六倍体小麦TaHKT2;2基因的克隆与多样性分析
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摘要
为深入研究小麦HKT基因家族其他成员的作用,利用同源序列克隆技术和六倍体小麦基因组BAC文库克隆了小麦TaHKT2;2基因。TaHKT2;2定位于小麦第7同源群,分别命名为TaHKT2;2-7A、TaHKT2;2-7B、TaHKT2;2-7D。TaHKT2;2与TaHKT2;1基因结构相同,均由3个外显子和2个内含子组成。在系统进化树上TaHKT2;2与TaHKT2;1聚类形成1个分支。TaHKT2;2-7A、TaHKT2;2-7B、TaHKT2;2-7D的基因编码区分别为1 602 bp、1 602 bp、1 596 bp,但利用RTPCR技术未能在8个六倍体小麦中克隆TaHKT2;2-7D c DNA。采用克隆测序技术分析了12个六倍体小麦中TaHKT2;2的自然多样性,结果表明,TaHKT2;2-7A的序列多样性高于TaHKT2;2-7B和TaHKT2;2-7D。TaHKT2;2仅见少数碱基发生改变,在TaHKT2;2 P-loop区无氨基酸变异。TaHKT2;2 5'侧翼序列保守性低于其编码区。TaHKT2;2在小麦驯化过程可能受到选择,属于驯化基因。
In order to study the roles of members of HKT gene family in wheat,TaHKT2; 2 was isolated by using homologous cloning strategy and screening genomic BAC library. TaHKT2; 2 genes were mapped on chromosomes 7 A,7 B,and 7 D,named as TaHKT2; 2-7 A,TaHKT2; 2-7 B,and TaHKT2; 2-7 D. TaHKT2; 2 and TaHKT2; 1 had the same genetic structure,composed of three exons and two introns,and formed a cluster with TaHKT2; 1 on phylogenetic tree of plant HKT transporters. The coding sequences of TaHKT2; 2-7 A,TaHKT2; 2-7 B,and TaHKT2; 2-7 D were 1 602,1 602,1 596 bp long,respectively,but TaHKT2; 2-7 D c DNA sequence was not isolated by RT-PCR in eight wheat varieties. The natural diversity of TaHKT2; 2 genes was analyzed by cloning and sequencing from 12 wheat varieties. The results showed that TaHKT2; 2-7 A was found to be more diverse than TaHKT2; 2-7 B and TaHKT2; 2-7 D. Only a few bases changed in the alleles of TaHKT2; 2 genes in wheat. No amino-acid natural variation lay in the P-loops of deduced protein sequences of all alleles of TaHKT2; 2 in 12 wheat varieties. The identity of coding sequences was much higher than that of 5' flanking regions of TaHKT2; 2 genes. TaHKT2; 2 might be selected over the course of wheat domestication and belonged to domestication gene.
In order to study the roles of members of HKT gene family in wheat,TaHKT2; 2 was isolated by using homologous cloning strategy and screening genomic BAC library. TaHKT2; 2 genes were mapped on chromosomes 7 A,7 B,and 7 D,named as TaHKT2; 2-7 A,TaHKT2; 2-7 B,and TaHKT2; 2-7 D. TaHKT2; 2 and TaHKT2; 1 had the same genetic structure,composed of three exons and two introns,and formed a cluster with TaHKT2; 1 on phylogenetic tree of plant HKT transporters. The coding sequences of TaHKT2; 2-7 A,TaHKT2; 2-7 B,and TaHKT2; 2-7 D were 1 602,1 602,1 596 bp long,respectively,but TaHKT2; 2-7 D c DNA sequence was not isolated by RT-PCR in eight wheat varieties. The natural diversity of TaHKT2; 2 genes was analyzed by cloning and sequencing from 12 wheat varieties. The results showed that TaHKT2; 2-7 A was found to be more diverse than TaHKT2; 2-7 B and TaHKT2; 2-7 D. Only a few bases changed in the alleles of TaHKT2; 2 genes in wheat. No amino-acid natural variation lay in the P-loops of deduced protein sequences of all alleles of TaHKT2; 2 in 12 wheat varieties. The identity of coding sequences was much higher than that of 5' flanking regions of TaHKT2; 2 genes. TaHKT2; 2 might be selected over the course of wheat domestication and belonged to domestication gene.
引文
[1]Schachtman D P,Schroeder J I.Structure and transport mechanism of a high-affinity potassium uptake transporter from higher plants[J].Nature,1994,370(6491):655-658.
[2]Rubio F,Gassmann W,Schroeder J I.Sodium-driven potassium uptake by the plant potassium transporter HKT1and mutations conferring salt tolerance[J].Science,1995,270:1660-1663.
[3]Gassman W,Rubio F,Schroeder J I.Alkali cation selectivity of the wheat root high-affinity potassium transporter HKT1[J].The Plant Journal,1996,10(5):869-882.
[4]Uozumi N,Kim E J,Rubio F,et al.The Arabidopsis HKT1gene homolog mediates inward Na+currents in Xenopus laevis oocytes and Na+uptake in Saccharomyces cerevisiae[J].Plant Physiology,2000,122:1249-1259.
[5]M?ller I S,Gilliham M,Jha D,et al.Shoot Na+exclusion and increased salinity tolerance engineered by cell typespecific alteration of Na+transport in Arabidopsis[J].The Plant Cell,2009,21(7):2163-2178.
[6]Berthomieu P,Conéjéro G,Nublat A,et al.Functional analysis of At HKT1 in Arabidopsis shows that Na+recirculation by the phloem is crucial for salt tolerance[J].The EMBO Journal,2003,22(9):2004-2014.
[7]Plett D,Safwat G,Gilliham M,et al.Improved salinity tolerance of rice through cell type-specific expression of At HKT1;1[J].PLo S One,2010,5(9):e12571.
[8]Sunarpi,Horie T,Motoda J,et al.Enhanced salt tolerance mediated by At HKT1 transporter-induced Na+unloading from xylem vessels to xylem parenchyma cells[J].The Plant Journal,2005,44(6):928-938.
[9]Horie T,Yoshida K,Nakayama H,et al.Two types of HKT transporters with different properties of Na+and K+transport in Oryza sativa[J].The Plant Journal,2001,27(2):129-138.
[10]Garciadeblás B,Senn M E,Ba1uelos M A,et al.Sodium transport and HKT transporters:The rice model[J].The Plant Journal,2003,34(6):788-801.
[11]Ren Z H,Gao J P,Li L G,et al.A rice quantitative trait locus for salt tolerance encodes a sodium transporter[J].Nature Genetics,2005,37(10):1141-1146.
[12]Jabnoune M,Espeout S,Mieulet D,et al.Diversity in expression patterns and functional properties in the rice HKT transporter family[J].Plant Physiology,2009,150(4):1955-1971.
[13]Yao X,Horie T,Xue S,et al.Differential sodium and potassium transport selectivities of the rice Os HKT2;1 and Os HKT2;2 transporters in plant cells[J].Plant Physiology,2010,152(1):341-355.
[14]Oomen R J,Benito B,Sentenac H,et al.HKT2;2/1,a K+-permeable transporter identified in a salt-tolerant rice cultivar through surveys of natural genetic polymorphism[J].The Plant Journal,2012,71(5):750-762.
[15]James R A,Davenport R J,Munns R.Physiological characterization of two genes for Na+exclusion in durum wheat,Nax1 and Nax2[J].Plant Physiology,2006,142(4):1537-1547.
[16]Huang S,Spielmeyer W,Lagudah E S,et al.A sodium transporter(HKT7)is a candidate for Nax1,a gene for salt tolerance in durum wheat[J].Plant Physiology,2006,142(4):1718-1727.
[17]Byrt C S,Platten J D,Spielmeyer W,et al.HKT1;5-like cation transporters linked to Na+exclusion loci in wheat,Nax2 and Kna1[J].Plant Physiology,2007,143:1918-1928.
[18]Munns R,James R A,Xu B,et al.Wheat grain yield on saline soils is improved by an ancestral Na+transporter gene[J].Nature Biotechnology,2012,30(4):360-364.
[19]James R A,Blake C,Byrt C S,et al.Major genes for Na+exclusion,Nax1 and Nax2(wheat HKT1;4 and HKT1;5),decrease Na+accumulation in bread wheat leaves under saline and waterlogged conditions[J].Journal of Experimental Botany,2011,62:2939-2947.
[20]李孟军,高欣娜,史占良,等.小麦SKC1-like基因的克隆及多样性研究[J].麦类作物学报,2012,32(4):597-602.
[21]李孟军,李亚青,张磊.小麦高亲和性钾转运蛋白基因(HKT1)多样性分析与染色体定位[J].华北农学报,2015,30(5):1-6.
[22]Platten J D,Cotsaftis O,Berthomieu P,et al.Nomenclature for HKT transporters,key determinants of plant salinity tolerance[J].Trends in Plant Science,2006,11(18):372-374.
[23]Maser P,Hosoo Y,Goshima S,et al.Glycine residues in potassium channel-like selectivity filters determine potassium selectivity in four-loop-per-subunit HKT transporters from plants[J].Proceedings of the National Academy of Sciences of the United States of America,2002,99(9):6428-6433.
[24]Yamasaki M,Tenaillon M I,Bi I V,et al.A large-scale screen for artificial selection in maize identifies candidate agronomic loci for domestication and crop improvement[J].The Plant Cell,2005,17(11):2859-2872.
[2]Rubio F,Gassmann W,Schroeder J I.Sodium-driven potassium uptake by the plant potassium transporter HKT1and mutations conferring salt tolerance[J].Science,1995,270:1660-1663.
[3]Gassman W,Rubio F,Schroeder J I.Alkali cation selectivity of the wheat root high-affinity potassium transporter HKT1[J].The Plant Journal,1996,10(5):869-882.
[4]Uozumi N,Kim E J,Rubio F,et al.The Arabidopsis HKT1gene homolog mediates inward Na+currents in Xenopus laevis oocytes and Na+uptake in Saccharomyces cerevisiae[J].Plant Physiology,2000,122:1249-1259.
[5]M?ller I S,Gilliham M,Jha D,et al.Shoot Na+exclusion and increased salinity tolerance engineered by cell typespecific alteration of Na+transport in Arabidopsis[J].The Plant Cell,2009,21(7):2163-2178.
[6]Berthomieu P,Conéjéro G,Nublat A,et al.Functional analysis of At HKT1 in Arabidopsis shows that Na+recirculation by the phloem is crucial for salt tolerance[J].The EMBO Journal,2003,22(9):2004-2014.
[7]Plett D,Safwat G,Gilliham M,et al.Improved salinity tolerance of rice through cell type-specific expression of At HKT1;1[J].PLo S One,2010,5(9):e12571.
[8]Sunarpi,Horie T,Motoda J,et al.Enhanced salt tolerance mediated by At HKT1 transporter-induced Na+unloading from xylem vessels to xylem parenchyma cells[J].The Plant Journal,2005,44(6):928-938.
[9]Horie T,Yoshida K,Nakayama H,et al.Two types of HKT transporters with different properties of Na+and K+transport in Oryza sativa[J].The Plant Journal,2001,27(2):129-138.
[10]Garciadeblás B,Senn M E,Ba1uelos M A,et al.Sodium transport and HKT transporters:The rice model[J].The Plant Journal,2003,34(6):788-801.
[11]Ren Z H,Gao J P,Li L G,et al.A rice quantitative trait locus for salt tolerance encodes a sodium transporter[J].Nature Genetics,2005,37(10):1141-1146.
[12]Jabnoune M,Espeout S,Mieulet D,et al.Diversity in expression patterns and functional properties in the rice HKT transporter family[J].Plant Physiology,2009,150(4):1955-1971.
[13]Yao X,Horie T,Xue S,et al.Differential sodium and potassium transport selectivities of the rice Os HKT2;1 and Os HKT2;2 transporters in plant cells[J].Plant Physiology,2010,152(1):341-355.
[14]Oomen R J,Benito B,Sentenac H,et al.HKT2;2/1,a K+-permeable transporter identified in a salt-tolerant rice cultivar through surveys of natural genetic polymorphism[J].The Plant Journal,2012,71(5):750-762.
[15]James R A,Davenport R J,Munns R.Physiological characterization of two genes for Na+exclusion in durum wheat,Nax1 and Nax2[J].Plant Physiology,2006,142(4):1537-1547.
[16]Huang S,Spielmeyer W,Lagudah E S,et al.A sodium transporter(HKT7)is a candidate for Nax1,a gene for salt tolerance in durum wheat[J].Plant Physiology,2006,142(4):1718-1727.
[17]Byrt C S,Platten J D,Spielmeyer W,et al.HKT1;5-like cation transporters linked to Na+exclusion loci in wheat,Nax2 and Kna1[J].Plant Physiology,2007,143:1918-1928.
[18]Munns R,James R A,Xu B,et al.Wheat grain yield on saline soils is improved by an ancestral Na+transporter gene[J].Nature Biotechnology,2012,30(4):360-364.
[19]James R A,Blake C,Byrt C S,et al.Major genes for Na+exclusion,Nax1 and Nax2(wheat HKT1;4 and HKT1;5),decrease Na+accumulation in bread wheat leaves under saline and waterlogged conditions[J].Journal of Experimental Botany,2011,62:2939-2947.
[20]李孟军,高欣娜,史占良,等.小麦SKC1-like基因的克隆及多样性研究[J].麦类作物学报,2012,32(4):597-602.
[21]李孟军,李亚青,张磊.小麦高亲和性钾转运蛋白基因(HKT1)多样性分析与染色体定位[J].华北农学报,2015,30(5):1-6.
[22]Platten J D,Cotsaftis O,Berthomieu P,et al.Nomenclature for HKT transporters,key determinants of plant salinity tolerance[J].Trends in Plant Science,2006,11(18):372-374.
[23]Maser P,Hosoo Y,Goshima S,et al.Glycine residues in potassium channel-like selectivity filters determine potassium selectivity in four-loop-per-subunit HKT transporters from plants[J].Proceedings of the National Academy of Sciences of the United States of America,2002,99(9):6428-6433.
[24]Yamasaki M,Tenaillon M I,Bi I V,et al.A large-scale screen for artificial selection in maize identifies candidate agronomic loci for domestication and crop improvement[J].The Plant Cell,2005,17(11):2859-2872.