【作者】 陈健妙；

【导师】 刘兆普； 秦松；

【作者基本信息】 南京农业大学 ， 应用海洋生物学， 2011， 博士

【摘要】 近年,麻疯树(Jatropha curcas L.)由于具有种子含油量高且油质优良,作为生物柴油原料得到特别关注,引发了大规模的投资和扩张种植,但如果麻疯树与粮食竞争用地或利用高碳土地,它便失去可持续发展的优势。目前,麻疯树作为新兴生物能源资源,当前对其研究主要集中在种子含油量、籽油特性、籽油成份以及用籽油生产生物柴油方面,而其基本农艺性状、该物种对生物和非生物胁迫的响应方面知识了解甚少。例如,麻疯树对盐胁迫的响应。尚未了解这些知识,意味着大规模种植不是没有社会经济和生态方面的风险。为了选出高产、耐盐的麻疯树生态型,我们测定了从十五个不同麻疯树生态型中初筛出的三个蒴果产量较高的生态型(南油1,2和3号)的种子及其籽油的理化特性,评估其作为生物柴油原料的潜力。然后,探讨不同生态型麻疯树对NaCl胁迫的生长和生理生化响应,以阐明麻疯树的耐盐性及其耐盐机理。继而进一步探讨了麻疯树对不同海水浓度的耐受性以及海水处理对麻疯树生长和籽油含油率和脂肪酸成份的影响。这些基础研究有助于筛选优质高产、耐盐麻疯树品系,为以后培育麻疯树耐盐品种奠定基础。主要研究结果如下：1、三种生态型麻疯树的种仁含油量均在60%以上,且籽油的脂肪酸主要由油酸(C18：1)、棕榈酸(C16：0)、亚油酸(C18：2)、硬脂酸(C18：0)和十七碳酸(C17：0)组成,三者的碳链长度均主要集中在C16-18,皆以不饱和脂肪酸为主,具有较好的生物柴油原料利用价值。其中,以南油3号种仁含油量和生物产量最高,其作为生物柴油原料的潜力最大。南油1、2号间的种仁含油量与生物产量无显著差异,但2号麻疯树的种子具有优良的结实性状、物理和遗传特性,利于降低生物柴油生产的成本。因此,其作为生物柴油原料的潜力比1号麻疯树的相对大。2、南油2、3号苗用不同浓度NaCl (0,50,100和200mmol-L"1)处理24天,叶片RWC皆与对照无显著差异,表明盐胁迫下麻疯树具有较强的保持水分平衡的能力。50mmol·L’1NaCl处理,南油2、3号麻疯树净光合速率Pn,PS Ⅱ的最大光化学效率(Fv/Fm)、叶片光合总面积与对照无显著差异,光合性能维持稳定,全株干重不受影响。100、200mmol·L-1NaCl处理,南油2、3号苗的全株干重皆比对照显著降低,3号苗降低的幅度较大,表明低盐50mmol·L-1NaCl对麻疯树生长无明显影响,中高盐胁迫下,南油2号抵抗盐胁迫的能力较3号苗强。3、南油2号麻疯树苗SOD活性在50mmol·L-1NaCl处理下,比对照显著增加,200mmol·L-1NaCl处理,比对照显著降低,而3号苗的SOD活性随着盐度的增加显著降低。南油2、3号苗的POD活性在50mmol·L-1NaCl处理,皆与对照无显著差异。随着盐度增加,2号苗的POD活性比对照显著增加,而3号苗的比对照显著减小但维持相对稳定。南油2、3号树苗的CAT活性在50mmol·L-1NaCl处理下,分别比对照显著增加58%和24%,随后变化趋势如SOD活性。以上结果表明,盐胁迫激活麻疯树叶片中抗氧化酶活性,与南油3号相比,2号苗抗氧化酶活性能更好地协调增加和维持得相对高。4、随着盐度的增加,两种麻疯树根、茎和叶中的Na+和Cl-含量呈递增趋势。50mmol·L-1NaCl处理,Na+和Cl-在茎和根中增加的幅度明显大于叶中的,且Na+和Cl-主要分布在茎和根的皮层以及茎的髓部,表明低盐胁迫下,麻疯树能将Na+和Cl-区域到茎和根的皮层和髓部细胞中,降低Na+和Cl-对叶片的伤害.200mmol·L-1NaCl处理,Na+和Cl-在叶片和茎中增加的幅度比根中的大,并主要把Na+和Cl-区隔到叶片主脉的皮层和木质部,降低Na+和Cl-对叶片栅栏和海绵织织的伤害,维持叶片光合能力。与南油3号相比,2号苗K+、Ca2+浓度降低的幅度相对小,因此能维持相对高的K+/Na+和Ca2+/Na+比,维持离子平衡的能力较3号苗强。5、50、200mmol·L-1NaCl处理麻疯树苗5天后,根中PM-H+-ATPase的水解活性、Vmax, Km、质子泵活性和pH梯度比对照显著增高,尤其在低盐处理。以翻译水平为主激活根部PM-H+-ATPase,很好地维持根部对N、P营养的吸收以及糖类的获取,促进根部生长和提高根冠比,提高麻疯树的抗盐性。盐处理下,叶片PM-H+-ATPase的Km值显著降低,提示盐胁迫下,麻疯树叶片PM-H+-ATPase明显增加了对底物ATP的亲和性,50mmol-L-1NaCl处理,质子泵活性维持稳定,主要归于脂质膜的改变,而200mmol-L-1NaCl处理质子泵活性的增加,则可能是由于弱酸介导激活质子转运。盐胁迫下,根和叶片中的V-H+-ATPase和V-H+-PPase也不同程度被激活。盐胁迫下,麻疯树根和叶中质膜H+-ATPase、V-H+-ATPase和V-H+-PPase的这些改变,是其对盐胁迫的一套适应机制。与南油3号苗相比,2号苗根和叶中的PM-H+-ATPase. V-H+-ATPase5V-H+-PPase活性被激活和维持得相对高,间接表明盐胁迫下,南油2号维持离子和营养平衡的能力、将Na+和Cl-区隔到液泡里的能力相对强,从而抵抗盐胁迫的能力相对强。2～5的结果表明,盐胁迫下,麻疯树能通过维持稳定的光化学活性和叶片水分状况以维持稳定的Pn；激活较高的抗氧化酶活性以维持膜稳定性；以翻译调控为主,激活根部PM-H+-ATPase以维持根部N、P和糖类等营养吸收和转运以及离子平衡；激活叶片V-H+-ATPase和V-H+-PPase以区域有毒离子到液泡里等策略来抵抗盐胁迫。与南油3号相比,南油2号具有较好的响应策略,故其耐盐性相对强。6、10%海水处理对南油2号麻疯树苗期生长无明显影响,但显著促进其营养期生长和单株结果产量；20%海水处理对营养期生和单株结果无明显影响,但降低其脂肪酸中亚麻酸含量,利于减少籽油酸败程度,从而提高生物柴油原料的品质。总而言之,三个高产麻疯树生态型(南油1、2和3号)的种仁含油量均在60%以上,具有较好的生物柴油原料利用价值。其中,以南油3、2号作为生物柴油原料的潜力相对大。南油2号麻疯树抗盐能力比3号的强。麻疯树能适应10%～20%海水处理,具有适应滩涂栽培的潜力,可进一步培育耐盐麻疯树品种,以作为海岸基干林带混交林中的生态经济型树种。更多还原

【Abstract】 In recent years, the physic nut (Jatropha curcas L.) has received special attention as a biodiesel feedstock, because of its high seed oil content and quality. Large-scale investments and expansions of physic nut plantations have been triggered. If the physic nut competes for land with food crops or high carbon stocks, its acclaimed sustainability advantages are lost. The seeds of physic nut plants represent a promising bio-energy source and current research is mainly focused on their oil content, characteristics and composition and their use and application to biodiesel production. Knowledge of the physic nut agronomic properties and the plant’s physiological responses to biotic and abiotic stress are not thoroughly understood. For example, there is limited information on how the physic nut responds to salt stress.These knowledge gaps imply that developing large scale plantations is not without socio-economic and ecological risk. In order to selected salt-tolerant, high yield physic nut ecotypes, we analyzed the chemical and physical characteristics of seeds and seed oils of three physic nut ecotypes (Nanyou1,2and3) with relatively high capsule yield, which were initially screened from fifteen physic nut ecotypes. Furthermore, In order to clarify the salt tolerance of physic nut and its salt tolerance mechanisms, we analyzed the growth and physiological and biochemical responses to salt stress between the Nanyou2and3. The Nanyou2was also exposed to different concentrations of seawater to clarify the seawater-tolerance in physic nut and the effect of sea water on physic nut growth, seed oil content and the fatty acid compositions. All these basic reasearch would help to develop strategies for improving understanding the physic nut plants response to salt stress, screen high yield and salt-tolerant strains of physic nut, cultivate for salt-tolerant cultivars in the future. The main results obtained were shown as follows:1. The kernel oil content of three physic nut ecotypes was more than60%. Moreover, the fatty acid compositions in three physic nut seed oils were mainly oleic acid (C18:1), palm itic acid (C16:0), linoleic acid (C18:2), stearic acid (C18:0) and margaric acid (C17:0), and were dominated by unsaturated fatty acids. Thus, they were all good biodiesel feedstocks. Among these three physic nut ecotypes, the kernel oil content and biological yield of the Nanyou3was highest. Therefore, it had the greatest potential for biodiesel feedstock. There was no significant difference in kernel oil content and biological yield between the Nanyou1and2. However, the Nanyou2was with excellent fruit characters and excellent physical and genetic characteristics, which were helpful for reducing the cost of biodiesel production. Thus, compared with the Nanyou1, the Nanyou2had a greater potential for biodiesel feedstock.2. Seedlings of Nanyou2and Nanyou3were subjected to different concentrations of NaCl (0,50,100and200mmol-L"1) treatment for24days. Compared with the control leaves, both the leaf RWC had no significant difference in two ecotypes, suggested that physic nut plants has a strong capacity to maintain cell water balance under salt stress. Under50mmol-L-1NaCl treatment, compared with the control, the Pn,Fv/Fm and the totally photosynthetic area had no significant difference, indicated the photosynthetic performance remained stable. Thus, the whole plant dry weight was not affected by50mmol-L"1NaCl in both Nanyou2and Nanyou3. Under100and200mmol·L-1NaCl treatment, both the dry weights of Nanyou2and Nanyou3were reduced significantly, especially that of Nanyou3, indicated50mmol·L-1NaCl had no significant effect on the physic nut growth. The Nanyou2had a strong capacity of resistance to salt stress than the Nanyou3under mid-high salt stress.3. The SOD activity of Nanyou2increased under50mmol-L-1NaCl treatment and decreased significantly under200mmol-L-1NaCl treatment, while that of Nanyou3decreased continuously with a significant degree. The POD activity in both Nanyou2and3maintained stable under50mmol-L"1NaCl treatment, with the increasing NaCl treatment, the POD activity in Nanyou2increased significantly, while that in Nanyou3significantly decreased but maitained stable. The CAT activities of both ecotypes increased significantly when treated with50mmol-L’1NaCl, and then showed similar trend as the SOD activities did in both ecotypes. These results suggested that the activity of antioxidant enzymes in physic nut seedling leaves could be activated under salt stress. Compared with the Nanyou3, the Nanyou2could maitain higher antioxidant enzyme activities and had a better ability to increase coordinately and remain higher activity.4.With the increasing NaCl concentration, the increasing trend of Na+and Cl-content in physic nut roots, stems and leaves was observed. The increase of Na+and Cl-content in physic nut shoots and roots was more conspicuous than that in leaves under50mmol-L-1NaCl treatment. Moreover, the Na+and Cl-were mainly distributed in the cortex of root and stem as well stem pith, so it could reduce the damage to the leaf. Under200mmol-L-1NaCl treatment, the increasing rate of Na+and Cl-content in leaves and stems was higher than that of roots, and the Na+and Cl-were mianly distributed to the cortex and xylem of the leaf main vein to reduce the injury for palisade and spongy tissue. Thus, the leaf could maintain photosynthetic capacity. Compared with the Nanyou3, the decreasing rate of K+and Ca2+in Nanyou2was lower, so it could maitain higher K+/Na+andCa2+/Na+ratio than the Nanyou3did. Thus, its ability to maintain ion balance was stronger than that of the Nanyou3.5. This study investigates the acclimation of PM-H+-ATPase of Nanyou2physic nut roots and leaves treated with0,50and200mmol-L-1NaCl for5days. Upon comparison with control roots, the PM H+-ATPase hydrolytic activity, Vmax, Km, H+-pumping activity and pH gradient potential across the plasma membrane were significantly higher in roots treated with NaCl, especially under mild salt stress. The translational activation of PM-H+-ATPase of physic nut roots helped to maintain nitrogen and phosphorus uptake as well as soluble sugar acquisition in root, thus promoting root growth and increasing root shoot ratio and increasing the salt tolerance of physic nut. Compared with the control leaves, with NaCl treatment, lower Km values for the PM H+-ATPase of leaves were observed, suggested that the affinity of PM H+-ATPase towards ATP increases as a function of salt treatment. The maintenance of H+transport under50mmol-L"1NaCl was attributed to a modification of the lipid membrane, and the increase in H+transport under200mmol-L-1NaCl could be because of an acid-mediated activation. Under salt stress, the activity of V-H+-ATPase and V-H+-PPase of roots and leaves was also activated in varying degrees in two physic nut ecotypes.These modulations of PM H+-ATPase, V-H+-ATPase and V-H+-PPase in the roots and leaves of physic nut, could represent a set of adaptive mechanisms to salinity. Compared with the Nanyou3, the Nanyou2maintained higher activity of theses enzymes in roots and leaves under salt stress, indirectly suggested that the ability of ionic and nutritional balance and the compartmentation Na+and Cl-into the vacuole was relatively stronger than that of the Nanyou3. Thus its ability to resist salt stress was relatively stronger than that of the Nanyou3.The results (2-5) indicate that young physic nut plants are able to cope with salt stress by maintaining stable integrity of the photochemical activity and stable leaf water status associated with stable Pn, maintaining higher antioxidant enzyme activities associated with stable membrane, activating root PM-H+-ATPase mainly through translational regulation and leaf PM-H+-ATPase mainly through transcriptional and/or post-translational regulation, associated with nutrients absorbtion and ion homeostasis, activating leaf V-H+-ATPase and V-H+-PPase associated with compartmentation the toxic ions and cytosolic pH homeostasis etc. These responses might represent a set of adaptive mechanisms employed by physic nut to cope with salt stressful conditions. Compared with the Nanyou3, the Nanyou2had better response strategies.6.10%seawater treatment had no significant effect on the growth of the Nanyou2seedling, but it enhanced the vegetative growth of the Nanyou2, and the seed production of per plant.20%sea water treatment had no significant effect on the vegetative growth and the production of per plant. However, it reduced the linolenic acid and linoleic acid content of the fatty acid, which helped to improve the quality of biodiesel feedstock.In conclusion, the kernel oil content of three physic nut ecotypes was more than60%. Thus, they were all good biodiesel feedstocks. Among these three physic nut ecotypes, the kernel oil content and biological yield of the Nanyou3was highest, and the Nanyou2was with excellent fruit characters and excellent physical and genetic characteristics, which were helpful for reducing the cost of biodiesel production.Therefore, the Nanyou3and2had the greater potential for biodiesel feedstock. Compared with the Nanyou3, the Nanyou2had a relatively high salt tolerance. Further research showed that physic nut can adapt to10%～20%sea water treatment. It has the potential to adapt to grow in the coastal area, so we can further cultivate the salt-tolerant physic nut cultivar, as the coastal mixed forest tree species for the ecological and economic use.更多还原