丹波黑大豆GmMATE2基因的克隆及功能鉴定
|
摘要
丹波黑大豆(Glycine max'Tamba')是一种耐铝植物,其耐铝机制是根尖分泌柠檬酸络合铝离子。为了发掘其优异的耐铝基因资源,本研究以丹波黑大豆铝胁迫转录组数据为基础,利用反转录PCR(reverse transcription PCR, RT-PCR)克隆了1个编码丹波黑大豆柠檬酸通道蛋白基因,命名为GmMATE2(GenBank No. MF497433.1)。该基因的CDS序列长度为1 674 bp,预测编码557个氨基酸,分子量为60.38 kD,等电点为9.54。GmMATE2蛋白不稳定性指数为28.38,是稳定蛋白质。氨基酸序列分析表明,GmMATE2含有9个跨膜结构、1个Polysacc_synt_C结构域及2个多药及毒性复合物排出转运蛋白(multidrug and toxic compound extrusion, MATE)家族特有的MatE结构。进化树分析表明,GmMATE2与蒺藜苜蓿(Medicago truncatula)、白车轴草(Trifolium repens)和鹰嘴豆(Cicer arietinum)的MATE蛋白亲缘关系较近。丹波黑大豆在铝胁迫(pH 4.5, 0.5 mmol/L CaCl2, 100μmol/L AlCl3)下,利用半定量RT-PCR分析表明,GmMATE2在根尖上调表达,24 h时表达量最高,根尖柠檬酸的分泌量随着时间的延长而增多,表明该基因参与铝胁迫反应。构建过表达载体pBI121-GmMATE2,利用农杆菌(Agrobacterium tumefaciens)介导的叶盘法转化烟草(Nicotiana tabacum),获得3株转基因烟草植株。对转基因烟草进行耐铝分析,在铝胁迫(pH 4.5, 0.5 mmol/L CaCl_2, 50μmol/L AlCl3)下,qRT-PCR分析表明,在转基因烟草中GmMATE2上调表达;转基因烟草根尖柠檬酸分泌量是野生型的2.56～3.79倍;转基因烟草根相对伸长量是野生型的1.91～3.45倍;转基因烟草根尖铬天青S染色较野生型浅。上述结果表明,丹波黑大豆GmMATE2是柠檬酸通道蛋白基因,能提高转基因烟草柠檬酸分泌量及耐铝性。本研究为改良铝敏感植物的耐铝性提供了基因资源。
Tamba Black Soybean(Glycine max 'Tamba') is an aluminum-resistant plant whose aluminum-resistant mechanism is that their root tips secrete the citric complex aluminum ions. In order to explore its excellent resistance to aluminum genetic resources, this study, with the foundation of transcriptome data about Tamba black soybean's aluminum stress, cloned one of Tamba black soybean's gene of citrate transporter by using reverse transcription PCR(RT-PCR) and this gene was named GmMATE2(GenBank No.MF497433.1). CDS of GmMATE2 was 1 674 bp in length and GmMATE2 encoded 557 amino acids with a relative molecular mass of 60.38 kD, the isoelectric point of 9.54. GmMATE2, whose instability index was28.38, was a stable protein. Amino acids sequence of GmMATE2 analysis showed that it contained nine transmembrane structures, one Polysacc_synt_C domain and two family-specific MatE structures of multidrug and toxic compound extrusion(MATE). Evolutionary tree analysis showed that GmMATE2 was closely related to MATE proteins of Medicago truncatula, Trifolium repens and Cicer arietinum. When Tamba black soybean subjected to aluminum stress(pH 4.5, 0.5 mmol/L CaCl2, 100 μmol/L AlCl3), the semi-quantitative RT-PCR analysis showed that GmMATE2 was up-regulated in the root tips, with the highest expression at 24 h and citric acid secretion in the root tips increased with time, which suggested that GmMATE2 was involved in response to the aluminum stress. The plant overexpression vector pBI121-GmMATE2 was constructed and 3 transgenic tobaccos(Nicotiana tabacun) were obtained by agrobacterium-mediated leaf disc method. When 3 transgenic tobaccos subjected to aluminum stress(pH 4.5, 0.5 mmol/L CaCl_2, 50 μmol/L AlCl3), qRT-PCR analysis indicated that GmMATE2 was up-regulated in transgenic tobaccos. The citric acid secretion of transgenic tobacco root tips were 2.56～3.79 times that of wild types. The relative elongation of transgenic tobacco roots were 1.91～3.45 times that of wild types. The root tips of transgenic tobaccos were more stained lightly with chrome azurine S than that of the wild types. All the results above indicated that GmMATE2 of Tamba black soybean was a citrate transporter gene, which could improve citric acid secretion and aluminum resistance of transgenic tobaccos. This study provides genetic resources for improving aluminum resistance of aluminum sensitive plants.
Tamba Black Soybean(Glycine max 'Tamba') is an aluminum-resistant plant whose aluminum-resistant mechanism is that their root tips secrete the citric complex aluminum ions. In order to explore its excellent resistance to aluminum genetic resources, this study, with the foundation of transcriptome data about Tamba black soybean's aluminum stress, cloned one of Tamba black soybean's gene of citrate transporter by using reverse transcription PCR(RT-PCR) and this gene was named GmMATE2(GenBank No.MF497433.1). CDS of GmMATE2 was 1 674 bp in length and GmMATE2 encoded 557 amino acids with a relative molecular mass of 60.38 kD, the isoelectric point of 9.54. GmMATE2, whose instability index was28.38, was a stable protein. Amino acids sequence of GmMATE2 analysis showed that it contained nine transmembrane structures, one Polysacc_synt_C domain and two family-specific MatE structures of multidrug and toxic compound extrusion(MATE). Evolutionary tree analysis showed that GmMATE2 was closely related to MATE proteins of Medicago truncatula, Trifolium repens and Cicer arietinum. When Tamba black soybean subjected to aluminum stress(pH 4.5, 0.5 mmol/L CaCl2, 100 μmol/L AlCl3), the semi-quantitative RT-PCR analysis showed that GmMATE2 was up-regulated in the root tips, with the highest expression at 24 h and citric acid secretion in the root tips increased with time, which suggested that GmMATE2 was involved in response to the aluminum stress. The plant overexpression vector pBI121-GmMATE2 was constructed and 3 transgenic tobaccos(Nicotiana tabacun) were obtained by agrobacterium-mediated leaf disc method. When 3 transgenic tobaccos subjected to aluminum stress(pH 4.5, 0.5 mmol/L CaCl_2, 50 μmol/L AlCl3), qRT-PCR analysis indicated that GmMATE2 was up-regulated in transgenic tobaccos. The citric acid secretion of transgenic tobacco root tips were 2.56～3.79 times that of wild types. The relative elongation of transgenic tobacco roots were 1.91～3.45 times that of wild types. The root tips of transgenic tobaccos were more stained lightly with chrome azurine S than that of the wild types. All the results above indicated that GmMATE2 of Tamba black soybean was a citrate transporter gene, which could improve citric acid secretion and aluminum resistance of transgenic tobaccos. This study provides genetic resources for improving aluminum resistance of aluminum sensitive plants.
引文
李金金,刘昂,王平,等.2014.铝胁迫下丹波黑大豆根尖细胞线粒体参与细胞凋亡的研究[J].农业生物技术学报,22(6):712-719.(Li J J,Liu A,Wang P,et al.2014.Root tip cell mitochondria involvement in programmed cell death induced by aluminum stress of Tamba black soybean(Glycine max)[J].Journal of Agricultural Biotechnology,22(6):712-719.)
钱绍方,陈丽梅,陈宣钦,等.2011.酸性土壤胁迫下丹波黑大豆和云南小黑豆生理特性研究[J].大豆科学,30(6):941-945.(Qian S F,Chen L M,Chen X Q,et al.2011.Physiological properties of soybean(Glycine max)tamba and Yunnanxiaoheidou under acid soil stress[J].Soybean Science,30(6):941-945.)
熊毅,李庆魁.1987.中国土壤[M].北京:科学出版社,pp.39.(Xiong Y,Li Q K.1987.Soils of China[M].Beijing:Science Press,pp.39.)
Brown M H,Paulsen I T,Skurray R A.2010.The multidrug efflux protein NorM is a prototype of a new family of transporters[J].Molecular Microbiology,31(1):394-395.
Degenhardt J,Larsen P B,Howell S H,et al.1998.Aluminum resistance in the Arabidopsis mutantalr-104 is caused by an aluminum-induced increase in rhizosphere pH[J].Plant Physiology,117(1):19-27.
Delhaize E,Hebb D M,Ryan P R.2001.Expression of a Pseudomonas aeruginosa citrate synthase gene in tobacco is not associated with either enhanced citrate accumulation or efflux[J].Plant Physiology,125(4):2059-2067.
Eticha D,Stass A,Horst W J.2005.Cell-wall pectin and its degree of methylation in the maize root-apex:Significance for genotypic differences in aluminium resistance[J].Plant,Cell&Environment,28(11):1410-1420.
Furukawa J,Yamaji N,Wang H,et al.2007.An aluminum-activated citrate transporter in barley[J].Plant and Cell Physiology,48(8):1081-1091.
Gruber B D,Ryan P R,Richardson A E,et al.2010.HvALMT1from barley is involved in the transport of organic anions[J].Journal of Experimental Botany,61(5):1455-1467.
Hong X,Zhang Q,Karyakin A A,et al.2010.Structure of a cation-bound multidrug and toxic compound extrusion transporter[J].Nature,467(7318):991.
Huang C F,Yamaji N,Chen Z,et al.2012.A tonoplast-localized half-size ABC transporter is required for internal detoxification of aluminum in rice[J].The Plant Journal,69(5):857-867.
Kochian L V,Pi?eros M A,Liu J,et al.2015.Plant adaptation to acid soils:The molecular basis for crop Aluminum resistance[J].Annual Review of Plant Biology,66(1):571-598.
Liu J,Li Y,Wang W,et al.2016.Genome-wide analysis of MATE transporters and expression patterns of a subgroup of MATE genes in response to aluminum toxicity in soybean[J].BMC Genomics,17(1):223.
Liu J,Magalhaes J V,Shaff J,et al.2009.Aluminum-activated citrate and malate transporters from the MATE and ALMT families function independently to confer Arabidopsis aluminum tolerance[J].The Plant Journal,57(3):389-399.
Magalhaes J V,Liu J,Guimaraes C T,et al.2007.A gene in the multidrug and toxic compound extrusion(MATE)family confers aluminum tolerance in Sorghum[J].Nature genetics,39(9):1156.
Ma J F,Hiradate S.2000.Form of aluminium for uptake and translocation in buckwheat(Fagopyrum esculentum Moench)[J].Planta,211(3):355-360.
Ma Z,Miyasaka S C.1998.Oxalate exudation by taro in response to Al[J].Plant Physiology,118(3):861-865.
Maron L G,Pi?eros M A,Guimar?es C T,et al.2010.Two functionally distinct members of the MATE(multi-drug and toxic compound extrusion)family of transporters potentially underlie two major aluminum tolerance QTLs in maize[J].The Plant Journal,61(5):728-740.
Melo J O,Lana U G P,Pi?eros M A,et al.2013.Incomplete transfer of accessory loci influencing SbMATE expression underlies genetic background effects for aluminum tolerance in Sorghum[J].The Plant Journal,73(2):276-288.
Min Y,Guo C L,Zhao X L,et al.2018.Adenosine 5'-monophosphate decreases citrate exudation and aluminium resistance in Tamba black soybean by inhibiting the interaction between 14-3-3 proteins and plasma membrane H+-ATPase[J].Plant Growth Regulation,84(2):285-292.
Morita Y,Kodama K,Shiota S,et al.1998.NorM,a putative multidrug efflux protein,of Vibrio parahaemolyticus and its homolog in Escherichia coli[J].Antimicrobial agents and chemotherapy,42(7):1778-1782.
Ofei-Manu P,Wagatsuma T,Ishikawa S,et al.2001.The plasma membrane strength of the root-tip cells and root phenolic compounds are correiated with Al tolerance in several common woody plants[J].Soil Science and Plant Nutrition,47(2):359-375.
Pi?eros M A,Can?ado G M A,Maron L G,et al.2008.Not all ALMT1-type transporters mediate aluminum-activated organic acid responses:The case of ZmALMT1-an anion-selective transporter[J].The Plant Journal,53(2):352-367.
Sasaki T,Yamamoto Y,Ezaki B,et al.2004.A wheat gene encoding an aluminum activated malate transporter[J].The Plant Journal,37(5):645-653.
Von Uexküll H R,Mutert E.1995.Global extent,development and economic impact of acid soils[J].Plant and soil,171(1):1-15.
Wehr J B,Blamey F P C,Hanna J V.2010.Hydrolysis and speciation of Al bound to pectin and plant cell wall material and its reaction with the dye chrome azurol S[J].Journal of Agricultural&Food Chemistry,58(9):5553-5560.
Xia J,Yamaji N,Kasai T,et al.2011.Plasma membrane-localized transporter for aluminum in rice[J].Proceedings of the National Academy of Sciences of the USA,107(43):18381-18385.
Yang X Y,Yang J L,Zhou Y,et al.2011.A de novo synthesis citrate transporter,Vigna umbellata multidrug and toxic compound extrusion,implicates in Al-activated citrate efflux in rice bean(Vigna umbellata)root apex[J].Plant,Cell&Environment,34(12):2138-2148.
Zhang J,Wei J,Li D,et al.2017.The role of the plasma membrane h+-atpase in plant responses to Aluminum toxicity[J].Frontiers in Plant Science,8:1757.
Zheng S J,Ma J F,Matsumoto H.1998.High aluminum resistance in buckwheat:I.Al-induced specific secretion of oxalic acid from root tips[J].Plant Physiology,117(3):745-751.
Zhou G,Delhaize E,Zhou M,et al.2013.The barley MATEgene,HvAACT1,increases citrate efflux and Al3+tolerance when expressed in wheat and barley[J].Annals of Botany,112(3):603-612.
Zhu X F,Wan J X,Sun Y,et al.2014.Xyloglucan endotransglucosylase-hydrolase17 interacts with xyloglucan endotransglucosylase-hydrolase31 to confer xyloglucan endotransglucosylase action and affect aluminum sensitivity in Arabidopsis[J].Plant Physiology,165(4):1566-1574.
钱绍方,陈丽梅,陈宣钦,等.2011.酸性土壤胁迫下丹波黑大豆和云南小黑豆生理特性研究[J].大豆科学,30(6):941-945.(Qian S F,Chen L M,Chen X Q,et al.2011.Physiological properties of soybean(Glycine max)tamba and Yunnanxiaoheidou under acid soil stress[J].Soybean Science,30(6):941-945.)
熊毅,李庆魁.1987.中国土壤[M].北京:科学出版社,pp.39.(Xiong Y,Li Q K.1987.Soils of China[M].Beijing:Science Press,pp.39.)
Brown M H,Paulsen I T,Skurray R A.2010.The multidrug efflux protein NorM is a prototype of a new family of transporters[J].Molecular Microbiology,31(1):394-395.
Degenhardt J,Larsen P B,Howell S H,et al.1998.Aluminum resistance in the Arabidopsis mutantalr-104 is caused by an aluminum-induced increase in rhizosphere pH[J].Plant Physiology,117(1):19-27.
Delhaize E,Hebb D M,Ryan P R.2001.Expression of a Pseudomonas aeruginosa citrate synthase gene in tobacco is not associated with either enhanced citrate accumulation or efflux[J].Plant Physiology,125(4):2059-2067.
Eticha D,Stass A,Horst W J.2005.Cell-wall pectin and its degree of methylation in the maize root-apex:Significance for genotypic differences in aluminium resistance[J].Plant,Cell&Environment,28(11):1410-1420.
Furukawa J,Yamaji N,Wang H,et al.2007.An aluminum-activated citrate transporter in barley[J].Plant and Cell Physiology,48(8):1081-1091.
Gruber B D,Ryan P R,Richardson A E,et al.2010.HvALMT1from barley is involved in the transport of organic anions[J].Journal of Experimental Botany,61(5):1455-1467.
Hong X,Zhang Q,Karyakin A A,et al.2010.Structure of a cation-bound multidrug and toxic compound extrusion transporter[J].Nature,467(7318):991.
Huang C F,Yamaji N,Chen Z,et al.2012.A tonoplast-localized half-size ABC transporter is required for internal detoxification of aluminum in rice[J].The Plant Journal,69(5):857-867.
Kochian L V,Pi?eros M A,Liu J,et al.2015.Plant adaptation to acid soils:The molecular basis for crop Aluminum resistance[J].Annual Review of Plant Biology,66(1):571-598.
Liu J,Li Y,Wang W,et al.2016.Genome-wide analysis of MATE transporters and expression patterns of a subgroup of MATE genes in response to aluminum toxicity in soybean[J].BMC Genomics,17(1):223.
Liu J,Magalhaes J V,Shaff J,et al.2009.Aluminum-activated citrate and malate transporters from the MATE and ALMT families function independently to confer Arabidopsis aluminum tolerance[J].The Plant Journal,57(3):389-399.
Magalhaes J V,Liu J,Guimaraes C T,et al.2007.A gene in the multidrug and toxic compound extrusion(MATE)family confers aluminum tolerance in Sorghum[J].Nature genetics,39(9):1156.
Ma J F,Hiradate S.2000.Form of aluminium for uptake and translocation in buckwheat(Fagopyrum esculentum Moench)[J].Planta,211(3):355-360.
Ma Z,Miyasaka S C.1998.Oxalate exudation by taro in response to Al[J].Plant Physiology,118(3):861-865.
Maron L G,Pi?eros M A,Guimar?es C T,et al.2010.Two functionally distinct members of the MATE(multi-drug and toxic compound extrusion)family of transporters potentially underlie two major aluminum tolerance QTLs in maize[J].The Plant Journal,61(5):728-740.
Melo J O,Lana U G P,Pi?eros M A,et al.2013.Incomplete transfer of accessory loci influencing SbMATE expression underlies genetic background effects for aluminum tolerance in Sorghum[J].The Plant Journal,73(2):276-288.
Min Y,Guo C L,Zhao X L,et al.2018.Adenosine 5'-monophosphate decreases citrate exudation and aluminium resistance in Tamba black soybean by inhibiting the interaction between 14-3-3 proteins and plasma membrane H+-ATPase[J].Plant Growth Regulation,84(2):285-292.
Morita Y,Kodama K,Shiota S,et al.1998.NorM,a putative multidrug efflux protein,of Vibrio parahaemolyticus and its homolog in Escherichia coli[J].Antimicrobial agents and chemotherapy,42(7):1778-1782.
Ofei-Manu P,Wagatsuma T,Ishikawa S,et al.2001.The plasma membrane strength of the root-tip cells and root phenolic compounds are correiated with Al tolerance in several common woody plants[J].Soil Science and Plant Nutrition,47(2):359-375.
Pi?eros M A,Can?ado G M A,Maron L G,et al.2008.Not all ALMT1-type transporters mediate aluminum-activated organic acid responses:The case of ZmALMT1-an anion-selective transporter[J].The Plant Journal,53(2):352-367.
Sasaki T,Yamamoto Y,Ezaki B,et al.2004.A wheat gene encoding an aluminum activated malate transporter[J].The Plant Journal,37(5):645-653.
Von Uexküll H R,Mutert E.1995.Global extent,development and economic impact of acid soils[J].Plant and soil,171(1):1-15.
Wehr J B,Blamey F P C,Hanna J V.2010.Hydrolysis and speciation of Al bound to pectin and plant cell wall material and its reaction with the dye chrome azurol S[J].Journal of Agricultural&Food Chemistry,58(9):5553-5560.
Xia J,Yamaji N,Kasai T,et al.2011.Plasma membrane-localized transporter for aluminum in rice[J].Proceedings of the National Academy of Sciences of the USA,107(43):18381-18385.
Yang X Y,Yang J L,Zhou Y,et al.2011.A de novo synthesis citrate transporter,Vigna umbellata multidrug and toxic compound extrusion,implicates in Al-activated citrate efflux in rice bean(Vigna umbellata)root apex[J].Plant,Cell&Environment,34(12):2138-2148.
Zhang J,Wei J,Li D,et al.2017.The role of the plasma membrane h+-atpase in plant responses to Aluminum toxicity[J].Frontiers in Plant Science,8:1757.
Zheng S J,Ma J F,Matsumoto H.1998.High aluminum resistance in buckwheat:I.Al-induced specific secretion of oxalic acid from root tips[J].Plant Physiology,117(3):745-751.
Zhou G,Delhaize E,Zhou M,et al.2013.The barley MATEgene,HvAACT1,increases citrate efflux and Al3+tolerance when expressed in wheat and barley[J].Annals of Botany,112(3):603-612.
Zhu X F,Wan J X,Sun Y,et al.2014.Xyloglucan endotransglucosylase-hydrolase17 interacts with xyloglucan endotransglucosylase-hydrolase31 to confer xyloglucan endotransglucosylase action and affect aluminum sensitivity in Arabidopsis[J].Plant Physiology,165(4):1566-1574.
© 2004-2018 中国地质图书馆版权所有 京ICP备05064691号 京公网安备11010802017129号 地址：北京市海淀区学院路29号 邮编：100083 电话：办公室：(+86 10)66554848；文献借阅、咨询服务、科技查新：66554700 |