低氧联合盐处理调控豆类芽菜低级磷酸肌醇富集机理

In this study, effects of hypoxia and salinity stress on germinating soybean and mung bean at different growth stages and portions of sprouts will be investigated systematically, in order to: select optimal partial pressure of oxygen, salt type and concentration for promoting accumulation of lower inositol phosphate, illustrate the functions of phytase and phosphoinositide-specific phospholipase C (PI-PLC) in phytic acid degradation and PI-PLC pathway for a contribution to bioaccumulation of lower inositol phosphate in higher plants, explore the influences of GA3 inhibitor and Ca2+ channel inhibitor in culture medium on activities of phytase and PI-PLC, as well as contents of phytic acid, PI(4,5)P2, and lower inositol phosphate, and clarify different regulatory mechanisms of endogenous GA3 and Ca2+ on phytase and PI-PLC gene expressions. Furthermore, proteomics technology will be applied for analyses of protein-gene expression and its relationship with lower inositol phosphate accumulation to reveal the bio-regulatory functions not only in the bioinformatics perspective, but also for scientific mechanisms at the bio-molecular level. This study has not been reported world-wide as yet. It will enrich scientific theory of vegetable sprout food, provide useful technological information for improving the healthy functions, and promote commercial production of edible vegetable sprouts and sustainable development of functional food industry in the future.

本研究以大豆和绿豆芽菜为试材，研究低氧联合盐处理对不同生长阶段及芽体各部位中低级磷酸肌醇积累的影响，旨在筛选有利于低级磷酸肌醇富集的适宜氧分压与盐种类及施用浓度，探明添加磷脂酰肌醇特异性磷脂酶C（PI-PLC）途径中限速酶PI-PLC专性抑制剂及芽菜中PI-PLC途径和植酸降解途径对低级磷酸肌醇富集的贡献。探索低氧联合盐处理时，GA3生物合成抑制剂和Ca2+通道抑制剂对芽菜中植酸酶和PI-PLC活性、植酸和磷脂酰4,5-二磷酸肌醇含量及低级磷酸肌醇富集的影响，探讨内源GA3和Ca2+对植酸酶、PI-PLC基因表达的调节机制。运用蛋白质组学技术分析差异表达蛋白性能，从生物信息学角度推测其生物调节功用，在分子水平上揭示芽菜中肌醇-3-磷酸(IP3)等低级磷酸肌醇的富集机理。该研究在国内、外尚无报道。因此，本课题的开展具有重要理论意义和应用价值。

本项目以大豆和绿豆芽苗为试材，采用低氧培育（15% O2）并添加CaCl2、乳酸钙、柠檬酸钠、乙酸钠和酒石酸钠盐等物质，筛选出了有利于植酸降解和低级磷酸肌醇富集的培养液组分，其中CaCl2、乳酸钙、乙酸钠处理可有效地富集低级磷酸肌醇；乳酸钙联合赤霉素（GA3）对大豆芽菜植酸降解具有叠加作用。优化获得0.27 mM乳酸钙喷淋处理大豆，避光条件下发芽4 d，其植酸降解效果显著，且提高了锌、铁元素的利用率。因此，用低浓度乳酸钙喷淋处理可促进大豆芽生长以及植酸降解，提高其营养品质。探讨了NaCl-CaCl2处理下PI-PLC途径对绿豆芽菜低级磷酸肌醇富集的影响，研究了NaCl、CaCl2及其联合处理与新霉素（PI-PLC途径抑制剂）共同处理的绿豆芽菜中植酸和低级磷酸肌醇含量、磷脂酶C（PLC）活性和PI-PLC基因表达量的变化。CaCl2、NaCl-CaCl2能有效地提高PI-PLC途径对IP3形成的贡献率；在NaCl、CaCl2和NaCl+CaCl2处理下，PI-PLC途径对绿豆芽菜Ins(1,4,5)P3的贡献率分别为23.11%、43.23%和37.37%。在柠檬酸钠、乙酸钠和酒石酸钠处理下绿豆发芽过程中InsP3、InsP4、InsP5和In（1,4,5）P3含量显著增加。三种钠盐处理下，PI-PLC途径In（1,4,5）P3富集的贡献率分别为66.4%、67.8%和61.15%。用0.27 mM乳酸钙联合1 μM GA3及10 mg/L多效唑（PAC，内源GA合成抑制剂），发现乳酸钙和GA3协同处理促进了大豆芽菜生长及植酸降解，同时磷元素利用率、植酸降解相关酶活力得到提高。通过比较Ca2+处理的大豆芽菜转录组和蛋白质组学研究，得到1,912个差异基因和460个差异蛋白，其中部分与磷酸肌醇代谢过程有关。绿豆芽菜经檬酸钠、乙酸钠和酒石酸钠处理后，鉴定出150个差异表达蛋白，这三种有机酸盐处理通过显著上调磷酸肌醇酶（P54928）和紫色酸性磷酸酶的表达丰度来促进植酸降解和低级磷酸肌醇富集。