王影;李慧;蔺经;杨青松;张绍铃;常有宏;

• 1:南京农业大学园艺学院
• 2:江苏省农业科学院果树研究所
• 3:江苏省高效园艺作物遗传改良重点实验室

摘要(Abstract)：

【目的】NHX基因亚家族为钠氢逆转运体,具有Na+/H+exchange(PF00999)蛋白结构域,广泛存在于多种物种中,主要参与植物响应盐胁迫过程离子平衡的重建。分析鉴定杜梨中NHX基因及其耐盐机制有助于实际生产中更为有效地利用这一砧木资源。【方法】结合已公布的梨基因组数据以及拟南芥相关数据,通过生物信息学手段,鉴定杜梨NHX基因家族成员;通过MEGA7.0软件进行序列比对以及进化分析;通过在线工具Pfam、SMART以及GSDS进行基因结构分析;通过定量PCR技术分析非生物胁迫下基因在不同组织中的表达特征;应用火焰石墨炉原子吸收光谱仪测定NaCl胁迫下杜梨不同组织中Na+与K+含量。【结果】成功鉴定出16个候选基因,其中有4个基因属于NHX亚家族,4个基因属于CHX亚家族,8个基因属于KEA亚家族。预测的候选基因均含有Na+/H+exchange功能域;NaCl胁迫下,预测的16个基因在叶片中的表达差异大,但在8 h以后均为负调控;在茎中,PEG6000处理下,PbNHX1的表达量显著增高,且高于同一时期NaCl胁迫下的表达水平,仅在48 h时低于同一时期NaCl胁迫下的表达水平;在根中,基因PbNHX12、PbNHX13、PbNHX14和PbNHX15在NaCl胁迫和PEG6000处理下在任何时间段均为负调控。【结论】在盐胁迫过程中,与拟南芥NHX基因进化关系最近的PbNHX1和PbNHX11呈现出负调控模式,与拟南芥KEA基因进化关系相近的PbNHX10和PbNHX7可能参与K+的运输。

关键词(KeyWords)： 杜梨;NHX基因家族;非生物胁迫;实时定量PCR

Abstract:

Keywords:

基金项目(Foundation): 国家自然科学基金(31372051,31772287);; 江苏省自然科学基金(BK20151361)

作者(Author): 王影;李慧;蔺经;杨青松;张绍铃;常有宏;

Email:

DOI: 10.13925/j.cnki.gsxb.20180347

参考文献(References)：

• [1] ALLAKHVERDIEV S I,SAKAMOTO A,NISHIYAMA Y,INABA M,MURATA N. Ionic and osmotic effects of NaCl-induced inactivation of photosystemsⅠandⅡin synechococcus sp[J]. Plant Physiology,2000,123(3):1047-1056.
• [2] KOVD A,VICTOR A. Loss of productive land due to salinization[J]. Ambio,1982,12(2):91-93.
• [3] FLOWERS T,TROKE P,YEO A. The mechanism of salt tolerance in halophytes[J]. Annual Review of Plant Physiology,1977,28(1):89-121.
• [4] WANG W,VINOCUR B,ALTMAN A. Plant responses to drought,salinity and extreme temperatures:towards genetic engineering for stress tolerance[J]. Planta,2003,218(1):3-6.
• [5]刘永富.梨砧木杜梨和BA29组培苗生根移栽体系的研究[D].保定:河北农业大学,2010.LIU Yongfu. Study on rooting and transplanting system of tissue culture seedlings of pear rootstock[D]. Baoding:Hebei Agricultural University,2010.
• [6]胡涛,张鸽香,郑福超,曹钰.植物盐胁迫响应的研究进展[J].分子植物育种,2018,16(9):3006-3015.HU Tao,ZHANG Gexiang,ZHENG Fuchao,CAO Yu. Advances in plant salt stress response[J]. Molecular Plant Breeding,2018,16(9):3006-3015.
• [7]谷俊,耿贵,李冬雪,於丽华.盐胁迫对植物各营养器官形态结构影响的研究进展[J].中国农学通报,2017,33(24):62-67.GU Jun,GENG Gui,LI Dongxue,YU Lihua. Research progress on the effect of salt stress on the morphological structure of various vegetative organs[J]. China agriculture Bulletin,2017,33(24):62-67.
• [8] TATTINI M,GUCCI R,CORADESCHI M A,PONZIIO C,EVERARD J D. Growth,gas exchange and ioncontent in olea europaea plants during salinity stress and subsequent relief[J].Physiologia Plantarum,1995,95(2):203-210.
• [9]常红军,秦毓茜.植物的盐胁迫生理[J].安阳师范学院学报,2006,40(5):149-152.CHANG Hongjun,QIN Yuqian. The salt stress physiology of plants[J]. Journal of Anyang Normal University,2006,40(5):149-152.
• [10]李彦,张英鹏,孙明.盐分胁迫对植物的影响及植物耐盐机理研究进展[J].中国农学通报,2008,24(1):258-265.LI Yan,ZHANG Yingpeng,SUN Ming. Research progress on the effect of salt stress on plants and the mechanism of salt tolerance in plants[J]. Chinese Agronomy Bulletin,2008,24(1):258-265.
• [11]曾红学,王俊.盐害生理及植物抗盐性[J].生物学通报,2006,40(1):1-3.ZENG Hongxue,WANG Jun. Salt stress physiology and salt resistance of plant physiology[J]. Bulletin of Biology,2006,40(1):1-3.
• [12] HAUSER F,HORIE T. A conserved primary salt tolerance mechanism mediated by HKT transporters:a mechanism for sodium exclusion and maintenance of high K+/Na+radio in leaves during salinity stress[J]. Plant,Cell&Environment,2010,33(4):552-565.
• [13] PARDO J M,CUBERO B,LEIDI E O,QUINTERO F J. Alkali cation exchangers:roles in cellular homeostasis and stress tolerance[J]. Journal of Experimental Botany,2006,57(5):1181-1199.
• [14] GORHAM J,JONES R G W,MCDONNELL E. Some mechanisms of salt tolerances in crop plants[J]. Plant Soil,1985,89(1/3):15-40.
• [15] BLUMWALD E,POOLE R J. Na+/H+antiport in isolated tonoplast vesicles from storage tissue of beta vulgaris[J]. Plant Physiology,1985,78(1):163-167.
• [16] APSE M P,AHARON G S,SNEDDEN W A,BLUMWALD E.Slat tolerance coferred by overexpression of a vacuolar Na+/H+antiport in arabidopsis[J]. Science,1999,285(5431):1256-1258.
• [17] SZE H,LI X,PALMGREN M G. Energization of plant cell membrances by H+-pumping ATPases regulation and biosynthesis[J]. The Plant Cell,1999,11(4):677-690.
• [18] SHI H,LEE B H,WU S J,ZHU J K. Overexpression of plant a plasma membrane Na+/H+antiporter gene improves salt tolerance in Arabidopsis thaliana[J]. Nature Biotechnology,2003,21(1):81-85.
• [19] ZHOU H S,QI K J,LIU X. Genome-wide identification and comparative analysis of the cation proton antiporters family in pear and four other rosaceae species[J]. Molecular Genetics and Genomics,2016,291(4):1727-1742.
• [20] 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(3):409-420.
• [21]邵红雨,孔广超,齐军仓,任丽彤,王瑞清,曹连莆.植物耐盐生理生化特性的研究发展[J].安徽农学通报,2006,12(9):51-53.SHAO Hongyu,KONG Guangchao,QI Juncang,REN Litong,WANG Ruiqing,CAO Lianpu. Research and development of physiological and biochemical characteristics under salt tolerant of plants[J]. Anhui Agricultural Science Bulletin,2006,12(9):51-53.
• [22] CHANG S,PURYEAR J,CAIRNEY J. A simple and efficient method for isolation RNA from pine trees[J]. Plant Molecular,2002,17(8):844-851.
• [23] GONZALEZ-MENDOZA D,QUIROZ M A,ZAPATA-PEREZ O. An improved method for the isolationof total RNA from avicennia germinans leaves[J]. Zeitschrift fur Naturforschung C,A Journal of Biosciences,2008,63(1/2):124-126.
• [24]马媛春.葡萄CPA1基因家族注释和功能分化及植物CPA1基因家族过表达的META分析[D].南京:南京农业大学,2014.MA Yuanchun. CPA1 gene family annotation and functional differentiation and META analysis of CPA1 gene family overexpression[D]. Nanjing:Nanjing Agricultural University,2014.
• [25] BASSIL E,TAJIMA H,LIANG Y C,OHTO M A,USHIJIMA K,NAKANO R,ESUMI T,COKU A,BELMONTE M,BLUMWALD E. The arabidopsis Na+/H+antiporters NHX1 and NHX2control vacuolar pH and K+homeostasis to regulate growth,flower development and reproduction[J]. The Plant Cell,2011,23:3482-3497.
• [26] CHOE S. Potassium channel structures[J]. Nature Reviews Neuroscience,2002,3(1):115-121.
• [27]郑晟.拟南芥K+/H+反向转运体AtKEA基因家族的生物学功能研究[D].兰州:甘肃农业大学,2014.ZHENG Sheng. Study on the biological function of the AtKEA gene family of K+/H+reverse transporter in Arabidopsis[D]. Lanzhou:Gansu Agricultural University,2014.
• [28] HARRIS C,FLIEGE L. Amiloride and the Na+/H+exchanger protein:mechanism and significance of innhibition of the Na+/H+exchanger(review)[J]. International Journal of Molecular Medicine,1999,3(3):315-336.
• [29] YUN C H,LITTLE P J,NATH S K,LEVINE S A,POUYSSEGUR J,TSE C M,DONOWITZ M. Leu143 in the putative fourth membrane spanning domain is critical for amiloride inhibition of an epithelial Na+/H+exchanger isoform(NHE-2)[J]. Biochemical and Biophysical Research Communications,1993,193(2):532-539.
• [30] BARRAGAN V,LEIDI E O,ANDRES Z,RUBIO L,DE LUCA A,FERNANDEZ J A,CUBERO B,PARDO J M. Ion exchangers NHX1 and NHX2 mediate active potassium uptake into vacuoles to regulate cell turgor and stomatal function in Arabidopsis[J]. The Plant Cell,2012,24:1127-1142.
• [31] BASSIL E,HIROMI T,LIANG Y C,OHTO M A,USHIJIMA K,NAHANO R,ESUMI T,COKU A,BELMOMTE M,BLUMWALD E. The arabidopsis Na+/H+antiporters NHX1 and NHX2control vacuolar pH and K+homeostasis to regulate growth,flower development,and reproduction[J]. The Plant Cell,2011,23(2):3482-3497.
• [32] ZHAO J,CHENG N H,MOTES C M,BLANCAFLOR E B,MOORE M,GONZALES N,PADMANABAN S,SZE H,WARD J M,HIRSCHI K D. AtCHX13 is a plasma membrane K^sup+^transporter[J]. Plant Physiology,2008,148(2):796-807.
• [33] LU Y X,CHANROJ S,ZULKIFLI L,JOHNSON M A,UOZUMI J N,CHEUNG A,SZE H. Pollen tubes lacking a pair of K+transporters fail to target ovules in arabidopsis[J]. The Plant Cell,2011,23(1):81-93.