【作者】 陆燕元；

【导师】 邓西平；

【作者基本信息】 西北农林科技大学 ， 植物学， 2010， 博士

【摘要】 甘薯淀粉含量高,增产潜力大,是世界第七大粮食作物,也是重要饲料和能源作物,对解决粮食短缺和能源危机具有重要意义。日益严峻的全球性的水分亏缺严重限制了甘薯的生长和产量。提高作物的抗逆性,实现有限水资源的高效利用和作物丰产成为了目前农业生产研究的热点和难点。因此,阐明干旱缺水条件下甘薯的生理生化适应机制,并通过基因工程手段培育耐旱新品种或对现有品种进行遗传改良,对增大粮食供给、提高甘薯的丰产潜力具有重要的理论和实践意义。基于此,本研究以转铜锌超氧化物歧化酶(Cu/Zn SOD)和抗坏血酸过氧化物酶(APX)基因甘薯(TS)和未转基因甘薯(NS)为试验材料,通过室内聚乙二醇(PEG 6000)介导的干旱胁迫处理试验,研究在不同程度的干旱及复水条件下转基因和未转基因甘薯光合系统参数、抗氧化防御系统以及根系水力导度的协同响应机制。随后,以室外盆栽控水的方式,进一步研究干旱及复水条件对甘薯生长、产量、收获指数以及水分利用效率的影响。主要研究结果如下:1、PEG介导的水分胁迫条件下,甘薯叶片的叶绿素含量(Chl)、光合速率(Pn)、气孔导度(Gs)、蒸腾速率(Tr)以及PSII最大光化学量子产量(Fv/Fm)均明显降低,且随胁迫时间的延长而持续降低。在水分胁迫初期的0-24 h期间,甘薯光合下降的主要原因是气孔导度下降导致的气孔限制,24 h后,非气孔限制成为主要影响因子。转基因甘薯叶片的Pn、Tr、Gs在各个胁迫处理中均比未转基因植株下降的幅度小,特别是在胁迫初期与未转基因植株之间差异显著。12和24 h胁迫后复水后,转基因和未转基因甘薯叶片的Pn、Tr、Gs值随复水时间的延长而持续升高,到复水后期(72 h),转基因植株基本能恢复到与对照无明显差异的(P>0.05)水平,而未转基因的恢复能力较弱。48和72 h胁迫后复水,各项光合参数回升缓慢,转基因和未转基因甘薯之间差异也不显著。说明转入的抗氧化酶基因在一定的干旱胁迫程度内不仅增强转基因甘薯在水分胁迫处理下叶片的光合能力,而且有效提高了其在复水过程中的修复能力。2、水分胁迫条件下转基因甘薯叶片质膜受伤害程度轻于未转基因植株,MDA含量、相对质膜透性和超氧阴离子含量提高的幅度明显低于未转基因植株;复水之后转基因植株叶片MDA和相对质膜透性也比未转基因植株下降较快,表明转入抗氧化酶基因不但提高了转基因植株忍耐氧化胁迫的能力,同时也增强了其修复能力。在水分胁迫下,甘薯叶片SOD和APX活性提高的幅度较大,而CAT活性则被抑制,复水后,SOD和APX活性呈现升高后降再升高的双峰变化趋势,CAT则呈缓慢回升的单峰趋势。在胁迫及复水过程中,转基因甘薯抗氧化酶活性均显著高于未转基因植株。说明转基因甘薯可能是通过显著增强这SOD和APX两个酶的活性,提高了其对水分胁迫的忍耐能力和修复作用。此外,当甘薯遭受的水分胁迫24 h或更长时诱导了甘薯“胁迫蛋白”的表达,可溶性蛋白含量提高,转基因甘薯的可溶性蛋白含量高于未转基因植株。可溶性蛋白可参与细胞渗透势的调节,对代谢酶和膜系统其保护作用,提高了甘薯对水分胁迫的抗性。3、通过对水分胁迫下甘薯幼苗根系导水性和抗氧化酶系统的研究表明,水分胁迫显著影响了甘薯幼苗根系的代谢活动,降低了根系的吸水能力。在水分胁迫初期,转基因甘薯导水率下降幅度显著低于为未基因植株,但水分胁迫持续48和72 h后,TS和NS根系水导均下降至近似于零。这可能是由于转入抗氧化酶基因通过显著增强抗氧化酶SOD、APX和CAT在水分胁迫条件下的表达,尤其是SOD、APX酶活性,以及提高了渗透调节物质的含量(可溶性蛋白含量),减轻了转基因幼苗植株在一定水分胁迫程度受到的氧化胁迫伤害,有效保护了根系的生理功能,使转基因甘薯根系吸水能力高于未转基因植株,复水之后也能够很快的恢复。但在严重的水分胁迫条件下,根系结构及功能受损严重,TS和NS根系导水率均近似于零,复水后也很难恢复。4、采用盆栽控水的方式,进一步研究转基因和未转基因甘薯在自然干旱条件下的形态及生理生化响应。与室内试验结果类似,土壤干旱处理使甘薯叶片的质膜受损,且受损程度与干旱胁迫程度成正比,但各水分胁迫条件下TS质膜受伤害程度均轻于NS。甘薯叶片SOD、APX和CAT抗氧化酶的变化也与室内结果类似,均在在土壤干旱处理下都有显著的升高,但随干旱程度加剧及复水进程,各抗氧化酶变化趋势不尽一致:SOD酶活性是随干旱程度的加大而进一步升高的,而APX和CAT在严重干旱条件下反而受到了抑制。复水之后,受到抑制的酶活性先升高,然后随复水时间的延长而逐渐降低,而SOD复水后呈逐渐降低的趋势。而且,抗氧化酶的活性在不同的干旱胁迫程度及复水下均高于未转基因植株。在土壤干旱条件下,甘薯主茎长度、分枝数的减少,但转基因甘薯主茎长度、分枝数以及绿叶数等均高于未转基因植株,说明前者可以减轻干旱胁迫对其造成的伤害,维持内在生理代谢活动的稳定,从而使植株生长受到的限制较轻。5、正常的灌水条件下转基因和未转基因甘薯的生物量及薯块产量均无明显差异。中度及重度干旱胁迫处理分别使转基因甘薯的地上生物量、总生物量分别降低了30.0和38.9%,而未转基因植株则分别降了38.2和47.8%,但TS和NS差异不显著,而两个干旱胁迫程度下TS根系生物量的增加幅度显著高于未转基因植株。干旱处理显著降低甘薯的薯块产量,在严重干旱处理下,甚至有部分植株根系不能膨大形成薯块,这可能是由于甘薯在干旱处理下把更多的光合产物分配转运至根系和冠部等原因。一定程度的干旱促进了甘薯水分利用效率的提高,但过于严重的干旱,则会降低WUE。转基因甘薯在生物水分利用效率上优于未转基因植株,但在产量水分利用效率上没有表现出优势。更多还原

【Abstract】 Sweet potato (pomoea batatas Lam), with the character of rich in starch and high-yield potential, is the world’s seventh food crop and important feed and energy resource. Therefore, to cope with these global crises over food and energy supplies as well as environmental problems, it is urgently required to develop new industrial crop varieties. Enhancing crop drought tolerance, improving crop yield and water use efficiency is a big challenge for agricultural industry. Increasingly global water deficit crisis has severely limited the growth and yield of sweet potato. Therefore, better understanding the adaptation mechanism of sweet potato to water stress and developing new varieties of drought tolerance or improving existing by genetic engineering technology have important theoretical and practical significance on the increased food supply and improve the yield potential of sweet potato. Based on this, overexpressing of Cu/Zn superoxide dismutase (Cu/Zn SOD) and ascorbate peroxidase (APX) gene of sweet potato (TS) and non-transgenic potato (NS) were used as the materials and were comparatively analyzed the collaborative response mechanism of photosynthetic parameters, antioxidant defense system and the root hydraulic conductivity under different degrees of drought stress and rewatering conditions, which was mediated by polyethylene 2 alcohol (PEG 6000). Subsequently, potted experiment was conducted to evaluate the response of growth, yield, and harvest index and water use efficiency in transgenic and non-transgenic sweet potato under soil drought and water conditions on potato. The major results are as follows:1. Under PEG-mediated water stress conditions, the chlorophyll content (Chl), photosynthetic rate (Pn), stomatal conductance (Gs), transpiration rate (Tr) and maximum photochemical yield of PSII (Fv / Fm) of leaves of sweet potato were significantly lower, and with the stress time and sustained decrease. During the first 0-24 h of water stress, photosynthetic decline of sweet potato is mainly due to stomatal limitation inducing by decreased stomatal conductance; More than 24 h, the non-stomatal limitation as the main factors. The decrease of Pn, Tr, Gs in transgenic lines in all stress treatments is smaller than that in non-transgenic plants, particularly during the shorter stress, the difference of TS and NS was significant. Rewatering from 12 and 24 h water stress, Pn, Tr, Gs values with rehydration time and continues to rise. After 72 h recovery, transgenic plants can basically return to the control level (P> 0.05), whereas NS plants was not. Rewatering from 48 and 72 h water stress, the photosynthetic parameters both in transgenic and non-transgenic lines recovered slowly, and there was no significant difference between of TS NS. The results suggested that introducing antioxidant enzyme gene to sweet potato not only enhance TS plants’drought tolerance, but also improve their repair capacity.2. Under water stress conditions, leaf plasma membrane of transgenic sweet potato was much less injured than non-transgenic plants: increased MDA content, relative membrane permeability and superoxide anion content was significantly lower than the rate of NS plants, and decreased rapidly once were released from drought stress conditions, indicating that overexpression of antioxidant enzyme gene in sweet potato enhance simultaneously the capacity of oxidative stress tolerance and recovery. Under water stress, SOD and APX activity in leaves of sweet potato improved greatly, and the CAT activity was inhibited. After rehydration, SOD and APX activity showed a double bimodal trend: increased first, then declined, and increased again, while CAT showed a gradual increase trend during rewatering period. In the process of stress and rewatering, antioxidant enzyme activities in transgenic potato were significantly higher than non- transgenic plants, suggesting that TS plants could increase their drought tolerance and repair capacity by enhancing expression of SOD and APX activity. In addition, 24 h or longer water stress treatment induced "stress protein" expression in sweet potato, such as soluble protein, which involved in the regulation of cell osmotic potential of metabolic enzymes and the protective role of membrane systems. TS plants showed higher soluble protein content than NS.3. Analysis the response of sweet potato seedling root water conductivity and the antioxidant enzyme system on water stress showed that water stress significantly affected the root metabolic activity, reduced root water uptake capacity. In the early stage of water stress, hydraulic conductivity of transgenic potato decrease was significantly lower than non-transgenic plants, but after 48 and 72 h water stress, TS and NS didn’t show a difference in root hydraulic conductivity: both of them were decreased to near zero. This may be due to, in a certain water stress degree, transgenic plants seedlings reduce the damage from oxidative stress by significantly enhancing the expression of antioxidant enzymes such as SOD, APX and CAT, especially SOD, APX activity, and increased osmolyte content (soluble protein). Therefore the root of the physiological function of the transgenic plants was effective protection; maintained root water uptake function. Moreover, they could were also be restored soon once was released from water stress condition. However, under severe water stress, root structure and function of both TS and NS plants were injured severely: root hydraulic conductivity rates declined to near zero and were very difficult to recovery after rehydration.4. Through potted experiment, further analysis the physiological response in transgenic and non-transgenic sweet potato under natural drought conditions. Similar as to laboratory test results, soil drought damaged to the leaves plasma membrane of sweet potato and the extent of damage is proportional to the degree of drought stress, however, the injury level of the plasma membrane in TS was lighter than NS under the water stress conditions. The response of SOD, APX and CAT antioxidant enzyme activities on water stress condition also was consistent to lab test results: was induced by water stress. However, several enzyme activities showed a difference response on different drought intensity and rewatering period. SOD activity was sustained increased with the increasing of drought stress, while APX and CAT were induced in moderate drought and then restrained by severe drought conditions. After rehydration, APX and CAT activities first increased first and then gradually decreased with the rehydration time, while SOD activities was gradually decreased after rewatering. In addition, the activity of antioxidant enzymes of in TS plants were higher than that in NS, under both of two drought stress treatments and succedent recovery period. Soil drought reduced main stem length, number of branches in sweet potato, but the length stem, branches and green leaves in TS were still higher than NS, indicating that TS can reduce the damage by drought stress to maintain the stability of the internal physiological metabolic activity, so that less restrictions on plant growth.5. Under well water conditions, the biomass and tuber yield didn’t show observable difference between transgenic and non-transgenic sweet potato. Compared to well water control, moderate stress reduced aboveground biomass of TS and NS by 30.0 and 38.9%, respectively, while were down by 38.2 and 47.8% under severe drought respectively. Although the reduction in TS was less than NS, the difference was not significant. Whereas, the increase of root biomass in TS was significantly higher than rate of NS plants,under both of moderate and severe drought conditions. Soil drought treatment significantly restrained the tuber yield development, practically under sever drought condition, even some plant roots can not form a tuber enlargement. This may due to, transgenic sweet potato distributed more photosynthetic products to the roots and crown under drought stress, as a result, the tuber yield decrease was steeper. A certain degree of drought contributed to the improvement of water use efficiency of sweet potato, but too severe drought, reduced WUE. Compared to NS, TS plants showed superiority in the biological WUE, but production WUE was not.更多还原