【作者】 张晶；

【导师】 尹永祺； 杨嘉；

【作者基本信息】 扬州大学 ， 农业硕士（专业学位）， 2025， 硕士

【摘要】 鸡爪谷（Eleusine coracana L.）是一种耐寒耐旱的特色杂粮作物,因其丰富的营养价值备受关注。鸡爪谷籽粒中富含蛋白质、膳食纤维和黄酮类化合物、多酚等生物活性成分。黄酮类化合物在人体中展现出抗炎、抗菌、抗氧化以及抗病毒等多种药理活性。因此提升鸡爪谷中黄酮含量并以其为原料开发营养保健食品受到关注。研究表明,植物籽粒在外源处理条件下发芽,尤其是硫酸锌（Zinc sulfate,ZnSO₄）或茉莉酸甲酯（Methyl jasmonate,Me JA）等处理能够促进植物体内黄酮的生物合成。水杨酸（Salicylic Acid,SA）和γ-氨基丁酸（γ-aminobutyric acid,GABA）作为重要的植物生长调节剂,不仅参与调控植物生长发育,增强非生物胁迫抗性,而且诱导次级代谢产物的合成。本文优化鸡爪谷发芽工艺,从生理代谢、酶活力和基因表达水平三个层面探究发芽及外源化合物处理调控鸡爪谷芽苗中黄酮生物合成及其生理代谢调控作用,以期为提高鸡爪谷的营养价值、拓展鸡爪谷原料的应用提供理论依据和技术支撑。主要研究结果如下:1、优化了鸡爪谷发芽富集黄酮的工艺。发芽时间、发芽温度和光照时长均显著影响鸡爪谷中黄酮含量（p<0.05）,通过响应面法优化得到最佳发芽条件为:发芽时间5.7 d,发芽温度31.0℃,光照时长17.4 h,在此条件下鸡爪谷芽苗中黄酮含量最高达7.10μg/株。2、研究了鸡爪谷发芽期间生理代谢和黄酮生物合成的变化。鸡爪谷在发芽5 d内,黄酮含量持续增加,3日龄和5日龄鸡爪谷芽苗中黄酮含量分别是籽粒的4.71和6.15倍。鸡爪谷发芽后总酚和花色苷含量显著增加（p<0.05）。鸡爪谷发芽过程中过氧化氢酶（Catalase,CAT）、过氧化物酶（Peroxidase,POD）、抗坏血酸过氧化物酶（Ascorbate peroxidase,APX）和超氧化物歧化酶（Superoxide dismutase,SOD）活力及其对应的基因表达量均显著提高（p<0.05）,表明发芽激活了鸡爪谷芽苗抗氧化系统。同时,发芽显著增强了3日龄和5日龄鸡爪谷芽苗中苯丙氨酸解氨酶（Phenylalanine ammonia-lyase,PAL）、肉桂酸4-羟化酶（Cinnamic acid 4-hydroxylase,C4H）和4-香豆素辅酶A连接酶（4-Coumarate:Co A Ligase,4CL）活力并上调了黄酮合成代谢相关基因（Ec PAL、Ec C4H和Ec4CL等）的相对表达量,表明发芽通过提高黄酮合成关键酶活力及合成相关基因的表达量促进了鸡爪谷芽苗中黄酮的合成积累。3、研究了ZnSO₄/Me JA处理对鸡爪谷芽苗生理代谢和黄酮富集的影响。筛选得到适宜富集鸡爪谷芽苗黄酮的处理条件为5 m M ZnSO₄和150μM Me JA。ZnSO₄/Me JA处理显著提高了鸡爪谷芽苗中黄酮含量（p<0.05）,在发芽6 d时,ZnSO₄/Me JA处理下黄酮含量分别为对照的1.21和1.22倍。鸡爪谷芽苗经ZnSO₄/Me JA处理后,H₂O₂、O₂^￣·和MDA含量增加,芽长和鲜重下降,表明ZnSO₄/Me JA对鸡爪谷芽苗造成了氧化损伤和生长抑制。ZnSO₄和Me JA处理均显著提高了CAT、POD和APX活力及相应基因的相对表达量（p<0.05）,增强了抗氧化能力。ZnSO₄处理显著提高PAL、4CL和C4H活力并上调Ec PAL、Ec C4H、Ec4CL等基因的相对表达量。Me JA显著提高PAL和C4H活力并上调Ec PAL、Ec C4H等基因的相对表达量。胁迫相关转录因子Ec MYB和Ec NAC在ZnSO₄/Me JA处理下均显著上调（p<0.05）。ZnSO₄和Me JA处理对鸡爪谷芽苗中黄酮合成的调控呈现一定的差异,但均通过增强黄酮合成关键酶活力及相关基因的相对表达量来促进黄酮的生物合成。4、研究了外源SA/GABA对ZnSO₄处理下鸡爪谷芽苗生理代谢和黄酮富集的影响。筛选得到ZnSO₄处理下适宜富集鸡爪谷芽苗黄酮的外源添加物及其作用浓度为50μM SA和1 m M GABA。外源SA/GABA均显著增加了ZnSO₄处理下鸡爪谷芽苗中黄酮含量并改善了芽苗的生长形态,但对生理代谢的调控有所不同。其中,SA显著降低了鸡爪谷芽苗中MDA、H₂O₂和O₂^￣·含量,而GABA显著降低了H₂O₂和O₂^￣·含量（p<0.05）。同时,SA显著增强了CAT、SOD和APX活力并上调Ec CAT、Ec SOD和Ec APX相对表达量,而GABA显著增强了CAT、SOD、APX和POD活力并上调Ec CAT、Ec SOD、Ec POD和Ec APX相对表达量（p<0.05）,表明外源SA/GABA均能通过增加抗氧化酶活力及其基因表达量,提高抗氧化能力。此外,ZnSO₄处理下,外源SA/GABA通过增强PAL和C4H活力及提高Ec PAL、Ec C4H等基因的相对表达量来促进黄酮积累（p<0.05）。结果表明,SA/GABA处理通过增强苯丙烷代谢途径中关键酶的活性及其基因表达水平,从而促进黄酮类化合物的富集。更多还原

【Abstract】 Finger millet（Eleusine coracana L.）is a hardy and drought-resistant speciality mixed grain crop that has attracted much attention for its rich nutritional value.The seeds of finger millet are rich in protein,dietary fibre,flavonoids,polyphenols and other bioactive components.Flavonoids have anti-inflammatory,antibacterial,antioxidant and antiviral pharmacological effects on the human body,so the enhancement of the flavonoid content of finger millet and its raw materials for the development of nutritious health food had received attention.Studies had shown that germination of plant seeds under exogenous treatments,especially ZnSO₄ or Methyl jasmonate（Me JA）,could promote flavonoid biosynthesis in plants.Salicylic acid（SA）andγ-aminobutyric acid（GABA）,as important plant growth regulators,were not only involved in the regulation of plant growth and development and enhancement of abiotic stress resistance,but also induced the synthesis of secondary metabolites.In this paper,we optimized the germination process of finger millet,and explored the mechanism of flavonoid biosynthesis and its physiological metabolism regulation in finger millet sprouts by germination and exogenous compounds treatment from the three levels of physiological metabolism,enzyme activity and gene expression,with a view to providing theoretical basis and technical support for improving the nutritional value of finger millet and expanding the application of finger millet as raw material.The main research results are as follows:1.The process of flavonoid accumulation by germination of finger millet was optimised.The germination time,germination temperature and illumination duration all significantly affected the flavonoid content of finger millet（p<0.05）,and the optimum germination conditions were optimised by response surface methodology as follows:germination time of5.7 d,germination temperature of 31.0°C,and illumination duration of 17.4 h.The flavonoid content of finger millet n sprouts was the highest of 7.10μg/sprout under these conditions.2.Changes in physiological metabolism and flavonoid biosynthesis during germination of finger millet were studied.The flavonoid content of finger millet continued to increase during 5 d of germination time,and the flavonoid content in 3-and 5-day-old finger millet sprouts was 4.71 and 6.15 times higher than that of seeds,respectively.The contents of total phenolic and anthocyanin increased significantly in the finger millet after germination（p<0.05）.The activities and gene expressions of catalase（CAT）,peroxidase（POD）,ascorbate peroxidase（APX）and superoxide dismutase（SOD）were significantly increased（p<0.05）during germination of finger millet germination,indicating that germination activated the antioxidant system of finger millet sprouts.Meanwhile,germination significantly enhanced the activities of phenylalanine ammonia-lyase（PAL）,cinnamic acid 4-hydroxylase（C4H）,and 4-coumarate:Co A ligase（4CL）in 3-and 5-day-old finger millet sprouts and up-regulated the relative expression of flavonoid anabolism-related genes（Ec PAL,Ec C4H,and Ec4CL,etc.）,suggesting that germination promotes flavonoid synthesis and accumulation in finger millet sprouts by increasing the activity of key flavonoid synthesis enzymes and the expression of synthesis-related genes.3.The effects of ZnSO₄/Me JA treatment on the physiological metabolism and flavonoid accumulation of finger millet sprouts were investigated.The appropriate conditions for flavonoid accumulation were 5 m M ZnSO₄ and 150μM Me JA,and ZnSO₄/Me JA treatment significantly increased the flavonoid content in the sprouts（p<0.05）,and the flavonoid content in the sprouts after ZnSO₄/Me JA treatment was 1.21 and 1.22 times higher than that of the control at the germination time for 6 d,respectively.The increase in the content of H₂O₂,O₂^￣·and MDA and the decrease in sprout length and fresh weight after ZnSO₄/Me JA treatment in finger millet sprouts indicated that ZnSO₄/Me JA caused oxidative damage and growth inhibition in finger millet sprouts.Both ZnSO₄ and Me JA treatments significantly increased the activities of CAT,POD and APX and the relative expression of the corresponding genes（p<0.05）.ZnSO₄ treatment significantly increased the activities of PAL,4CL and C4H and up-regulated the relative expression of genes such as Ec PAL,Ec C4H,and Ec4CL,etc.Me JA significantly increased the activities of PAL and C4H and up-regulated the relative expression of genes such as Ec PAL,and Ec C4H.The stress-related transcription factors Ec MYB and Ec NAC were both significantly up-regulated by ZnSO₄/Me JA（p<0.05）.The regulation of flavonoid synthesis in finger millet sprouts by ZnSO₄ and Me JA treatments showed some differences,but both promoted flavonoid biosynthesis by increasing the activity of the key enzymes for flavonoid synthesis and the relative expression of related genes.4.The effects of exogenous SA/GABA on the physiological metabolism and flavonoid accumulation in ZnSO₄-treated finger millet sprouts were investigated.The exogenous additions of 50μM SA and 1 m M GABA were screened to be suitable for flavonoid enrichment in ZnSO₄-treated finger millet sprouts,and the concentrations of these additives were 50μM SA and 1 m M GABA,respectively.Both exogenous SA/GABA significantly increased the content of flavonoid and improved the growth morphology of finger millet sprouts under ZnSO₄ treatment,but the regulation of physiological metabolism was different.Among them,SA significantly decreased the contents of MDA,H₂O₂,and O₂^￣·in finger millet sprouts,while GABA significantly decreased the contents of H₂O₂,and O₂^￣·（p<0.05）.Meanwhile,SA significantly enhanced the activities of CAT,SOD and APX and up-regulated relative expression of Ec CAT,Ec SOD and Ec APX,while GABA significantly enhanced the activities of CAT,SOD,APX and POD and up-regulated the relative expression of Ec CAT,Ec SOD,Ec POD and Ec APX（p<0.05）,indicating that both exogenous SA/GABA could improve antioxidant capacity and alleviate oxidative damage by ZnSO₄ treatment by increasing the activities and gene expression of antioxidant enzyme.In addition,under ZnSO₄treatment,exogenous SA/GABA promoted flavonoid accumulation by enhancing the activities of PAL and C4H and increasing the relative expression of Ec PAL and Ec C4H genes（p<0.05）,suggesting that SA/GABA promotes flavonoid enrichment by further activating of the activity and gene expression of key enzymes of the phenylpropanoid pathway.更多还原