【作者】 伍小兵；

【导师】 邓西平；

【作者基本信息】 西北农林科技大学 ， 植物学， 2010， 硕士

【摘要】 本研究针对甘薯在北方推广种植中常遇到的苗期干旱导致存活率低以及常遭受冷害这两个关键性问题,我们以前人的关于H₂O₂在植株根系生长和抗逆性的方面以及转入抗氧化酶基因在植株抗逆性的作用为指导,用转Cu/Zn SOD和APX基因甘薯幼苗以及其非转基因对照植株为实验材料,采用水培和盆栽相结合的方法,研究了外源H₂O₂对幼苗进行根部处理能否促进甘薯幼苗不定根的生长以及同时运用外源H₂O₂对甘薯幼苗盆栽植株进行叶片喷施处理和转入抗氧化酶基因这两种策略能否解决甘薯的抗冷性问题,得到如下主要结果:1.将不同浓度的外源H₂O₂以及其清除剂抗坏血酸（AsA）加入未转基因甘薯幼苗的水培液中进行6天的实验处理。结果显示:（1）较低浓度的H₂O₂（0.5 mmoL/L和2.5mmoL/L）处理,促进了甘薯幼苗不定根系的生长,处理组每株根系的总重量、根数目、平均根长、总根表面积以及单根活力均显著高于对照,其中0.5mmoL/L处理组诱导作用最为明显;当浓度达到5mmoL/L时,则表现出显著的抑制作用,幼苗不定根系的生长几乎全部受到了抑制,而且幼根还受到了严重的伤害;用4.0 mmol/L和10.0 mmoL/L的AsA处理组也都表现出显著的抑制作用,不但不定根系的形成完全受到抑制,还阻碍了幼苗茎截面处的损伤修复;而先用2.5 mmoL/L H₂O₂处理3天后再用4.0mmoL/L AsA处理3天实验组则表现出一定的根系伸长抑制特性,但根系数目显著增多。（2）进一步分析叶片损伤程度、光合色素以及内源H₂O₂含量变化情况,发现内源H₂O₂、MDA含量在各个处理之间没有显著差异,但均高于对照组,而光合色素在各个处理组间变化情况与植株的根系生长状况呈现出一致性。2.以转Cu/Zn SOD和APX基因甘薯苗以及其非转基因对照植株为实验材料,研究了其在5℃冷胁迫一夜（12h）后植株的冷后恢复情况以及用1.0 mmoL/L H₂O₂于冷胁迫前4h预处理对其抗冷性增强的问题。结果显示:（1）在短时间（12h）冷胁迫后的很短时间（2h）内,非转基因植株表现出APX,SOD,CAT这几种抗氧化酶活性以及类胡萝卜素这一非酶类抗氧化剂含量的显著降低,膜透性显著升高,光合色素以及光合电子传递链受到破坏,光合速率显著下降;在经历一天多的室温恢复后,植株的上述几种抗氧化酶活性以及类胡萝卜素含量都得到显著提升,而膜透性继续增加,光合电子传递链得到完全修复,光合色素却显著下降,但光合作用得到了较大程度的恢复。（2）在冷胁迫后,转基因植株较对非转基因对照植株上述几种抗氧化酶和类胡萝卜素表现出了更高的活性和含量,较低的膜透性,具有较强的光合色素以及光合电子传递系统的保护能力,因而其光合速率相对较强,显示出了相对较强的抗冷胁迫能力。（3）用1.0 mmoL/L H₂O₂预处理组的植株较未预处理组在冷胁迫后的恢复过程中,表现出了较强的上述抗氧化酶活性的恢复能力,较低的膜损伤物质MDA的产生量以及较低的膜通透性,且在冷处理后明显表现出较良好的生长状况,因而得出用1.0mmoL/L H₂O₂预处理植株能够显著提高甘薯幼苗的冷后恢复能力,增强其抗冷性。3.从上述两实验结果的得出:（1）可以通过对幼苗根系进行外源H₂O₂处理来促进甘薯幼苗不定根系的大量形成和快速生长,其处理的最适浓度为0.5mmoL/L;（2）通过转入Cu/Zn SOD和APX基因能够增强甘薯幼苗的抗冷性,且在冷害到来前用1.0mmoL/L H₂O₂预处理植株亦能够使甘薯幼苗的抗冷性得到提高,而同时运用这两种策略则能够更显著地提高植株的抗冷性。更多还原

【Abstract】 When we try to extend sweetpotato planting area to the north of china,We often faced two main key problems, these are drought-induced low survival rate and confronted with chilling stress.We take the former studies,which on the H₂O₂ in plant root growth and stress resistance as well as the role of transfer antioxidant enzyme gene in plants to stress resistance, as a guide, Using transgenic sweet potato that expressing both Cu/Zn SOD and APX in chloroplasts and its non-transgenic control plant as materials, to study on whether the adding of exogenous H₂O₂ in seedlings culture solution to promote the adventitious roots growth, and also on whether the use of exogenous H₂O₂ sprayed on the leaves of potted sweet potato seedling as well as the transfer antioxidant enzyme gene strategy could resolve these chilling resistance problem of sweetpotato.And then we got these results as following:1. In the experiment, varies concentrations of exogenous hydrogen peroxide as well as a hydrogen peroxide scavenger ascorbic acid were added into the sweetpotato seedlings’culture solution to study the effect of H₂O₂ on adventitious roots and leaves growth of sweetpotato seedling. Results showed that:（1） When sweetpotato seedling cultured in lower concentrations H₂O₂ solutions （0.5mmol/L and 2.5 mmol/L） had display an inducement on adventitious root formation and growth, the total root weight, root number, total root length and total root surface area per seedling, as well as root activity of were significantly higher than the control which added nothing, especially in 0.5 mmol/L H₂O₂ treatment; As the concentration of H₂O₂ reached to 5 mmol/L, almost the whole growth of adventitious roots were significant inhibited, roots were seriously damaged also; While added with AsA in the culture solution also showed a significant inhibition on adventitious roots growth, neither the concentration was 4.0 mmol/L nor 10.0 mmol/L, the formation of adventitious roots has been completely inhibited, the damage-reparation on the stem cross-section of the cutting seedlings has also been hindered; However, in the treatment with 4 mmol/L AsA added after treated with 2.5 mmol/L H₂O₂ for three days, the elongation of adventitious roots was inhibited to a certain degree.（2） It has been found that there has a consistency between the growth status of adventitious roots and the leaves growth status, leaves MDA content, leaves water content as well as the changes in the photosynthetic pigments by further analyze the leaves physiology metabolism status, but no consistency has been discovered between the concentrations of endogenous H₂O₂ in leaves and the exogenous H₂O₂ concentrations which added in culture solution. In conclusion, the adventitious roots growth could be induced by exogenous H₂O₂ below the injury concentration （5.0 mmol/L） and it could be reversed by AsA treatment, it also showed that a certain concentration of accumulated endogenous H₂O₂ is indispensable for the plants in the course of adventitious roots formation. H₂O₂ effect on leaves’physiological process was result from the effects it did on roots growth status first, and then the growth status of roots affect the leaves growth status. The best inducement on adventitious roots and leaves growth was found in 0.5 mmol/L H₂O₂ treatment during our experiment.2. We take transgenic potted sweetpotato that expressing both Cu/Zn SOD and APX in chloroplasts and its non-transgenic control plant as materials, study on the recoverability of them after one night （12h） chilling stress at 5℃, and also the enhancement of chilling resistance when treated them with 1.0 mmol/L H₂O₂ before chilling stress. The results showed that:（1） Soon after a short time （12h） chilling stress, the activity of anti-oxidative enzymes as SOD, AXP and CAT in non-transgenic sweet potato decreased significantly and so did the content of carotenoid, but the membrane permeability increased significantly, photosynthetic pigment and photosynthetic electron transport chain were damaged, so the photosynthetic rate reduced; while after one day recovery in room temperature, the activities of above mentioned anti-oxidative enzymes and the content of carotenoid all increased significantly, membrane permeability also increased continuously, and photosynthetic electron transport chain was repaired completely while photosynthetic pigment decreased significantly, but the photosynthesis recovered to a large extent.（2） After chilling stress, compared with non-transgenic sweet potato, it could be observed that in transgenic sweet potato, the activities of above mentioned anti-oxidative enzymes and the content of caroternoid were higher, membrane permeability was lower, have a stronger ability to protect photosynthetic pigment and photosynthetic electron transport chain, therefore the photosynthetic rate was relatively higher, so a stronger chilling resistance was showed in transgenic sweet potato.（3） It could also be observed in the recovery process of the plants that had been treated with 1.0 mmol/L H₂O₂ compared with non-treatments before chilling stress, the recovery capabilities of the anti-oxidative enzymes were stronger, the content of MDA and membrane permeability were lower, and the growth status after chilling was better, therefore it could be concluded that the pretreatment with 1.0 mmol/L H₂O₂ could improve the recovery capability of the plant after chilling and enhance the chilling resistance.3. From results of the above two experiment, we can obtained: （1） When treated root with exogenous H₂O₂, it can promote the formation of sweetpotato seedling adventitious roots to a large number and accelerate root elongation growth, the optimal induce concentration is 0.5mmoL / L; Through transfer the Cu/Zn SOD and APX genes in sweetpotato can enhance its chilling resistance, and sprayed with 1.0mmoL /L H₂O₂ on leaves before the arrival of cold weather could also be able to improve plant chilling resistance , further more,combined theses two strategies would be more significant to improve the cold resistance of plants.更多还原