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蜈蚣藻多糖对水稻种子抗盐作用研究
刘宏
Subtype硕士
Thesis Advisor李鹏程
2019-05
Degree Grantor中国科学院大学
Place of Conferral中国科学院海洋研究所
Degree Name工程硕士
Keyword蜈蚣藻多糖,抗盐,水稻种子,分子量
Abstract

水稻(Oryza sativa L.)是一种盐敏感型粮食作物,盐胁迫会抑制水稻生长。我国土地盐渍化较为严重,其中有近20%的水稻遭受不同程度的盐胁迫,而水稻种子在萌发阶段抗逆能力弱,盐胁迫后,生长将受到抑制,最终导致减产。现阶段研究表明,海藻多糖可以用作肥料促进植物生长,提高植物抗逆性。海藻多糖的抗盐活性可能与分子量相关,但其作用机制尚不明确。因此,我们选择了红藻门(Rhodophyta)中的蜈蚣藻(Grateloupia filicina)为例,研究蜈蚣藻多糖对盐胁迫下水稻种子的影响,为环境友好型农作物抗盐制剂研发以及海藻资源在农业方面的应用提供一定的理论基础。

首先,对蜈蚣藻多糖的降解方法进行研究。比较了传统水浴加热降解和微波辅助降解的降解效率,利用降解速度更快的微波辅助法进行降解,并比较了不同的温度、pH值、过氧化氢浓度、微波功率对降解多糖分子量的影响。结果显示,通过升温(70 oC-90 oC)和降低pHpH 3-pH 1)能够显著加快降解速度,而改变过氧化氢浓度(1%-3%)和微波功率(300 W-900 W),降解效果不明显。根据各反应条件对蜈蚣藻多糖分子量的影响,我们制备得到了4种不同分子量   40.8 kDa22.6 kDa5.1 kDa3.0 kDa)的蜈蚣藻多糖,并对全部多糖样品进行理化性质分析和结构表征。结果显示,多糖的总糖含量为52.4%-65.1%,硫酸根含量为13.5%-26.1%,蛋白含量0.17%-0.95%,单糖组成以半乳糖为主。红外光谱图显示降解并未破坏主要糖链结构,扫描电镜呈现了高分子量多糖为紧密连接状态,低分子量为松散镂空状态。

利用制备的不同分子量蜈蚣藻多糖对水稻种子进行处理,同时,以文献中报道具有较好抗盐活性的浒苔多糖和壳聚糖作对比,结果显示,降解后分子量为40.8 kDa的蜈蚣藻多糖LGFP-1在种子发芽势、根长、活力指数等方面优于其他多糖。在此基础上,以不同浓度的LGFP-1对水稻种子进行处理,结果发现     0.1 mg/mL多糖处理组水稻根长和活力指数等方面优于其他浓度。并进一步从水稻生理生化层面检测LGFP-1对水稻抗盐代谢产物的影响。结果显示0.1 mg/mLLGFP-1能够显著提升水稻脯氨酸含量,提高SODPOD的活性,从而缓解盐胁迫对水稻的渗透胁迫和氧化胁迫。

最后,为了进一步探究多糖的作用机理,我们将多糖与FITC连接,利用激光共聚焦显微镜观察水稻对多糖的吸收情况,结果显示,多糖在各组织中分布差异明显,水稻的根和幼苗都有荧光现象,根荧光强度更强,能明显看到细胞壁的荧光成像,幼苗的荧光成弥散的点状,而子叶无荧光现象;同时在分子水平上,验证了蜈蚣藻多糖对水稻3个抗盐特征基因调控的影响。荧光定量PCR结果显示,多糖能够上调基因OsCLC1OsCLC2OsSOS1,尤其是OsSOS1的表达,由此证明蜈蚣藻多糖LGFP-1可以作用于水稻,并在分子水平上调离子区域化抗盐基因,进一步解释了蜈蚣藻多糖对水稻种子抗盐调节的机理。

Other Abstract

Rice (Oryza sativa L.) is a salt-sensitive crop which could be suppressed by salt stress. In China, nearly 20% rice is affected by salinization land which is the key factor to decrease yield. For seed germination, it is very important for the weak resistance in defending salt stress, which could depress rice growth and development. A large number of studies have shown that seaweed polysaccharides could be used as fertilizer to improve plant resistance, and molecular weight might have an influence on the effect but mechanisms are not clear. So, we chose the Grateloupia filicina from Rhodophyta to degrade different Mws polysaccharides and studied their effects for rice seeds under salt stress, which could provide more theory for environmentally friendly anti-salt additive and polysaccharides high value utilization.

First, we compared the traditional heating degradation and microwave irradiation degradation method, and studied the degraded parameters such as temperature, pH, H2O2 concentration and microwave power. In this research, improving temperature (70 oC-90 oC) and decreasing pH (pH 3-pH 1) could accelerate degrading, but the effects of enhancing H2O2 concentration (1%-3%) and microwave power        (300 W-900 W) were limited. Finally, polysaccharides with different Mws (LGFP-1: 40.8 kDa, LGFP-2: 22.6 kDa, LGFP-3: 5.1 kDa, LGFP-4: 3.0 kDa) were prepared. The chemical characterization and structure were tested. Briefly, the content of sugar, sulfate, and protein was 52.4%-65.1%, 13.5%-26.1% and 0.17%-0.95% separately, and monosaccharide was mainly composed of galactose. The infrared spectrogram showed that the degradation did not destroy the main sugar structure, and the scanning electron microscopy showed the low molecular weight polysaccharide with more clearly loose texture.

Therefore, different molecular weight polysaccharides were applied to rice seeds under salt stress, and chitosan and Enteromorpha polysaccharides were compared. Through seed germination quality, LGFP-1 was chosen for the high seed germination potential, root length and vigor index. Furthermore, optimum concentration was decided at 0.1 mg/mL for the best germination quality. Next, the effects on physiological and biochemical were studied and the result showed that LGFP-1 could significantly improve proline content and the activity of SOD and POD which could alleviate the osmotic and oxidative stress under salt stress. For further study on the mechanism, FITC was linked to LGFP-1 and the result from confocal laser scanning microscope showed polysaccharide could be absorbed by root and shoot with different distribution. From the microscope, the fluorescence was more obvious in root than shoot. It’s easy to find FITC fluorescence was in root epidermis cells, and fluorescence in shoot was sparse, and there was no fluorescence in cotyledon. At the same time, the effect on anti-salt gene was studied. LGFP-1 could significantly increase OsCLC1, OsCLC2 and OsSOS1 expression, especially OsSOS1 that is responsible for the ion regionalization of salt resistant, which suggested LGFP-1 could enhance rice seed anti-salt activity by regulating genes.

Language中文
Document Type学位论文
Identifierhttp://ir.qdio.ac.cn/handle/337002/156837
Collection实验海洋生物学重点实验室
Recommended Citation
GB/T 7714
刘宏. 蜈蚣藻多糖对水稻种子抗盐作用研究[D]. 中国科学院海洋研究所. 中国科学院大学,2019.
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