發(fā)布時(shí)間：2018-05-22 11:22

  本文選題：水稻(Oryza + sativa　； 參考：《浙江大學(xué)》2016年博士論文

【摘要】：稻米中的功能性脂質(zhì)具有改善稻米品質(zhì)及人類健康的重要功能,近年來逐漸受到廣泛關(guān)注。然而,磷脂這類具有重要營養(yǎng)價(jià)值的稻米脂質(zhì),由于測定技術(shù)的制約,至今不曾被充分開發(fā)利用。稻米胚乳中磷脂的溶血形式-溶血磷脂(LPLs)能與淀粉結(jié)合形成直鏈淀粉-脂質(zhì)復(fù)合物,從而顯著影響稻米食用品質(zhì)及淀粉特性。雖已有研究明確了稻米胚乳中LPLs的變異范圍,但全面的LPLs譜分析、基因型×環(huán)境互作對(duì)LPLs積累的影響、LPLs與稻米食用品質(zhì)關(guān)系、LPLs與磷素代謝關(guān)系等基礎(chǔ)研究依然欠缺。闡明稻米LPLs的積累規(guī)律、遺傳控制及其調(diào)控機(jī)理,對(duì)稻米品質(zhì)改良與消費(fèi)指導(dǎo)具有重要意義。因此,有必要詳細(xì)鑒定稻米磷脂組分及其變異規(guī)律并解析其遺傳機(jī)理,為今后發(fā)掘、選育符合消費(fèi)者需求和食品安全的高營養(yǎng)價(jià)值新品種提供理論依據(jù)。本研究圍繞稻米淀粉磷脂展開,建立了一種快速、高效的用于提取并定量測定稻米LPLs含量的方法。同時(shí),開展了稻米中LPLs的遺傳多樣性及其與稻米淀粉特性、水稻籽粒磷代謝的潛在關(guān)系研究。主要結(jié)果總結(jié)如下：1.建立了一種水溶性正丙醇提取,結(jié)合液相色譜-質(zhì)譜(HPLC-MS)用于一步測定稻米淀粉LPLs的方法。結(jié)果表明,不同稻米碾磨方法對(duì)于淀粉LPLs的提取及最終測定結(jié)果影響較小。用75%正丙醇(8 mL/0.15 g)在1000C下加熱2 h來提取LPLs的簡單一步法與復(fù)雜多步法的提取效果相近。優(yōu)化了HPLC-MS檢測方法,能在15 min中同時(shí)鑒定到10種主要的淀粉LPLs成分。該方法需樣量低,可用于大規(guī)模水稻樣品的淀粉總LPLs及其組分的定量分析,也可用于其他作物的淀粉LPLs分析。2.稻米中淀粉LPLs各組分含量在20份水稻品種中顯著不同,表明稻米淀粉LPLs具有廣泛的遺傳多樣性。稻米淀粉LPLs的主要成分是溶血磷脂酰膽堿(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)�？�?cè)苎�磷脂酰膽堿、總磷脂酰乙醇胺和總?cè)苎�磷脂含量分別為4727.1-7685.2 μg/g,882.8-1809.5μg/g,5609.8-9401.1μg/g。盡管方差分析表明環(huán)境和基因型-環(huán)境互作對(duì)LPLs積累具有顯著影響,但這兩種效應(yīng)分別僅占總變異的0.7-38.9%和1.8-6.6%。相關(guān)性分析發(fā)現(xiàn),LPCI4：0與LPE14：0,LPCI6：0與LPE16：0,LPC18：1與LPE18：1,LPC18：2與LPE18：2,LPC18：3與LPE18：3呈顯著正相關(guān),表明LPLs在合成過程中可能是先合成脂肪酸鏈再連接一定比例的氨基醇。聚類分析表明,20份水稻能被分為三類,而主成分分析同樣表明這些水稻可分為三類,其中第一、二成分能分別解釋57.7%和16.2%的總變異。初步關(guān)聯(lián)分析共鑒定到22個(gè)控制LPLs合成的可能相關(guān)位點(diǎn)(P0.001),分布于除第3和7染色體外所有染色體。初步找到可能的淀粉磷脂合成相關(guān)基因3個(gè),重新測定了這些基因的序列,設(shè)計(jì)了基因標(biāo)簽標(biāo)記,為進(jìn)一步研究稻米淀粉LPLs代謝及遺傳分析提供候選基因。3.稻米的糊化特性和熱學(xué)特性是稻米蒸煮食用品質(zhì)和工業(yè)應(yīng)用的重要評(píng)價(jià)指標(biāo),分析了稻米LPLs各組分與糊化特性和熱學(xué)特性的相關(guān)關(guān)系。結(jié)果表明,冷膠粘度(CPV)、崩解值(BD)、回復(fù)值(CS)等糊化特性與LPLs存在顯著相關(guān)性。除焓變外,熱學(xué)特性一般與LPLs無顯著相關(guān)性。通過偏相關(guān)分析排除表觀直鏈淀粉含量(AAC)的影響后,發(fā)現(xiàn)CPV與個(gè)別LPLs組分仍然存在顯著正相關(guān),BD、CS和個(gè)別LPLs組分仍然呈顯著負(fù)相關(guān)。這些結(jié)果表明,稻米淀粉LPLs能以獨(dú)立的或與直鏈淀粉結(jié)合的方式,影響稻米糊化特性。4.提高磷素(P)吸收和利用效率有助于改善稻米營養(yǎng)與品質(zhì),然而水稻籽粒中磷素運(yùn)輸與分配的分子機(jī)理尚不清晰。利用5份低植酸水稻(low phytic acid,lpa)突變體來探究水稻種子中植酸、溶血磷脂等主要的含磷化合物及無機(jī)磷的變化。結(jié)果表明,lpa突變體的植酸、植酸磷含量顯著降低,而無機(jī)磷含量明顯升高。OsST和OsMIK基因突變導(dǎo)致水稻籽粒中個(gè)別LPLs組分和相應(yīng)的溶血磷脂-磷含量(LPL-P)顯著減少。與之相反,OsMRP5和Os2-PGK基因突變則引起除LPC14：0外,其他LPLs組分及相應(yīng)的LPL-P含量顯著增加。以KBNT-lpa和嘉禾218雜交后代9個(gè)株系為材料,用于驗(yàn)證Os2-PGK基因?qū)�LPLs合成與積累的影響。結(jié)果證明,9個(gè)株系中l(wèi)pa突變體的總?cè)苎�磷脂含量仍然高于正常株系的含量。該結(jié)果表明,水稻植酸和磷脂的生物合成過程存在某種交互聯(lián)系,而這種聯(lián)系很可能是通過籽粒中無機(jī)磷的分配與再分配來實(shí)現(xiàn)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.
【學(xué)位授予單位】：浙江大學(xué)
【學(xué)位級(jí)別】：博士
【學(xué)位授予年份】：2016
【分類號(hào)】：S511
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本文編號(hào)：1921858