厚藤ASR基因克隆及功能初步分析
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摘要
通过对厚藤(Ipomoea pes-caprae(Linn.)Sweet.)cDNA文库的筛选,获得了一个编码厚藤ASR(ABA-stress-ripening)基因的全长cDNA,命名为IpASR。研究结果显示,IpASR编码区全长648 bp,共编码215个氨基酸;蛋白质等电点为5.42,分子量为24.57 k D。通过在酵母中表达,发现IpASR能够提高转基因酵母的耐盐性及抗氧化能力。进一步以厚藤成年植株及幼苗为材料进行实时荧光定量PCR分析,结果表明,IpASR基因在厚藤成年植株各组织中广泛表达;高盐、甘露醇胁迫和ABA处理可诱导该基因在厚藤幼苗中的表达。结合GFP融合蛋白的亚细胞定位和生物信息学分析,发现IpASR蛋白为核蛋白,推测IpASR基因参与了厚藤生长发育的调控,并可响应ABA和非生物胁迫的诱导。
We focused on the full-length cDNA encoding ASR protein isolated from a Ipomoea pes-caprae( Linn.) Sweet. c DNA library. Results showed the coding region of IpASR cDNA was 648 bp,encoding a 215 amino acid protein with a molecular weight of 24. 57 kD and isoelectric point of 5. 42. By ectopic expression of IpASR in yeast,we found that IpASR improved the salt and H_2O_2 tolerance of transgenic yeast strains. Using adult plants and seedlings of I. pes-caprae with or without abiotic stress and ABA treatment,real-time RT-PCR analysis showed that IpASR was widely expressed in different adult organs in I. pes-caprae.The IpASR transcript was induced under abiotic stress and ABA treatment. The subcellular localization assay combining bioinformatics analysis showed that IpASR was a nucleoprotein.These results suggest that IpASR might play an important role in the regulation of I. pes-caprae growth and development,and therefore respond to environmental abiotic stress and the ABA signal pathway.
We focused on the full-length cDNA encoding ASR protein isolated from a Ipomoea pes-caprae( Linn.) Sweet. c DNA library. Results showed the coding region of IpASR cDNA was 648 bp,encoding a 215 amino acid protein with a molecular weight of 24. 57 kD and isoelectric point of 5. 42. By ectopic expression of IpASR in yeast,we found that IpASR improved the salt and H_2O_2 tolerance of transgenic yeast strains. Using adult plants and seedlings of I. pes-caprae with or without abiotic stress and ABA treatment,real-time RT-PCR analysis showed that IpASR was widely expressed in different adult organs in I. pes-caprae.The IpASR transcript was induced under abiotic stress and ABA treatment. The subcellular localization assay combining bioinformatics analysis showed that IpASR was a nucleoprotein.These results suggest that IpASR might play an important role in the regulation of I. pes-caprae growth and development,and therefore respond to environmental abiotic stress and the ABA signal pathway.
引文
[1]Wang HY,Wang HL,Shao HB,Tang XL.Recent advances in utilizing transcription factors to improve plant abiotic stress tolerance by transgenic technology[J].Front Plant Sci,2016,7:67.
[2]Ahanger MA,Akram NA,Ashraf M,Alyemeni MN,Wijaya L,Ahmad P.Plant responses to environmental stressesfrom gene to biotechnology[J].Ao B Plants,2017,9:25.
[3]Carrari F,Fernie AR,Iusem ND.Heard it through the grape vine?ABA and sugar cross-talk:the ASR story[J].Trends Plant Sci,2004,9:57-59.
[4]Wang LZ,Hu W,Feng JL,Yang XY,Huang QJ,et al.Identification of the ASR gene family from Brachypodium distachyon and functional characterization of Bd ASR1 in response to drought stress[J].Plant Cell Rep,2016,35:1221-1234.
[5]Iusem ND,Bartholomew DM,Hitz WD,Scolnik PA.Tomato(Lycopersicon esculentum)transcript induced by water deficit and ripening[J].Plant Physiol,1993,102:1353-1354.
[6]Rossi M,Iusem ND.Sequence of Asr2,a member of a gene family from Lycopersicon esculentum encoding chromosomal proteins:Homology to an intron of the polygalacturonase gene[J].DNA Seq,1995,5:225-227.
[7]Rossi M,Lijavetzky D,Bernacchi D,Hopp HE,Iusem N.Asr genes belong to a gene family comprising at least three closely linked loci on chromosome 4 in tomato[J].Mol Gen Genet,1996,252:489-492.
[8]Frankel N,Carrari F,Hasson E,Iusem ND.Evolutionary history of the Asr gene family[J].Gene,2006,378:74-83.
[9]Fischer I,Camus-Kulandaivelu L,Allal F,Stephan W.Adaptation to drought in two wild tomato species:the evolution of the Asr gene family[J].New Phytol,2011,190:1032-1044.
[10]González RM,Iusem ND.Twenty years of research on Asr(ABA-stress-ripening)genes and proteins[J].Planta,2014,239:941-949.
[11]Battaglia M,Olvera-Carrillo Y,Garciarrubio A,Campos F,Covarrubias AA.The enigmatic LEA proteins and other hydrophilins[J].Plant Physiol,2008,148:6-24.
[12]Tardieu F,Parent B,Caldeira CF,Welcker C.Genetic and physiological controls of growth under water deficit[J].Plant Physiol,2014,164:1628-1635.
[13]Dai JR,Liu B,Feng DR,Liu HY,He YM,et al.Mp Asr encodes an intrinsically unstructured protein and enhances osmotic tolerance in transgenic Arabidopsis[J].Plant Cell Rep,2011,30:1219-1230.
[14]Jha B,Lal S,Tiwari V,Yadav SK,Agarwal PK.The Sb ASR-1 gene cloned from an extreme halophyte Salicornia brachiata enhances salt tolerance in transgenic tobacco[J].Mar Biotechnol,2012,14:782-792.
[15]Hu YX,Yang X,Li XL,Yu XD,Li QL.The Sl ASR gene cloned from the extreme halophyte Suaeda liaotungensis K.enhances abiotic stress tolerance in transgenic Arabidopsis thaliana[J].Gene,2014,549:243-251.
[16]Li JR,Dong Y,Li C,Pan YL,Yu JJ.Si ASR4,the target gene of Si ARDP from Setaria italica,improves abiotic stress adaption in plants[J].Front Plant Sci,2017,7:2053.
[17]欧阳蒲月,刘楠,张伟伟,王俊,简曙光.海滩植物厚藤(Ipomoea pescaprae)的生物学及生理生态特性[J].湖南科技大学学报(自然科学版),2011,26(4):117-121.Ouyang PY,Liu N,Zhang WW,Wang J,Jian SG.Biological and eco-physiological characteristics of a beach plant Ipomoea pescaprae[J].Journal of Hunan University of Science and Technology:Natural Science Edition,2011,26(4):117-121.
[18]Zhou Y,Yin XC,Duan RJ,Hao GP,Guo JC,Jiang XY.Sp AHA1 and Sp SOS1 coordinate in transgenic yeast to improve salt tolerance[J].PLo S One,2015,10:e0137447.
[19]Quintero FJ,Blatt MR,Pardo JM.Functional conservation between yeast and plant endosomal Na+/H+antiporters[J].FEBS Lett,2000,471:224-228.
[20]Miryeganeh M,Takayama K,Tateishi Y,Kajita T.Longdistance dispersal by sea-drifted seeds has maintained the global distribution of Ipomoea pes-caprae subsp.brasiliensis(Convolvulaceae)[J].PLo S One,2014,9:e91836.
[21]Mishra A,Tanna B.Halophytes:potential resources for salt stress tolerance genes and promoters[J].Front Plant Sci,2017,8:829.
[22]Hincha DK,Thalhammer A.LEA proteins:IDPs with versatile functions in cellular dehydration tolerance[J].Biochem Soc Trans,2012,40:1000-1003.
[23]Yamada T,Ichimura K,Kanekatsu M,van Doorn WG.Gene expression in opening and senescing petals of morning glory(Ipomoea nil)flowers[J].Plant Cell Rep,2007,26:823-835.
[24]Kim IS,Kim YS,Yoon HS.Rice ASR1 protein with reactive oxygen species scavenging and chaperone-like activities enhances acquired tolerance to abiotic stresses in Saccharomyces cerevisiae[J].Mol Cells,2012,33:285-293.
[25]Arenhart RA,Schunemann M,Bucker Neto L,Margis R,Wang ZY,Margis-Pinheiro M.Rice ASR1 and ASR5 are complementary transcription factors regulating aluminium responsive genes[J].Plant Cell Environ,2016,39:645-651.
[2]Ahanger MA,Akram NA,Ashraf M,Alyemeni MN,Wijaya L,Ahmad P.Plant responses to environmental stressesfrom gene to biotechnology[J].Ao B Plants,2017,9:25.
[3]Carrari F,Fernie AR,Iusem ND.Heard it through the grape vine?ABA and sugar cross-talk:the ASR story[J].Trends Plant Sci,2004,9:57-59.
[4]Wang LZ,Hu W,Feng JL,Yang XY,Huang QJ,et al.Identification of the ASR gene family from Brachypodium distachyon and functional characterization of Bd ASR1 in response to drought stress[J].Plant Cell Rep,2016,35:1221-1234.
[5]Iusem ND,Bartholomew DM,Hitz WD,Scolnik PA.Tomato(Lycopersicon esculentum)transcript induced by water deficit and ripening[J].Plant Physiol,1993,102:1353-1354.
[6]Rossi M,Iusem ND.Sequence of Asr2,a member of a gene family from Lycopersicon esculentum encoding chromosomal proteins:Homology to an intron of the polygalacturonase gene[J].DNA Seq,1995,5:225-227.
[7]Rossi M,Lijavetzky D,Bernacchi D,Hopp HE,Iusem N.Asr genes belong to a gene family comprising at least three closely linked loci on chromosome 4 in tomato[J].Mol Gen Genet,1996,252:489-492.
[8]Frankel N,Carrari F,Hasson E,Iusem ND.Evolutionary history of the Asr gene family[J].Gene,2006,378:74-83.
[9]Fischer I,Camus-Kulandaivelu L,Allal F,Stephan W.Adaptation to drought in two wild tomato species:the evolution of the Asr gene family[J].New Phytol,2011,190:1032-1044.
[10]González RM,Iusem ND.Twenty years of research on Asr(ABA-stress-ripening)genes and proteins[J].Planta,2014,239:941-949.
[11]Battaglia M,Olvera-Carrillo Y,Garciarrubio A,Campos F,Covarrubias AA.The enigmatic LEA proteins and other hydrophilins[J].Plant Physiol,2008,148:6-24.
[12]Tardieu F,Parent B,Caldeira CF,Welcker C.Genetic and physiological controls of growth under water deficit[J].Plant Physiol,2014,164:1628-1635.
[13]Dai JR,Liu B,Feng DR,Liu HY,He YM,et al.Mp Asr encodes an intrinsically unstructured protein and enhances osmotic tolerance in transgenic Arabidopsis[J].Plant Cell Rep,2011,30:1219-1230.
[14]Jha B,Lal S,Tiwari V,Yadav SK,Agarwal PK.The Sb ASR-1 gene cloned from an extreme halophyte Salicornia brachiata enhances salt tolerance in transgenic tobacco[J].Mar Biotechnol,2012,14:782-792.
[15]Hu YX,Yang X,Li XL,Yu XD,Li QL.The Sl ASR gene cloned from the extreme halophyte Suaeda liaotungensis K.enhances abiotic stress tolerance in transgenic Arabidopsis thaliana[J].Gene,2014,549:243-251.
[16]Li JR,Dong Y,Li C,Pan YL,Yu JJ.Si ASR4,the target gene of Si ARDP from Setaria italica,improves abiotic stress adaption in plants[J].Front Plant Sci,2017,7:2053.
[17]欧阳蒲月,刘楠,张伟伟,王俊,简曙光.海滩植物厚藤(Ipomoea pescaprae)的生物学及生理生态特性[J].湖南科技大学学报(自然科学版),2011,26(4):117-121.Ouyang PY,Liu N,Zhang WW,Wang J,Jian SG.Biological and eco-physiological characteristics of a beach plant Ipomoea pescaprae[J].Journal of Hunan University of Science and Technology:Natural Science Edition,2011,26(4):117-121.
[18]Zhou Y,Yin XC,Duan RJ,Hao GP,Guo JC,Jiang XY.Sp AHA1 and Sp SOS1 coordinate in transgenic yeast to improve salt tolerance[J].PLo S One,2015,10:e0137447.
[19]Quintero FJ,Blatt MR,Pardo JM.Functional conservation between yeast and plant endosomal Na+/H+antiporters[J].FEBS Lett,2000,471:224-228.
[20]Miryeganeh M,Takayama K,Tateishi Y,Kajita T.Longdistance dispersal by sea-drifted seeds has maintained the global distribution of Ipomoea pes-caprae subsp.brasiliensis(Convolvulaceae)[J].PLo S One,2014,9:e91836.
[21]Mishra A,Tanna B.Halophytes:potential resources for salt stress tolerance genes and promoters[J].Front Plant Sci,2017,8:829.
[22]Hincha DK,Thalhammer A.LEA proteins:IDPs with versatile functions in cellular dehydration tolerance[J].Biochem Soc Trans,2012,40:1000-1003.
[23]Yamada T,Ichimura K,Kanekatsu M,van Doorn WG.Gene expression in opening and senescing petals of morning glory(Ipomoea nil)flowers[J].Plant Cell Rep,2007,26:823-835.
[24]Kim IS,Kim YS,Yoon HS.Rice ASR1 protein with reactive oxygen species scavenging and chaperone-like activities enhances acquired tolerance to abiotic stresses in Saccharomyces cerevisiae[J].Mol Cells,2012,33:285-293.
[25]Arenhart RA,Schunemann M,Bucker Neto L,Margis R,Wang ZY,Margis-Pinheiro M.Rice ASR1 and ASR5 are complementary transcription factors regulating aluminium responsive genes[J].Plant Cell Environ,2016,39:645-651.