【作者】 金丽虹；

【导师】 田坚；

【作者基本信息】 长春理工大学 ， 材料物理与化学， 2011， 博士

【摘要】 利用氩离子(Ar+)激光(波长分别为457、465、472、477、488、496、502、514nnm,功率密度=5.7mW/mm2)辐照高异黄酮品种大豆的萌发胚芽1、3、5、7min,通过对比分析水培激光辐照组(L)与对照组(ck)幼苗期的子叶和叶片的光合特性、蛋白质代谢、异黄酮含量和抗氧化性等差异,系统地讨论了大豆幼苗经Ar+激光辐照后生物学性能的变化规律。旨在较全面、深入了解低功率Ar+激光对生物组织的相互作用机理,为选育高异黄酮含量大豆提供理论和实验依据。完成的工作主要包括以下几个方面：1.大豆异黄酮含量与品质性状的相关性研究对来源不同的七个品种大豆中的6种异黄酮组分(染料木素、染料木苷、大豆苷元、大豆苷、黄豆黄素和黄豆黄苷)、蛋白质(清蛋白、谷蛋白、球蛋白、醇蛋白、7S球蛋白、11S球蛋白和总蛋白质)、粗脂肪和5种脂肪酸(软脂酸、硬脂酸、油酸、亚油酸和亚麻酸)进行定量分析。结果表明,不同品种大豆的异黄酮含量与蛋白质、脂肪含量相关性有所差异。其中,(1)6种大豆异黄酮分别与清蛋白、11S球蛋白、总蛋白质、蛋脂总量呈程度不同的正相关性,与谷蛋白、球蛋白和醇蛋白呈负相关性；(2)6种大豆异黄酮与亚油酸、亚麻酸均呈正相关,且与亚油酸的正相关性显著；(3)染料木素、染料木苷与大豆粗脂肪呈负相关性,而大豆苷元、大豆苷、黄豆黄素和黄豆黄苷与大豆粗脂肪呈正相关性。2.Ar+激光辐照对大豆幼苗生理生化指标的影响低功率Ar+激光辐照吉农19大豆种子胚芽后,其浸泡液电导率、含水速率,幼苗的株高、叶绿素、光合碳同化产物、可溶性蛋白质、热稳定性蛋白质、总游离氨基酸含量和碳酸酐酶(CA)、内源蛋白酶活性均有别于对照组(ck),激光辐照可以影响质膜、幼苗株高以及光合同化作用、蛋白质代谢。结果表明：457～502nm激光辐照可有效地修复了水胁迫下大豆种子质膜损伤,降低细胞内物质外流；在32组激光辐照组中,502nm激光辐照5min可有效地促进叶绿素的合成(10.09%)；在提高Chla/Chlb比值(16.78%)、CA活性(118.25%)、热稳定性蛋白质含量(47.47%)、游离氨基酸含量(12.33%)的同时,降低了内源蛋白酶活性(34.85%)和幼苗株高(1.43%)；即激光辐照可以在加速大豆幼苗光合碳同化产物、蛋白质代谢的同时,加速幼苗体内代谢进程。3.Ar+激光辐照对大豆幼苗异黄酮代谢的影响利用HPLC法对Ar+激光辐照组大豆幼苗子叶和叶片中染料木素、染料木苷、大豆苷元、大豆苷、黄豆黄素和黄豆黄苷6种异黄酮组分含量进行测定,其结果表明：32组激光辐照组中,50%幼苗子叶、15.6%幼苗叶片中6种异黄酮组分含量均高于对照组,其中以502nm激光辐照3min效果最为明显,大豆幼苗子叶、叶片中6种异黄酮物质合成的增加幅度分别为44.06%和117.46%：Ar~+激光辐照提高了大豆幼苗中染料木素、染料木苷、大豆苷元、大豆苷含量的同时,降低了黄豆黄素、黄豆黄苷含量,有效地提高了大豆幼苗生理活性强异黄酮物质比例；激光辐照影响了异黄酮合成前体物质苯丙氨酸(Phe)含量及异黄酮生物合成途径苯丙氨酸转氨酶(PAL)、肉桂酸羟化酶(C4H)、辅酶A连接酶(4CL)、异黄酮合成酶(SIF)活性,从而促进了异黄酮生物合成。4.Ar~+激光辐照对大豆幼苗抗氧化性的影响吉农19萌发大豆种子胚芽经Ar+激光辐照后,其幼苗子叶和叶片中超氧化岐化酶(SOD)、过氧化物酶(POD)、过氧化氢酶(CAT)三种酶类抗氧化剂活性,抗坏血酸(AsA)、类胡萝卜素(Car)、脯氨酸(Pro)、紫外吸收物、还原型谷胱甘肽(GSH)非酶抗氧化物含量和膜脂过氧化产物之一丙二醛(MDA)含量均有别于对照组(ck),激光辐照可影响大豆幼苗酶类抗氧化剂活性及非酶类抗氧化剂含量。结果表明：激光辐照可以显著增加大豆幼苗SOD、CAT、POD活性及AsA、Car、Pro和紫外吸收物含量,降低叶片GSH、子叶Pro含量,提高了子叶GSH、叶片Pro含量；激光波长、辐照时间是影响幼苗抗氧化能力的两个因素,502nm激光辐照3min分别降低了子叶、叶片过氧化水平49.64%和65.56%,而496nm激光辐照1min分别降低了子叶、叶片过氧化水平73.27%和78.21%。因此,Ar+激光辐照大豆种子胚芽可以提高其幼苗的抗氧化性。更多还原

【Abstract】 The germinating germ of soybean with high isoflavone content were irradiated by 5.7mW/mm2 Ar+ laser with wavelenth of 457,465,472,477,488,496,502,514 nm for different times of 1,3,5 and 7 minute(s). By comparing the differences of their biological characteristics between the experimental group (L) and the control (ck), such as seedling cotyledon and leaf photosynthesis, protein metabolism, isoflavone content and antioxidation, some systematic studies had been done about the biological effects before and after the irradiation. The aim was to fully understand biological stimulation mechanism of the low-power Ar+ laser irradiated on biomass and to provide some theoretical and experimental basis on breeding soybean with high isoflavone content by way of laser irradiation. Completed work includes the following main aspects:1. Study on correlations between isoflavones content and quality of soybeanIsoflavone components (genistein, genistin, daidzein, daidzin, glycitein and glycitin), proteins (albumin, glutelin, globulin, gliadin,7S globulin,11S globulin and total protein), crude fat and five fatty acids (palmitic acid, stearic acid, oleic acid, linoleic acid and linolenic acid) in seven different soybean germplasms were used for quantitative analysis. The results showed that:there were different correlations between isoflavone content and protein content and oil content in different soybean germplasms. Among them, (1) the six isoflavone components content had different significant positive correlations with the content of albumin, 11S globulin, total protein, total fat, and negative correlations with the content of glutelin, globulin, gliadin respectively. (2) the six soybean isoflavone components were positively correlated with linoleic acid and linolenic acid significantly, especially linoleic acid. (3) genistein, genistin were negatively correlated with soybean fat, while daidzein, daidzin, glycitein and glycitin had some positive correlations with soybean fat. After quality analysis of seven different soybean germplasms, Jinong 19, in which the total content of six isoflavones were 2437.28μg/g, was selected as an experimental material for the study of biological effects with Ar+ laser irradiation on soybean seedlings.2. Effect of Ar+ laser irradiation on the soybean seedlings in physiological and biochemical propertiesWhen the germ of the soybean seed (named as Jinong 19) were being irradiated by a low-power Ar+ laser, its conductivity of the soaking solution, moisture content, plant height, carbonic anhydrase (CA) activity, chlorophyll, photoassimilate, endogenous protease activity, soluble protein, the heat-stable protein, total free amino acids content behaved remarkable difference from the control group (ck). Laser irradiation could affect the plasma membrane, plant height and photosynthetic assimilation, protein metabolism. The results showed that: 457-502nm laser irradiation could effectively repair the plasma membrane injuries of soybean seeds under water stress, reduce the outflow of intracellular material. And among 32 sets of laser irradiation groups (L), laser irradiation with 502nm for 5 minutes could be effectively in promoting chlorophyll synthesis (10.09%), raising the Chla/Chlb ratio (16.78%), CA activity (118.25%), heat-stable protein content (47.47%) and total free amino acids content (12.33%), while reducing the endogenous protease activity (34.85%) and plant height (1.43%). Laser irradiation accelerates the product of soybean seedling photoassimilate, protein metabolism, at the same time, accelerates the process of metabolism of the seedlings.3. Effect of Ar+ laser irradiation on soybean seedlings in isoflavones metabolismHPLC method was applied in the measure of content of the six kinds of isofavone: genistein, genistin, daidzein, daidzin, glycitein, glycitin in the soybean seedlings cotyledons and leaves after Ar+ laser irradiation. The results showed that:among 32 irradiation groups, the content of 6 isoflavone components in seedling cotyledons and in leaves were 50% and 15.6% higher than that in ck respectively. The irradiation with 502nm for 3 minutes could be effective in promoting 6 isoflavone synthesis in soybean seedling cotyledons and leaves. And the increase rates were 44.06% and 117.46% respectively. There were certain constraints between biosynthesis system of the glycitein and glycitin, The activity of PAL, C4H,4CL, SIF and Phe content in soybean seedlings were the influencing factors of the level of the isoflavone content, while laser irradiation could change the content of Phe, which was the precursors substance of isoflavone, and the activity of related enzymes in biosynthesis. The impact differs from the laser wavelength and irradiation time. Phe level is the main factor, while the PAL, C4H,4CL, SIF activity have positive correlations with isoflavone content.4. Effect of Ar+ laser irradiation on soybean seedlings in antioxidationThe activity of super oxide dismutase (SOD), peroxidase (POD), catalase (CAT), the content of ascorbic acid (AsA), carotenoids (Car), proline(Pro), UV-absorbing compounds, glutathione(GSH) and malondialdehyde (MDA) in cotyledons and leaves of Jinong 19 seedlings were different from the control group (ck) after the germinating soybean seeds germ were irradiated by Ar+ laser. Antioxidant enzyme activity and non-enzyme antioxidant content of soybean seedlings changed evidently. The results showed that:the activity of SOD, CAT, POD and the content of AsA, Car, Pro, and UV-absorbing compounds in soybean seedlings were significantly increased. The content of GSH in leaves, Pro in cotyledons were reduced, while the content of GSH in cotyledons, Pro in leaves increased. Laser wavelength and irradiation time were two main factors which could affect antioxidant capacity. Laser wavelength of 477,488,496 and 502nm and the laser irradiation time of 5,7 minutes could reduce the peroxidation of seedlings. Therefore, antioxidation of soybean seedling could be improved by Ar+ laser irradiation on seed germ.更多还原