杜梨PbNHX1基因的克隆、表达分析及功能验证
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摘要
【目的】克隆1个杜梨Na~+/H~+逆向转运蛋白基因PbNHX1,并对其序列特征、表达特点及功能进行研究。【方法】采用RT-PCR和PCR克隆PbNHX1的c DNA和DNA序列,利用生物信息学方法进行序列分析,定量PCR检测其在非生物胁迫下转录水平变化,酵母互补试验检测PbNHX1基因的离子转运能力。【结果】杜梨PbNHX1基因c DNA编码区长1 704 bp,对应基因组DNA序列长3 594 bp,由13个外显子和12个内含子组成,编码蛋白含567个氨基酸。系统进化树显示,PbNHX1位于液泡膜型Na~+/H~+逆向转运蛋白分支上,与杨树液泡膜型Na~+/H~+逆向转运蛋白Pt NHX1.3基因亲缘关系较近。正常生长条件下,PbNHX1在杜梨叶片中表达量高于根。施加200 mmol·L-1Na Cl、10%(φ)PEG6000或100μmol·L-1ABA后,PbNHX1在叶片中的转录水平持续上升;其在根中的表达量先升后降,表达高峰依次出现在处理后6、3和6 h。PbNHX1的转入可恢复Na Cl、KCl和潮霉素B对nhx1缺失酵母菌株AXT3的生长抑制,转基因酵母细胞中Na~+和K~+含量显著增加。【结论】PbNHX1具有植物NHXs基因家族的固有特征,对盐碱、渗透胁迫和ABA处理均存在转录响应,转入该基因能够提高酵母nhx1缺失突变体AXT3对盐胁迫的耐受能力,部分恢复其对阳离子的转运功能,从而促进Na~+、K~+积累。
【Objective】Pyrus betulaefolia is one of the main rootstocks for pear P. betulaefolia as a rootstock can effectively reduce Na~+accumulation in the root, inhibit the Na~+transportation to the scion, and improve the salt tolerance of pear varieties. In order to reveal the molecular mechanism of salt tolerance and provide experimental basis for the further research, we researched the Na~+/H~+antiporter protein in P.betulaefolia. One PbNHX1 gene was cloned from this species and its sequence characteristics and expression characteristics were analyzed.【Methods】The sodium/hydrogen exchanger 1-like gene(XM_018651314.1) in Pyrus×bretschneideri(Chinese white pear) was used as the electronic probe to search the transcriptome database of P. betulaefolia Na Cl-treated seedlings. Then, one transcript Pbr017120 was obtained for designing gene-specific primers. Indeed, its c DNA and DNA sequences were isolated using RT-PCR and PCR techniques. And the physical and chemical properties of PbNHX1 protein were analyzed by Protparam online software. The introns and exons of PbNHX1 genes were analyzed using Gene Structure Display Server. The signature motifs were analyzed by MEME software and the phylogenetic tree was built by MEGA 6.0. The expression condition of PbNHX1 under the abiotic stresses, such as 200 mmol·L-1 Na Cl, 10%(φ) PEG6000 and 100 μmol·L-1 ABA, were analyzed by quantitative PCR(q RT-PCR). Finally, yeast complementary experiments with a salt sensitive yeast mutant AXT3 were performedto verify the functions of PbNHX1 gene. After solid and liquid cultivation, the growth status of transgenicyeast was detected. Their total Na~+and K~+contents were also tested by a flame-graphite furnace atomicabsorption spectrometer.【Results】The c DNAs of PbNHX1 was 1 704 bp, and its DNA was 3 594 bp,which included 13 exons and 12 introns. This gene encoded a protein containing 567 amino acids, its rela-tive molecular weight and isoelectric point(p I) was 62.179 ku and 5.55, respectively. Moreover, the PbNHX1 elements consisted of C2 861 H4 437 N669 O805 S21. The phylogenetic tree showed that PbNHX1 was located inthe branch of vacuolar Na~+/H~+antiporter, which was far from the plasma membrane Na~+/H~+antiporter geneof Arabidopsis At NHX7 or rice Os NHX8, and was closely related to poplar vacuolar Na~+/H~+antiporter gene Pt NHX1.3 or Arabidopsis At NHX2. The expression level of the PbNHX1 was higher in the leaves than thatin its roots under normal growth conditions. After treatment with 200 mmol·L-1 Na Cl, PbNHX1 transcrip-tional level obviously increased both in the roots and in the leaves. For example, PbNHX1 expression levelincreased firstly and then decreased in the roots once the seedlings were treated with salt. Its expressionpeak appeared at 6 h. At that time, the amount of PbNHX1 transcription was 2.4 times higher than that ofthe control. Then, its expression level began to decrease closely to the original level at 24 h. On the onehand, the expression level of PbNHX1 kept on rising in the leaves after the treatment of salt. In the case of10% PEG6000, PbNHX1 expression level increased firstly and then decreased in the roots. Its expressionpeak appeared at 3 h, which was 1.2 times higher than that of the control. After that time, its expressionlevel turned to fall down and recovered closely to the original level at 24 h. In the leaves, the expressionlevel of PbNHX1 continued to rise when the salt existed and it was 22.6 times higher than that of the con-trol at 24 h. After 100 μmol·L-1 ABA treatment, PbNHX1 expression level increased firstly and then de-creased in the roots. Its expression peak appeared at 6 h, which was 1.6 times higher than that of the con-trol. Then its transcription declined closely to the original level at 24 h. In the leaves, the expression levelof PbNHX1 increased during the treatment period, which was 8.0 times higher than that of the control at24 h. These results indicated that PbNHX1 was regulated significantly in the leaves under different abiot-ic stress. The results of YPD solid culture showed that transform of PbNHX1 could recover the growth inhi-bition of Na Cl, KCl and hygromycin B to the nhx1 mutant yeast strain AXT3 when the cells were treatedwith 20 mg·L-1 hygromycin B, 20-50 mmol·L-1 Na Cl or 0.5-1.00 mol·L-1 KCl. Meanwhile, the results ofliquid culture showed that transform of PbNHX1 reduced the growth inhibition of the mutant strain AXT3 to Na Cl and KCl when the cells were treated with 50-100 mmol·L-1 Na Cl or 0.5-1.0 mol·L-1 KCl. Fur-thermore, the contents of Na~+and K~+significantly increased in PbNHX1 transgenic yeast cells comparedwith the mutant strain without this gene when 20 mmol·L-1 Na Cl presence.【Conclusion】Our results haveshowed that PbNHX1 gene belong to the NHX gene family of P. betulaefoli, which has the inherent charac-teristics of plant NHXs family. This gene response to Na Cl, osmotic and ABA stresses. Transfer of the PbNHX1 gene can increase the salt tolerance of the nhx1 mutant yeast strain AXT3 and partly recover its iontransport capacity and facilitate the accumulation of the Na~+and K~+ions.
【Objective】Pyrus betulaefolia is one of the main rootstocks for pear P. betulaefolia as a rootstock can effectively reduce Na~+accumulation in the root, inhibit the Na~+transportation to the scion, and improve the salt tolerance of pear varieties. In order to reveal the molecular mechanism of salt tolerance and provide experimental basis for the further research, we researched the Na~+/H~+antiporter protein in P.betulaefolia. One PbNHX1 gene was cloned from this species and its sequence characteristics and expression characteristics were analyzed.【Methods】The sodium/hydrogen exchanger 1-like gene(XM_018651314.1) in Pyrus×bretschneideri(Chinese white pear) was used as the electronic probe to search the transcriptome database of P. betulaefolia Na Cl-treated seedlings. Then, one transcript Pbr017120 was obtained for designing gene-specific primers. Indeed, its c DNA and DNA sequences were isolated using RT-PCR and PCR techniques. And the physical and chemical properties of PbNHX1 protein were analyzed by Protparam online software. The introns and exons of PbNHX1 genes were analyzed using Gene Structure Display Server. The signature motifs were analyzed by MEME software and the phylogenetic tree was built by MEGA 6.0. The expression condition of PbNHX1 under the abiotic stresses, such as 200 mmol·L-1 Na Cl, 10%(φ) PEG6000 and 100 μmol·L-1 ABA, were analyzed by quantitative PCR(q RT-PCR). Finally, yeast complementary experiments with a salt sensitive yeast mutant AXT3 were performedto verify the functions of PbNHX1 gene. After solid and liquid cultivation, the growth status of transgenicyeast was detected. Their total Na~+and K~+contents were also tested by a flame-graphite furnace atomicabsorption spectrometer.【Results】The c DNAs of PbNHX1 was 1 704 bp, and its DNA was 3 594 bp,which included 13 exons and 12 introns. This gene encoded a protein containing 567 amino acids, its rela-tive molecular weight and isoelectric point(p I) was 62.179 ku and 5.55, respectively. Moreover, the PbNHX1 elements consisted of C2 861 H4 437 N669 O805 S21. The phylogenetic tree showed that PbNHX1 was located inthe branch of vacuolar Na~+/H~+antiporter, which was far from the plasma membrane Na~+/H~+antiporter geneof Arabidopsis At NHX7 or rice Os NHX8, and was closely related to poplar vacuolar Na~+/H~+antiporter gene Pt NHX1.3 or Arabidopsis At NHX2. The expression level of the PbNHX1 was higher in the leaves than thatin its roots under normal growth conditions. After treatment with 200 mmol·L-1 Na Cl, PbNHX1 transcrip-tional level obviously increased both in the roots and in the leaves. For example, PbNHX1 expression levelincreased firstly and then decreased in the roots once the seedlings were treated with salt. Its expressionpeak appeared at 6 h. At that time, the amount of PbNHX1 transcription was 2.4 times higher than that ofthe control. Then, its expression level began to decrease closely to the original level at 24 h. On the onehand, the expression level of PbNHX1 kept on rising in the leaves after the treatment of salt. In the case of10% PEG6000, PbNHX1 expression level increased firstly and then decreased in the roots. Its expressionpeak appeared at 3 h, which was 1.2 times higher than that of the control. After that time, its expressionlevel turned to fall down and recovered closely to the original level at 24 h. In the leaves, the expressionlevel of PbNHX1 continued to rise when the salt existed and it was 22.6 times higher than that of the con-trol at 24 h. After 100 μmol·L-1 ABA treatment, PbNHX1 expression level increased firstly and then de-creased in the roots. Its expression peak appeared at 6 h, which was 1.6 times higher than that of the con-trol. Then its transcription declined closely to the original level at 24 h. In the leaves, the expression levelof PbNHX1 increased during the treatment period, which was 8.0 times higher than that of the control at24 h. These results indicated that PbNHX1 was regulated significantly in the leaves under different abiot-ic stress. The results of YPD solid culture showed that transform of PbNHX1 could recover the growth inhi-bition of Na Cl, KCl and hygromycin B to the nhx1 mutant yeast strain AXT3 when the cells were treatedwith 20 mg·L-1 hygromycin B, 20-50 mmol·L-1 Na Cl or 0.5-1.00 mol·L-1 KCl. Meanwhile, the results ofliquid culture showed that transform of PbNHX1 reduced the growth inhibition of the mutant strain AXT3 to Na Cl and KCl when the cells were treated with 50-100 mmol·L-1 Na Cl or 0.5-1.0 mol·L-1 KCl. Fur-thermore, the contents of Na~+and K~+significantly increased in PbNHX1 transgenic yeast cells comparedwith the mutant strain without this gene when 20 mmol·L-1 Na Cl presence.【Conclusion】Our results haveshowed that PbNHX1 gene belong to the NHX gene family of P. betulaefoli, which has the inherent charac-teristics of plant NHXs family. This gene response to Na Cl, osmotic and ABA stresses. Transfer of the PbNHX1 gene can increase the salt tolerance of the nhx1 mutant yeast strain AXT3 and partly recover its iontransport capacity and facilitate the accumulation of the Na~+and K~+ions.
引文
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[17]OKUBO M,FURUKAWA Y,SAKURATANI T.Growth,flowering and leaf properties of pear cultivars grafted on two Asian pear rootstock seedlings under Na Cl irrigation[J].Scientia Horticulture,2000,85(1/2):91-101.
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[19]LI H,LIN J,YANG Q S,LI X G,CHANG Y H.Comprehensive analysis of differentially expressed genes under salt stress in pear(Pyrus betulaefolia)using RNA-Seq[J].Plant Growth Regulation,2017,82(10):409-420.
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[2]RODRIGUEZ-ROSALES M P,GALVEZ F J,HUERTAS R,ARANDA M N,BAGHOUR M,CAGNAC O,VENEMA K.Plant NHX cation/proton antiporters[J].Plant Signaling&Behavior,2009,4(4):265-276.
[3]HAMADA A,SHONO M,XIA T,OHTA M,HAYASHI Y,TANAKA A,HAYAKAWA T.Isolation and characterization of a Na+/H+antiporter gene from the halophyte Atriplex gmelini[J].Plant Molecular Biology,2001,46(1):35-42.
[4]SHI H Z,ZHU J K.Regulation of expression of the vacuolar Na+/H+antiporter gene At NHX1 by salt stress and abscisic acid[J].Plant Molecular Biology,2002,50(3):543-550.
[5]LIU Q L,XU K D,ZHONG M,PAN Y Z,JIANG B B,LIU G L,JIA Y.Cloning and characterization of a novel vacuolar Na+/H+antiporter gene(Dgnhx1)from chrysanthemum[J].Plos One,2013,8(12):e83702.
[6]ZHANG H,LIU Y X,CHAPMAN S,LOVE A J,XU Y,XIA T.A newly isolated Na+/H+antiporter gene,Dm NHX1,confers salt tolerance when expressed transiently in Nicotiana benthamiana or stably in Arabidopsis thaliana[J].Plant Cell Tissue and Organ Culture,2012,110(2):189-200.
[7]WU C G,YANG G D,MENG Q W,ZHENG C C.The cotton Gh NHX1 gene encoding a novel putative tonoplast Na+/H+antiporter plays an important role in salt stress[J].Plant Cell Physiology,2004,45(5):600-607.
[8]TANG R,LI C,XU K,DU Y H,XIA T.Isolation,functional characterization,and expression pattern of a vacuolar Na+/H+antiporter gene Tr NHX1 from Trifolium repens L.[J].Plant Molecular Biology Reporter,2010,28(1):102-111.
[9]MISHRA S,ALAVILLI H,LEE B H,PANDA S K,SAHOO L.Cloning and characterization of a novel vacuolar Na+/H+antiporter gene(Vu NHX1)from drought hardy legume,cowpea for salt tolerance[J].Plant Cell Tissue and Organ Culture,2015,120(1):19-33.
[10]MISHRA S,BEHURA R,AWASTHI J P,DEY M,SAHOO D,BHOWMIK S S D,PANDA S K,SAHOO L.Ectopic overexpression of a mungbean vacuolar Na+/H+antiporter gene(Vr NHX1)leads to increased salinity stress tolerance in transgenic Vigna unguiculata L.walp[J].Molecular Breeding,2014,34(3):1345-1359.
[11]LIANG M X,LIN M M,LIN Z Y,ZHAO L,ZHAO G M,LI Q,YIN X Z.Identification,functional characterization,and expression pattern of a Na Cl-inducible vacuolar Na+/H+antiporter in chicory(Cichorium intybus L.)[J].Plant Growth Regulation,2015,75(3):605-614.
[12]SUN M H,MA Q J,LIU X,ZHU X P,HU D P,HAO Y J.Molecular cloning and functional characterization of Md NHX1 reveals its involvement in salt tolerance in apple calli and Arabidopsis[J].Scientia Horticulturae,2017,215:126-133.
[13]QUINTERO F J,BLATT M R,PARDO J M.Functional conservation between yeast and plant endosomal Na+/H+antiporters1[J].Febs Letters,2000,471(2/3):224-228.
[14]GOUIAA S,KHOUDI H,LEIDI E O,PARDO J M,MASMOUDI K.Expression of wheat Na+/H+antiporter TNHXS1 and H+-pyrophosphatase TVP1 genes in tobacco from a bicistronic transcriptional unit improves salt tolerance[J].Plant Molecular Biology,2012,79(1/2):137-155.
[15]WU C X,GAO X H,KONG X Q,ZHAO Y X,ZHANG H.Molecular cloning and functional analysis of a Na+/H+antiporter gene Th NHX1 from a halophytic plant Thellungiella halophila[J].Plant Molecular Biology Reporter,2009,27(1):1-12.
[16]MATSUMOTO K,TAUMURA F,CHUN J P,IKEDA T,IMANISHI K,TANABE K.Enhancement in salt tolerance of Japanese pear by using Pyrus betulaefolia rootstock[J].Horticulture Research,2007,6(1):47-52.
[17]OKUBO M,FURUKAWA Y,SAKURATANI T.Growth,flowering and leaf properties of pear cultivars grafted on two Asian pear rootstock seedlings under Na Cl irrigation[J].Scientia Horticulture,2000,85(1/2):91-101.
[18]许园园,蔺经,李晓刚,李慧,常有宏.杜梨Pb CBL10基因表达与启动子功能分析[J].果树学报,2014,31(6):1024-1031.XU Yuanyuan,LIN Jin,LI Xiaogang,LI Hui,CHANG Youhong.Expression of the Pb CBL10 gene and functional analysis of its promoter in pear plants(Pyrus betulaefolia)[J].Journal of Fruit Science,2014,31(6):1024-1031.
[19]LI H,LIN J,YANG Q S,LI X G,CHANG Y H.Comprehensive analysis of differentially expressed genes under salt stress in pear(Pyrus betulaefolia)using RNA-Seq[J].Plant Growth Regulation,2017,82(10):409-420.
[20]BAILEY T,ELKAN C.Fitting a mixture model by expectation maximization to discover motifs in biopolymers[C]//Seattle,USA:Proceedings of the Second International Conference on Intelligent Systems for Molecular Biology(ISMB-94),1994.
[21]TAMURA K,PETERSON D,PETERSON N,STECHER G,NEI M,KUMAR S.Molecular evolutionary genetics analysis using maximum likelihood,evolutionary distance,and maximum parsimony methods[J].Molecular Biology and Evolution,2011,28:2731-2739.
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