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烟草转 AER/DHAR 基因及 AER-DHAR 双基因抗旱耐盐性分析
吴 茜
Subtype硕士
Thesis Advisor殷俐娜
2015-05
Degree Grantor中国科学院研究生院
Place of Conferral北京
Keyword2-烯醛还原酶 脱氢抗坏血酸还原酶 活性氧 氧化伤害
Abstract

在逆境胁迫下,植物体内的活性氧会大量积累,一方面这些性质活泼的 ROS 可
通过直接修饰 DNA、RNA、蛋白质以及碳水化合物等,给细胞造成伤害;另一方面,
大量的活性氧会引发脂质过氧化的链式反应,产生大量的醛。醛是一种在 ROS 下游
所产生的,比 ROS 具有更强攻击力的一类物质,能够使膜的完整性遭到破坏,并造
成细胞毒害。因此氧化伤害可以分为上游的活性氧伤害和下游的活性醛基伤害。在活
性氧下游伤害中产生的含有 α , β-不饱和双键的 2-烯醛毒性更强,而 2-烯醛还原酶
(2-alkenal reductase,AER)可以催化烯醛基中不饱和双键的加氢还原,生成饱和的
醛,然后再由其它的醛还原酶作用生成二氧化碳和水,从而极大地减轻了醛的毒害作
用。
本研究探索是否可以利用 2-烯醛还原酶(AER)来清除氧化伤害下游醛自由基达
到提高植物的抗旱和耐盐能力,以及其提高植物抗旱抗盐能力和氧化伤害上游清除基
因 DHAR 的区别,验证同时清除植物氧化伤害的上游和下游是否可以更好的提高植
物的抗旱抗盐能力。本研究以超表达拟南芥 2-alkenal reductase(AER)基因烟草、超
表达拟南芥 DHAR 基因烟草、转双基因烟草(AER-DHAR)和野生型烟草(SR)及
空载体转化株系(VECT)为研究材料。在室内通过模拟干旱和盐胁迫,对其抗旱耐
盐性进行分析,测定了干旱胁迫和复水后以及 200mM NaCl 的盐胁迫下各个烟草株系
的生物量、光合速率、叶绿素荧光参数、叶绿素含量、MDA、H 2 O 2 含量和 Na + 含量
等指标。得到如下主要结果:
(1)干旱胁迫后各转基因烟草株系的生物量、叶绿素含量、净光合速率、PSII 最
大光化学效率及 H 2 O 2 的清除能力均显著高于野生型和空载体株系;复水之后,烟草
植株的各项生理指标都得到一定程度的恢复,而转基因株系相比于野生型和空载体株
系恢复迅速,恢复能力更强。说明转 AER,DHAR 以及转 AER-DHAR 均通过提高植
物抗氧化能力来提高植物的抗旱能力。(2)盐处理后转基因烟草的地上部分生物量、叶绿素含量和光合速率都显著高于
野生型和空载体株系,而 MDA 和 H 2 O 2 的含量明显低于对照植株,此外,各转基因
株系中的 Na + 含量也显著低于野生型和空载体株系。结果表明超表达 AER,DHAR
以及转 AER-DHAR 均显著提高植物的抗盐能力。其提高植物的抗盐能力归结为通过
提高植物的抗氧化能力来减轻植物的氧化伤害和通过提高植物生长速率减轻了其离
子毒害。
(3)通过清除氧化伤害下游(超表达 AER)和清除氧化伤害上游(超表达 DHAR)
一样显著提高植物的抗旱抗盐能力。超表达双基因 AER-DHAR 烟草并没有表现出比
单基因 AER/DHAR 烟草更好的抗旱抗盐能力。
综合以上结果表明,通过清除氧化伤害的下游醛伤害可以提高植物的抗旱抗盐能
力。本研究也进一步明确 AER 在抗氧化系统中的作用,证明了可以通过清除活性氧
下游醛自由基来提高植物的抗旱耐盐能力,明确完整的抗氧化系统在提高植物抗旱抗
盐中的作用及抗氧化伤害上下游之间的关系。
关键词:2-烯醛还原酶;脱氢抗坏血酸还原酶;活性氧;氧化伤害

Other Abstract

There will be a substantial accumulation of ROS in plants under adversity stress. On
the one hand, ROS can damage to cells by modifying DNA, RNA, proteins and
carbohydrates. On the other hand, a large number of reactive oxygen species can cause a
chain reaction of lipid peroxidation, then produce large amounts of aldehydes. Aldehyde is
the product of downstream of ROS. It has more harmful than the ROS substances. It can
destroy the integrity of the membrane and cause poison to cells. So the oxidative damage
can be divided into upstream of the active oxygen damage and downstream of the active
aldehyde group. 2-olefine aldehyde are more toxic which contains α , β-unsaturated double
bond in the downstream. 2-alkenal reductase can catalytic hydrogenation of unsaturated
double bond of olefin aldehyde to generate saturated aldehydes, then generates carbon
dioxide and water by other aldehyde reductase, then greatly reduced the toxicity of
formaldehyde.
In this study, we investigated whether can use 2 - olefine aldehyde reductase (AER)
to clear the aldehyde in downstream of oxidation damage to improve drought resistance
and salt resistance of tobacco, and verify whether clear the upstream and downstream of
oxidative damage at the same time can improve the drought resistance and salt resistance
of plants preferably. Transgenic tobacco plants with overexpressing 2-alkenal reductase,
overexpressing dehydroascorbate reductase, wild-type tobacco plants ( SR ) and
vector-transformed control line(VECT)were used to measure the biomass, photosynthetic
rate, chlorophyll fluorescence parameters, contents of chlorophyll, MDA, H 2 O 2 and Na +
under drought stress, rehydration and salt stress. We got these results as following:
(1)The results showed that the biomass, contents of chlorophyll, photosynthetic rate,  chlorophyll fluorescence parameters and the capability for scavenging H 2 O 2 of the
transgenic tobacco plants were significantly higher than wild-type tobacco plants and
vector-transformed control line under drought treatment. After rehydration, the
physiological indexes of tobacco recovered. The recovery capability of transgenic lines is
better than that of wild-type and vector-transformed control line. It turned out that the
transgenic tobacco plants with overexpression AER, DHAR and AER - DHAR by
increasing antioxidant capacity to improve drought resistance ability of plants.
(2)The overall growth situation and photosynthetic rate of transgenic tobacco are
significantly better than the wild-type under salt stress. Meanwhile, the aboveground
biomass, chlorophyll content and photosynthetic rate were all markedly higher than
wild-type plants and vector-transformed control line. However, the content of MDA and
H 2 O 2 of transgenic plants were obviously lower than wild-type plants. In addition, the
content of Na + of transgenic plants were observably lower wild-type plants and
vector-transformed control line. The results showed that over-expression AER, DHAR and
AER - DHAR were significantly improve the salt resistance of tobacco.
(3)We obtained that eliminating the downstream (over-expression AER) and
upstream(over-expression DHAR) of oxidative damage can prominently enhance the
drought resistance and salt resistance of plants. The drought resistance and salt resistance
of overexpression of AER-DHAR gene of tobacco is not better than single gene
AER/DHAR of transgenic tobacco plants.
From the above results, we can obtain that overexpression of AER and DHAR gene
could enhance drought and salt tolerance in transgenic tobacco plants by eliminating the
aldehyde in downstream of oxidation damage. We comprehend the role of AER in the
antioxidant system. Further more, we can understand the role of antioxidant system in
improving the drought resistance and salt resistance of plants.
Key words: 2-alkenal reductase, dehydroascorbate reductase, ROS, Oxidative damage.

Language中文
Document Type学位论文
Identifierhttp://ir.iswc.ac.cn/handle/361005/9037
Collection水保所知识产出(1956---)
Recommended Citation
GB/T 7714
吴 茜. 烟草转 AER/DHAR 基因及 AER-DHAR 双基因抗旱耐盐性分析[D]. 北京. 中国科学院研究生院,2015.
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