低氧与热激调控芸薹属芽苗菜异硫氰酸酯富集机理研究

This project is designed to investigate the impacts of hypoxia, heating and their combined stresses on myrosinase (MYR) activity and isothiocyanates (ITCs) content of germinated broccoli (Brassica oleracea var.italica) and cabbage (Brassica oleracea var. capitata) sprouts. ITCs are the hydrolyed products of glucosinolates with anticarcinogenic activities. MYR is the key enzyme for glucosinolate hydrolyzation into ITCs. Additional effects of sulfur-containing amino acid and sulfate supplied to the sprouts during germination, jasmonic acid (JA), abscisic acid (ABA) and Ca2+ inhibitors under the combined hypoxia-heating treatment on MYR and epithiospecifier protein (ESP) activities and ITCs accumulation, as well as the optimal O2 ratio of the atmosphere and air temperature for ITCs formation will be evaluated in this study as well. Eluciating the differences of MYR gene expression among endogenous JA, ABA and Ca2+/calmodulin will enable to explore the regulatory mechanism of ITCs accumulation in Brassica sprouts. It will be of benefit to improving MYR activity while inhibiting ESP activity in order to enhance ITCs formation. This study has not yet been reported world-wide. The proposed project will not only develop food-science theory of vegetable sprouts, but also provide useful technical information for improving their healthy functions to promote industrial development of edible vegetable sprouts in the future.

本课题以青花菜和甘蓝等芸薹属植物的芽苗菜为试材，在培育时添加含硫氨基酸或硫酸盐等物质，探索有利于芽苗菜富集异硫氰酸酯的效果；研究低氧和热激胁迫及其联合处理对不同生长阶段的芽苗菜、芽体各部位中黒芥子酶（MYR）活性、硫苷定向水解产物(ITCs)含量变化的影响，优化ITCs富集量最高时的氧分压与热激温度；研究低氧胁迫下热激处理时，茉莉酸(JA)抑制剂、脱落酸(ABA)抑制剂、Ca2+抑制剂对芽苗菜中MYR活性、上皮硫特异蛋白(ESP)活性和ITCs富集量的影响，探明内源JA、ABA和钙调蛋白对MYR基因表达的差异，从分子水平上揭示芽苗菜ITCs富集与调控机理，将有助于在逆境胁迫条件下提高芽苗菜MYR活性，钝化ESP活性，从而增强ITCs定向富集效果。该研究在国内外尚无报道。它既可深化芽苗类蔬菜食品的科学理论，又可为增强其保健功能提供技术依据，推动芽苗菜食品产业的工业化发展。

本项目以西兰花和甘蓝等芸薹属植物的芽苗菜为试材，在培育时添加含硫氨基酸或硫酸盐等物质，筛选出了有利于芽苗菜富集硫苷和异硫氰酸酯的培养液组分，其中ZnSO4处理可有效地富集西兰花芽苗硫苷和异硫氰酸盐(ITCs)。研究了低氧和热激胁迫及其联合处理对不同生长阶段的芽苗菜、芽体各部位中黒芥子酶（MYR）活性、硫苷定向水解产物ITCs含量变化的影响，优化出了ITCs富集量最高时的氧分压与热激温度。热激和低氧或联合处理显著上调了硫苷合成基因Elong、CYP83A1和FMOGS-OX1的表达，从而使西兰花芽苗中3-丁烯基硫苷（GNA）、萝卜硫苷（GRA）和4-甲硫基丁基硫苷（GER）及总硫苷得以积累。研究了茉莉酸(JA)抑制剂、脱落酸(ABA)、Ca2+对芽苗菜中MYR活性、上皮硫特异蛋白(ESP)活性和ITCs富集量的影响，西兰花芽苗在100 μM JA处理下，GRA和总硫苷含量分别较对照提高了2.01倍和2.53倍，同时提高了MYR活性，但是对ESP活性无影响；在甘蓝芽苗中， JA（100 μM）显著提高了硫苷合成和ITCs形成相关基因表达、以及MYR活性和吲哚族硫苷含量；ABA处理同样显著地提高了西兰花和甘蓝芽苗中MYR活性及其基因表达量和硫苷含量；外源Ca2+除了有效地促进芽苗菜生长外，还增加了MYR基因表达和激活了MYR活性。这些结果表明在热激、低氧、激素和钙处理下，通过提高硫苷合成关键酶基因表达、MYR活性和硫苷合成来促进ITCs的累积。蛋白质组学分析发现，热激降低了西兰花芽苗中S-腺苷甲硫氨酸合成酶1（SAM1）、S-腺苷甲硫氨酸合成酶2（SAM2）和S-腺苷甲硫氨酸合成酶4（SAM4）的表达量，提高了3-异丙基苹果酸脱氢酶（IPMDH）、3-异丙基苹果酸脱氢酶大亚基（IIL1）和3-异丙基苹果酸脱氢酶小亚基3（IPMDS3）的蛋白表达，降低了ESP蛋白和腈特异蛋白2（NSP2）的表达，从而有利于异硫氰酸盐的形成。JA和ABA显著提高了西兰花芽苗中脂肪族硫苷合成相关转录因子MYB28及相关基因Elong、CYP79F1、CYP83A1、UGT74B1、ST5b、FMOGS-OX1和AOP2的表达，从而增加了硫苷和ITCs的积累。