【作者】 于翠；

【导师】 吕德国；

【作者基本信息】 沈阳农业大学 ， 果树学， 2009， 博士

【摘要】 山楂属植物的黄酮代谢物种类丰富多样,某些黄酮代谢物具有医疗或保健作用,为人类提供丰富的药物,对人类的生活具有重要作用。鉴于根系是植物体内黄酮代谢的启动部位,因此本研究以山楂（Crataegus pinnatifida Bge.）为试材,对不同生育期山楂根系黄酮成分及与黄酮合成代谢相关物质（可溶性糖和蛋白质）以及相关酶活性的变化规律进行研究;同时,研究了不同小分子有机物（谷氨酸、葡萄糖、甘露醇、草酸、L-苯丙氨酸和L-精氨酸）、缺素（氮、磷、钾、钙、镁、锌、铁）、生长调节剂（ABA、GA₃）、盐胁迫以及根系浸提液等对山楂根系黄酮代谢的调控。主要结果如下:1.山楂不同器官产生的黄酮化合物的种类和数量均不同。根系中总黄酮含量高于根系分泌液。山楂根系和根系分泌液中的黄酮成分差异较大,根系中的黄酮成分的保留时间大多集中在30 min～40 min,以亲脂性黄酮成分为主,多为游离的苷元;而根系分泌液中黄酮成分保留时间大多集中在5 min～30 min,以亲水性黄酮成分为主,多为苷类。2.不同生育期山楂各器官的黄酮成分与含量不同,新梢停长期根系分泌液中黄酮成分与含量最高,并检测到3种已知黄酮成分,分别为牡荆素鼠李糖苷、金丝桃苷和槲皮素。展叶期与新梢迅速生长期根系分泌液中检测到2种已知黄酮成分,分别为牡荆素鼠李糖苷和槲皮素;而落叶期检测到2种,分别为芦丁和金丝桃苷。各生育期山楂根系中均检测到金丝桃苷。3.葡萄糖、谷氨酸、L-精氨酸、L-苯丙氨酸处理山楂根系及根系分泌液中黄酮成分与含量高于对照,且谷氨酸处理黄酮成分最丰富。HPLC分析表明,400 mg·L^-1谷氨酸处理根系分泌液中黄酮成分最大峰面积为25号峰,其他处理最大峰面积黄酮成分均为1号峰牡荆素鼠李糖苷或9号峰（为一未知峰）;小分子有机物处理根系分泌液中均检测到牡荆素鼠李糖苷,草酸处理除外。各处理根系最大峰面积黄酮成分为12号或45号峰,均为未知峰。4.缺氮、缺磷处理山楂根系中的总黄酮含量高于缺镁、缺锌、缺铁处理及对照,而根系分泌液中总黄酮含量则与之相反。缺镁、缺锌与缺铁处理山楂根系分泌液中黄酮成分多于对照,且均检测出牡荆素鼠李糖苷,芦丁,牡荆素,金丝桃苷和槲皮素,其中缺锌处理黄酮成分最丰富。各缺素处理山楂根系分泌液中最大峰面积黄酮成分均与对照不同,对照为10号峰,缺氮处理为12号峰,缺磷处理为25号峰,缺钾处理为16号峰,缺钙处理为26号峰,缺镁处理为20号峰,缺锌处理为15号峰,均为未知峰,而缺铁处理为3号峰牡荆素的峰面积最大。各处理根系最大峰面积黄酮成分为45号或12号或43号峰,均为未知峰。5.ABA处理山楂根系中的总黄酮含量高于GA₃处理和对照,而根系分泌液中的总黄酮含量低于GA₃处理和对照,呈负相关。GA₃处理根系分泌液中检测出已知黄酮成分为芦丁、金丝桃苷和槲皮素,ABA处理为牡荆素鼠李糖苷与金丝桃苷,而对照为牡荆素鼠李糖苷。GA₃和ABA处理根系分泌液中最大峰面积黄酮成分均为10号,而对照为9号峰。GA₃、ABA及对照处理根系中最大峰面积黄酮成分均为12号峰。6.山楂根系分泌液中总黄酮含量以0.1%盐胁迫处理最高,而根系中总黄酮含量则与之相反,以0.3%盐胁迫处理最高。各浓度盐处理根系分泌液中均检测到牡荆素鼠李糖苷和牡荆素,而对照只检测到牡荆素鼠李糖苷。0.1%与0.5%盐处理根系分泌液中最大峰面积黄酮成分均为1号峰牡荆素鼠李糖苷,而CK和0.3%盐处理最大峰面积黄酮成分均为9号峰,为一未知峰。各处理与对照根系中最大峰面积黄酮成分均为12号峰。7.根系浸提液处理提高了山楂根系分泌液中总黄酮的含量,而降低了根系中总黄酮含量。对照与10 g·L^-1根系浸提液处理山楂根系分泌液中均只检测出牡荆素鼠李糖苷,而100 g·L^-1根系浸提液处理检测到牡荆素鼠李糖苷、芦丁、牡荆素和金丝桃苷。对照与10 g·L^-1根系浸提液处理根系分泌液中最大峰面积黄酮成分均为9号峰,而100 g·L^-1根系浸提液处理为14号峰,均为未知峰。对照、10 g·L^-1与100 g·L^-1根系浸提液处理根系最大峰面积黄酮成分均为12号峰。100 g·L^-1系浸提液处理山楂根际微生物种群结构变化较剧烈,且与CK和10 g·L^-1根系浸提液处理相反。8.可溶性糖等光合作用初级产物变化规律与总黄酮相似;黄酮合成代谢关键酶PAL活性与黄酮的积累变化规律相似,呈现出一定的正相关性;而PPO活性与黄酮积累变化趋势相反,随着山楂黄酮含量的增加而降低。不同处理山楂根系呼吸途径不同,但大多以TCA循环为主。更多还原

【Abstract】 Crataegus spp.contains many kinds of flavonoids which have medical or healthy effects and play an important role in human life.In this study,the flavonoids,flavonoids metabolic-related substances（soluble sugar and protein）and enzyme activities were studied at different growth periods using hawthorn（Crataegus pinnatifida Bge.）as materials.At the same time,the effects of different organic matters（glutamic acid,glucose,mannine,oxalic acid,L-phenylalanine and L-arginine）,mineral nutrition（nitrogen,phosphorus,potassium, calcium,magnesium,zinc,iron）,growth regulators（ABA,GA₃）,salt stress as well as water extracts of hawthorn roots on fiavonoids metabolites were studied.The main results are as follows:1.The types and quantities of fiavonoids generated from roots and root exudates of hawthorn are different.The total fiavonoids content of roots is higher than root exudates.The fiavonoids contained in roots and root exudates have a significant difference.The retention time of flavonoids in roots is concentrated in 30～40 min and the main flavonoids are lipophilic,and most of them are free aglycone.The retention time of flavonoids in root exudates is concentrated in 5～30 min and the main fiavonoids are hydrophilic,and most of them are glycosides.2.The flavonoids and content in different growth periods of hawthorn were different. The total fiavonoids content of root exudates was the highest in withholding growing periods of shoot,and three fiavonoids were detected,which were rhamnosylvitexin,hyperoside and quercetin.Two flavonoids were detected in leaf-expansion period and fast growing period of shoot,which were rhamnosylvitexin and quercetin.Two flavonoids were detected in defoliation period,which were rutin and hyperoside.Hyperoside was detected in roots in each growth periods.3.The flavonoids and total flavonoids content of roots and root exudates were higher than control in exogenous glucose,glutamic acid,L-arginine and L-phenylalanine treatments in hawthorn.The maximum peak area of fiavonoids is peak 25 in 400 mg·L^-1glutamic acid treatment,while other treatments was peak 1（rhamnosylvitexin）or peak 9（unknown peak）. Rhamnosylvitexin was detected in root exudates of each organic matters treatment,except for the oxalic acid treatment.The flavonoid of maximum peak area of roots in each treatment is peak 12 or peak 45,which was unknown peak. 4.The total flavonoids content of roots in nitrogen deficiency and phosphorus deficiency treatments were higher than magnesium deficiency,zinc deficiency,iron deficiency treatments and control in hawthorn,while the flavonoids content of root exudates was lower. The flavonoids of magnesium deficiency,zinc deficiency and iron deficiency treatments were higher than control,and consisted of rhamnosylvitexin,rutin,vitexin,hyperoside and quercetin.The flavonoids were more riches in zinc deficiency treatment.The flavonoids of maximum peak area in mineral deficient treatments were different compared to control,and control was peak 10,and nitrogen deficiency treatment,phosphorus deficiency treatment, potassium deficiency treatment,calcium deficiency treatment,magnesium deficiency treatment,and zinc deficiency treatment were peak 12,25,16,26,20,and 15,separately,and all of these peaks were unknown peak,while iron deficiency treatment was peak 3 that was vitexin.The flavonoids of maximum peak area in roots were peak 45,12 or 43 that was unknown peak.5.The total flavonoids content of roots in ABA treatment was higher than GA3 treatment and control,while that of in root exudates was lower than GA₃ treatment and control and showing negative correlation.Rutin,hyperoside and quercetin were detected in root exudates of GA₃ treatment,while that of in ABA treatment was rhamnosylvitexin and hyperoside,and control only had rhamnosylvitexin.The flavonoids of maximum peak area in ABA and GA₃ treatments were peak 10,while that of in control was peak 9.The flavonoid of maximum peak area of roots in all growth regulator treatments and control was peak 12.6.The total flavonoids content of root exudates was the highest in 0.1%salt stress treatment,while that of in root was different compared to 0.1%salt stress treatment,and 0.3% salt stress treatment was the most.The rhamnosylvitexin and vitexin were detected in all salt stress treatments,while the control only consisted of rhamnosylvitexin.The flavonoids of maximum peak area of root exudates in 0.1%and 0.5%salt stress treatments was peak 1 （rhamnosylvitexin）,while in CK and 0.3%salt stress treatments was peak 9 that was unknown peak.The flavonoid of maximum peak area of roots in all salt stress treatments and control was peak 12.7.The total flavonoids content of root exudates was higher in water extract of hawthorn roots treatment compared to control,while the total flavonoids content of root was lower.10 g·L^-1water extract of hawthorn roots treatment and control consisted of rhamnosylvitexin, while 100 g·L^-1water extract of hawthorn roots treatment consisted of rhamnosylvitexin,rutin, vitexin and hyperoside.The maximum peak area flavonoids of root exudates in 10 g·L^-1water extract of hawthorn roots treatment and control was peak 9,while in 100 g·L^-1water extract of hawthorn roots treatment was peak 14 that was unknown peak.The flavonoid of maximum peak area of roots in all treatments and control was peak 12.The quantity of rhizosphere microbes in 100 g·L^-1water extract of hawthorn roots treatment changed more severe and contrary to the 10 g·L^-1water extract of hawthorn roots treatment and control.8.The flavonoids are secondary metabolites,whose formation is based on photosynthesis,and the changes of primary metabolites of photosynthesis such as soluble sugar and protein was similar to flavonoids.The changes of PAL activity were similar to the accumulation of flavonoids showing a positive correlation,while the PPO activity decreased with the increasing of flavonoids content showing a negative correlation.The root respiration pathways in all treatments were different and TCA was the main root respiration pathways in most of treatments.更多还原