【作者】 腊贵晓；

【导师】 方萍；

【作者基本信息】 浙江大学 ， 植物营养学， 2009， 博士

【摘要】 芥蓝（Brassica alboglabra L.）是我国特有的芸薹属蔬菜,原产于中国南方,因营养丰富、味道鲜美而受到消费者欢迎。最近研究发现,芥蓝富含抗癌次生代谢物质芥子油苷,因而,作为抗癌蔬菜备受国内、日本及欧美关注。芥子油苷是以氨基酸为前体的含氮、硫次生代谢物,其含量及组分不仅受遗传、环境和气候条件等因子影响,也受大量及微量营养元素供应状况的制约。芥蓝的生长及高产对氮素营养需求较大,但是,如何通过氮素营养的调控,在确保高产的同时提高芥子油苷含量目前还知之甚少。为此,本文以芥蓝为材料,在对芥蓝芥子油苷的提取、分离鉴定方法进行优化的基础上,研究了供氮水平、氮素形态、氨基酸（甲硫氨酸和色氨酸）叶面喷施以及环境CO₂与供氮水平互作对芥蓝芥子油苷组分和含量的影响,取得主要结果如下:（1）通过比较HPLC不同流动相及柱长对芥子油苷组分的分离效果,筛选出以乙腈/水体系为流动相和长柱（250mm×4.6mm）等有利于芥子油苷分离的优化条件。通过比较不同提取温度、提取剂甲醇浓度、提取时间及料液比等对芥蓝菜薹芥子油苷的提取效果,发现提取温度和料液比对总芥子油苷、总脂肪族芥子油苷和总吲哚族芥子油苷的提取量均有影响显著,确定75℃,料液比1:45（g:mL）为优化提取条件。在10种不同基因型芥蓝菜薹中共分离检测到11种芥子油苷组分,其中脂肪族芥子油苷7种,吲哚族芥子油苷4种,未检出芳香族芥子油苷。（2）水培试验结果表明,供氮水平(5mmol N L^-1～30mmol N L^-1)对芥蓝的地上部生物量、菜薹总芥子油苷、总脂肪族及均有显著或极显著影响,但是对总吲哚族芥子油苷含量影响不显著。尽管低氮水平(5mmol N L^-1)下,芥蓝菜薹总芥子油苷含量最高,但芥蓝的生长因氮素营养缺乏而明显受阻;供氮水平提高到15mmol N L^-1时,芥蓝地上部生物量最高并有较高的芥子油苷含量;供氮水平提高到20～30mmol N L^-1,芥蓝地上部生物量并不再显著增加,而且菜薹芥子油苷含量却显著降低。低氮水平下(5mmol N L^-1)的芥蓝菜薹高芥子油苷含量可能缘于生物量过小所导致的浓缩效应;而高氮水平下(20～30mmol N L^-1)芥子油苷含量降低的原因,可能在于过高的供氮水平使芥蓝氮素吸收量显著增加而降低了硫的吸收比例,进而导致植株体内过高的氮硫比（7左右）,因而可能不利于芥子油苷的合成和积累。（3）芥蓝不同NH₄⁺-N/NO₃^--N比的水培试验结果表明,100%铵态氮处理的芥蓝植株矮小,并出现萎蔫和氨毒现象,收获时无膨大菜薹生成,部分植株死亡,生物量显著低于其余各处理。除100%铵态氮处理外,不同NH₄⁺-N/NO₃^--N比处理对芥蓝菜薹中硝酸盐含量、总芥子油苷、总脂肪族芥子油苷和总吲哚族芥子油苷含量影响显著,对生物量并无显著影响。硝酸盐含量随着铵态氮比例的增加而显著降低。其中50%铵态氮处理的总芥子油苷、总脂肪族芥子油苷含量最高;75%铵态氮处理的总吲哚族芥子油苷含量最高。可见,适当的NH₄⁺-N/NO₃^--N比有利于芥子油苷的合成和积累。虽然,NH₄⁺-N/NO₃^--N比对菜薹全氮影响显著,但菜薹全硫含量及氮硫比却无显著变化,可见NH₄⁺-N/NO₃^--N比对芥子油苷含量的影响可能不是由于植株氮硫比的改变而引起的,其影响机制有待进一步研究。（4）表面活性剂辅助的甲硫氨酸、色氨酸喷施芥蓝基质栽培试验结果表明,与喷清水对照相比,甲硫氨酸、色氨酸单独喷施及表面活性剂辅助的色氨酸叶面喷施,对芥蓝菜薹总芥子油苷、总脂肪族芥子油苷和总吲哚族芥子油苷含量及芥蓝产量均无显著影响。表面活性剂辅助的甲硫氨酸叶面喷施显著提高了芥蓝菜薹总芥子油苷、总脂肪族芥子油苷含量,尤其是3-丁烯基芥子油苷含量,而对总吲哚族芥子油苷含量无显著影响。说明有效供给甲硫氨酸能明显促进脂肪族芥子油苷的合成和积累,而辅之以表面活性剂能增进叶面对甲硫氨酸的吸收效果。（5）CO₂浓度(350和800μL L^-1)与供氮水平(5、10和20mmol N L^-1)两因子芥蓝水培试验结果表明,无论CO₂浓度高低,低氮(5mmol N L^-1)水平下,芥蓝生长明显受阻;中、高氮水平(10～20mmol N L^-1)下芥蓝地上部及菜薹鲜重均显著增加,而且CO₂浓度升高对此具有显著的增进作用。芥蓝菜薹总芥子油苷、总脂肪族芥子油苷含量随供氮水平的提高显著降低,而吲哚族芥子油苷含量反而所提高。中、低氮水平下CO₂浓度升高有利于芥蓝菜薹总芥子油苷、总脂肪族芥子油苷含量的提高,高氮水平下CO₂浓度升高的增进作用并不显著。其原因可能在于CO₂浓度升高使芥蓝菜薹全C含量及C/N比增加,而全N及全S含量显著降低,导致中、低氮水平下的N/S比显著降低,可能更有利于脂肪族芥子油苷的合成与积累;高氮水平下的N/S比并无显著变化,仍处于较高的水平（6左右）,因此可能对脂肪族芥子油苷的合成与积累也无显著影响。更多还原

【Abstract】 Chinese kale（Brassica alboglabra L.） is a kind of Chinese native Brassica vegetables,which was consumed originally in southern China for its rich nutrition and delicious taste and now has been gaining popular in whole China and Japan as well as Europe and America due to its high content of glucosinolates（GSs） with anti-carcinogenic function.GSs are secondary metabolites derived from amino acids. The concentration and composition of GSs in plant tissue is not only determined by genetic,environmental and seasonal factors,but also by the amount and form of macro- and micro-nutrient elements supplied.The growth and high biomass accumulation of Chinese kale required a large amount of nitrogen nutrition. However it is still unclear whether the GS concentration could be increased by improving nitrogen fertilizing strategy.To reveal this issue,after modifying the methods for GS separation and extraction from Chinese kale bolting stems,the effects of nitrogen levels,nitrogen forms in culture solution,and top dressing of methionine and tryptophan with or without surfactant as well as the interactions between CO₂ concentration and N levels on the GS concentration based on hydroponic and artificial medium culture experiments were investigated.The main results were as follows:（1） To improve the procedure of GSs separation by HPLC,the different mobile phases and different lengths of C18 column were compared.The results showed that desulpho-GS compounds could be completely separated by HPLC with C18 column of 250 mm×4.6 mm and the mobile phase of the acetonitrile/water system.The concentration of GSs extracted from Chinese kale under different extract temperatures,methanol concentrations of extract solution,extract times,and ratios of sample to extract solution were compared.The results revealed that the concentration of the total GSs,the total aliphatic GSs as well as the total indolyl GSs extracted were significantly affected by ratio of sample to solution and extract temperature.The optimal extracting temperature and ratio of sample to extract solution were 75℃and 1:45（g:mL）,respectively.And 11 GS compounds were detected in 10 genotypes of Chinese kale bolting stems,including 7 aliphatic and 4 indolyl GSs,while no aromatic GS was identified.（2） The results of the hydroponics experiment with five nitrogen（N） levels of 5 mmol L^-1 to 30 mmol L^-1 showed that the biomass of Chinese kale and the concentrations of total GSs,total aliphatic GSs and total indolyl GSs were significantly affected by N levels in culture solution.Although the highest concentration of total GSs was obtained at the lowest N level of 5 mmol L^-1,the growth of Chinese kale was badly suppressed because of nitrogen deficiency.The highest biomass and relative higher total GS concentration were gained at middle N level of 15 mmol L^-1.The biomass did not significantly increase while the total GS concentration was obviously decreased under the higher N levels of 20～30 mmol L^-1 compared to that at 15 mmol N L^-1 treatment.It suggested that the highest GS concentration at lowest N level of 5 mmol L^-1 maybe caused by the "concentration effect" of low biomass,while the lowest GS concentration at higher N levels of 20～30 mmol N L^-1 perhaps resulted from the imbalance of N/S ratio due to over uptake of nitrogen.（3） The hydroponics experiment with 5 ratios of ammonium（NH₄⁺-N） to nitrate （NO₃^--N） from 100/0 to 0/100 was conducted for Chinese kale.It is found that Chinese kale supplied with complete NH₄⁺-N grown very slowly and appeared obvious ammonium toxic symptom with partial plants died.Besides no bolting stem formed the lowest biomass was observed in this treatment.Except for the biomass of Chinese kale,the concentrations of total GSs,total aliphatic GSs and total indolyl GSs were all significantly different among the other four treatments supplied with partial or complete NO₃^--N.Nitrate content sharply decreased with increasing percentages of ammonium.The highest concentration of total GSs and total aliphatic GSs in bolting stems were detected at the treatment supplied with 50%NH₄⁺-N and the highest total indolyl GS concentration was achieved at the treatment with 75% NH₄⁺-N,which indicated that the appropriate ratio of ammonium to nitrate in nutrient solution may be helpful for the GS synthesis and accumulation in Chinese kale bolting stems.The sulfur content and N/S ratio but not nitrogen content in bolting stems were not significantly affected by the ratio of ammonium to nitrate, which indicated that the differences of GS concentrations among the treatments with different ratio of ammonium to nitrate was not caused by the change of N/S ratio. Detailed mechanism of ammonium to nitrate ratio on the GS concentration in Chinese kale needs to be further explored.（4） The artificial medium culture experiment was carried out to investigate the top dressing effects of methionine and tryptophan with or without surfactant on the GS concentrations in Chinese kale bolting stems.The results revealed that top dressing of methionine,tryptophan and tryptophan with surfactant did not affect the concentrations of total GSs,total aliphatic and total indolyl GSs as well as the biomass.Top dressing of tryptophan combined with surfactant significantly increased the total GSs and the total aliphatic GS concentrations,especially the concentration of gluconapin,but not for total indolyl concentration.It implied that methionine absorbed from leaves surface may promote the synthesis and accumulation of GSs and the surfactant could assistant the absorption of methionine through the Chinese kale leaves covered with a heavy layer of waxiness.（5） The results of the hydroponic experiment with two CO₂ concentrations of 350 and 800μL L^-1 and three N levels of 5,10 and 20 mmol L^-1 showed that at both normal and high CO₂ concentration,the growth of Chinese kale was suppressed at the lowest N level and the biomass was increased significantly as N level increased. Furthermore,enrichment of CO₂ concentration considerably enhanced the biomass under higher three nitrogen levels and fresh weight of bolting stems at N levels of 10 and 20 mmol L^-1.The concentrations of total GSs and total aliphatic GSs were decreased significantly as nitrogen level increased,while the total indolyl GS concentration increased.Enrichment of CO₂ concentration from 350 to 800μL L^-1 elevated concentrations of total GSs and total aliphatic GSs under low and middle N levels,but not at high N level.The C content and C/N ratios of Chinese kale bolting stems were increased as CO₂ enriched under all three nitrogen levels,while both the N content and the S contents decreased.The N/S ratios decreased at lower N levels of 5 and 10 mmol L^-1 because the degree of N content reduced was larger than that of S content,which may be one of the reasons why the total aliphatic GS concentrations under lower N levels with lower N/S ratio were higher than that at the higher nitrogen level when CO₂ elevated.更多还原