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2025年8月6日 星期三
  2015, Vol. 23 Issue (12): 1576-1587    
  研究论文与报告 本期目录 | 过刊浏览 | 高级检索 |
超量表达烟草高亲和钾离子转运体蛋白基因(NtHAK1)提高烟草盐胁迫能力
秦利军1,宋拉拉2,赵丹2,赵德刚3
1. 贵州大学农业生物工程研究院
2.
3. 贵州大学农业生物工程重点实验室
Overexpression of Nicotiana tabacum High-affinity Potassium Ion Transporter Protein Gene(HAK1) Improves the Salt-tolerance in Tobacco(N. tabacum) Plants
1, 1, 1, 2
1.
2. Guizhou Key Lab of Agricultural Bioengineering,Guizhou University
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摘要 高亲和钾离子转运体蛋白(high-affinity potassium ion transporter protein, HAK)在植物盐害胁迫过程中起重要调控作用。本研究分析了盐胁迫对超量表达烟草(Nicotiana tabacum) HAK1植株种子发芽率(germination rate)、发芽势(germination potential)和发芽指数(germination index)以及盐害对烟苗(4~5叶期)叶绿素(chlorophyll, Chl)、丙二醛(malondialdehyde, MDA)含量和超氧化物歧化酶(superoxide dismutase, SOD)、过氧化物酶 (peroxidase, POD)活性的影响。结果表明,当Na+浓度低于50 mmol/L时,转基因和野生型植株的种子发芽率、发芽势和发芽指数不受影响;Na+浓度为100和150 mmol/L时,超量表达烟株种子萌发率、萌发势和发芽指数分别为81.6%、85.0%、42.6%和63.9%、78.8%、20.2%,均显著高于野生型植株(P<0.05)。盐胁迫处理后, 野生型植株Chl含量从处理前(0 d)的2.25 mg/g (FW)降至处理5 d时的1.31 mg/g (FW),降幅为41.8%,而转基因植株的降幅平均为36.5%,其降幅显著低于野生型植株;盐胁迫处理5 d时,野生型植株MDA含量为105 nmol/g (FW),显著高于超量表达植株[96.52 nmol/g (FW)] (P<0.05)。同时,NaCl胁迫处理引起了超量表达NtHAK1植株和野生型植株的抗氧化酶(antioxidant enzymes, AOEs)活性显著增加(P<0.05),超量表达植株的超氧化物歧化酶(superoxide dismutase, SOD)和过氧化氢酶(catalase, CAT) 平均酶活增幅为38.8和58.1%,而野生型植株为34.2%和54.6%。对超量表达及野生型植株钠钾吸收相关基因表达分析表明,盐胁迫3 d时超量表达植株的高亲和钾离子转运体蛋白基因HAK1、钾外流通道蛋白(potassium outward rectifier channel protein)基因TORK1和液泡Na+/H+逆向运输蛋白(vacuolar Na+/H+ antiporter protein)基因NHX1的表达量分别为野生型植株的3.85、1.79和1.69倍。另外,超量表达植株根、叶组织的Na/K比值分别为0.110和0.106,而野生型植株Na/K比分别为0.147和0.135,均显著高于前者(P<0.05)。本研究为后续培育耐盐烟草新种质和进一步研究HAK1基因介导的耐盐机制提供参考资料。
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秦利军
宋拉拉
赵丹
赵德刚
关键词 烟草高亲和钾离子转运体蛋白基因(NtHAK1)盐胁迫生理指标Real-time PCR烟草    
Abstract:The high-affinity potassium ion transporter protein (HAK) plays an important role in improving the salt-tolerance in plants. In this study, we investigated the effect of salt stress on germination rate (Gr), germination potential (Gp), and germination index (Gi) in seeds of NtHAK1-overexpression plants and on the content of chlorophyll (Chl) and malondialdehyde (MDA), the activity of superoxide dismutase (SOD), peroxidase (POD), catalase (CAT) in NtHAK1-overexpressing tobacco plantlets (4 to 5 leaf stage). The results showed there was no siginificant effect on Gr, Gp, and Gi in the seeds of overexpressing and wild-type tobaccos under 50 mmol/L NaCl, but siginificant difference appeared between overexpressing plants and wild-type plants with 81.6% for Gr, 85.0% for Gp, and 42.6% for Gi in transgenic tobaccos under 100 mmol/L NaCl and 63.9% for Gr, 78.8% for Gp, and 20.2% for Gi in transgenic tobaccos under 150 mmol/L NaCl, which both were significantly higher than that in wild-type tobaccos (P<0.05). The Chl content decreased from 2.25 mg/g (FW) before salt treatment to 1.31 mg/g(FW) at 5 d after salt treatment in wild-type plantlets and the decreasing amplitude of Chl content was 41.8%, while the average value of decreasing amplitude was 36.5% in transgenic tobaccos, which was significantly lower than the former. At 5 d after salt treatment, the MDA content was 105 nmol/g(FW) in non-transgenic plants, while it was 96.52 nmol/g(FW) in overexpressing plants. Meanwhile, the activities of antioxidant enzymes (AOEs), including SOD and CAT, were significantly up-regulated in NtHAK1-overexpression tobaccos with the increasing amplitude of 38.8% for SOD activity and 58.1% for CAT activity, while it was 34.2% for SOD and 54.6% for CAT in wild-type ones. The results from quantitative Real-time PCR for Na+/K+ absorption-related genes at 3 d after salt treatment in overexpressing and wild-type tobacco showed the relative expression levels of HAK1, potassium outward rectifier channel protein (TORK1), and vacuolar Na+/H+ antiporter protein (NHX1) were significantly up-regulated in NtHAK1- overexpression tobacco plants compared with wild-type ones. They were 3.85 folds for HAK1, 1.79 folds for TORK1, and 1.69 folds for NHX1 higher than those in wild-type tobaccos. In addition, the ratio of Na to K in different plant tissues demonstrated that it was 0.110 in root tissue and 0.106 in leaf tissue of transgenic plants respectively, while it was 0.147 for root tissue and 0.135 for leaf tissue in wild-type plants. The ratio of Na to K of different tissues in transgenic tobaccos was significantly lower (P<0.05) than that in wild-type ones. This study provids the plant materials for breeding the salt-tolerant tobaccos and further studying the mechanism of HAK1-mediated salt-tolerance in transgenic plants.
Key wordshigh-affinity potassium ion transporter protein(NtHAK1)    Salt-stress    physiological indexes    qRT-PCR    Tobacco
收稿日期: 2015-04-13      出版日期: 2015-10-11
基金资助:中国烟草总公司贵州省公司科技专项“基因组学指导的烟草定向突变育种新技术研究及应用;国家转基因生物新品种培育科技重大专项'转基因生物的“基因删除”和“基因拆分”技术研究'
通讯作者: 赵德刚     E-mail: degangzhao@yahoo.com
引用本文:   
秦利军 宋拉拉 赵丹 赵德刚. 超量表达烟草高亲和钾离子转运体蛋白基因(NtHAK1)提高烟草盐胁迫能力[J]. , 2015, 23(12): 1576-1587.
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http://journal05.magtech.org.cn/Jwk_ny/CN/     或     http://journal05.magtech.org.cn/Jwk_ny/CN/Y2015/V23/I12/1576
 
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