【作者】 孙世芹；

【导师】 阎秀峰；

【作者基本信息】 东北林业大学 ， 生态学， 2005， 博士

【摘要】 喜树（Camptotheca acuminata）是中国特有树种,因其次生代谢产物喜树碱具有显著的抗癌和抗反转录病毒活性而备受关注。本文研究了不同氮素水平和形态下砂培喜树幼苗生长发育过程中氮素及喜树碱代谢的动态变化过程,分析了喜树幼苗氮素及喜树碱代谢对环境氮素营养的响应,总结和探讨了提高喜树幼苗幼叶中喜树碱含量的氮素营养条件。研究结果可为喜树次生代谢与环境关系的研究提供基础资料,对于以获取高含量喜树碱为目的的喜树培育等生产实践也有一定的指导意义。研究的主要结果如下: 1.施氮显著提高了喜树幼苗生物量,16 mmol·L^-1为适合喜树幼苗生长的供氮水平。氮供给不足时,喜树幼苗的根系优先生长,以增强养分吸收能力。施氮也明显提高喜树幼苗的氮浓度,叶片的氮浓度最高,随氮素水平增加的变化幅度也最大,说明施氮促进幼苗对氮的吸收,并且优先分配给叶片。营养液中NO₃^--N比例的增加有利于喜树幼苗生长,NH₄⁺-N/NO₃^--N为25/75时幼苗生物量最大,NO₃^--N为唯一氮源时又下降,单一NH₄⁺-N下生物量显著降低。同样,NH₄⁺-N/NO₃^--N为25/75时喜树幼苗吸收更多的氮,植株体内养分积累也最多。 2.随着氮素供给的增加,喜树幼苗叶绿体色素、可溶性蛋白含量、叶片硝酸还原酶（NR）和谷氨酰胺合成酶（GS）活性显著增加（根中酶活性远低于叶片）,但继续提高(32 mmol·L^-1)时喜树幼苗并未积累更多的可溶性蛋白。叶片NR和GS活性在处理中前期较高,而在后期相对下降。营养液中NO₃^--N比例增加可显著诱导叶片的NR活性,NH₄⁺-N/NO₃^--N为50/50的GS活性最大,而NH₄⁺-N/NO₃^--N为25/75的最有利于叶绿体色素和可溶性蛋白的合成与积累。 3.喜树幼苗幼叶的喜树碱含量随氮素水平的增加而明显减小,氮素水平为4mmol·L^-1时幼叶的喜树碱含量最高（6.72‰）,是16 mmol·L^-1的1.1倍。适当的低氮胁迫能够显著地增加喜树幼苗幼叶的喜树碱含量。NH₄⁺-N/NO₃^--N为25/75时最有利于喜树碱在幼叶中的合成和积累,且在处理的前30天呈上升趋势而随后下降。短期NH₄⁺-N/NO₃^--N为25/75的处理可诱导幼叶中高含量的喜树碱。 4.喜树幼苗的色氨酸合酶（TSB）活性以幼叶最高,茎皮、成叶和根依次减弱,并且与喜树碱含量呈现良好的器官对应性。色氨酸脱羧酶（TDC）活性的最高部位为茎皮,其次为幼叶,成叶和根的很低,与喜树碱含量缺乏器官对应性。幼叶的TSB活性随时间进程先升后降再趋于平稳。后期在不同浓度氮素处理间差异不显著。茎皮的TDC活性随时间进程持续下降,且随着氮素水平降低而明显增加,氮素水平为4 mmol·L^-1更多还原

【Abstract】 Camptotheca acuminata, a tree species native to China, has gained great attention for its secondary metabolites camptothecin （CPT） which has remarkable anticancer and antiretrovirus activities. The change of nitrogen and CPT metabolism during the development of the seedlings supplied with different nitrogen concentrations and forms were studied by sand culture, and its responses to environmental nitrogen were analyzed. The nitrogen nutrition conditions to increase the CPT concentration in seedlings young leaves were also summarized and discussed. The results may provide some basic information for the research of the relationship between secondary metabolism in C. acuminata and its environment, and they also have some guidance significance in the practice of C. acuminata cultivation in order to gain high CPT concentration. The main results are as follows.1. Increase nitrogen supply significantly increased biomass of C. acuminata seedlings, 16 mmol.L^-1 may be the optimal nitrogen concentration for the seedlings growth. When the seedlings were supplied with deficient nitrogen, the growth of roots was preferential to absorb more nitrogen nutrition. Increase nitrogen supply significantly increased nitrogen concentration in the seedlings, and leaf nitrogen concentration was highest and had the most change extent with the increase of nitrogen supply, which suggest increase nitrogen supply may enhance the seedlings to absorb more nitrogen nutrition and preferentially partition to the leaves. Increase NO3-N ratio in nutrition solution was of advantage to the seedlings growth, the biomass was highest when NH₄⁺-N/NO₃^--N ratio was 25/75, and decreased when NO₃^--N was the sole nitrogen source, and significantly reduced when the seedlings were supplied only with NH₄⁺-N. Similarly, the seedlings absorbed more nitrogen nutrition and accumulated the most nutrition when NH₄⁺-N/NO₃^--N ratio was 25/75.2. Pigments, soluble protein, nitrate reductase （NR） and glutamine synthetase （GS） activities in leaves significantly increased with the increase of nitrogen supply （enzyme activity in roots was far lower than that in leaves）, but excessive nitrogen supply (32 mmol . L^-1) didn’t result in more soluble protein in the seedlings. NR and GS activities in leaves were relatively higher during early and middle stage of treatment, while relatively declined during late stage of treatment. Increase NO₃^--N ratio in nutrition solution may significantly induced leaf NR activity, the highest GS activity presented when NH₄⁺-N/NO₃^--N ratio was 50/50, while the optimal nitrogen conditions （NH₄⁺-N/NO₃^--N ratio was 25/75） was the most advantageous for pigments and soluble protein accumulation.3. CPT concentration in seedlings young leaves obviously declined with the increase of nitrogen supply, and it was the highest （6.72‰） when nitrogen supply was 4 mmol . L^-1, equal to 1.1 times of that when nitrogen supply was 16 mmol . L^-1. Proper deficient nitrogen stress can significantly increase the CPT concentration in seedlings young leaves. When NH₄⁺-N/NO₃^-1-N ratio was 25/75, CPT accumulation in young leaves displayed the best advantagesand increased in the early 30 days of treatment and then declined. A short-term treatment that NH4+-N/NO3-N ratio was 25/75 may gain high CPT concentration in young leaves.4. Tryptophan synthase （TSB） activity was the highest in young leaves and decreased sequentially in the stem bark, the mature leaves and the roots, which paralleled to CPT concentration. Tryptophan decarboxylase （TDC） activity was the highest in the stem bark, then in the young leaves, and the lowest in the mature leaves and the roots, which didn’t parallel to CPT concentration. TSB activity increased and then declined to constant level with the time course. There was no significant difference between different nitrogen concentration treatments at late stage of treatment. TDC activity in stem barks decreased continuously along with treatment days increasing, and presented obvious increase with decrease nitrogen supply, and it was the highest when nitrogen supply was 4 mmol ? L"1. There was some parallelism between TDC activity in stem barks and CPT concentration in young leaves. TDC activity in the stem bark was the highest when NH/-N/NCV-N ratio was 25/75, and the change of TDC activity paralleled to CPT concentration in young leaves.To sum up, 16 mmol ? L"1 may be the optimal nitrogen concentration for the growth of C. acuminata seedlings, whease CPT concentration in young leaves was the highest when nitrogen supply was 4 mmol ? L"1. The optimal nitrogen conditions （NH4+-N/NO3-N ratio was 25/75） for seedlings growth was also the best advantageous for CPT accumulation. Therefore, it is an efficient method to increase CPT concentration in young leaves that proper short-term nitrogen deficient stress was conducted after a period of vigorous growth.更多还原