槟榔AcAAP3基因cDNA克隆及其组织表达特性分析
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摘要
【目的】克隆槟榔氨基酸通透酶(AAP3)基因(AcAAP3),分析其生物学信息,并检测其组织表达特性,为探究AAP3在氨基酸转运及氮素利用机制提供理论依据。【方法】利用反转录PCR(RT-PCR)从槟榔品种热研1号中扩增AcAAP3基因cDNA序列,利用生物信息学方法对其编码蛋白的理化性质、亲疏水性、亚细胞定位、功能域及二、三级结构等进行预测分析,并利用实时荧光定量PCR检测AcAAP3基因在槟榔根、叶、茎、雄花和雌花中的表达特异性。【结果】克隆获得AcAAP3基因cDNA序列全长为1440 bp,编码479个氨基酸,蛋白理论分子量为52.834 kD,等电点(pI)为8.76,具有9个跨膜结构域,定位在细胞质膜上,为疏水性非分泌蛋白,具有1个保守的PF01490结构域(氨基酸转运结构域Aa_trans),属于氨基酸转运蛋白家族和APC超家族成员,其二级结构主要由α螺旋(45.72%)和不规则卷曲(34.66%)构成。AcAAP3蛋白氨基酸序列与椰枣(XP008799058.1)、油棕(XP010912574.1)、香蕉(XP009404321.1)和菠萝(XP020107563.1)AAPs蛋白的氨基酸序列相似性分别为91%、90%、84%和83%,说明不同植物的AAPs蛋白具有高度保守性。植物AAPs蛋白家族可分成两个亚类,即单子叶亚类和双子叶亚类,其中,AcAAP3蛋白与单子叶亚类的椰枣(XP008799058.1)和油棕(XP010912574.1)AAPs的亲缘关系较近。AcAAP3基因在槟榔各组织均有表达,表达量依次为根>叶>茎>雄花>雌花。【结论】AcAAP3基因具有明显的组织特异性,其编码蛋白可能在槟榔从外界吸收氨基酸及其体内氨基酸转运中发挥重要作用。
【Objective】Amino acid permeases(AAP3)gene(AcAAP3)was cloned from Areca catechu L.,its basic biological information and expression characteristics was analyzed to provide theoretical basis for further study on exploring the mechanism of amino acids transportation and nitrogen utilization of AAP3.【Method】cDNA sequence of AcAAP3 gene was amplified from A. catechu variety Reyan 1 using reverse transcription PCR(RT-PCR)method. Through bioinformatics analysis,the protein encoded by the gene was initially analyzed for physicochemical properties,hydrophilic/hydrophobic,subcellular localization,conserved domain,secondary and tertiary structures.Expression specificity of Ac AAP3 in root,leaf,stem,male flower and female flower were studied by real-time fluorescent quantitative PCR(qPCR).【Result】The results showed that the cDNA sequence of AcAAP3 gene was 1440 bp in length and encoded 479 amino acids. The theoretical molecular weight of AcAAP3 protein was about 52.834 kD,with a isoelectric point(pI)of 8.76. The encoded protein belonged to the amino acid transporter family and APC super-family with a conserved PF01490 domain(amino acid transporter domain,Aa_trans),which had nine transmembrane domain. AcAAP3 was located in plasma membrane and was a hydrophobic non-secretory protein. Its secondary structure was mainly composed of α helix(45.72%)and random coil(34.66%). The similarity of protein AcAAP3 amino acid sequences with Phoenix dactylifera(XP008799058.1),Elaeis guineensis(XP010912574.1),Musa acuminate(XP009404321.1)and Ananas comosus(XP020107563.1)were 91%,90%,84% and 83%,respectively,indicating that the AAPs protein amino acid sequence was highly conserved. Plant AAPs protein family could be divided into two sub-categories,namely monocotyledonous subclass and dicotyledonous subclass. The relationship between AcAAP3 protein and monocotyledonous subclass plants P. dactylifera(XP008799058.1)and E. guineensis(XP010912574.1)was close. AcAAP3 expressed in different tissues of A. catechu,and presented root>leaf>stem>male flower>female flower.【Conclusion】AcAAP3 gene has obvious tissue specificity expression. Its encoded protein may play a key role in the absorption of external amino acids and transportation of it in A. catechu.
【Objective】Amino acid permeases(AAP3)gene(AcAAP3)was cloned from Areca catechu L.,its basic biological information and expression characteristics was analyzed to provide theoretical basis for further study on exploring the mechanism of amino acids transportation and nitrogen utilization of AAP3.【Method】cDNA sequence of AcAAP3 gene was amplified from A. catechu variety Reyan 1 using reverse transcription PCR(RT-PCR)method. Through bioinformatics analysis,the protein encoded by the gene was initially analyzed for physicochemical properties,hydrophilic/hydrophobic,subcellular localization,conserved domain,secondary and tertiary structures.Expression specificity of Ac AAP3 in root,leaf,stem,male flower and female flower were studied by real-time fluorescent quantitative PCR(qPCR).【Result】The results showed that the cDNA sequence of AcAAP3 gene was 1440 bp in length and encoded 479 amino acids. The theoretical molecular weight of AcAAP3 protein was about 52.834 kD,with a isoelectric point(pI)of 8.76. The encoded protein belonged to the amino acid transporter family and APC super-family with a conserved PF01490 domain(amino acid transporter domain,Aa_trans),which had nine transmembrane domain. AcAAP3 was located in plasma membrane and was a hydrophobic non-secretory protein. Its secondary structure was mainly composed of α helix(45.72%)and random coil(34.66%). The similarity of protein AcAAP3 amino acid sequences with Phoenix dactylifera(XP008799058.1),Elaeis guineensis(XP010912574.1),Musa acuminate(XP009404321.1)and Ananas comosus(XP020107563.1)were 91%,90%,84% and 83%,respectively,indicating that the AAPs protein amino acid sequence was highly conserved. Plant AAPs protein family could be divided into two sub-categories,namely monocotyledonous subclass and dicotyledonous subclass. The relationship between AcAAP3 protein and monocotyledonous subclass plants P. dactylifera(XP008799058.1)and E. guineensis(XP010912574.1)was close. AcAAP3 expressed in different tissues of A. catechu,and presented root>leaf>stem>male flower>female flower.【Conclusion】AcAAP3 gene has obvious tissue specificity expression. Its encoded protein may play a key role in the absorption of external amino acids and transportation of it in A. catechu.
引文
李慧杰,齐家兴,单冬冬,王羽,王爽,崔喜艳.2017.转AAP1基因玉米游离氨基酸与可溶性蛋白质含量变化关系的研究[J].玉米科学,25(1):39-44.[Li H J,Qi J X,Shan D D,Wang Y,Wang S,Cui X Y.2017.Relationship between free amino acid and soluble protein content in transgenic AAP1 maize[J].Journal of Maize Sciences,25(1):39-44.]
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彭波,孔冬艳,黄亚娟,贾洋洋,马晨夕,宋慧,孙艳芳,何璐璐,庞瑞华,宋晓华,李慧龙,柳琳,黄雅琴,周棋赢,李金涛,宋世枝.2018.水稻OsAAP6基因结构与功能的生物信息学分析[J].西南农业学报,31(1):429-436.[Peng B,Kong D Y,Huang Y J,Jia Y Y,Ma C X,Song H,Sun Y F,He L L,Pang R H,Song X H,Li H L,Liu L,Huang Y Q,Zhou Q Y,Li J T,Song S Z.2018.Bioinformatics analysis of Os AAP6 gene structure and function in rice[J].Southwest China Journal of Agricultural Sciences,31(1):429-436.]
宋奇超,曹凤秋,巩元勇,程晓园,毕昕媛,刘来华.2012.高等植物氨基酸吸收与转运及生物学功能的研究进展[J].植物营养与肥料学报,18(6):1507-1517.[Song Q C,Cao F Q,Gong Y Y,Cheng X Y,Bi X Y,Liu L H.2012.Current research progresses of amino acids uptake,transport and their biological roles in higher plants[J].Plant Nutrition and Fertilizer Science,18(6):1507-1517.]
王莹,史振声,王志斌,李凤海.2008.植物对氨基酸的吸收利用及氨基酸在农业中的应用[J].中国土壤与肥料,(1):6-11.[Wang Y,Shi Z S,Wang Z B,Li F H.2008.Absorption and utilization of amino acids by plant and application of amino acids on agriculture[J].Soil and Fertilizer Sciences in China,(1):6-11.]
Amtmann A,Armengaud P.2009.Effects of N,P,K and S on metabolism:New knowledge gained from multilevel analysis[J].Current Opinion in Plant Biology,12(3):275-283.
Hunt E,Gattolin S,Newbury H J,Bale J S,Tseng H M,Barrett D A,Pritchard J.2010.A mutation in amino acid permease AAP6 reduces the amino acid content of the Arabidopsis sieve elements but leaves aphid herbivores unaffected[J].Journal of Experimental Botany,61(1):55-64.
Lee Y H,Foster J J,Voll L M,Weber A P,Tegeder M.2010.AAP1 transports uncharged amino acids into roots of Arabidopsis[J].Plant Journal for Cell&Molecular Biology,50(2):305-319.
Li R H,Jiang X T,Jin D H,Dhaubhadel S,Bian S M,Li XY.2015.Identification of 14-3-3 family in common bean and their response to abiotic stress[J].PLoS One,10(11):e0143280.
Lu K,Wu B W,Wang J,Zhu W,Nie H P,Qian J J,Huang WT,Fang Z M.2018.Blocking amino acid transporter OsAAP3 improves grain yield by promoting outgrowth buds and increasing tiller number in rice[J].Plant Biotechnology Journal,16(10):1710-1722.
Okumoto S,Koch W,Tegeder M,Fischer W N,Biehl A,Leister D,Stierhof Y D,Frommer W B.2004.Root phloemspecific expression of the plasma membrane amino acid proton co-transporter AAP3[J].Journal of Experimental Botany,55(406):2155-2168.
Peng B,Kong H L,Li Y B,Wang L Q,Zhong M,Sun L,Gao G J,Zhang Q L,Luo L J,Wang G W,Xie W B,Chen JX,Yao W,Peng Y,Lei L,Lian X M,Xiao J H,Xu C G,Li X H,He Y Q.2014.OsAAP6 functions as an important regulator of grain protein content and nutritional quality in rice[J].Nature Communications,5(1):5847.
Peng W,Liu Y J,Wu N,Sun T,He X Y,Gao Y X,Wu C J.2015.Areca catechu L.(Arecaceae):A review of its traditional uses,botany,phytochemistry,pharmacology and toxicology[J].Journal of Ethnopharmacology,164:340-356.
Rolletschek H,Hosein F,Miranda M,Heim U,Gotz K P,Schlereth A,Borisjuk L,Saalbach I,Wobus U,Weber H.2005.Ectopic expression of an amino acid transporter(VfAAP1)in seeds of Vicia narbonensis and pea increases storage proteins[J].The Plant Physiology,137(4):1236-1249.
Sanders A,Collier R,Trethewy A,Gould G,Sieker R,Tegeder M.2010.AAP1 regulates import of amino acids into developing Arabidopsis embryos[J].The Plant Journal,59(4):540-552.
Santiago J P,Tegeder M.2016.Connecting source with sink:The role of Arabidopsis AAP8 in phloem loading of amino acids[J].Plant Physiology,171(1):508-521.
Svennerstam H,Ganeteg U,N?sholm T.2008.Root uptake of cationic amino acids by Arabidopsis depends on functional expression of amino acid permease 5[J].New Phytologist,180(3):620-630.
Tan Q M,Aleel K G,Pelissier H C,Rentsch D,Tegeder M.2008.Characterization and expression of French bean amino acid transporter PvAAP1[J].The Plant Science,174(3):348-356.
Tegeder M,Rentsch D.2010.Uptake and partitioning of amino acids and peptides[J].Molecular Plant,3(6):997-1011.
Zhang L Z,Tan Q M,Lee R,Trethewy A,Lee Y H,Tegeder M.2010.Altered xylem-phloem transfer of amino acids affects metabolism and leads to increased seed yield and oil content in Arabidopsis[J].Plant Cell,22(11):3603-3620.
Zhao D Q,Zhou C H,Sheng Y L,Liang G H,Tao J.2011.Molecular cloning and expression of phytoene synthase,lycopene beta-cyclase,and beta-carotene hydroxylase genes in persimmon(Diospyros kaki L.)fruits[J].Plant Molecular Biology Reporter,29(2):345-351.
马清,管超,夏曾润,王茜,王锁民.2015.高等植物氮素转运蛋白研究进展[J].兰州大学学报(自然科学版),51(2):217-227.[Ma Q,Guan C,Xia Z R,Wang Q,Wang S M.2015.Membrane proteins mediating nitrogen transport in higher plants[J].Journal of Lanzhou University(Natural Sciences),51(2):217-227.]
彭波,孔冬艳,黄亚娟,贾洋洋,马晨夕,宋慧,孙艳芳,何璐璐,庞瑞华,宋晓华,李慧龙,柳琳,黄雅琴,周棋赢,李金涛,宋世枝.2018.水稻OsAAP6基因结构与功能的生物信息学分析[J].西南农业学报,31(1):429-436.[Peng B,Kong D Y,Huang Y J,Jia Y Y,Ma C X,Song H,Sun Y F,He L L,Pang R H,Song X H,Li H L,Liu L,Huang Y Q,Zhou Q Y,Li J T,Song S Z.2018.Bioinformatics analysis of Os AAP6 gene structure and function in rice[J].Southwest China Journal of Agricultural Sciences,31(1):429-436.]
宋奇超,曹凤秋,巩元勇,程晓园,毕昕媛,刘来华.2012.高等植物氨基酸吸收与转运及生物学功能的研究进展[J].植物营养与肥料学报,18(6):1507-1517.[Song Q C,Cao F Q,Gong Y Y,Cheng X Y,Bi X Y,Liu L H.2012.Current research progresses of amino acids uptake,transport and their biological roles in higher plants[J].Plant Nutrition and Fertilizer Science,18(6):1507-1517.]
王莹,史振声,王志斌,李凤海.2008.植物对氨基酸的吸收利用及氨基酸在农业中的应用[J].中国土壤与肥料,(1):6-11.[Wang Y,Shi Z S,Wang Z B,Li F H.2008.Absorption and utilization of amino acids by plant and application of amino acids on agriculture[J].Soil and Fertilizer Sciences in China,(1):6-11.]
Amtmann A,Armengaud P.2009.Effects of N,P,K and S on metabolism:New knowledge gained from multilevel analysis[J].Current Opinion in Plant Biology,12(3):275-283.
Hunt E,Gattolin S,Newbury H J,Bale J S,Tseng H M,Barrett D A,Pritchard J.2010.A mutation in amino acid permease AAP6 reduces the amino acid content of the Arabidopsis sieve elements but leaves aphid herbivores unaffected[J].Journal of Experimental Botany,61(1):55-64.
Lee Y H,Foster J J,Voll L M,Weber A P,Tegeder M.2010.AAP1 transports uncharged amino acids into roots of Arabidopsis[J].Plant Journal for Cell&Molecular Biology,50(2):305-319.
Li R H,Jiang X T,Jin D H,Dhaubhadel S,Bian S M,Li XY.2015.Identification of 14-3-3 family in common bean and their response to abiotic stress[J].PLoS One,10(11):e0143280.
Lu K,Wu B W,Wang J,Zhu W,Nie H P,Qian J J,Huang WT,Fang Z M.2018.Blocking amino acid transporter OsAAP3 improves grain yield by promoting outgrowth buds and increasing tiller number in rice[J].Plant Biotechnology Journal,16(10):1710-1722.
Okumoto S,Koch W,Tegeder M,Fischer W N,Biehl A,Leister D,Stierhof Y D,Frommer W B.2004.Root phloemspecific expression of the plasma membrane amino acid proton co-transporter AAP3[J].Journal of Experimental Botany,55(406):2155-2168.
Peng B,Kong H L,Li Y B,Wang L Q,Zhong M,Sun L,Gao G J,Zhang Q L,Luo L J,Wang G W,Xie W B,Chen JX,Yao W,Peng Y,Lei L,Lian X M,Xiao J H,Xu C G,Li X H,He Y Q.2014.OsAAP6 functions as an important regulator of grain protein content and nutritional quality in rice[J].Nature Communications,5(1):5847.
Peng W,Liu Y J,Wu N,Sun T,He X Y,Gao Y X,Wu C J.2015.Areca catechu L.(Arecaceae):A review of its traditional uses,botany,phytochemistry,pharmacology and toxicology[J].Journal of Ethnopharmacology,164:340-356.
Rolletschek H,Hosein F,Miranda M,Heim U,Gotz K P,Schlereth A,Borisjuk L,Saalbach I,Wobus U,Weber H.2005.Ectopic expression of an amino acid transporter(VfAAP1)in seeds of Vicia narbonensis and pea increases storage proteins[J].The Plant Physiology,137(4):1236-1249.
Sanders A,Collier R,Trethewy A,Gould G,Sieker R,Tegeder M.2010.AAP1 regulates import of amino acids into developing Arabidopsis embryos[J].The Plant Journal,59(4):540-552.
Santiago J P,Tegeder M.2016.Connecting source with sink:The role of Arabidopsis AAP8 in phloem loading of amino acids[J].Plant Physiology,171(1):508-521.
Svennerstam H,Ganeteg U,N?sholm T.2008.Root uptake of cationic amino acids by Arabidopsis depends on functional expression of amino acid permease 5[J].New Phytologist,180(3):620-630.
Tan Q M,Aleel K G,Pelissier H C,Rentsch D,Tegeder M.2008.Characterization and expression of French bean amino acid transporter PvAAP1[J].The Plant Science,174(3):348-356.
Tegeder M,Rentsch D.2010.Uptake and partitioning of amino acids and peptides[J].Molecular Plant,3(6):997-1011.
Zhang L Z,Tan Q M,Lee R,Trethewy A,Lee Y H,Tegeder M.2010.Altered xylem-phloem transfer of amino acids affects metabolism and leads to increased seed yield and oil content in Arabidopsis[J].Plant Cell,22(11):3603-3620.
Zhao D Q,Zhou C H,Sheng Y L,Liang G H,Tao J.2011.Molecular cloning and expression of phytoene synthase,lycopene beta-cyclase,and beta-carotene hydroxylase genes in persimmon(Diospyros kaki L.)fruits[J].Plant Molecular Biology Reporter,29(2):345-351.