【作者】 王金英；

【导师】 郑金贵；

【作者基本信息】 福建农林大学 ， 作物遗传育种， 2009， 博士

【摘要】 MT(melatonin,中文译为褪黑素)是一种吲哚类色胺(N-乙酰-5-甲氧基色胺),最早是从牛的松果体中发现的。褪黑素是一种生物进化保留分子,广泛存在于包括人类在内的动物、植物和原核生物中。MT是目前已知的抗氧化作用最强的内源性自由基清除剂,而且通过食用含有褪黑素的食物,可使其进入动物和人体中,起到清除体内自由基、提高机体免疫功能、延缓衰老和改善睡眠等功效。以玉米(Zea mays L.)、水稻(Oryza sativa L.)、燕麦(Avena sativa L.)作为试验材料,研究内容和目的是:研究探索出一种农作物籽粒中褪黑素的有效分离、提取方法和建立测定作物籽粒中有效成分褪黑素的高效液相色谱法分析方法;分析测定玉米、水稻、燕麦三种作物种间、品种间籽粒中褪黑素含量及其差异;研究在不同种植环境下作物籽粒中褪黑素含量的变化,研究筛选出褪黑素含量高而且稳定的作物种质资源;研究提高玉米籽粒褪黑素含量的外源调控诱导配套技术。主要研究结果如下:1.建立测定作物籽粒中褪黑素的高效液相色谱法-荧光检测(HPLC-FD)分析方法,结果表明,褪黑素在0.01~1.20 mg/kg浓度范围内,线性关系良好,其标准曲线的回归方程为Y=643436X-2446.1,R2=0.9998,平均回收率达94.11%。所需测定条件:Alltima C18, 4.6mm×250mm不锈钢柱为色谱柱,50mM Na2HPO4 /H 3PO4缓冲液(PH4.5):甲醇=50:50为流动相,流速为1ml/min,进样量10μL,激发光波长为280nm,发射光波长为348 nm。研究出玉米籽粒中褪黑素分离、提取效率较高的方法,超声功率为800W左右,超声时间30-60min的破碎籽粒处理,可使籽粒细胞中的褪黑素有效成份得到较为充分释放并溶于甲醇溶剂中。2.研究测定132份玉米品种的籽粒、145份水稻品种的糙米和35份燕麦品种的籽粒中褪黑素含量。结果表明,玉米品种平均含量为96.5 ng/g,变幅为0～2034 ng/g,MT最高玉米品种YM056,为2034 ng/g,是玉米平均MT水平的21.08倍;燕麦平均为98.7 ng/g,变幅为0～568 ng/g,含量最高的燕麦品种是OAT07,为568 ng/g,是燕麦平均MT水平的5.75倍;水稻平均为16.0 ng/g,变幅为0～264 ng/g,MT最高水稻品种SD001,为264 ng/g,是水稻平均MT水平的16.5倍。表现出不同作物种间、同一种作物不同品种间MT含量都存在很大的差异。3.分析了小米(Setaria italica)、荞麦(Fagopyrum esculentum)、黄豆(Glycine mdx Merrill)、绿心豆(Glycine max var)、莲子(Nelumro nucifera)等10种杂粮和豆类作物籽粒的褪黑素含量,其中,绿心豆含褪黑素302 ng/g,莲子为67 ng/g、小米为25 ng/g,其它七种作物褪黑素含量都低于20 ng/g。4.水稻糙米种皮颜色与MT含量具有相关性,即红色米的糙米和黑色米的糙米中MT含量显著高于白色米的糙米中的MT含量。测试的红色米和黑色米的糙米品种共22份,其中11份(占50%)糙米品种含有MT,平均含量为134.18 ng/g;测试白色米糙米品种123份,其中14份(占11.34%)白色米糙米品种含有MT,平均含量为60.50 ng/g。5.研究筛选出MT含量高且三年重复种植含量稳定的玉米品种三份:YM115(906.0 ng/g)、YM046(792.0 ng/g)和YM-N01(644.0 ng/g);水稻品种中MT含量高且在三季种植下含量稳定的有八份:SD001(249.67 ng/g)、SD075(249.33 ng/g)、SD103(190.33 ng/g)、SD093(197.00 ng/g)、SD025(189.33 ng/g)、SD067(170.00 ng/g)、SD125(157.00 ng/g)、SD119(142.67 ng/g)。6.选取经测定含有MT的水稻品种15份分别于早季和晚季种植,结果得出,早、晚季种植的糙米中平均MT含量分别为132.3 ng/g和144.2 ng/g,经显著性比较,晚稻糙米平均MT含量显著高于早稻。7.玉米苗期(六叶一心)进行外源诱导处理,诱导5d后分别取玉米叶片测定MT含量,结果表明,用3号配方(诱导物ID01、浓度为150mg/kg)和4号配方(诱导物ID01、浓度为300mg/kg),对供试6个玉米品种的诱导效果均较佳,平均提高玉米叶片MT含量分别达50.59%和69.05%。而其它配方诱导效果只对特定的品种有显著效果,如1号配方(诱导物ID01、浓度为5mg/kg)和2号配方(诱导物ID01、浓度为50mg/kg)对玉米品种YM148、YM115和YM139效果显著;5号配方(诱导物ID02、浓度166.7 mg/kg)对玉米品种YM056提高23.08%;6号配方(诱导物ID02、浓度1667 mg/kg)和7号配方(诱导物ID02、浓度10000mg/kg)对玉米品种YM056、YM049和YM115有显著效果,而对其它品种效果并不显著。玉米苗期进行外源诱导处理,成熟时玉米籽粒中MT含量与对照差异均不显著。8.玉米灌浆期进行外源诱导处理,于成熟期时(诱导处理15d后)分别测定玉米籽粒、玉米苞叶和叶片中的MT含量,结果表明:(1)不同配方对玉米籽粒MT含量的影响:7号配方(诱导物ID02,浓度10000 mg/kg)、8号配方(诱导物ID02,浓度25000 mg/kg)、6号配方(诱导物ID02,浓度1667 mg/kg)可使4份玉米品种籽粒平均MT含量分别提高15.43%、10.21%和9.86%;不同配方对不同玉米品种的诱导效果不同,玉米品种YM139用10号配方、11号配方、9号配方、4号配方、3号配方、7号配方、6号配方诱导处理效果显著,分别提高25.20%、24.28%、22.06%、16.48%、13.01%、12.29%、10.74%;YM115用11号配方、10号配方、7号配方诱导后,分别提高12.44%、11.77%和11.08%;YM056用7号配方和6号配方诱导分别提高13.57%和12.05%;YM049用7号配方和6号配方诱导分别提高20.27%和10.75%。(2)不同配方对玉米苞叶MT含量的影响:3号配方(诱导物ID01,浓度150mg/kg)、4号配方(诱导物ID01,浓度300mg/kg)可使玉米苞叶MT平均含量分别提高15.44%和10.70%。其中,3号配方对玉米品种YM115、YM139、YM056和YM049苞叶MT含量分别提高19.89%、17.73%、17.11%和11.28%;2号配方(诱导物ID01,浓度50 mg/kg)使玉米品种YM115和YM139苞叶MT含量分别提高15.94%和13.01%;4号配方使YM056苞叶MT含量提高16.08%;8号配方使YM139苞叶MT含量提高10.33%。(3)不同配方对玉米叶片MT含量的影响:4号配方、3号配方和2号配方均能提高玉米叶片MT含量,平均含量分别提高18.71%、18.48%和9.63%。9.玉米不同器官,MT含量相差很大,含量高低顺序:籽粒>苞叶>叶片>根。其中,籽粒含量大大高于苞叶和叶片,苞叶和叶片含量相差不大,根部MT含量很低,用HPLC检测不到。更多还原

【Abstract】 MT (short for Melatoninm,褪黑素in Chinese ) is a indole-trytamine(N-Acetyl-5-hydroxy tryptamine). It was initially found in the pineal of bovine. MT is a highly conserved molecule during bio-evolution, which exists extensively in animals including human, in plants, and in prokaryotes. It was approved that MT is an endogenous free radical scavenger, which is the most powerful antioxidant so far. More importantly, MT in food inevitably enters animals and human through eating the food with MT and plays efficacies in purging inner free radical, enhancing body immunity, delaying aging and improving sleep, etc.The maize(Zea mays L.), rice(Oryza sativa L.) and oat(Avena sativa L.) were utilized as the materials of the experiment, and the content and purpose of this study were as follows: Seeking for an effective method on melatonin isolating and extracting from crop seeds, and setting up a high performance liquid chromatography (thereafter abbreviated as HPLC) analysis method for determining the content of the active ingredient-melatonin in crop seeds; To determine MT content in the seeds of maize, rice and oat and to analyze its differences among various species and varieties; Study on MT content variation in crop seeds under different planting environments, from which to select crop germplasm resources with high and steady MT content; Study on the exogenous inductions method which could improve MT content in maize seeds.The main results of this study were as follows:1. To establish a HPLC- FD(whose full name was Fluorescence Detection) method for determining the content of MT in crop seeds. results were showed that the linearity relationship for MT was perfect within 0.01~1.20 mg/kg. The regression equation was Y=643436X-2446.1 and R2=0.9998, the average recovery rate was 94.11%. determination terms were required in this method: Alltima C18 , 4.6mm×250mm was as chromatographic column. The mobile phase was constituted by 50mM Na2HPO4 /H 3PO4 buffer(PH4.5):methanol=50:50, at a flow rate of 1.0ml/min,10μL extracted samples were injected for the analyses. The excitation wavelength was 280nm and the emission wavelength was 348 nm.Studied out the method which isolated and extracted effectively MT from maize seeds, that is, seeds was crushed by 800W or so supersonic power and 30-60min supersonic time. This method could effectively make MT release from seed cells and dissolve into methanol solvent.2. To determine MT level in seeds of 132 maize varieties, in unpolished rice of 145 rice varieties and in seeds of 35 oat varieties. The results were: mean content of MT in maize varieties was 96.5ng/g, range was 0～2034ng/g, variety with highest content of MT (2034ng/g) was YM056, which was 21.08 times of mean MT content in maize; mean content of MT in oat varieties was 98.7ng/g, range was 0～568ng/g, variety with highest content of MT (568ng/g) was OAT07, which was 5.75 times of mean MT content in oat; mean content of MT in rice varieties was 16.0ng/g, range was 0～264ng/g, variety with highest content of MT (264ng/g) was SD001, which was 16.5 times of mean MT content in rice. Results above showed that content of MT differed greatly among various crop species and varieties.3. To analyse MT content of seed in 10 kinds of minor cereals and legume crops such as millet(Setaria italica), buckwheat(Fagopyrum esculentum), soybean(Glycine mdx Merrill), black bean(Glycine max var), lotus seed(Nelumro nucifera), etc. Results: MT content was 302ng/g in black bean(Glycine max var), was 67 ng/g in lotus seed(Nelumro nucifera) and was 25ng/g, in millet(Setaria italica). MT content in the other 7 crops were all lower than 20 ng/g.4. There was a correlation between different seed coat colors of unpolished rice and MT content of them, that is, MT content in red (or black) unpolished rice was significantly higher than that in white unpolished rice. 22 red (or black) unpolished rice were analyzed, MT was present in 11 of them (50%), mean content of MT was 134.18ng/g; 123 white unpolished rice were analyzed, MT was present in 14 of them (11.34%), mean content of MT was 60.50ng/g.5. To select 3 maize varieties with high MT content and steadily in 3 years repeat: YM115(906.0 ng/g), YM046(792.0ng/g )and YM-N01(644.0ng/g), and 8 rice varieties with high MT content and steadily in 3 seasons planting repeat: SD001 (249.67ng/g)、SD075(249.33ng/g)、SD103(190.33ng/g)、SD093(197.00ng/g)、SD025 ( 189.33ng/g )、SD067 ( 170.00ng/g )、SD125 ( 157.00ng/g )、SD119(142.67ng/g).6. Selecting 15 rice varieties with high MT content, which were planted in early season and late season of year 2006 respectively. Results were that, mean MT content in unpolished rice planted in early and late season were 132.3ng/g and 144.2ng/g accordingly, and mean content of MT in unpolished rice planted in late season was significantly higher than that in early season , by significance comparison.7. Maize was sprayed by exogenous inductions, during seeding stage (leaves 6, core 1), and leaves of the maize was tooken to test the MT content after 5 days induction, results indicates that, as for 6 varieties of maize, with inducing formula No.3(inducer ID01, concentration 150mg/kg) and inducing formula No.4(inducer ID01, concentration 300mg/kg), effects of both were fine, MT content in maize leaves were improved averagely 50.59% and 69.05% respectively. And other induce treatments only had significant effects on given varieties. For example, inducing formula No.1(inducer ID01, concentration 5mg/kg) and inducing formula No.2(inducer ID01, concentration 50mg/kg) had significant effects on maize varieties YM148,YM115 and YM139; inducing formula No.5(inducer ID02, concentration 166.7mg/kg) could improve MT content in maize variety YM056 23.08%; inducing formula No.6(inducer ID01, concentration 1667mg/kg) and inducing formula No.7(inducer ID02, concentration 10000mg/kg) had significant effects on maize varieties YM056,YM049 and YM115, however, had non-significant effect on other varieties.Maize was sprayed by exogenous inductions, during seeding stage, MT content in maize seeds of maturity was all non-significantly differed from CK.8. maize during filling stage was Induced by exogenous inductions, and to determine MT contents in maize seeds, maize bracts and leaves in maturity stage (15d after inducing treatment), the results showed as follows:(1) Impacts on MT content in maize seeds by exogenous inductions: inducing formula No.7(inducer ID02 and concentration 10000mg/kg), inducing formula No.8(inducer ID02 and concentration 25000mg/kg), inducing formula No.6(inducer ID02 and concentration 1667mg/kg) could improve mean contents of MT in 4 maize varieties seeds to 15.43%,10.21% and 9.86% respectively;various maize varieties had different inducing effects, maize variety YM139 had significant inducing treatment effects with formula No.10, No.11, No.9, No.4, No.3, No.7, No.6, improved 25.20%, 24.28%, 22.06%, 16.48%, 13.01%, 12.29%, 10.74% accordingly;after inducing treatment with formula No.11, No.10, No.7, MT content in variety YM115 improved 12.44%, 11.77%, 11.08% accordingly;after inducing treatment with formula No.7, No.6, MT content in variety YM056 improved 13.57% and 12.05%, accordingly;after inducing treatment with formula No.7 and No.6, MT content in variety YM049 improved 20.27% and 10.75% respectively;(2) Impact on MT content in maize bracts by exogenous inductions: inducing formula No.3 (inducer ID01 and concentration 150 mg/kg), inducing formula No.4 (inducer ID01 and concentration 300 mg/kg) could increase mean content of MT in maize bracts 15.44% and 10.70% respectively. Of which, formula No.3 inducing treatment to maize varieties YM115, YM139, YM056 and YM049 could improve MT content in maize bracts 19.89%, 17.73%, 17.11% and 11.28%; inducing treatment to maize varieties YM115 and YM139 with inducing formula No.2 (inducer ID01 and concentration 50mg/kg) could improve MT content in maize bracts 15.94% and 13.01% accordingly; inducing treatment to maize variety YM056 with inducing formula No.4 could improve MT content in maize bracts 16.08%; inducing treatment to maize variety YM139 with inducing formula No.8 could improve MT content in maize bracts 10.33%;(3) Impact on MT content in maize leaves by exogenous inductions: all of 3 inducing treatments with inducing formula No.4, No.3, No.2 could improve MT content in maize leaves, mean contents of MT was improved 18.71%, 18.48% and 9.63% respectively.9. MT contents in different organs of maize were greatly different, MT content was, orderly, seeds>bracts>leaves>roots. Of which, MT content in seeds was higher greatly than that in bracts and leaves, there was no significant difference between bracts and leaves, MT content in roots was too low to be detected with HPLC.更多还原