巨尾桉EuGR1基因的克隆及其低温胁迫下的表达特性分析
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摘要
本研究通过反转录PCR方法克隆了巨尾桉谷胱甘肽还原酶(Glutathione reductase,简称GR)基因,该基因定位于巨尾桉细胞质、长度为1485 bp,在NCBI申请基因注册(GenBank Accession Number KU904639)。构建了pET-EuGR1原核表达载体,酶活检测表明转化菌株GR活性较高。利用实时定量PCR分析巨尾桉EuGR1的时空表达特性,结果表明:EuGR1的表达量随着巨尾桉叶片的成熟而降低,在幼叶中表达量最大,近根部的叶片表达量最低;在巨尾桉组培苗中,EuGR1在茎中表达量最大,叶中表达量较低,根中表达量最低。通过转基因抗寒巨尾桉温室苗研究EuGR1基因在低温胁迫下的表达模式,发现常温和低温胁迫下耐寒性强的株系其EuGR1的表达量较高(如P40、P41、P52),耐寒性弱的株系其EuGR1的表达量较低(如P36、F44、F76),说明巨尾桉EuGR1的表达量与植株的抗寒能力具有相关性。随着低温胁迫时间的延长,EuGR1的表达量在36 h后出现降低趋势,表明EuGR1在桉树耐低温胁迫的前期发挥作用较大。
Glutathione reductase(GR) catalyzes the conversion of oxidized glutathione to reduced glutathione(GSH). EuGR1(GenBank Accession Number: KU904639) is located in the Eucalyptus grandis×E.ophylla cytoplasm containing 1485 bp nucleotides and encoding 495 amino acids. A prokaryotic expression vector of pET-EuGR1 was constructed and showed high GR activity in E. coli BL21. Quantitative PCR was used to analyze the spatiotemporal expression of EuGR1 in eucalyptus. The results showed that the expression of EuGR1 was the highest in the young leaves of eucalyptus seedlings and decreased with the maturity of leaves. In the tissue culture of eucalyptus the expression of EuGR1 was the highest in the stem, followed by the leaf, and the expression in the root was the lowest. The expression of EuGR1 gene in the cold-resistant transgenic eucalyptus was studied under low temperature stress. The result showed that plants with strong cold tolerance under normal temperature and low temperature stress had higher expression of EuGR1(such as P40, P41, P52) than the plants with weak cold tolerance(such as P36, F44, F76), indicating that the expression level of EuGR1 in eucalyptus was associated with cold resistance. However, the expression of EuGR1 kept decreasing in the tested plants at low temperature treatment after 36 h suggesting that cytoplasmic EuGR1 might play an important role in the early stage of low temperature stress tolerance in Eucalyptus.
Glutathione reductase(GR) catalyzes the conversion of oxidized glutathione to reduced glutathione(GSH). EuGR1(GenBank Accession Number: KU904639) is located in the Eucalyptus grandis×E.ophylla cytoplasm containing 1485 bp nucleotides and encoding 495 amino acids. A prokaryotic expression vector of pET-EuGR1 was constructed and showed high GR activity in E. coli BL21. Quantitative PCR was used to analyze the spatiotemporal expression of EuGR1 in eucalyptus. The results showed that the expression of EuGR1 was the highest in the young leaves of eucalyptus seedlings and decreased with the maturity of leaves. In the tissue culture of eucalyptus the expression of EuGR1 was the highest in the stem, followed by the leaf, and the expression in the root was the lowest. The expression of EuGR1 gene in the cold-resistant transgenic eucalyptus was studied under low temperature stress. The result showed that plants with strong cold tolerance under normal temperature and low temperature stress had higher expression of EuGR1(such as P40, P41, P52) than the plants with weak cold tolerance(such as P36, F44, F76), indicating that the expression level of EuGR1 in eucalyptus was associated with cold resistance. However, the expression of EuGR1 kept decreasing in the tested plants at low temperature treatment after 36 h suggesting that cytoplasmic EuGR1 might play an important role in the early stage of low temperature stress tolerance in Eucalyptus.
引文
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[12]Yu X,Pasternak T,Eiblmeier M,et al.Plastid-localized glutathione reductase 2 regulated glutathione redox status is essential for Arabidopsis root apical meristem maintenance[J].Plant Cell,2013,25(11):4451-4468.
[13]赵艳玲,韩瑶,靳玉蕾.过表达EuCuZnSOD提高巨尾桉的耐寒性研究[J].广西植物,2018,38(1):101-108.
[2]Shintaro M,Oshiyuki M,Daichi M,et al.Regulation of reactive oxygen species-mediated abscisic acid signaling in guard cells and drought tolerance by glutathione[J].Frontiers in Plant Science,2013,4:472.
[3]Anderson J V,Chevone B I,Hess J L.Seasonal variation in the antioxidant system of eastern white pine needles[J].Plant Physiology,1992,98:501-508.
[4]Wildi B,Lutz C.Antioxidant composition of selected high alpine plant species from different altitudes[J].Plant Cell&Environment,1996,9:138-146.
[5]Walker M A,Mc Kersie B D.Role of the ascorbate glutathione antioxidant system in chilling resistance of tomato[J].Plant Physiology,1993,141:234-239.
[6]Badiani M,Paolacci A R,Fusari A,et al.Non-optimal growth temperatures and antioxidants in the leaves of Sorghum bicolor(L.)Moench.:II.Short-term acclimation[J].Plant Physiology,1997,151:409-421.
[7]Kocsy G,Szalai G,Vagujfalvi A,et al.Genetic study of glutathione accumulation during cold hardening in wheat[J].Planta,2000,210(2):295-301.
[8]Kocsy G,Von Ballmoos P,Rüegsegger A,et al.Increasing the glutathione content in a chilling-sensitive maize genotype using safeners increased protection against chilling-induced injury1[J].Plant Physiology,2001,127(3):1147-1156.
[9]Christine H,Foyer NS,Sophie P,et al.Overexpression of glutathione reductase but not glutathione synthetase leads to increases in antioxidant capacity and resistance to photoinhibition in poplar trees[J].Plant Physiology,1995,109:1047-1057.
[10]赵艳玲,姚婕.巨尾桉铜分子伴侣蛋白EuCCS基因的克隆及表达分析[J].华侨大学学报(自然科学版),2017,38(3):374-378.
[11]Deng Z,Zhao M,Liu H,et al.Molecular cloning,expression profiles and characterization of a glutathione reductase in Hevea brasiliensis[J].Plant Physiology&Biochemistry,2015,96:53-63.
[12]Yu X,Pasternak T,Eiblmeier M,et al.Plastid-localized glutathione reductase 2 regulated glutathione redox status is essential for Arabidopsis root apical meristem maintenance[J].Plant Cell,2013,25(11):4451-4468.
[13]赵艳玲,韩瑶,靳玉蕾.过表达EuCuZnSOD提高巨尾桉的耐寒性研究[J].广西植物,2018,38(1):101-108.