茶树CsMDHAR基因的克隆与非生物胁迫响应分析
|
摘要
基于茶树转录组数据,以龙井43茶树cDNA为模板,克隆得到开放阅读框(ORF)长度为1 305 bp,编码434个氨基酸的茶树单脱氢抗坏血酸还原酶基因,命名为CsMDHAR。蛋白序列特征和基因表达模式分析表明,CsMDHAR蛋白含有FAD结合功能域,属于FAD依赖的吡啶核苷酸-硫基氧化还原酶(Pyr-redox-2)家族。该蛋白有两个无序化区域,而且包含有32个磷酸化位点,理论相对分子量为47.21 kDa,pI为5.99,属于亲水性蛋白;CsMDHAR蛋白二级结构以α-螺旋和随机卷曲为主。通过PlantCARE和PLACE对启动子上游调控元件进行分析预测,结果显示,CsMDHAR基因启动子上游1 000 bp中含有多个与光、激素以及植物抗逆有关的顺式作用元件。利用荧光定量PCR方法检测龙井43和迎霜的CsMDHAR、CsAO和CsAPX在4种不同非生物胁迫下的表达水平,结果表明,在低温(4℃)胁迫下,两个茶树品种的CsMDHAR、CsAO和CsAPX的表达均受抑制,且品种间的差异较小;在高温(38℃)和干旱(200 g·L-1 PEG)胁迫下龙井43中的CsMDHAR表达均上调,分别在8 h和2 h时达到最大值,为对照的1.49倍和1.85倍;盐(200 mmol·L-1 NaCl)胁迫下,CsAO和CsAPX在两种茶树品种中的表达量变化趋势相似,但变化幅度不同,可能与品种间不同的抗逆性有关。
In this study, a MDHAR gene(CsMDHAR) was cloned from Camellia sinensis cv. ‘Longjing 43' based on the transcriptome data of tea plant. Sequence analysis shows that the open reading frame length of CsMDHAR was 1 305 bp, encoding 434 amino acids with a molecular weight of approximately 47.21 kDa and the theoretical isoelectric point of 5.99. CsMDHAR was a hydrophilic protein, including two unordered regions and 32 phosphorylation sites. CsMDHAR belonged to Pyr-redox-2 super-family containing a highly conserved region called FAD domain, and mainly composed of α-helix and random coil. PlantCARE and PLACE database prediction analysis suggest that there were many cis-elements related to light, hormones and stress resistance in the 1 000 bp upstream region of CsMDHAR gene. The expression profiles of CsMDHAR, CsAO and CsAPX in tea cv ‘Longjing 43' and ‘Yingshuang' under high temperature, low temperature, drought, and salt treatments were detected by qRT-PCR. The results indicate that the expression profiles of CsMDHAR, CsAO and CsAPX were suppressed under 4℃, and there were no significantly differences in ‘Longjing 43' and ‘Yingshuang'. However, the expression profiles of CsMDHAR gene were upregulated under 38℃ or 200 g·L-1 PEG treatments in ‘Longjing 43', with the highest 2.5 and 5 times of the control at 8 h and 2 h, respectively. In addition, the expression trends of CsAO and CsAPX in both cultivars were similar under NaCl(200 mmol·L-1) treatment, but the variation ranges were different, which might be related to the different stress response in tea plant.
In this study, a MDHAR gene(CsMDHAR) was cloned from Camellia sinensis cv. ‘Longjing 43' based on the transcriptome data of tea plant. Sequence analysis shows that the open reading frame length of CsMDHAR was 1 305 bp, encoding 434 amino acids with a molecular weight of approximately 47.21 kDa and the theoretical isoelectric point of 5.99. CsMDHAR was a hydrophilic protein, including two unordered regions and 32 phosphorylation sites. CsMDHAR belonged to Pyr-redox-2 super-family containing a highly conserved region called FAD domain, and mainly composed of α-helix and random coil. PlantCARE and PLACE database prediction analysis suggest that there were many cis-elements related to light, hormones and stress resistance in the 1 000 bp upstream region of CsMDHAR gene. The expression profiles of CsMDHAR, CsAO and CsAPX in tea cv ‘Longjing 43' and ‘Yingshuang' under high temperature, low temperature, drought, and salt treatments were detected by qRT-PCR. The results indicate that the expression profiles of CsMDHAR, CsAO and CsAPX were suppressed under 4℃, and there were no significantly differences in ‘Longjing 43' and ‘Yingshuang'. However, the expression profiles of CsMDHAR gene were upregulated under 38℃ or 200 g·L-1 PEG treatments in ‘Longjing 43', with the highest 2.5 and 5 times of the control at 8 h and 2 h, respectively. In addition, the expression trends of CsAO and CsAPX in both cultivars were similar under NaCl(200 mmol·L-1) treatment, but the variation ranges were different, which might be related to the different stress response in tea plant.
引文
[1]陈宗懋.饮茶与健康的起源和历史[J].中国茶叶, 2018,40(10):5-7.
[2] Wang W, Xin H, Wang M, et al. Transcriptomic analysis reveals the molecular mechanisms of drought-stress-induced decreases in Camellia sinensis leaf quality[J]. Frontiers in Plant Science, 2016, 7(795):385. DOI:10.3389/fpls.2016.00385.
[3] Gill S S, Tuteja N. Reactive oxygen species and antioxidant machinery in abiotic stress tolerance in crop plants[J]. Plant Physiology and Biochemistry, 2010, 48(12):909-930.
[4] Cervilla L M, Blasco B, Ríos J J, et al. Oxidative stress and antioxidants in tomato(Solanum lycopersicum)plants subjected to boron toxicity[J]. Annals of Botany, 2007,100(4):747-756.
[5] Garchery C, Gest N, Do P T, et al. A diminution in ascorbate oxidase activity affects carbon allocation and improves yield in tomato under water deficit[J]. Plant Cell&Environment,2013, 36(1):159-175.
[6] Alvina Grace L, Doherty C J, Bernd M R, et al. CIRCADIAN CLOCK-ASSOCIATED 1 regulates ROS homeostasis and oxidative stress responses[J]. Proceedings of the National Academy of Sciences of the United States of America, 2012,109(42):17129-17134.
[7] Wang Z, Xiao Y, Chen W. Increased vitamin C content accompanied by an enhanced recycling pathway confers oxidative stress tolerance in Arabidopsis[J]. Journal of Integrative Plant Biology, 2010, 52(4):400-409.
[8] Khalil O A K, Vellosa J C R, Quadros A U D, et al.Curcumin antifungal and antioxidant activities are increased in the presence of ascorbic acid[J]. Food Chemistry. 2012,133(3):1001-1005.
[9] Mi Y L, Pulla R K, Park J M, et al. Over-expression of L-gulono-γ-lactone oxidase(GLOase)gene leads to ascorbate accumulation with enhanced abiotic stress tolerance in tomato[J]. In Vitro Cellular&Developmental Biology-Plant, 2012, 48(5):453-461.
[10]陈莉,辛海波,李晓艳,等.百合MDHAR基因的克隆与表达分析[J].林业科学. 2010, 46(9):178-181.
[11]邹礼平.番茄抗坏血酸生物合成与代谢途径中相关酶基因的克隆与调控[D].武汉:华中农业大学, 2005.
[12]邓春婷.番茄抗坏血酸代谢相关酶基因DHAR1和MDHAR1在抗逆方面的研究[D].武汉:华中农业大学,2009.
[13]刘巍.小麦抗条锈病基因Yr10及防卫相关基因TaMDHAR4的功能分析[D].杨凌:西北农林科技大学,2014.
[14]李佼.茶树抗坏血酸生物合成相关酶基因的克隆与表达分析[D].杨凌:西北农林科技大学, 2013.
[15]庞磊.茶树抗坏血酸过氧化物酶(APX)基因克隆及逆境胁迫下的表达特性与生理响应[D].合肥:安徽农业大学,2012.
[16] Wu Z J, Li X H, Liu Z W, et al. De novo assembly and transcriptome characterization:novel insights into catechins biosynthesis in Camellia sinensis[J]. BMC Plant Biology,2014, 14(1):277. DOI:10.1186/s12870-014-0277-4.
[17] Kumar S, Stecher G, Tamura K. MEGA7:molecular evolutionary genetics analysis version 7.0 for bigger datasets[J]. Molecular Biology&Evolution, 2016, 33(7):1870-1874.
[18] Wu Z J, Tian C, Jiang Q, et al. Selection of suitable reference genes for qRT-PCR normalization during leaf development and hormonal stimuli in tea plant(Camellia sinensis)[J]. Scientific Reports, 2016, 6:19748. DOI:10.1038/srep19748.
[19] Li H, Liu Z, Wu Z J, et al. Differentially expressed protein and gene analysis revealed the effects of temperature on changes in ascorbic acid metabolism in harvested tea leaves[J]. Horticulture Research, 2018, 5(1):65. DOI:10.1038/s41438-018-0070-x.
[20] Livak K J, Schmittgen T D. Analysis of relative gene expression data using real-time quantitative PCR and the 2-DDCT method[J].Methods, 2001, 25(4):402-408.
[21] Gasteiger E, Gattiker A, Hoogland C, et al. ExPASy:the proteomics server for in-depth protein knowledge and analysis[J]. Nucleic Acids Research, 2003, 31(13):3784-3788.
[22] Haroldsen V M, Chi-Ham C L, Shashank K, et al.Constitutively expressed DHAR and MDHAR influence fruit,but not foliar ascorbate levels in tomato[J]. Plant Physiology&Biochemistry, 2011, 49(10):1244-1249.
[23] Mande S S, Sarfaty S, Allen M D, et al. Protein-protein interactions in the pyruvate dehydrogenase multienzyme complex:dihydrolipoamide dehydrogenase complexed with the binding domain of dihydrolipoamide acetyltransferase[J]. Structure, 1996, 4(3):277-286.
[24] Park A K, Kim I S, Do H, et al. Structure and catalytic mechanism of monodehydroascorbate reductase, MDHAR,from Oryza sativa L. japonica[J]. Scientific Reports, 2016,6:33903. DOI:10.1038/srep33903.
[25] Urao T, Yamaguchi-Shinozaki K, Urao S, et al. An Arabidopsis myb homolog is induced by dehydration stress and its gene product binds to the conserved MYB recognition sequence[J]. The Plant Cell, 1993, 5(11):1529-1539.
[26] Stephan W, Franziska T, Kamy S, et al. CONSTANS and the CCAAT box binding complex share a functionally important domain and interact to regulate flowering of Arabidopsis[J].The Plant Cell, 2006, 18(11):2971-2984.
[27]林源秀,顾欣昕,吴宛玲,等.草莓FaMDHAR和FaGR的克隆与表达分析[J].园艺学报, 2015, 42(7):1241-1250.
[28]马玉华.逆境胁迫对苹果抗坏血酸代谢相关酶活性及基因表达的影响[D].杨凌:西北农林科技大学, 2008.
[29] Zhang Y Y, Li H X, Shu W B, et al. RNA interference of a mitochondrial APX gene improves vitamin C accumulation in tomato fruit[J]. Scientia Horticulturae, 2011, 129(2):220-226.
[30] Zhang Y, Li H, Shu W, et al. Suppressed expression of ascorbate oxidase gene promotes ascorbic acid accumulation in tomato fruit[J]. Plant Molecular Biology Reporter, 2011,29(3):638-645.
[2] Wang W, Xin H, Wang M, et al. Transcriptomic analysis reveals the molecular mechanisms of drought-stress-induced decreases in Camellia sinensis leaf quality[J]. Frontiers in Plant Science, 2016, 7(795):385. DOI:10.3389/fpls.2016.00385.
[3] Gill S S, Tuteja N. Reactive oxygen species and antioxidant machinery in abiotic stress tolerance in crop plants[J]. Plant Physiology and Biochemistry, 2010, 48(12):909-930.
[4] Cervilla L M, Blasco B, Ríos J J, et al. Oxidative stress and antioxidants in tomato(Solanum lycopersicum)plants subjected to boron toxicity[J]. Annals of Botany, 2007,100(4):747-756.
[5] Garchery C, Gest N, Do P T, et al. A diminution in ascorbate oxidase activity affects carbon allocation and improves yield in tomato under water deficit[J]. Plant Cell&Environment,2013, 36(1):159-175.
[6] Alvina Grace L, Doherty C J, Bernd M R, et al. CIRCADIAN CLOCK-ASSOCIATED 1 regulates ROS homeostasis and oxidative stress responses[J]. Proceedings of the National Academy of Sciences of the United States of America, 2012,109(42):17129-17134.
[7] Wang Z, Xiao Y, Chen W. Increased vitamin C content accompanied by an enhanced recycling pathway confers oxidative stress tolerance in Arabidopsis[J]. Journal of Integrative Plant Biology, 2010, 52(4):400-409.
[8] Khalil O A K, Vellosa J C R, Quadros A U D, et al.Curcumin antifungal and antioxidant activities are increased in the presence of ascorbic acid[J]. Food Chemistry. 2012,133(3):1001-1005.
[9] Mi Y L, Pulla R K, Park J M, et al. Over-expression of L-gulono-γ-lactone oxidase(GLOase)gene leads to ascorbate accumulation with enhanced abiotic stress tolerance in tomato[J]. In Vitro Cellular&Developmental Biology-Plant, 2012, 48(5):453-461.
[10]陈莉,辛海波,李晓艳,等.百合MDHAR基因的克隆与表达分析[J].林业科学. 2010, 46(9):178-181.
[11]邹礼平.番茄抗坏血酸生物合成与代谢途径中相关酶基因的克隆与调控[D].武汉:华中农业大学, 2005.
[12]邓春婷.番茄抗坏血酸代谢相关酶基因DHAR1和MDHAR1在抗逆方面的研究[D].武汉:华中农业大学,2009.
[13]刘巍.小麦抗条锈病基因Yr10及防卫相关基因TaMDHAR4的功能分析[D].杨凌:西北农林科技大学,2014.
[14]李佼.茶树抗坏血酸生物合成相关酶基因的克隆与表达分析[D].杨凌:西北农林科技大学, 2013.
[15]庞磊.茶树抗坏血酸过氧化物酶(APX)基因克隆及逆境胁迫下的表达特性与生理响应[D].合肥:安徽农业大学,2012.
[16] Wu Z J, Li X H, Liu Z W, et al. De novo assembly and transcriptome characterization:novel insights into catechins biosynthesis in Camellia sinensis[J]. BMC Plant Biology,2014, 14(1):277. DOI:10.1186/s12870-014-0277-4.
[17] Kumar S, Stecher G, Tamura K. MEGA7:molecular evolutionary genetics analysis version 7.0 for bigger datasets[J]. Molecular Biology&Evolution, 2016, 33(7):1870-1874.
[18] Wu Z J, Tian C, Jiang Q, et al. Selection of suitable reference genes for qRT-PCR normalization during leaf development and hormonal stimuli in tea plant(Camellia sinensis)[J]. Scientific Reports, 2016, 6:19748. DOI:10.1038/srep19748.
[19] Li H, Liu Z, Wu Z J, et al. Differentially expressed protein and gene analysis revealed the effects of temperature on changes in ascorbic acid metabolism in harvested tea leaves[J]. Horticulture Research, 2018, 5(1):65. DOI:10.1038/s41438-018-0070-x.
[20] Livak K J, Schmittgen T D. Analysis of relative gene expression data using real-time quantitative PCR and the 2-DDCT method[J].Methods, 2001, 25(4):402-408.
[21] Gasteiger E, Gattiker A, Hoogland C, et al. ExPASy:the proteomics server for in-depth protein knowledge and analysis[J]. Nucleic Acids Research, 2003, 31(13):3784-3788.
[22] Haroldsen V M, Chi-Ham C L, Shashank K, et al.Constitutively expressed DHAR and MDHAR influence fruit,but not foliar ascorbate levels in tomato[J]. Plant Physiology&Biochemistry, 2011, 49(10):1244-1249.
[23] Mande S S, Sarfaty S, Allen M D, et al. Protein-protein interactions in the pyruvate dehydrogenase multienzyme complex:dihydrolipoamide dehydrogenase complexed with the binding domain of dihydrolipoamide acetyltransferase[J]. Structure, 1996, 4(3):277-286.
[24] Park A K, Kim I S, Do H, et al. Structure and catalytic mechanism of monodehydroascorbate reductase, MDHAR,from Oryza sativa L. japonica[J]. Scientific Reports, 2016,6:33903. DOI:10.1038/srep33903.
[25] Urao T, Yamaguchi-Shinozaki K, Urao S, et al. An Arabidopsis myb homolog is induced by dehydration stress and its gene product binds to the conserved MYB recognition sequence[J]. The Plant Cell, 1993, 5(11):1529-1539.
[26] Stephan W, Franziska T, Kamy S, et al. CONSTANS and the CCAAT box binding complex share a functionally important domain and interact to regulate flowering of Arabidopsis[J].The Plant Cell, 2006, 18(11):2971-2984.
[27]林源秀,顾欣昕,吴宛玲,等.草莓FaMDHAR和FaGR的克隆与表达分析[J].园艺学报, 2015, 42(7):1241-1250.
[28]马玉华.逆境胁迫对苹果抗坏血酸代谢相关酶活性及基因表达的影响[D].杨凌:西北农林科技大学, 2008.
[29] Zhang Y Y, Li H X, Shu W B, et al. RNA interference of a mitochondrial APX gene improves vitamin C accumulation in tomato fruit[J]. Scientia Horticulturae, 2011, 129(2):220-226.
[30] Zhang Y, Li H, Shu W, et al. Suppressed expression of ascorbate oxidase gene promotes ascorbic acid accumulation in tomato fruit[J]. Plant Molecular Biology Reporter, 2011,29(3):638-645.