from total RNA of sugarcane roots. The full length of the SsHAK2 gene was 2 798 bp, and contained a complete open reading frame of 2 352 bp encoding a protein with 784 amino acids. The molecular weight of SsHAK2 was 87.602 k D and the isoelectric point of 8.85, as the alkaline protein. It contained twelve transmembrane segmentand(S1~S12) and the possibility of subcellular localization in plasma membrane was 80%. The deduced polypeptide of SsHAK2 had three transmembrane domains, including potassium transporter domain and amino acid transporter domain. The homology analysis indicated that SsHAK2 shared sequence homology with other members of HAK family in plant, such as Zea mays, Oryza sativa, Hordeum vulgare, with the level of sequence identity ranging from 52% to 95%. The changes in expression of SsHAK2 under low K, drought and salt stresses were detected by q PCR analysis. Under low K stress condition, the gene had the highest inducible expression level at 96 hours, which was 1.70-fold than that of control. Under salt stress, the expression level began to increase rapidly after 48 hours stress of salt and also reached the highest at 96 hours or 4.37-fold than that of control. Under drought stress, the gene had the highest level at 12 hours or 4.07-fold than that of control, and then dropped to 1.69-fold at 24 hours. Afterwards from 24 to 48 hours, it was up-regulated and after 48 hours became down-regulated rapidly, by 96 hours which had the lowest inducible expression level or only 1/4 of that in control. It suggests that SsHAK2 might take part in responses to various stress in addition to low K. This study has established a foundation for future research on understanding the molecular mechanism of K uptake in sugarcane.
引文
艾兴辉.2013.小麦转运蛋白基因Ta BASS2和Ta HAK11的功能研究[D].博士学位论文,山东大学,导师:夏光敏pp.79-80.(Ai X H.2013.Functional Study of transport er genes Ta BASS2 and Ta HAK11 in wheat,Dissertation for Ph.D.,Shandong University,Supervisor:Xia G.M.pp.79-80.)
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黄莹,曾巧英,敖俊华,等.2013.钾水平对甘蔗氮、磷、钾养分吸收利用的影响[J].广东农业科学,40(10):50-53(Huang Y,Zeng Q Y,Ao J H,et al.2013.Effects of dif ferent potassium levels on nutrient absorption and utili zation of N,P,K in sugarcane[J].Guangdong Agricultur al Science,40(10):50-53.)
李奇伟,敖俊华,卢颖林,等.2012.甘蔗及其近缘属植物钾效率差异研究[J].广东农业科学,39(21):29-32.(Li QW,Ao J H,Lu Y L,et al.2012.Differences of potassi um efficiency among different genotypes of sugarcan and its related genera[J].Guangdong Agricultural Sci ence,39(21):29-32.)
宋毓峰,张良,董连红,等.2013.植物KUP/HAK/KT家族钾转运体研究进展[J].中国农业科技导报,15(6):92-98(Song Y F,Zhang L,Dong L H,et al.2013.Research progress on KUP/HAK/KT potassium transporter fami ly in plant[J].Journal of Agricultural Science and Tech nology,15(6):92-98.)
孙洪荣,秦利军,赵丹,等.2013.超量表达Nt HKA基因烟草对不同光辐射的光合响应[J].贵州农业科学,41(12)24-27.(Sun H R,Qin L J,Zhao D,et al.2013.Photosyn thesis response of overexpressed Nt HAK1 gene tobacco to various optical radiation[J].Guizhou Agricultural Sci ences,41(12):24-27.)
Ahn S J,Shin R,Schachtman D P.2004.Expression of KTKUP genes in Arabidopsis and the role of root hairs in K+uptake[J].Plant Physiology,134(3):1135-1145.
Ba?uelos M A,Garciadeblas B,Cubero B,et al.2002.Inven tory and functional characterization of the HAK potassi um transporters of rice[J].Plant Physiology,130(2)784-795.
Epstein E,Rains D W,Elzam O E.1963.Resolution of dua mechanisms of potassium absorption by barley roots[J]Proceedings of the National Academy of Sciences of th USA,49(5):684-692.
Famoso A N,Clark R T,Shaff J E,et al.2010.Developmen of a novel aluminum tolerance phenotyping platform used for comparisons of cereal aluminum tolerance and investigations into rice aluminum tolerance mechanism[J].Plant Physiology,153(4):1678-1691.
Gierth M,M?ser P,Schroeder J I.2005.The potassium trans porter At HAK5 functions in K+deprivation-induced high-affinity K+uptake and AKT1 K+channel contribu tion to K+uptake kinetics in Arabidopsis roots[J].Plan Physiology,137(3):1105-1114.
Maathuis F J M.2006.The role of monovalent cation trans porters in plant responses to salinity[J].Journal of Exper imental Botany,57(5):1137-1147.
M?ser P,Thomine S,Schroeder J I,et al.2001.Phylogeneti relationships within cation transporter families of Arabi dopsis[J].Plant Physiology,126(4):1646-1667.
Santa-Maria G E,Rubio F,Dubcovsky J,et al.1997.Th HAKl gene ofbarley is a member of a large gene family and encodes a high-affinity potassium transporter[J]Plant Cell,9:2281-2289.
Shin R,Schachtman D P.2004.Hydrogen peroxide mediate plant root cell response to nutrient deprivation[J].Pro ceedings of the National Academy of Sciences of th USA,101(23):8827-8832.
Su H,Golldack D,Zhao C S,et al.2002.The expression o HAK-type K+transporters is regulated in response to sa linity stress in common ice plant[J].Plant Physiology129(4):1482-1493.
Takahashi R,Nishio T,Ichizen N,et al.2007.High affinity K+transporter Pha HAK5 is expressed only in salt-sensi tive reed plants and shows Na+permeability under Na Cstress[J].Plant Cell Reports,26(9):1673-1679.
Véry A A,Sentenac H.2003.Molecular mechanisms and reg ulation of K+transport in higher plants[J].Plant Biolo gy.54:575-603.
Wang Y H,Garvin D F,Kochian L V.2002.Rapid induction of regulatory and transporter genes in response to phos phorus,potassium,and iron deficiencies in tomato roots.Evidence for cross talk and root/rhizosphere-medi ated signals[J].Plant Physiology,130(3):1361-1370.
Yang X,Ji J,Wang G,Yang S,et al.2011.Over-expressing Salicornia europaea(Se NHX1)gene in tobacco im proves tolerance to salt[J].African Journal of Biotech nology,10(73):16452-16460.
Zeng Q,Ling Q,Fan L,et al.2015.Transcriptome profiling of sugarcane roots in response to low potassium stres[J].PLo S One,10(5):e0126306.
常红军,刘兰霞.2006.植物抗旱分子机理研究进展[J].安徽农业科学,34(18):4509-4510.(Chang H J,Liu L X2006.Research on the advance of Molecular Mecha nism in Plants Drought tolerance[J].Journal of Anhu Agricultural Sciences,34(18):4509-4510.)
黄莹,曾巧英,敖俊华,等.2013.钾水平对甘蔗氮、磷、钾养分吸收利用的影响[J].广东农业科学,40(10):50-53(Huang Y,Zeng Q Y,Ao J H,et al.2013.Effects of dif ferent potassium levels on nutrient absorption and utili zation of N,P,K in sugarcane[J].Guangdong Agricultur al Science,40(10):50-53.)
李奇伟,敖俊华,卢颖林,等.2012.甘蔗及其近缘属植物钾效率差异研究[J].广东农业科学,39(21):29-32.(Li QW,Ao J H,Lu Y L,et al.2012.Differences of potassi um efficiency among different genotypes of sugarcan and its related genera[J].Guangdong Agricultural Sci ence,39(21):29-32.)
宋毓峰,张良,董连红,等.2013.植物KUP/HAK/KT家族钾转运体研究进展[J].中国农业科技导报,15(6):92-98(Song Y F,Zhang L,Dong L H,et al.2013.Research progress on KUP/HAK/KT potassium transporter fami ly in plant[J].Journal of Agricultural Science and Tech nology,15(6):92-98.)
孙洪荣,秦利军,赵丹,等.2013.超量表达Nt HKA基因烟草对不同光辐射的光合响应[J].贵州农业科学,41(12)24-27.(Sun H R,Qin L J,Zhao D,et al.2013.Photosyn thesis response of overexpressed Nt HAK1 gene tobacco to various optical radiation[J].Guizhou Agricultural Sci ences,41(12):24-27.)
Ahn S J,Shin R,Schachtman D P.2004.Expression of KTKUP genes in Arabidopsis and the role of root hairs in K+uptake[J].Plant Physiology,134(3):1135-1145.
Ba?uelos M A,Garciadeblas B,Cubero B,et al.2002.Inven tory and functional characterization of the HAK potassi um transporters of rice[J].Plant Physiology,130(2)784-795.
Epstein E,Rains D W,Elzam O E.1963.Resolution of dua mechanisms of potassium absorption by barley roots[J]Proceedings of the National Academy of Sciences of th USA,49(5):684-692.
Famoso A N,Clark R T,Shaff J E,et al.2010.Developmen of a novel aluminum tolerance phenotyping platform used for comparisons of cereal aluminum tolerance and investigations into rice aluminum tolerance mechanism[J].Plant Physiology,153(4):1678-1691.
Gierth M,M?ser P,Schroeder J I.2005.The potassium trans porter At HAK5 functions in K+deprivation-induced high-affinity K+uptake and AKT1 K+channel contribu tion to K+uptake kinetics in Arabidopsis roots[J].Plan Physiology,137(3):1105-1114.
Maathuis F J M.2006.The role of monovalent cation trans porters in plant responses to salinity[J].Journal of Exper imental Botany,57(5):1137-1147.
M?ser P,Thomine S,Schroeder J I,et al.2001.Phylogeneti relationships within cation transporter families of Arabi dopsis[J].Plant Physiology,126(4):1646-1667.
Santa-Maria G E,Rubio F,Dubcovsky J,et al.1997.Th HAKl gene ofbarley is a member of a large gene family and encodes a high-affinity potassium transporter[J]Plant Cell,9:2281-2289.
Shin R,Schachtman D P.2004.Hydrogen peroxide mediate plant root cell response to nutrient deprivation[J].Pro ceedings of the National Academy of Sciences of th USA,101(23):8827-8832.
Su H,Golldack D,Zhao C S,et al.2002.The expression o HAK-type K+transporters is regulated in response to sa linity stress in common ice plant[J].Plant Physiology129(4):1482-1493.
Takahashi R,Nishio T,Ichizen N,et al.2007.High affinity K+transporter Pha HAK5 is expressed only in salt-sensi tive reed plants and shows Na+permeability under Na Cstress[J].Plant Cell Reports,26(9):1673-1679.
Véry A A,Sentenac H.2003.Molecular mechanisms and reg ulation of K+transport in higher plants[J].Plant Biolo gy.54:575-603.
Wang Y H,Garvin D F,Kochian L V.2002.Rapid induction of regulatory and transporter genes in response to phos phorus,potassium,and iron deficiencies in tomato roots.Evidence for cross talk and root/rhizosphere-medi ated signals[J].Plant Physiology,130(3):1361-1370.
Yang X,Ji J,Wang G,Yang S,et al.2011.Over-expressing Salicornia europaea(Se NHX1)gene in tobacco im proves tolerance to salt[J].African Journal of Biotech nology,10(73):16452-16460.
Zeng Q,Ling Q,Fan L,et al.2015.Transcriptome profiling of sugarcane roots in response to low potassium stres[J].PLo S One,10(5):e0126306.