稻米溶血磷脂的鉴定、遗传多样性及其与品质的关系研究

发布时间：2018-05-22 11:22

  本文选题：水稻(Oryza + sativa　； 参考：《浙江大学》2016年博士论文

【摘要】：稻米中的功能性脂质具有改善稻米品质及人类健康的重要功能,近年来逐渐受到广泛关注。然而,磷脂这类具有重要营养价值的稻米脂质,由于测定技术的制约,至今不曾被充分开发利用。稻米胚乳中磷脂的溶血形式-溶血磷脂(LPLs)能与淀粉结合形成直链淀粉-脂质复合物,从而显著影响稻米食用品质及淀粉特性。虽已有研究明确了稻米胚乳中LPLs的变异范围,但全面的LPLs谱分析、基因型×环境互作对LPLs积累的影响、LPLs与稻米食用品质关系、LPLs与磷素代谢关系等基础研究依然欠缺。阐明稻米LPLs的积累规律、遗传控制及其调控机理,对稻米品质改良与消费指导具有重要意义。因此,有必要详细鉴定稻米磷脂组分及其变异规律并解析其遗传机理,为今后发掘、选育符合消费者需求和食品安全的高营养价值新品种提供理论依据。本研究围绕稻米淀粉磷脂展开,建立了一种快速、高效的用于提取并定量测定稻米LPLs含量的方法。同时,开展了稻米中LPLs的遗传多样性及其与稻米淀粉特性、水稻籽粒磷代谢的潜在关系研究。主要结果总结如下：1.建立了一种水溶性正丙醇提取,结合液相色谱-质谱(HPLC-MS)用于一步测定稻米淀粉LPLs的方法。结果表明,不同稻米碾磨方法对于淀粉LPLs的提取及最终测定结果影响较小。用75%正丙醇(8 mL/0.15 g)在1000C下加热2 h来提取LPLs的简单一步法与复杂多步法的提取效果相近。优化了HPLC-MS检测方法,能在15 min中同时鉴定到10种主要的淀粉LPLs成分。该方法需样量低,可用于大规模水稻样品的淀粉总LPLs及其组分的定量分析,也可用于其他作物的淀粉LPLs分析。2.稻米中淀粉LPLs各组分含量在20份水稻品种中显著不同,表明稻米淀粉LPLs具有广泛的遗传多样性。稻米淀粉LPLs的主要成分是溶血磷脂酰胆碱(LPC)16：0(3009.7-4697.8μg/g)、LPC 18：2(836.6-2182.3 μg/g)和溶血磷脂酰乙醇胺(LPE)16：0(625.7-1139.8 gg/g)、LPE 18：2(170.6-481.6μg/g)。总溶血磷脂酰胆碱、总磷脂酰乙醇胺和总溶血磷脂含量分别为4727.1-7685.2 μg/g,882.8-1809.5μg/g,5609.8-9401.1μg/g。尽管方差分析表明环境和基因型-环境互作对LPLs积累具有显著影响,但这两种效应分别仅占总变异的0.7-38.9%和1.8-6.6%。相关性分析发现,LPCI4：0与LPE14：0,LPCI6：0与LPE16：0,LPC18：1与LPE18：1,LPC18：2与LPE18：2,LPC18：3与LPE18：3呈显著正相关,表明LPLs在合成过程中可能是先合成脂肪酸链再连接一定比例的氨基醇。聚类分析表明,20份水稻能被分为三类,而主成分分析同样表明这些水稻可分为三类,其中第一、二成分能分别解释57.7%和16.2%的总变异。初步关联分析共鉴定到22个控制LPLs合成的可能相关位点(P0.001),分布于除第3和7染色体外所有染色体。初步找到可能的淀粉磷脂合成相关基因3个,重新测定了这些基因的序列,设计了基因标签标记,为进一步研究稻米淀粉LPLs代谢及遗传分析提供候选基因。3.稻米的糊化特性和热学特性是稻米蒸煮食用品质和工业应用的重要评价指标,分析了稻米LPLs各组分与糊化特性和热学特性的相关关系。结果表明,冷胶粘度(CPV)、崩解值(BD)、回复值(CS)等糊化特性与LPLs存在显著相关性。除焓变外,热学特性一般与LPLs无显著相关性。通过偏相关分析排除表观直链淀粉含量(AAC)的影响后,发现CPV与个别LPLs组分仍然存在显著正相关,BD、CS和个别LPLs组分仍然呈显著负相关。这些结果表明,稻米淀粉LPLs能以独立的或与直链淀粉结合的方式,影响稻米糊化特性。4.提高磷素(P)吸收和利用效率有助于改善稻米营养与品质,然而水稻籽粒中磷素运输与分配的分子机理尚不清晰。利用5份低植酸水稻(low phytic acid,lpa)突变体来探究水稻种子中植酸、溶血磷脂等主要的含磷化合物及无机磷的变化。结果表明,lpa突变体的植酸、植酸磷含量显著降低,而无机磷含量明显升高。OsST和OsMIK基因突变导致水稻籽粒中个别LPLs组分和相应的溶血磷脂-磷含量(LPL-P)显著减少。与之相反,OsMRP5和Os2-PGK基因突变则引起除LPC14：0外,其他LPLs组分及相应的LPL-P含量显著增加。以KBNT-lpa和嘉禾218杂交后代9个株系为材料,用于验证Os2-PGK基因对LPLs合成与积累的影响。结果证明,9个株系中lpa突变体的总溶血磷脂含量仍然高于正常株系的含量。该结果表明,水稻植酸和磷脂的生物合成过程存在某种交互联系,而这种联系很可能是通过籽粒中无机磷的分配与再分配来实现的。
[Abstract]:Functional lipids in rice have been paid more and more attention to the improvement of rice quality and human health. However, rice lipids, such as phospholipids, which have important nutritional value, have not been fully exploited to the present. The lysophosphatidylcholine (LPLs) of phosphatidylcholine in rice embryo milk is not fully exploited and used. Starch combined with amylose - lipid complex, which significantly affects rice quality and starch properties. Although the variation range of LPLs in rice endosperm has been clarified, the overall LPLs spectrum analysis, the effect of genotype x environmental interaction on the accumulation of LPLs, the relationship between LPLs and rice food quality, the relationship between LPLs and phosphorus metabolism, and so on It is important to clarify the regularity of accumulation of rice LPLs, the genetic control and its regulation mechanism, which is of great significance to the improvement of rice quality and the guidance of consumption. Therefore, it is necessary to identify the composition of rice phospholipid and its variation law and analyze its genetic mechanism in order to find out the high nutritional value that meets the needs of consumers and food safety in the future. The new variety provides a theoretical basis. This study revolves around rice starch phospholipids, and establishes a rapid, efficient method for extracting and quantifying LPLs content in rice. At the same time, the genetic diversity of LPLs in rice and its potential relationship with rice starch properties and rice grain phosphor metabolites are studied. The main results are as follows: 1. A water soluble positive propanol extraction method combined with liquid chromatography mass spectrometry (HPLC-MS) was established for one step determination of rice starch LPLs. The results showed that different rice milling methods had little effect on the extraction and final determination of starch LPLs. The simple one step method and complex of extracting LPLs with 75% normal propanol (8 mL/0.15 g) under 1000C was heated to 2 h. The extraction effect of multistep method is similar. The HPLC-MS detection method is optimized, and 10 major starch LPLs components can be identified in 15 min. The method needs low sample size, which can be used for quantitative analysis of total starch LPLs and its components in large scale rice samples, and can also be used in the LPLs analysis of starch LPLs components in.2. rice of other crops. A significant difference was found in 20 rice varieties, indicating that rice starch LPLs has extensive genetic diversity. The main components of rice starch LPLs are lysophosphatidylcholine (LPC) 16:0 (3009.7-4697.8, g/g), LPC 18:2 (836.6-2182.3 mu g/g) and lysophosphatidyl ethanolamine (LPE) 16:0. The content of lysophosphatidyl choline, total phosphatidyl ethanolamine and total lysophosphatidylcholine was 4727.1-7685.2, 882.8-1809.5, g/g, 5609.8-9401.1 micron g/g., although variance analysis showed that environment and genotype environment interaction had significant influence on LPLs accumulation, but these two effects only accounted for the total variation of 0.7-38.9% and 1.8-6.6%. correlation analysis, respectively. It was found that LPCI4:0 and LPE14:0, LPCI6:0 and LPE16:0, LPC18:1 and LPE18:1, LPC18:2 and LPE18:2, LPC18:3 and LPE18:3 showed significant positive correlation, indicating that LPLs in the synthesis process may be the first synthesis of fatty acid chain and then a certain proportion of amino alcohols. Cluster analysis showed that 20 rice could be divided into three types, and the principal component analysis was the same. The sample shows that these rice can be divided into three types, of which first, second components can explain the total variation of 57.7% and 16.2% respectively. Preliminary association analysis has identified 22 possible related loci (P0.001) for controlling LPLs synthesis, distributed in all chromosomes except third and seventh staining in vitro, and preliminarily found 3 related genes related to starch phospholipid synthesis. In order to further study the metabolism and genetic analysis of rice starch LPLs, the gene label markers were designed to provide a candidate gene for rice starch metabolism and genetic analysis. The gelatinization and thermal properties of.3. rice were important evaluation indexes for the cooking quality and industrial application of rice rice, and the correlation between the components of rice LPLs and the gelatinization characteristics and the thermal characteristics was analyzed. The results showed that there was a significant correlation between the gelatinization properties of the viscosity (CPV), the disintegration value (BD), the recovery value (CS) and the LPLs. In addition to the enthalpy, there was no significant correlation between the thermal properties and LPLs. After partial correlation analysis excluded the apparent amylose content (AAC), it was found that there was still significant positive correlation between CPV and some LPLs components, BD, CS and AAC. A significant negative correlation was still found in the LPLs components. The results showed that rice starch LPLs could be independent or combined with amylose, affecting the rice gelatinization property,.4., and improving the absorption and utilization of phosphorus (P), which could help improve the nutrition and quality of rice. However, the molecular mechanism of phosphorus transport and distribution in rice grains was not clear. 5 Low phytic acid (LPA) mutants were used to explore the changes in phytic acid, phosphatidic phosphatidic and inorganic phosphorus in rice seeds. The results showed that the content of phytic acid in the LPA mutant and the phosphorus content of phytic acid significantly decreased, while the content of inorganic phosphorus increased significantly and the mutation of.OsST and OsMIK genes resulted in the individual LPLs components in the rice grain and the individual LPLs components in the rice seed. The corresponding lysophosphatidin phosphorus content (LPL-P) decreased significantly. On the contrary, the OsMRP5 and Os2-PGK gene mutations caused a significant increase in other LPLs components and corresponding LPL-P content except LPC14:0. 9 strains of KBNT-lpa and Jiahe 218 hybrid progenies were used as materials to verify the effect of Os2-PGK based on LPLs synthesis and accumulation. The results showed that 9 The total lysophosphatidic content of the LPA mutant in the strain was still higher than that of the normal strain. The results showed that there was some interaction between the phytic acid and the phospholipid biosynthesis process in rice, which was probably realized by the distribution and redistribution of inorganic phosphorus in the grain.
【学位授予单位】：浙江大学
【学位级别】：博士
【学位授予年份】：2016
【分类号】：S511
，

本文编号：1921858