【作者】 刘伟；

【导师】 朱端卫；

【作者基本信息】 华中农业大学 ， 植物营养， 2010， 博士

【摘要】 菊花是菊花(Chrysanthemum morifolium Ramat.)的头状花序,在我国中医药宝库及中医治疗史上占有重要地位。目前,随着中药材GAP的发展和人们对传统药材与健康关系的认识,菊花传统的栽培方式在逐渐地改进和提高,使其种植、生产更加趋向科学化。矿质营养元素是菊花栽培生长过程中不可或缺的因素,施用过多或者缺乏都会对菊花的生长不利,正确的施用不仅可提高菊花产量,同时还会改善其品质。本课题采用盆栽试验的方式,研究大量营养元素缺乏对菊花生长状态及次生代谢过程的影响,利用HPLC和FTIR的方法对菊花整个生育期、不同部位的次生代谢产物以及生长状态进行综合研究调查,结果为我国中药材GAP和探明药用植物次生代谢机制提供理论依据。主要研究结果如下：1.不同生育期缺氮会明显降低菊花产量,且生殖期缺氮越严重则减产幅度越大；全生育期缺氮会提高菊花头状花序与叶片中的总黄酮以及可溶性总糖含量；缺氮在影响黄酮含量的同时会影响其溶解性,即可能影响黄酮的种类；各个处理菊花的中、微量营养元素含量与相对应的黄酮含量有重要关系,其中Ca与黄酮含量在三个处理中均呈显著性相关,但处理间存在较大差别。2.HPLC图谱表明,全生育期缺氮会导致菊花叶片中的次生代谢物种类、含量出现变化,且这些变化与菊花的生育期有关；FTIR结果表明,不同生育期缺氮对菊花叶片和头状花序的图谱影响主要发生在1516和1325cm-1处,即药用菊花的第二特征区段(黄酮类化合物特征区段)。3.全生育期缺氮对菊花的生长及侧枝上的头状花序发育有严重影响。全生育期缺氮可以提高菊花在生殖期功能叶片的蒸腾速率、气孔导度和细胞间CO2浓度,但同时却降低了光合速率；全生育期缺氮促进菊花新根的生成,但根的整体发育略低于对照；根部发育以生殖期缺氮最差,表明菊花在营养生长期以后根部在一定条件下还可继续发育。4.缺氮对菊花叶片和头状花序中肉桂酸含量无影响,但可提高花序中对香豆酸的含量；全生育期缺氮可以提高苯丙氨酸在叶中的含量,但生殖期缺氮却能使其在头状花序中的含量提高；主成分分析(PCA)结果显示,不同生育期缺氮条件下,菊花的黄酮及酚类化合物的主代谢途径与正常施肥条件下不同,在正常生长条件下,这一代谢途径由底物和PAL酶决定,生殖期缺氮却由底物和PAL、4CL决定,而全生育期缺氮,则由C4H和4CL决定。5.不同生育期缺磷会明显降低菊花产量,生殖期缺磷越严重则减产幅度越大；全生育期缺磷会增加花与叶片黄酮、可溶性总糖含量；缺磷首先会导致花中黄酮含量的增加,即在生殖生长期,低磷胁迫首先会出现在菊花的生殖器官中,进而再影响到营养器官；中、微量营养元素在各处理中与黄酮的含量之间的回归方程随着生育期及施磷时期而变化,Ca与黄酮含量在三个处理中均呈显著性相关。6.不同生育期缺磷对菊花不同器官的FTIR图谱基本无影响,缺磷不会导致菊花内部新的活性基团产生；全生育期缺磷会增加菊花各器官HPLC图谱中某些峰的强度；缺磷对菊花功能叶片的影响主要表现在生殖生长期；不同生育期缺磷均会导致菊花中黄酮类物质的含量增加。7.缺磷降低菊花植株侧枝及侧蕾的发育,从而降低菊花产量；缺磷对菊花叶绿素a、b影响不大,但在营养生长期,不同生育期缺磷却能提高菊花功能叶片的蒸腾速率、气孔导度和光合作用,且生殖期缺磷还能提高叶片的胞间CO2浓度；在生殖生长期,不同生育期缺磷降低菊花的蒸腾速率、气孔导度以及胞间CO2浓度,但不同生育期缺磷提高了菊花叶片的光合作用；全生育期缺磷促进菊花根部的发育,但生殖期缺磷降低其根部的发育；不同生育期缺磷均能提高菊花根部黄酮含量。8.不同生育期缺磷条件下菊花植株中黄酮类化合物与苯丙氨酸的回归方程为Y=-294.46X+150.66(R2=0.9205,P<0.01)(对照)和Y=42.62X+2.49(R2=0.9564,P<0.01)(全生育期缺磷)；在正常施肥条件下,酚类化合物的主代谢过程中分两个主要部分,分别为主成分1(苯丙氨酸、PAL、肉桂酸和对香豆酸)(88.17%)和主成分2(4CL和C4H)(9.64%)；而在全生育期缺磷条件下的代谢主成分也为两个,分别为主成分1(苯丙氨酸、肉桂酸、对香豆酸以及C4H)(81.46%)和主成分2(PAL)(18.53%)；缺磷对酚类化合物的影响主要体现在PAL和C4H两个酶的活性方面。9.缺钾明显降低菊花头状花序中黄酮的含量,降幅达31.4%；各营养元素含量与黄酮含量有重要关系,但是其之间的回归方程不尽一致；缺钾可以降低菊花功能叶片中的蛋白质含量,但对头状花序中的蛋白质含量无影响；全生育期缺钾可以明显提高叶片及花序中的可溶性糖含量,与对照相比分别提高55.9%、221%和21.1%。10.在缺钾条件下,菊花FTIR图谱与对照无明显差别,而且在不同的生育期,菊花功能叶片的FTIR图谱也无差异,说明缺钾不会影响到菊花及其叶片中功能成分的变化；在缺钾条件下,菊花功能叶片的HPLC图谱与对照有一定差异,在缺钾条件下,HPLC图谱中的各物质种类和含量都有一定的变化,但是差异不明显；菊花叶片和花序之间的HPLC图谱差异较大。11.缺钾也会影响菊花侧枝及侧蕾的发育,从而降低菊花的产量；不同生育期缺钾对菊花叶片的光合作用影响不一致,缺钾可以提高菊花营养期叶片的蒸腾速率、气孔导度；在生殖期叶片中,不同生育期缺钾均能降低叶片的蒸腾速率、气孔导度以及细胞间CO2浓度,但能使叶片的光合作用上升；缺钾使菊花根的生成能力降低。12.全生育期缺钾明显降低菊花中的黄酮和绿原酸含量；缺钾条件下苯丙氨酸、肉桂酸、对香豆酸与黄酮含量之间的回归方程分别为Y=285.4118X2-286.1689X+79.8951(R2=0.9375,P<0.05),Y=2.4368X+3.0380(R2=0.9809,P<0.001)和Y=0.2476X+5.5720(R2=0.9929,P<0.0001)(Y，黄酮含量；X,分别为苯丙氨酸、肉桂酸、对香豆酸)；主成分分析结果显示,缺钾条件下,黄酮类化合物合成主途径中的主要两个影响因素分别占总信息的88.36%和10.57%,而施钾条件下的主要决定因素分别占总信息的88.71%和9.64%。更多还原

【Abstract】 Juhua is the flower of Chrysanthemum morifolium Ramat., a traditional Chinese herbal medicine plant and it occupies an important position in the Chinese medicine enterprises. Nowadays, the cultivation methods of C. morifolium are gradually changing and improving with the development of GAP and the people’s awareness of the relationship between traditional medicine and health, therefore, the cultivation trends to much more scientific. Macroelements are integrant for the growth and metabolism of C. morifolium, its rational application can improve not only yield but also characteristic of the plant. This study is to research the effects of macroelements deficiency on the growth, primary and secondary metabolism, and investigate the secondary metabolites with HPLC and FTIR approaches in the different organs and diverse growth stages of C. morifolium. The purpose of this issue is to provide theoretical basis for the GAP and the secondary metabolism research. Main results are as follow.1. N deficiency in different life stages could decrease the yield of C. morifolium, and more decrease of the N application, more reduce of the yield. N deficiency in whole life stage could increase the flavonoid and soluble sugar contents both in leaves and flowers. Moreover, N deficiency could affect the solubility of the flavonoid. The content of middle-microelements significantly related to the flavonoid, for instance, among them Ca related positively to the flavonoids in three different treatments.2. N deficiency could influence the kinds and contents of secondary metabolites in HPLC figure print of leaves, and that related to the life stage of C. morifolium. And the results of FTIR show that the effect of N deficiency in different life stages was at the wavenumber of 1516 and 1325 cm-1, which was the characteristic area of flavonoids.3. N deficiency in whole life stage affected the growth of plant, and the effect was also found in the development process of the lateral branch. N deficiency improved the transpiration rate, stomatal conductance and intercellylar CO2 concentration, but decreased the potosythetic rate in the same time. In addition, N deficiency can accelerate the development of new roots, but that still lower than CK, the growth of root in N2 (0.2 g N/kg soil) was growth of the worst in three treatments, which suggest that the root could develop extremely in the reproductive stage.4. N deficiency could not affect the content of cinnamic acid both in leaves and flowers, but it could improve the content of P-coumaric acid in flower. N1(0.05 g N/kg soil) could improve the phenylalanine in leaves, while N2 (0.2 g N/kg soil) could improve it in flowers. The results of PCA show that the flavonoid synthesis process was different with CK compared to N1 (0.05 g N/kg soil) and N2 (0.2 g N/kg soil) treatments. The main flavonoid synthesis process was documented by PAL under normal condition, however, the dominance was changed to PAL and 4CL (C4H and 4CL) under N2 (N1) condition.5. P deficiency in different life stages could decrease the yield of C. morifolium, and more decrease of the P application, more reduce of the yield. P1 (0.05 g P2O5/kg soil) could increase the content of flavonoid and soluble sugar in leaves and flowers. P deficiency lead to the improvement of flavonoids in flowers, and then the effect appeared in the leaves in reproductive stage. The content of nutrients significantly related to the flavonoid, for instance, Ca positively related to the flavonoids in three treatments.6. P deficiency could not affect the FTIR figure print of C. morifolium. The effect of P deficiency appeared in the reproductive stage in HPLC figure print, and P deficiency in different life stages increased the content of flavonoids.7. P deficiency decreased the growth of lateral branch and the development of buds in these branches, which reduced to the decreasing of the yield. P deficiency could not affect the content of chlorophyll, but increase the transpiration rate, stomatal conductance and of leaves in vegetable life stage. In reproductive stage, P deficiency decreased the transpiration rate, stomatal conductance and intercellylar CO2 concentration, but increased the potosythetic rate in the same time. P1 (0.05 g P2O5/kg soil) accelerated the development of root, which opposite to P2 (0.1 g P2O5/kg soil). P deficiency could increase the content of flavonoids in root of C. morifolium.8. P deficiency could influence the secondary metabolites in phenolic synthesis pathway in plant of C. morifolium, and the regression equation between flavonoid and phenylalanine content was Y=-294.46X+150.66 (R2=0.9205, P<0.01) in CK and Y =42.62X+2.49 (R2=0.9564, P<0.01) in P deficient treatment (Y, flavonoid content; X, phenylalanine content). There were two principal components to control the phenolic major synthesis process, that was principal 1 (phenylalanine, PAL, cinnamic acid and p-coumaric acid) (88.17%) and principal 2 (4CL, C4H) (9.64%) domination under normal growth of C. morifolium. However, under P deficiency condition, the principal components were principal 1 (phenylalanine, cinnamic acid and p-coumaric acid and C4H) (81.46%) and principal 2 (PAL)(18.53%)domination, respectively. The influence of P deficiency on phenolic major synthesis pathway related to the change of PAL and C4H activities. 9. K deficiency decreased the flavonoid content by 31.4% in flower of C. morifolium. The microelements related to the flavonoid significantly, but the regression equation was different with each other. K deficiency decreased the protein content in leaves, but did not affect the protein in flowers. K1 (0.05 g K2O/soil) increased the soluble sugar in leaves obviously by 55.9%,22.1% and 21.1%.10. The FTIR figure print of flowers and leaves were same in different K deficience condition, which suggested that the K deficiency did not affect the function group both in flower and leaves. The HPLC figure print was different in leaves in different life stages, but the difference was not significantly. The HPLC figure print of leaf was significantly different with flower.11. K deficiency decreased the growth of lateral branch and the development of buds in these branches, and then decreased the yield of the plant. K deficiency increased the transpiration rate and stomatal conductance in leaves of vegetative life stage, however, in reproductive stage, K deficiency decreased the transpiration rate, stomatal conductance and intercellylar CO2 concentration, but increased the potosythetic rate. K deficiency decreased the development of the root.12. K deficiency decreased the flavonoid and chlorogenic acid contents slightly in flower of C. morifolium. The regression equations between phenylalanine, cinnamic acid, p-coumaric acid and flavonoid content were Y=285.4118X2-286.1689X+79.8951(R2 =0.9375, P<0.05), Y=2.4368X+3.0380 (R2=0.9809, P<0.001) and Y=0.2476X+ 5.5720 (R2=0.9929, P<0.0001) under K deficiency (Y, flavonoid content; X, phenylalanine, cinnamic acid and P-coumaric acid, respectively). Principal component analysis illustrate that K deficiency did not influence the preliminary flavonoid synthesis pathway. There were two major principals from chorismate to coumaryl Co A synsthesis process and the substrate was the domination principal in both K deficient (88.36% and 10.57%) and sufficient treatments (88.71% and 9.64%).更多还原