茶树体胚APX基因克隆及其在体胚发生过程中的表达与酶活性分析
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摘要
以铁观音茶树体胚为材料,克隆了茶树抗坏血酸过氧化物酶基因CsAPX(Genbank登录号为MG799534.1)。CsAPX基因包含一个长度为753 bp的开放阅读框(ORF),共编码250个氨基酸,并对其进行相关生物信息学分析。APX酶活性测定结果表明在球形胚至子叶胚阶段APX酶活性呈现下降趋势,并在子叶胚阶段到达最低值,随着体胚进一步的分化,在子叶胚、成熟胚和萌发胚阶段,茶树APX酶活性不断上升,总体呈"先降后升"的趋势。在体胚发生过程中CsAPX基因的表达分析显示,CsAPX基因在茶树体胚发生的5个阶段均存在显著差异表达,总体呈"升—降—升"的趋势,其中在萌发胚阶段的CsAPX基因的表达水平显著高于其他4个阶段,推测CsAPX基因在体胚发生过程中的萌发胚阶段发挥着重要的作用。
The ascorbate peroxidase gene CsAPX(GenBank accession number: MG799534.1) was cloned from the somatic embryo of Camellia sinensis cultivar ‘Tieguanyin'. The CsAPX gene contained an open reading frame(ORF) of753 bp in length, which would encode a total of 250 amino acids, and bioinformatics analysis about CsAPX gene was also employed. The total APX activity was measured during the whole somatic embryogenesis and the results showed that the total APX enzyme activity presented a downward trend in the globular embryo to cotyledonary embryo stage,and reached the lowest value in the cotyledon embryo stage. With the further differentiation of the embryo cells, the total APX enzyme activity increased continuously during the cotyledonary embryo stage, the embryo maturation stage and the germinating stage, and showed an overall trend of "decreasing first and then rising". The expression of CsAPX was significantly different between the 5 stages of the somatic embryogenesis, showing an overall trend of "up-downup" with a significantly higher expression level in the germinating stage than in the other 4 embryo stages. It was speculated that CsAPX played an important role in the germinating stage.
The ascorbate peroxidase gene CsAPX(GenBank accession number: MG799534.1) was cloned from the somatic embryo of Camellia sinensis cultivar ‘Tieguanyin'. The CsAPX gene contained an open reading frame(ORF) of753 bp in length, which would encode a total of 250 amino acids, and bioinformatics analysis about CsAPX gene was also employed. The total APX activity was measured during the whole somatic embryogenesis and the results showed that the total APX enzyme activity presented a downward trend in the globular embryo to cotyledonary embryo stage,and reached the lowest value in the cotyledon embryo stage. With the further differentiation of the embryo cells, the total APX enzyme activity increased continuously during the cotyledonary embryo stage, the embryo maturation stage and the germinating stage, and showed an overall trend of "decreasing first and then rising". The expression of CsAPX was significantly different between the 5 stages of the somatic embryogenesis, showing an overall trend of "up-downup" with a significantly higher expression level in the germinating stage than in the other 4 embryo stages. It was speculated that CsAPX played an important role in the germinating stage.
引文
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[19]赵姗姗,郭玉琼,潘一斌,等.铁观音茶树叶片总RNA提取方法研究[J].龙岩学院学报,2015,33(2):78-81.
[20]朱晨,张舒婷,常笑君,等.茶树几丁质酶基因的克隆及其在干旱胁迫下的表达分析[J].热带作物学报,2017,38(5):894-902.
[21]孙云,江春柳,赖钟雄,等.茶树鲜叶抗坏血酸过氧化物酶活性的变化规律及测定方法[J].热带作物学报,2008,29(5):562-566.
[22]Hao X Y,Horvath D,Chao W,et al.Identification and Evaluation of Reliable Reference Genes for Quantitative Real-Time PCR Analysis in Tea Plant(Camellia sinensis(L.)O.Kuntze)[J].International Journal of Molecular Sciences,2014,15(12):22155-22172.
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[24]Wei C L,Yang H,Wang S B,et al.Draft genome sequence of Camellia sinensis var.sinensis provides insights into the evolution of the tea genome and tea quality[J].Proceedings of the National Academy of Sciences of the United States of America,2018,115(18):E4151-E4158.
[25]程文翰.棉花(Gossypium hirsutum L.)体细胞胚胎发生的生理及分子机制研究[D].石河子:石河子大学,2016.
[26]Zhang S G,Han S Y,Yang W H,et al.Changes in H2O2content and antioxidant enzyme gene expression during the somatic embryogenesis of Larix leptolepis[J].Plant Cell,Tissue and Organ Culture,2009,100(1):21-29.
[27]Luo J P,Jiang S T,Pan L J.Enhanced somatic embryogenesis by salicylic acid of Astragalus adsurgens Pall.:relationship with H2O2 production and H2O2-metabolizing enzyme activities[J].Plant Science,2001,161(1):125-132.
[28]盛德策,李凤兰,刘忠华.植物体细胞胚发生的细胞生物学研究进展[J].西北植物学报,2008,28(1):204-215.
[29]李娜,陆海.植物抗坏血酸过氧化物酶基因家族研究进展[J].成都大学学报(自然科学版),2011,30(2):97-101.
[30]王凤华.龙眼体细胞胚胎发生过程中的基因差别表达[D].福州:福建农林大学,2003.
[31]Mittler R,Feng X,Cohen M.Post-transcriptional suppression of cytosolic ascorbate peroxidase expression during pathogen-induced programmed cell death in tobacco[J].The Plant Cell,1998,10(3):461-473.
[32]Yoshimura K,Yabuta Y,Ishikawa T,et al.Identification of a cis element for tissue-specific alternative splicing of chloroplast ascorbate peroxidase pre-m RNA in higher plants[J].The Journal of Biological Chemistry,2002,277(43):40623-40632.
[33]Erickson S L,Lykke-Andersen J.Cytoplasmic m RNP granules at a glance[J].Journal of Cell Science,2011,124(Pt 3):293-297.
[2]Mittler R,Lam E,Shulaev V,et al.Signals controlling the expression of cytosolic ascorbate peroxidase during pathogen-induced programmed cell death in tobacco[J].Plant Molecular Biology,1999,39(5):1025-1035.
[3]Pandey S,Fartyal D,Agarwal A,et al.Abiotic stress tolerance in plants:myriad roles of ascorbate peroxidase[J].Frontiers in Plant Science,2017,8:581.
[4]Davletova S,Rizhsky L,Liang H J,et al.Cytosolic ascorbate peroxidase 1 is a central component of the reactive oxygen gene network of Arabidopsis[J].The Plant Cell,2005,17(1):268-281.
[5]罗丹.拟南芥中响应草酸胁迫的APX1基因及TMS1基因的功能分析[D].福州:福建农林大学,2012.
[6]Shi W M,Muramoto Y,Ueda A,et al.Cloning of peroxisomal ascorbate peroxidase gene from barley and enhanced thermotolerance by overexpressing in Arabidopsis thaliana[J].Gene,2001,273(1):23-27.
[7]Dunajska-Ordak K,Skorupa-K?aput M,Kurnik K,et al.Cloning and expression analysis of a gene encoding for ascorbate peroxidase and responsive to salt stress in beet(Beta vulgaris)[J].Plant Molecular Biology Reporter,2014,32(1):162-175.
[8]Chao J Q,Zhang S X,Chen Y Y,et al.Cloning,heterologous expression and characterization of ascorbate peroxidase(APX)gene in laticifer cells of rubber tree(Hevea brasiliensis Muell.Arg.)[J].Plant Physiology and Biochemistry,2015,97:331-338.
[9]Tian K H,Pan C,Yang Y F,et al.Differential expression of the ascorbate oxidase multigene family of Camellia sinensis in response to stress[J].The Journal of Horticultural Science and Biotechnology,2019,94(2):160-170.
[10]方智振,赖钟雄,赖呈纯,等.龙眼体细胞胚发生中期的蛋白质组学研究[J].中国农业科学,2011,44(14):2966-2979.
[11]李惠华,赖钟雄.植物抗坏血酸过氧化物酶研究进展(综述)[J].亚热带植物科学,2006,35(2):66-69.
[12]Belmonte M F,Macey J,Yeung E C,et al.The effect of osmoticum on ascorbate and glutathione metabolism during white spruce(Picea glauca)somatic embryo development[J].Plant Physiology and Biochemistry,2005,43(4):337-346.
[13]Jariteh M,Ebrahimzadeh H,Niknam V,et al.Developmental changes of protein,proline and some antioxidant enzymes activities in somatic and zygotic embryos of persian walnut(Juglans regia L.)[J].Plant Cell,Tissue and Organ Culture,2015,122(1):101-115.
[14]Khan M A,Abbasi B H,Ali H,et al.Temporal variations in metabolite profiles at different growth phases during somatic embryogenesis of Silybum marianum L.[J].Plant Cell,Tissue and Organ Culture,2015,120(1):127-139.
[15]邵巍.龙眼胚性培养物胞质型apx基因克隆及其表达研究[D].福州:福建农林大学,2008.
[16]王凤华,赖钟雄,郑金贵,等.龙眼体细胞胚胎发生过程的基因差异表达[J].农业生物技术学报,2005,13(6):703-708.
[17]Stasolla C,Van Z L,Egertsdotter U,et al.The effects of polyethylene glycol on gene expression of developing white spruce somatic embryos[J].Plant Physiology,2003,131(1):49-60.
[18]郭玉琼,黄道斌,常笑君,等.铁观音茶树体胚发生及其内源激素变化研究[J].应用与环境生物学报,2018,24(5):1-15.
[19]赵姗姗,郭玉琼,潘一斌,等.铁观音茶树叶片总RNA提取方法研究[J].龙岩学院学报,2015,33(2):78-81.
[20]朱晨,张舒婷,常笑君,等.茶树几丁质酶基因的克隆及其在干旱胁迫下的表达分析[J].热带作物学报,2017,38(5):894-902.
[21]孙云,江春柳,赖钟雄,等.茶树鲜叶抗坏血酸过氧化物酶活性的变化规律及测定方法[J].热带作物学报,2008,29(5):562-566.
[22]Hao X Y,Horvath D,Chao W,et al.Identification and Evaluation of Reliable Reference Genes for Quantitative Real-Time PCR Analysis in Tea Plant(Camellia sinensis(L.)O.Kuntze)[J].International Journal of Molecular Sciences,2014,15(12):22155-22172.
[23]Xia E H,Zhang H B,Sheng J,et al.The tea tree genome provides insights into tea flavor and independent evolution of caffeine biosynthesis[J].Molecular Plant,2017,10(6):866-877.
[24]Wei C L,Yang H,Wang S B,et al.Draft genome sequence of Camellia sinensis var.sinensis provides insights into the evolution of the tea genome and tea quality[J].Proceedings of the National Academy of Sciences of the United States of America,2018,115(18):E4151-E4158.
[25]程文翰.棉花(Gossypium hirsutum L.)体细胞胚胎发生的生理及分子机制研究[D].石河子:石河子大学,2016.
[26]Zhang S G,Han S Y,Yang W H,et al.Changes in H2O2content and antioxidant enzyme gene expression during the somatic embryogenesis of Larix leptolepis[J].Plant Cell,Tissue and Organ Culture,2009,100(1):21-29.
[27]Luo J P,Jiang S T,Pan L J.Enhanced somatic embryogenesis by salicylic acid of Astragalus adsurgens Pall.:relationship with H2O2 production and H2O2-metabolizing enzyme activities[J].Plant Science,2001,161(1):125-132.
[28]盛德策,李凤兰,刘忠华.植物体细胞胚发生的细胞生物学研究进展[J].西北植物学报,2008,28(1):204-215.
[29]李娜,陆海.植物抗坏血酸过氧化物酶基因家族研究进展[J].成都大学学报(自然科学版),2011,30(2):97-101.
[30]王凤华.龙眼体细胞胚胎发生过程中的基因差别表达[D].福州:福建农林大学,2003.
[31]Mittler R,Feng X,Cohen M.Post-transcriptional suppression of cytosolic ascorbate peroxidase expression during pathogen-induced programmed cell death in tobacco[J].The Plant Cell,1998,10(3):461-473.
[32]Yoshimura K,Yabuta Y,Ishikawa T,et al.Identification of a cis element for tissue-specific alternative splicing of chloroplast ascorbate peroxidase pre-m RNA in higher plants[J].The Journal of Biological Chemistry,2002,277(43):40623-40632.
[33]Erickson S L,Lykke-Andersen J.Cytoplasmic m RNP granules at a glance[J].Journal of Cell Science,2011,124(Pt 3):293-297.