马尾松水通道蛋白PmPIP1基因克隆及在干旱胁迫下的表达分析
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摘要
克隆马尾松Pinus massoniana水通道蛋白(AQP),并对其生物信息学与干旱胁迫表达模式进行分析。采用逆转录聚合酶链式反应(RT-PCR)以及互补脱氧核糖核酸(c DNA)末端快速扩增(RACE)方法克隆马尾松水通道蛋白基因。采用实时荧光定量聚合酶链式反应(q RT-PCR)分析其在干旱胁迫下的响应模式。结果克隆到一个马尾松水通道蛋白基因,命名为Pm PIP1(Gen Bank登录号为KF582038)。此基因c DNA全长序列为1 301 bp,包括867 bp的完整开放阅读框,99 bp的5′末端非翻译区和335 bp的3′末端非翻译区。编码288个氨基酸残基,分子量为30.86 k D,等电点(p I)8.48。Pm PIP1与菠菜Spinacia oleracea(2b5f A)水通道蛋白结构相似。Pm PIP1含有6个跨膜区,具有膜内在蛋白(MIP)家族信号序列、高等植物高度保守序列HINPAVTFG和2个天门冬酰胺-脯氨酸-丙氨酸(NPA)保守肽段。Pm PIP1基因属质膜内在蛋白(PIPs)亚族,与挪威云杉Picea abies水通道蛋白基因亲缘关系最近,同源性达95%。q RT-PCR表明Pm PIP1受干旱胁迫诱导表达。马尾松Pm PIP1的克隆丰富了植物AQP基因的资料库,同时推测Pm PIP1基因可能参与了马尾松干旱胁迫过程。
The aquaporin(AQP) gene plays an important role in plants adapting to abiotic stresses. To predict the AQP gene function and provide basal data for mechanism of Pinus massoniana's drought resistance, the sequence characteristics of the AQP gene from P. massoniana were analyzed and its expression profiling was studied after drought-stress treatment. The AQP gene was cloned using reverse transcription-polymerase chain reaction(RT-PCR) and rapid-amplification of complementary deoxyribonucleic acid(c DNA) ends(RACE).The expression of the AQP gene was then performed using quantitative reverse transcriptase-polymerase chain reaction(q RT-PCR). The full-length c DNA of the AQP gene from P. massoniana, designated Pm PIP1 with a registered number in Gen Bank KF582038, was obtained. Results of the sequence analysis showed that the size of Pm PIP1 was 1 301 bp, containing an 867 bp open reading frame that encoded 288 amino acid residues with30.86 k Da molecular weight and an 8.48 isoelectric point, a 99 bp 5′ terminal untranslated regions(UTR),and a 335 bp 3′ terminal UTR. The Pm PIP1 3D structure had a strong similarity to Spinacia oleracea(2b5f A).Pm PIP1 exhibited a typical structure with six transmembrane domains, and had the consensus sequence HINPAVTFG of membrane intrinsic protein(MIP) family and two highly conserved peptides Asn-Pro-Ala(NPA).The evolutionary analysis revealed that Pm PIP1 shared a 95% identity with Picea abies and belonged to PIPs.The Pm PIP1 expression patterns with drought conditions showed that drought did induce Pm PIP1. In conclusion, cloning of the Pm PIP1 gene from P. massoniana enriched the plant aquaporin gene database, and the q RT-PCR analysis indicated that the Pm PIP1 gene may be involved in the response related to drought stress.
The aquaporin(AQP) gene plays an important role in plants adapting to abiotic stresses. To predict the AQP gene function and provide basal data for mechanism of Pinus massoniana's drought resistance, the sequence characteristics of the AQP gene from P. massoniana were analyzed and its expression profiling was studied after drought-stress treatment. The AQP gene was cloned using reverse transcription-polymerase chain reaction(RT-PCR) and rapid-amplification of complementary deoxyribonucleic acid(c DNA) ends(RACE).The expression of the AQP gene was then performed using quantitative reverse transcriptase-polymerase chain reaction(q RT-PCR). The full-length c DNA of the AQP gene from P. massoniana, designated Pm PIP1 with a registered number in Gen Bank KF582038, was obtained. Results of the sequence analysis showed that the size of Pm PIP1 was 1 301 bp, containing an 867 bp open reading frame that encoded 288 amino acid residues with30.86 k Da molecular weight and an 8.48 isoelectric point, a 99 bp 5′ terminal untranslated regions(UTR),and a 335 bp 3′ terminal UTR. The Pm PIP1 3D structure had a strong similarity to Spinacia oleracea(2b5f A).Pm PIP1 exhibited a typical structure with six transmembrane domains, and had the consensus sequence HINPAVTFG of membrane intrinsic protein(MIP) family and two highly conserved peptides Asn-Pro-Ala(NPA).The evolutionary analysis revealed that Pm PIP1 shared a 95% identity with Picea abies and belonged to PIPs.The Pm PIP1 expression patterns with drought conditions showed that drought did induce Pm PIP1. In conclusion, cloning of the Pm PIP1 gene from P. massoniana enriched the plant aquaporin gene database, and the q RT-PCR analysis indicated that the Pm PIP1 gene may be involved in the response related to drought stress.
引文
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[2]MAUREL C,REIZER J,SCHROEDER J I,et al.The vacuolar membrane protein gamma-TIP creates water specific channels in Xenopus oocytes[J].EMBO J,1993,12(6):2241-2247.
[3]LUDEWIG U,DYNOWSKI M.Plant aquaporin selectivity:where transport assays,computer simulations and physiology meet[J].Cell Mol Life Sci,2009,66(19):3161-3175.
[4]PIE N'KOWSKA,J R,KOSICKA E,WOJTKOWSKA M,et al.Molecular identification of first putative aquaporins in snails[J].J Membrane Biol,2014,247(3):239-252.
[5]邹智,王丹华,莫业勇,等.橡胶死皮相关液泡型水通道蛋白基因TIP1的克隆与序列分析[J].安徽农业科学,2013,41(36):13851-13854.ZOU Zhi,WANG Danhua,MO Yeyong,et al.Molecular cloning and sequence analysis of a tonoplast aquaporin gene TIP1 associated with tapping panel dryness in Hevea brasiliensis[J].J Anhui Agric Sci,2013,4 1(36):13851-13854.
[6]ALEXANDERSSON E,FRAYSSE L,SJ魻VALL-LARSEN S,et al.Whole gene family expression and drought stress regulation of aquaporins[J].Plant Mol Biol,2005,59(3):469-484.
[7]LIAN Hongli,YU Xin,YE Qin,et al.The role of aquaporin RWC3 in drought avoidance in rice[J].Plant Cell Physiol,2004,45(4):481-489.
[8]LI Guowei,PENG Yanhui,YU Xin,et al.Transport functions and expression analysis of vacuolar membrane aquaporins in response to various stresses in rice[J].J Plant Physiol,2008,165(18):1879-1888.
[9]JOHANSON U,KARLSSON M,JOHANSSON I,et al.The complete set of genes encoding major intrinsic proteins in Arabidopsis provides a framework for a new nomenclature for major intrinsic protein in plants[J].Plant Physiol,2001,126(4):1358-1369.
[10]CHAUMONT F,BARRIEU F,WOJCIK E,et al.Aquaporins constitute a large and highly divergent protein family in maize[J].Plant Physiol,2001,125(3):1206-1215.
[11]SUGA S,IMAGAWA S,MAESHIMA M.Specificity of the accumulation of m RNAs and proteins of the plasma membrane and tonoplast aquaporins in radish organs[J].Planta,2001,212(2):294-304.
[12]丁贵杰,周志春,王章荣,等.马尾松纸浆用材林培育与利用[M].北京:中国林业出版社,2006:111.
[13]谌红辉,丁贵杰.马尾松造林密度效应研究[J].林业科学,2004,40(1):92-98.CHEN Honghui,DING Guijie.Study on planting density effects for Masson pine plantation[J].Sci Silv Sin,2004,40(1):92-98.
[14]施积炎,丁贵杰,袁小凤.不同家系马尾松苗木水分参数的研究[J].林业科学,2004,40(3):51-55.SHI Jiyan,DING Guijie,YUAN Xiaofeng.Studies on water parameters in Pinus massoniana seedlings of different families[J].Sci Silv Sin,2004,40(3):51-55.
[15]徐向华,丁贵杰.马尾松适应低磷胁迫的生理生化响应[J].林业科学,2006,42(9):24-28.XU Xianghua,DING Guijie.Physiological and biochemical responses of Pinus massoniana to low phosphorus stress[J].Sci Silv Sin,2006,42(9):24-28.
[16]何佩云,丁贵杰,谌红辉.连栽马尾松人工林土壤肥力比较研究[J].林业科学研究,2011,24(3):357-362.HE Peiyun,DING Guijie,CHEN Honghui.Comparison on soil fertilities of Masson pine plantations of different generations[J].For Res,2011,24(3):357-362.
[17]丁贵杰.马尾松人工纸浆材林采伐年龄初步研究[J].林业科学,2000,36(1):15-20.DING Guijie.The preliminary study on optimum cutting age of pulpwood stand for Masson pine plantation[J].Sci Silv Sin,2000,36(1):15-20.
[18]谌红辉,丁贵杰,温恒辉,等.造林密度对马尾松林分生长与效益的影响研究[J].林业科学研究,2011,24(4):470-475.CHEN Honghui,DING Guijie,WEN Henghui,et al.Effects of planting density on growth and economic benefit of Masson pine plantation[J].For Res,2011,24(4):470-475.
[19]王冰梅,郭晋隆,叶冰莹,等.马尾松银松素合酶基因启动子区的克隆及特征分析[J].亚热带农业研究,2008,4(4):292-296.WANG Bingmei,GUO Jinlong,YE Bingying,et al.Molecular cloning and analysis of pinosylvin synthase gene promoter from Pinus massoniana[J].Subtrop Agric Res,2008,4(4):292-296.
[20]李擎天,赵宇,周绮云,等.珠眉海棠水通道蛋白Mz PIP1;1基因的克隆与表达分析[J].中国农业大学学报,2012,17(3):63-68.LI Qingtian,ZHAO Yu,ZHOU Qiyun,et al.Cloning and expression analysis of Mz PIP1;1 gene from Malus zumi Mats[J].J China Agric Univ,2012,17(3):63-68.
[21]CUI Xianghuan,HAO Fushun,CHEN Hui,et al.Expression of the Vicia faba Vf PIP1 gene in Arabidopsis thaliana plants improves their drought resistance[J].J Plant Res,2008,121(2):207-214.
[22]ZHOU Shiyi,HU Wei,DENG Xiaomin,et al.Overexpression of the wheat aquaporin gene,Ta AQP7,e nhances drought tolerance in transgenic tobacco[J].PLo S One,2012,7(12):e52439.doi:10.1371/journal.pone.0052439.
[23]PFAFFL M W.A new mathematical model for relative quantification in real-time RT-PCR[J].Nucleic Acids Res,2001,29(9):2002-2007.
[24]王垠,刘关君,阎秀峰,等.西伯利亚蓼Ps PIP1基因的克隆及其在Na HCO3胁迫下的表达[J].遗传,2008,30(12):1621-1628.WANG Yin,LIU Guanjun,YAN Xiufeng,et al.Cloning of Ps PIP1 gene from Polygonum sibiricum Laxm.and analysis of its expression in response to Na HCO3[J].Hereditas,2008,30(12):1621-1628.
[25]JOHANSSON I,KARLSSON M,JOHANSON U,et al.The role of aquaporins in cellular and whole plant water balance[J].Biochim Biophys Acta Biomembr,2000,1465(1/2):324-342.
[26]HENZLER T,STEUDLE E.Reversible closing of water channels in charainternodes provides evidence for acomposite transport mode of the plasma membrance[J].J Exp Bot,1995,46(2):199-209.
[27]艾雪,褚怀亮,李雪芹,等.山核桃水通道蛋白Cc PIP同源基因克隆与表达分析[J].福建林学院学报,2009,29(3):252-257.AI Xue,CHU Huailiang,LI Xueqin,et al.The analysis of Carya carthayensis plasma membrane intrinsic protein gene Cc PIP clone and expression[J].J Fujian Coll For,2009,29(3):252-257.
[28]林江波,王伟英,邹晖,等.中国水仙水通道蛋白基因c DNA克隆、序列分析与表达[J].福建农业学报,2011,26(3):371-376.LIN Jiangbo,WANG Weiying,ZOU Hui,et al.Cloning,sequence analysis and expression of c DNA encoding aquaporin Nt PIP1 of Narcissus tazetta var.chinensis[J].Fujian J Agric Sci,2011,26(3):371-376.
[29]CHAUMONT F,MOSHELION M,DANIELS M J.Regulation of plant aquaporin activity[J].Biol Cell,2005,97(10):749-764.
[30]LI Liang,WANG Weiqi,HUANG Shan,et al.Cloning and function analysis of a plasma membrane intrinsic protein gene,Gm PIP in soybean(Glycine max L.Merr)[J].Australian J Crop Sci,2014,8(5):738-746.
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