杜梨干旱胁迫相关水通道蛋白基因的筛选与克隆
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摘要
以杜梨实生苗为材料,采用15%PEG 6000进行模拟干旱处理,利用qRTPCR、同源重组、亚细胞定位等方法,研究了不同水通道蛋白基因在15%PEG 6000胁迫条件下表达水平的变化趋势,筛选了响应干旱胁迫相关的水通道蛋白基因,并对其进行克隆和分析,以期为梨抗旱基因筛选提供参考依据。结果表明:AQPs在杜梨的根、茎、叶中均有表达,且随着胁迫时间的延长,大部分AQPs在根系中的表达水平呈先上升后下降的趋势,其中PIP2;2基因在胁迫处理3h后,相对表达量最高;从杜梨根系中克隆获得PbPIP2;2,该基因包含一个完整的846bp的开放阅读框,编码281个氨基酸。氨基酸序列比对分析结果表明,PbPIP2;2与其它近缘物种具有一致的保守功能域,其中与苹果MdPIP2;2、白梨PcPIP2;2相似度最高。洋葱表皮亚细胞定位发现PbPIP2;2基因定位在质膜上。
The seedlings of Pyrus betulifolia Bunge were used as materials,drought treatment was simulated by adding 15% PEG 6000 in the nutrient solution.The changes of expression levels of different aquaporin genes under 15% PEG6000 stress were studied by qRT-PCR,homologous recombination and subcellular localization methods,in order to provide reference for selection of drought resistant genes in pear.The results showed that multiple aquaporin genes were expressed in root,stem and leaf,and the expression levels of most of the aquaporin genes in root increased and then declined with the prolongation of stress time.Among them,PIP2;2 gene had the highest relative expression level after 3hours stress treatment.Then PIP2;2 gene was cloned from root of Pyrus betulifolia Bunge and the gene contained a complete open reading frame of 846 bp encoding 281 amino acids.The alignment of the amino acid sequence showed that PbPIP2;2 had a highly conserved functional domain similar to other related species,with the highest similarity with MdPIP2;2 and PcPIP2;2.By onion epidermis subcellular positioning analysis,we observed that the protein encoded by PbPIP2;2 was localized on the plasma membrane.
The seedlings of Pyrus betulifolia Bunge were used as materials,drought treatment was simulated by adding 15% PEG 6000 in the nutrient solution.The changes of expression levels of different aquaporin genes under 15% PEG6000 stress were studied by qRT-PCR,homologous recombination and subcellular localization methods,in order to provide reference for selection of drought resistant genes in pear.The results showed that multiple aquaporin genes were expressed in root,stem and leaf,and the expression levels of most of the aquaporin genes in root increased and then declined with the prolongation of stress time.Among them,PIP2;2 gene had the highest relative expression level after 3hours stress treatment.Then PIP2;2 gene was cloned from root of Pyrus betulifolia Bunge and the gene contained a complete open reading frame of 846 bp encoding 281 amino acids.The alignment of the amino acid sequence showed that PbPIP2;2 had a highly conserved functional domain similar to other related species,with the highest similarity with MdPIP2;2 and PcPIP2;2.By onion epidermis subcellular positioning analysis,we observed that the protein encoded by PbPIP2;2 was localized on the plasma membrane.
引文
[1]孙继亮.土壤水分对梨树叶片光合生理特性及果实品质的影响[D].南京:南京农业大学,2011.
[2]赵昌杰,刘松忠,张强.果树对干旱胁迫的响应研究进展[J].中国果树,2011(4):60-62.
[3]吴雪,杜长霞,杨冰冰,等.植物水通道蛋白研究综述[J].浙江农林大学学报,2015,32(5):789-796.
[4]徐娜.番茄SlMIP基因的克隆与耐盐性分析[D].北京:中国农业科学院,2013.
[5] GRONDIN A,MAULEON R,VADEZ V,et al.Root aquaporins contribute to whole plant water fluxes under drought stress in rice(Oryza sativa L.)[J].Plant Cell&Environment,2016,39(2):347-365.
[6]李仁.番茄水通道蛋白在干旱胁迫及果实成熟过程中的功能分析[D].北京:中国农业大学,2015.
[7] LI J,BAN L,WEN H,et al.An aquaporin protein is associated with drought stress tolerance[J].Biochemical&Biophysical Research Communications,2015,459(2):208-213.
[8]罗庆.枳水通道蛋白PIP和TIP基因家族成员的克隆及其在硼胁迫下的表达分析[D].武汉:华中农业大学,2015.
[9]杨乐.桃水通道蛋白编码基因PpTIP4.1超表达与不同形态氮素配合使用对尿素吸收的影响[D].青岛:山东农业大学,2012.
[10]PENG Y,LIN W,CAI W,et al.Overexpression of a Panax ginsengtonoplast aquaporin alters salt tolerance,drought tolerance and cold acclimation ability in transgenic Arabidopsis plants[J].Planta,2007,226(3):729-740.
[11]MAHDIEH M,MOSTAJERAN A,HORIE T,et al.Drought stress alters water relations and expression of PIP-type aquaporin genes in Nicotiana tabacum plants[J].Plant&Cell Physiology,2008,49(5):801-813.
[12]DING L,GAO C,LI Y,et al.The enhanced drought tolerance of rice plants under ammonium is related to aquaporin(AQP)[J].Plant Science,2015,234:14-21.
[13]HEINEN R B,BIENERT G P,COHEN D,et al.Expression and characterization of plasma membrane aquaporins in stomatal complexes of Zea mays[J].Plant Molecular Biology,2014,86(3):335-350.
[14]ZHAO H B,ZHANG Z B,PING X.Enhanced aquaporin activity of two different genotypes of drought-resistant wheat(Triticum aestivum L.)cultivars facilitate their adaptation to drought stress[J].Journal of Food Agriculture&Environment,2010,8(2):1158-1161.
[15]何勇清,方佳,余敏芬,等.植物质膜内在水通道蛋白PIPs的分子生物学研究进展[J].浙江农林大学学报,2012,29(3):446-452.
[16]冉昆,孙晓莉,张勇,等.杜梨质膜水孔蛋白基因PbPIP1的克隆与表达分析[J].植物生理学报,2016(6):868-876.
[17]宗宇,孙萍,牛庆丰,等.中国北方野生杜梨分布现状及其形态多样性评价[J].果树学报,2013(6):918-923.
[18]董星光,曹玉芬,王昆,等.中国3个主要梨砧木资源木质部导管分子结构及分布比较[J].植物学报,2015,50(2):227-233.
[19]余歆.水孔蛋白与植物对干旱和低温胁迫响应关系的研究[D].北京:中国科学院,2004.
[20]颜培玲.野生毛葡萄质膜水通道蛋白基因的克隆与功能验证[D].贵阳:贵州大学,2015.
[21]颜培玲,潘学军,张文娥.野生毛葡萄水通道蛋白基因VhPIP1的克隆及其在干旱胁迫下的表达分析[J].园艺学报,2015,42(2):221-232.
[2]赵昌杰,刘松忠,张强.果树对干旱胁迫的响应研究进展[J].中国果树,2011(4):60-62.
[3]吴雪,杜长霞,杨冰冰,等.植物水通道蛋白研究综述[J].浙江农林大学学报,2015,32(5):789-796.
[4]徐娜.番茄SlMIP基因的克隆与耐盐性分析[D].北京:中国农业科学院,2013.
[5] GRONDIN A,MAULEON R,VADEZ V,et al.Root aquaporins contribute to whole plant water fluxes under drought stress in rice(Oryza sativa L.)[J].Plant Cell&Environment,2016,39(2):347-365.
[6]李仁.番茄水通道蛋白在干旱胁迫及果实成熟过程中的功能分析[D].北京:中国农业大学,2015.
[7] LI J,BAN L,WEN H,et al.An aquaporin protein is associated with drought stress tolerance[J].Biochemical&Biophysical Research Communications,2015,459(2):208-213.
[8]罗庆.枳水通道蛋白PIP和TIP基因家族成员的克隆及其在硼胁迫下的表达分析[D].武汉:华中农业大学,2015.
[9]杨乐.桃水通道蛋白编码基因PpTIP4.1超表达与不同形态氮素配合使用对尿素吸收的影响[D].青岛:山东农业大学,2012.
[10]PENG Y,LIN W,CAI W,et al.Overexpression of a Panax ginsengtonoplast aquaporin alters salt tolerance,drought tolerance and cold acclimation ability in transgenic Arabidopsis plants[J].Planta,2007,226(3):729-740.
[11]MAHDIEH M,MOSTAJERAN A,HORIE T,et al.Drought stress alters water relations and expression of PIP-type aquaporin genes in Nicotiana tabacum plants[J].Plant&Cell Physiology,2008,49(5):801-813.
[12]DING L,GAO C,LI Y,et al.The enhanced drought tolerance of rice plants under ammonium is related to aquaporin(AQP)[J].Plant Science,2015,234:14-21.
[13]HEINEN R B,BIENERT G P,COHEN D,et al.Expression and characterization of plasma membrane aquaporins in stomatal complexes of Zea mays[J].Plant Molecular Biology,2014,86(3):335-350.
[14]ZHAO H B,ZHANG Z B,PING X.Enhanced aquaporin activity of two different genotypes of drought-resistant wheat(Triticum aestivum L.)cultivars facilitate their adaptation to drought stress[J].Journal of Food Agriculture&Environment,2010,8(2):1158-1161.
[15]何勇清,方佳,余敏芬,等.植物质膜内在水通道蛋白PIPs的分子生物学研究进展[J].浙江农林大学学报,2012,29(3):446-452.
[16]冉昆,孙晓莉,张勇,等.杜梨质膜水孔蛋白基因PbPIP1的克隆与表达分析[J].植物生理学报,2016(6):868-876.
[17]宗宇,孙萍,牛庆丰,等.中国北方野生杜梨分布现状及其形态多样性评价[J].果树学报,2013(6):918-923.
[18]董星光,曹玉芬,王昆,等.中国3个主要梨砧木资源木质部导管分子结构及分布比较[J].植物学报,2015,50(2):227-233.
[19]余歆.水孔蛋白与植物对干旱和低温胁迫响应关系的研究[D].北京:中国科学院,2004.
[20]颜培玲.野生毛葡萄质膜水通道蛋白基因的克隆与功能验证[D].贵阳:贵州大学,2015.
[21]颜培玲,潘学军,张文娥.野生毛葡萄水通道蛋白基因VhPIP1的克隆及其在干旱胁迫下的表达分析[J].园艺学报,2015,42(2):221-232.