硒、硫竞争对青花菜中萝卜硫素积累的调控机制研究

Broccoli is a Selenium enriched vegetable and enriched selenium compounds which have anti-cancer effects, there are high sulforaphane content in broccoli which origin from sulfur, the selenium and sulfur have similar chemical characteristics and metabolic pathways. It is difficult to understand how the selenium and sulfur competitive effects on the synthesis and metabolism of sulforaphane in broccoli. On the basis of over-expression and RNAi- expression of SMT gene in broccoli, the contents of selenium, sulfur, methionine, selenomethionine, selenocysteine, gulcoraphanin, sulforaphane and the related gene expression character were determined after the combined treatment with selenium and sulfur; On the basis of over-expression, RNAi- expression and vacuole-targeting expression of MY gene in broccoli, the activity, temporal and spatial expression character of myrosinase were determined after the combined treatment with selenium and sulfur. The feedback character of myrosinase integrate gulcoraphanin –myrosinase – sulforaphane in order to increase sulforaphane content induced by selenium-sulfur interaction effect, clarify the character of selenium-sulfur interaction physiology character during sulforaphane synthesis by metabolism process analysis, suggest the new idea for study on the mechanization of selenium-sulfur-interaction physiology and heredity character during sulforaphane synthesis in broccoli, and provide the theory evidence for high-sulforaphane-content broccoli breeding and quality production system.

青花菜具有很强的富硒作用并产生有防癌作用的硒化合物，同时可经硫代谢途径生成具有防癌作用的萝卜硫素。硒和硫具有相似的化学特征和代谢途径，硒、硫如何竞争性地影响青花菜中萝卜硫素的合成代谢一直是困扰科学家的问题。本项目拟以项目组获得的超表达、抑制表达转硒甲基转移酶基因青花菜植株为材料，分别给予其硒、硫组合处理，分析硒处理青花菜前后底物（硒、硫、甲硫氨酸、硒甲硫氨酸、硒半胱氨酸、萝卜硫苷）、产物（萝卜硫素）及相关基因表达的变化规律；同时以获得的超表达、抑制表达转黑芥子酶基因青花菜植株和拟构建的黑芥子酶基因液泡定位表达青花菜植株为材料，外源给予硒、硫组合处理，分析黑芥子酶基因时空表达差异和活性变化特征，循着底物—关键酶—产物的代谢途径实现明确硒、硫竞争如何调控底物（萝卜硫苷）合成以及黑芥子酶如何协调两者之间关系以促进萝卜硫素合成的研究目标，揭示硒、硫竞争调控青花菜萝卜硫素合成过程的代谢生理基础。

外源硒处理影响青花菜叶片和花球中的总硒、甲硫氨酸、和硒甲基硒代半胱氨酸含量，并由此影响硫代葡萄糖苷的合成，进一步分析发现硒处理能显著提高黑芥子酶基因表达及酶活性。硒处理差异基因的KEGG富集分析结果表明，硒处理青花菜后，青花菜植物-病原互作、淀粉和蔗糖代谢通路富集的基因数最多，硫代谢通路富集的差异基因也较多。其中在半胱氨酸和甲硫氨酸代谢途径富集基因71个、硫代谢基因39个、氮代谢基因17个、硫代葡萄糖苷基因4个。在甲硫氨酸向脂肪族硫代葡萄糖苷、吲哚族硫代葡萄糖苷代谢通路上有上调基因6个，在脂肪族硫代葡萄糖苷代谢通路上有下调基因1个。在硒代半胱氨酸向硒代甲硫氨酸代谢通路上有上调基因8个，在硒代甲硫氨酸向甲基硒醇代谢等通路上有下调基因7个。.研究发现BoSMT表达量水平在各个器官的规律与青花菜中硫代葡萄糖苷和萝卜硫素含量的分配规律一致，且BoSMT基因对外源硒处理的响应具有浓度依赖性。获得了BoSMT超量及BoSMT干涉的青花菜植株，硒未处理的超量和干涉植株中总硫苷和吲哚族硫苷含量未发生显著的变化，而转基因株系中的脂肪族硫苷含量显著低于野生型；与对照组（清水处理）相比，野生型青花菜在外源亚硒酸钠处理后总硫苷含量没有明显改变，而超量和干涉株系中的总硫苷含量均有显著的上升趋势；在外源硒处理后，超量株系中总硫苷含量要显著高于干涉株系和野生型青花菜中硫苷含量。说明在外源硒处理下，BoSMT基因有效缓解了硒-硫竞争代谢引起的总硫苷含量的降低。.研究发现BoMY在青花菜各个检测器官中均有所表达，其中，花球的表达量最高，而BoMY基因不响应低浓度的亚硒酸钠胁迫处理。获得了BoMY、BoFAMA超量及BoMY干涉的青花菜转化植株，发现BoFAMA正向调控了BoMY基因的转录水平，从而激活了黑芥子酶的活性，同时AtFAMA转录水平受阻的情况下，显著抑制了拟南芥AtTGG1的表达量，并最终造成了黑芥子酶活性的显著下降。证实了FAMA通过介导MY基因的转录活性，从而影响黑芥子酶活性和萝卜硫素的积累。