枇杷果实EjNADP-ME2基因及启动子克隆与表达分析
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摘要
以‘解放钟’和‘白梨’果实为材料,研究基于转录组筛选出的表达量较高且在2个品种中有差异的EjNADP-ME2(Unigene0001461)基因,采用RACE和RT-PCR技术成功克隆EjNADP-ME2的cDNA全长。系统进化树分析表明,EjNADP-ME2与苹果的NADP-ME基因关系最近。生物信息学预测结果显示,EjNADP-ME2蛋白应该位于细胞质中。用染色体步移法成功克隆了EjNADP-ME2的启动子序列,序列预测该基因启动子区域含有水杨酸、茉莉酸、脱落酸响应的顺式作用元件,此外还有厌氧诱导元件、MYB和MYC参与黄化和干旱诱导的结合位点MBS。荧光定量PCR分析表明,EjNADP-ME2基因的表达量变化趋势在2个品种中大致相同,均呈现出先降低后升高又降低的趋势,且该基因在低酸品种的表达量稍低于高酸品种。相关性分析结果显示,低酸品种EjNADP-ME2基因转录水平与苹果酸含量呈显著负相关。本研究的结果为进一步探究EjNADP-ME2基因在苹果酸代谢途径中的功能奠定坚实基础。
Fruits of a high-acid cultivar 'Jiefangzhong' and a low-acid cultivar ‘Baili' were used as the materials to study the EjNADP-ME2(Unigene0001461) gene whose expression level was relatively high yet different between the two cultivars as shown by transcriptomic data. Its full-length cDNA was successfully cloned by RACE and RT-PCR. Phylogenetic analysis showed that EjNADP-ME2 was closely related to the NADP-ME gene of apples. Bioinformatics analysis predicted that the EjNADP-ME2 protein should be located in the cytoplasm. The promoter of the gene was successfully cloned by chromosome walking and predicted that contained cis-acting elements responsive to salicylic acid, jasmonic acid, abscisic acid, anaerobic induction, and sites for MYB, MYC and MBS(yellowing and drought induction) binding. Real-time PCR analysis showed that the trends were similar in the expression of the gene between the two cultivars, showing a lower, higher and lower pattern at the earlier, middle and later stages of the fruit development, respectively. Correlation analysis suggested that EjNADP-ME2 expression was negatively correlated to malic acid content in the fruit of the low acid cultivar 'Baili', but not such in the other cultivar. The results of this study would provide starting materials for further exploration of the function of the EjNADP-ME2 gene in the metabolism of malic acid in the fruit of loquat.
Fruits of a high-acid cultivar 'Jiefangzhong' and a low-acid cultivar ‘Baili' were used as the materials to study the EjNADP-ME2(Unigene0001461) gene whose expression level was relatively high yet different between the two cultivars as shown by transcriptomic data. Its full-length cDNA was successfully cloned by RACE and RT-PCR. Phylogenetic analysis showed that EjNADP-ME2 was closely related to the NADP-ME gene of apples. Bioinformatics analysis predicted that the EjNADP-ME2 protein should be located in the cytoplasm. The promoter of the gene was successfully cloned by chromosome walking and predicted that contained cis-acting elements responsive to salicylic acid, jasmonic acid, abscisic acid, anaerobic induction, and sites for MYB, MYC and MBS(yellowing and drought induction) binding. Real-time PCR analysis showed that the trends were similar in the expression of the gene between the two cultivars, showing a lower, higher and lower pattern at the earlier, middle and later stages of the fruit development, respectively. Correlation analysis suggested that EjNADP-ME2 expression was negatively correlated to malic acid content in the fruit of the low acid cultivar 'Baili', but not such in the other cultivar. The results of this study would provide starting materials for further exploration of the function of the EjNADP-ME2 gene in the metabolism of malic acid in the fruit of loquat.
引文
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[2]陈发兴,刘星辉,陈立松.枇杷果肉有机酸组分及有机酸在果实内的分布[J].热带亚热带植物学报,2008,16(3):236-243.
[3]Etienne A,Génard M,Lobit P,et al.What controls fleshy fruit acidity?A review of malate and citrate accumulation in fruit cells[J].Journal of Experimental Botany,2013,64(6):1451-1469.
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[7]Laporte M M,Shen B,Tarczynski M C.Engineering for drought avoidance:expression of maize NADP-malic enzyme in tobacco results in altered stomatal function[J].Journal of Experimental Botany,2002,53(369):699-705.
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[11]Zhang Y,Li P,Cheng L.Developmental changes of carbohydrates,organic acids,amino acids,and phenolic compounds in‘Honeycrisp’apple flesh[J].Food Chemistry,2010,123(4):1013-1018.
[12]Saradhuldhat P,Paull R E.Pineapple organic acid metabolism and accumulation during fruit development[J].Scientia Horticulturae,2007,112(3):297-303.
[13]Iwakura M,Hattori J,Arita Y,et al.Studies on regulatory functions of malic enzymes[J].Biochemical Journal(Tokyo),1979,85:1355-1365.
[14]Chi W,Yang J,Wu N,et al.Four rice genes encoding NADPmalic enzyme exhibit distinct expression profiles[J].Bioscience,Biotechnology,and Biochemistry,2004,68:1865-1874.
[15]李明,姚玉新,刘志,等.苹果果实中细胞质型苹果酸酶基因(NADP-ME)的克隆与表达分析[J].中国农学通报,2007,23(7):95-100.
[16]Chen F X,Liu X H,Chen L S.Ethychlozate reduces acidity of loquat(Eriobotrya japonica)fruit[J].Scientia Horticulturae,2010,124(3):331-337.
[17]Chen F X,Liu X H,Chen L S.Developmental changes in pulp organic acid concentration and activities of acid-metabolising enzymes during the fruit development of two loquat(Eriobotrya japonica Lindl.)cultivars differing in fruit acidity[J].Food Chemistry,2009,114(2):657-664.
[18]Tao P,Guo W,Li B,et al.Genome-wide identification,classification,and analysis of NADP-ME family members from12 crucifer species[J].Molecular Genetics&Genomics,2016,291(3):1167-1180.
[19]Wheeler M C,Tronconi M A,Drincovich M F,et al.Acomprehensive analysis of the NADP-malic enzyme gene family of Arabidopsis[J].Plant Physiology,2005,139(1):39-51.
[20]Yao Y X,Li M,Liu Z,et al.Molecular cloning of three malic acid related genes Md PEPC,Md VHA-A,Mdcy ME,and their expression analysis in apple fruits[J].Scientia Horticulturae,2009,122(3):404-408.
[21]Etienne A,Moing A,Dirlewanger E,et al.Isolation and characterization of six peach c DNAs encoding key proteins in organic acid metabolism and solute accumulation:involvement in regulating peach fruit acidity[J].Physiologa Plantarum,2002,114:259-270.
[22]Mu X,Wang P,Du J,et al.Comparison of fruit organic acids and metabolism-related gene expression between Cerasus humilis(Bge.)Sok and Cerasus glandulosa(Thunb.)Lois[J].PLoS ONE,2018,13(4):e0196537.
[23]Yang L T,Chen L S.Expression of six malate-related genes in pulp during the fruit development of two loquat(Eriobotrya japonica)cultivars differing in fruit acidity[J].African Journal of Biotechnology,2011,4314:1684-5315.
[2]陈发兴,刘星辉,陈立松.枇杷果肉有机酸组分及有机酸在果实内的分布[J].热带亚热带植物学报,2008,16(3):236-243.
[3]Etienne A,Génard M,Lobit P,et al.What controls fleshy fruit acidity?A review of malate and citrate accumulation in fruit cells[J].Journal of Experimental Botany,2013,64(6):1451-1469.
[4]陈发兴,刘星辉,林华影,等.离子交换色谱法测定枇杷果实和叶片中的有机酸[J].福建农林大学学报(自然科学版),2004,33(2):195-199.
[5]Edwards G E,Andreo C S.NADP-malic enzyme from plants[J].Phytochemistry,1992,31(6):1845-1857.
[6]Chi W,Yang J,Wu N,et al.Four rice genes encoding NADPmalic enzyme exhibit distinct expression profiles[J].Bioscience,Biotechnology,and Biochemistry,2004,68(9):1865-1874.
[7]Laporte M M,Shen B,Tarczynski M C.Engineering for drought avoidance:expression of maize NADP-malic enzyme in tobacco results in altered stomatal function[J].Journal of Experimental Botany,2002,53(369):699-705.
[8]van Doorsseleare J,Villaroel R,von Montagu M,et al.Nucleotide sequence of a c DNA encoding malic enzyme from poplar[J].Plant Physiology,1991,96:1385-1386.
[9]Yoshida M.Genétical studies on the fruit quality of peach varieties.I.Acidity[J].Bulletin of the Horticultural Research Station(in Japanese),1970(A9):1-15.
[10]Franke K E,Adams D O.Cloning a full-length c DNA for malic enzyme(EC 1.1.1.40)from grape berries[J].Plant Physiology,1995,107:1009-1010.
[11]Zhang Y,Li P,Cheng L.Developmental changes of carbohydrates,organic acids,amino acids,and phenolic compounds in‘Honeycrisp’apple flesh[J].Food Chemistry,2010,123(4):1013-1018.
[12]Saradhuldhat P,Paull R E.Pineapple organic acid metabolism and accumulation during fruit development[J].Scientia Horticulturae,2007,112(3):297-303.
[13]Iwakura M,Hattori J,Arita Y,et al.Studies on regulatory functions of malic enzymes[J].Biochemical Journal(Tokyo),1979,85:1355-1365.
[14]Chi W,Yang J,Wu N,et al.Four rice genes encoding NADPmalic enzyme exhibit distinct expression profiles[J].Bioscience,Biotechnology,and Biochemistry,2004,68:1865-1874.
[15]李明,姚玉新,刘志,等.苹果果实中细胞质型苹果酸酶基因(NADP-ME)的克隆与表达分析[J].中国农学通报,2007,23(7):95-100.
[16]Chen F X,Liu X H,Chen L S.Ethychlozate reduces acidity of loquat(Eriobotrya japonica)fruit[J].Scientia Horticulturae,2010,124(3):331-337.
[17]Chen F X,Liu X H,Chen L S.Developmental changes in pulp organic acid concentration and activities of acid-metabolising enzymes during the fruit development of two loquat(Eriobotrya japonica Lindl.)cultivars differing in fruit acidity[J].Food Chemistry,2009,114(2):657-664.
[18]Tao P,Guo W,Li B,et al.Genome-wide identification,classification,and analysis of NADP-ME family members from12 crucifer species[J].Molecular Genetics&Genomics,2016,291(3):1167-1180.
[19]Wheeler M C,Tronconi M A,Drincovich M F,et al.Acomprehensive analysis of the NADP-malic enzyme gene family of Arabidopsis[J].Plant Physiology,2005,139(1):39-51.
[20]Yao Y X,Li M,Liu Z,et al.Molecular cloning of three malic acid related genes Md PEPC,Md VHA-A,Mdcy ME,and their expression analysis in apple fruits[J].Scientia Horticulturae,2009,122(3):404-408.
[21]Etienne A,Moing A,Dirlewanger E,et al.Isolation and characterization of six peach c DNAs encoding key proteins in organic acid metabolism and solute accumulation:involvement in regulating peach fruit acidity[J].Physiologa Plantarum,2002,114:259-270.
[22]Mu X,Wang P,Du J,et al.Comparison of fruit organic acids and metabolism-related gene expression between Cerasus humilis(Bge.)Sok and Cerasus glandulosa(Thunb.)Lois[J].PLoS ONE,2018,13(4):e0196537.
[23]Yang L T,Chen L S.Expression of six malate-related genes in pulp during the fruit development of two loquat(Eriobotrya japonica)cultivars differing in fruit acidity[J].African Journal of Biotechnology,2011,4314:1684-5315.