大豆种质和培养条件对大豆愈伤组织积累异黄酮的影响
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摘要
大豆异黄酮(Soybean isoflavones,SI)是大豆等豆科植物生长过程中形成的一类次生代谢物。近年来研究表明,大豆异黄酮具有多种生物活性和药理功效,是次生代谢产物的研究热点之一。目前,大豆异黄酮主要从大豆粕、豆胚及加工过程的废水中提取,提取效率较低,在自然界中资源有限,为此国内外相继开展了植物组织培养合成大豆异黄酮的工作。
本实验与HPLC相比较,通过优化电泳条件和采用內标分析,改进了测定大豆异黄酮含量的高效毛细管电泳方法;探讨了大豆种质异黄酮含量差异与其愈伤组织中异黄酮积累的相互关系,明确了组织培养过程中异黄酮的积累时期和关键酶PAL的动态变化,并在此基础上探索了影响愈伤组织积累异黄酮的培养条件。主要实验结果如下:
1.通过优化电泳条件和采用內标分析,改进了测定大豆异黄酮含量的高效毛细管电泳方法(HPCE)。最终电泳条件为:含有5%甲醇的40mmol/L硼砂溶液(pH10.0)为电泳缓冲液,压力进样20s,柱温25℃、分离电压16.5kV,检测波长为254nm,以鹰嘴豆芽素A作为內标物。在此条件下,6种异黄酮组分的峰面积和迁移时间的相对标准偏差(RSD)分别在0.5%～2.4%之间和1.8%～3.8%之间,加样回收率在97.2%～103.3%之间,RSD值均小于4.0%,6种异黄酮组分可在21min內完全分离并具有良好的线性关系。HPCE与HPLC对大豆异黄酮分离均较好,测定结果无明显差异。HPCE是一种检测异黄酮成分较为理想的方法。
2.不同大豆品种诱导形成的愈伤组织,其生物量、异黄酮含量和产量的差异均达极显著水平;大豆品种与外植体的互作对愈伤组织生物量、异黄酮含量和产量的影响均达极显著水平;不同外植体诱导的愈伤组织之间,其异黄酮含量和产量的差异极显著,但生物量的差异不显著。相关分析表明,不同大豆品种(整粒、胚轴和子叶)异黄酮含量与其愈伤组织(分别由胚轴和子叶诱导)的异黄酮含量和产量均呈显著正相关,由此可见大豆品种异黄酮含量与其愈伤组织的异黄酮含量和产量密切相关,选育异黄酮组培专用型品种具有种质遗传基础。实验结果还表明,大豆品种来源、播期类型和异黄酮含量高低对其愈伤组织生长和异黄酮积累有重要影响。由建阳秋大豆胚轴诱导的愈伤组织,其生物量和异黄酮产量均较高,分别为1.24g/Flask(瓶)和13.16mg/Flask(瓶),适合作为诱导愈伤组织积累大豆异黄酮的材料。
3.由愈伤组织生长和大豆异黄酮积累的动态变化可知,愈伤组织生物量和大豆异黄酮产量的增长趋势基本上一致,呈现“S”形曲线特征,但子叶和胚轴诱导产生的愈伤组织间存在差异。在整个愈伤组织培养过程中,PAL活性呈双峰型变化,大豆异黄酮积累之前先有一个酶活性增加过程,酶活化与最终产物的出现存在短暂的滞后时间,PAL酶活性对愈伤组织异黄酮合成存在重要影响。由此可见,在培养过程中愈伤组织大豆异黄酮的积累与初级代谢有密切联系,PAL是组织培养合成大豆异黄酮的一个主要的限速酶。
4.培养条件对大豆愈伤组织生物量、异黄酮含量和产量的影响:
(1)光照时间变化对大豆愈伤组织异黄酮的合成具有显著影响。在黑暗和光照交替处理下(8H/16h),愈伤组织的异黄酮含量最高为11.09(mg/g),同时异黄酮产量也最高,为12.04(mg/Flask)。结果表明,光照和黑暗处理相结合可有效促进愈伤组织生长和异黄酮的积累。
(2)蔗糖浓度变化显著影响愈伤组织的生长和异黄酮积累,增大蔗糖浓度可以促进大豆愈伤组织中异黄酮积累,其中4%蔗糖浓度处理效果较好,异黄酮含量和产量分别为2%处理的1.30和1.61倍;
(3)大豆愈伤组织对2,4-D、NAA和6-BA处理存在明显的浓度效应,2,4-D浓度为1.5mg/L时效果较好,能够实现愈伤组织生物量和异黄酮含量的同步增长;6-BA浓度为0.4mg/L时能显著促进愈伤组织生长和异黄酮积累;
(4)苯丙氨酸添加浓度对愈伤组织生长和异黄酮积累有显著影响。浓度升高有利于愈伤组织异黄酮的积累,但对愈伤组织生物量有较大抑制作用。苯丙氨酸添加的较适浓度在0.120mmol/L左右,在该浓度下愈伤组织异黄酮产量最高,为12.36mg/瓶,是对照的1.07倍。
(5)水杨酸(SA)作为诱导子对大豆愈伤组织异黄酮积累和愈伤组织生长诱导作用明显,当SA浓度为120μmol/L时,异黄酮含量和产量都达最高水平,分别是对照的1.35和1.58倍。
Soybean isoflavones are a large class of secondary metabolites found mostly in legumes. Recently, the isoflavones which have been associated with several health benefits, have been the one of the hottest topics in secondary metabolites. Isoflavones are usually extracted from soybean seeds and the waste water of the processing, so isoflavones resource is relatively limited. The use of plant cell cultures for large-scale production of active compounds is of interest from scientific and economical points of view. Many secondary metabolites have been reported to be produced by plant cell cultures. So soybean cell cultures can be used to provide high concentrations of bioactive isoflavones.
Compared with HPLC, HPCE was improved to detect the contents of isoflavones with the method of internal standard analyze. The effect of soybean varieties and the explants on the growth of callus and accumulation of isoflavnones has been investigated. Meantime, dynamic changes of the accumulation of isoflavnones and the activity of PAL (EC 4.3.1.5) have been studied. Then the culture conditions which influence the synthesis of isoflavones in soyben callus has been explored. The main results were as follows:
1. High performance capillary electrophoresis was improved to quickly analyze contents of isoflavones in soybean seed and callus. The biochanin was used as an internal standard. Electrophoresis conditions were as follows: a capillary tube with 40mmol/L sodium borate containing 5% methanol as the running buffer(pH10.0) and an applied voltage of 16.5kV, at 25℃, detected at a wavelength of 254 nm. Under the optimum conditions, 6 kinds of isoflavones were separated successfully within 21 minutes. The corrected peak areas of isoflavones increased linearly with components of isoflavones, and the recoveries were ranged from 97.2%～103.3%. The analytical results demonstrate the method is simple, quick and well reproducible, and can be used for the determination of isoflavones.
2. There were significant differences of the biomass, content and yield of isoflavones among soybean callus from difference varieties and the interplay between variety and soybean callus. There were significant differences of the content and yield of isoflavones, but no significant difference of the biomass among the calluses from different explants. It was found that there was very significant positive correlation between the content of isoflavones of different varieties and the content and the yield of isoflavones in soybean callus. In a word, the content of isoflavones in difference varieties has significant effect on accumulation of isoflavones in soybean callus. By the experiments in this paper, there always has high contents and yield of isoflanoves in soybean callus from Jianyangqiudadou hypocotyls. So it can be used as a potential material for producing isoflavones in the future.
3. The growth of soybean callus was approximately consistent with the accumulation of isoflavones by studying the dynamic changes of the accumulation of isoflavnones and the activity of PAL (EC 4.3.1.5). The shape of dynamic trends like a "S" curve, but there was little difference among the different explants. So there was close connection between accumulation of isoflavones and primary metabolism. During the entire course of culture of callus, the activity of PAL changed in a double-wave. The activity of PAL was activated before the accumulation of isoflavones. There was a short lag phase between activation of PAL and synthesis of isoflavones. Taken together, the activity of PAL has significant affect on the synthesis of isoflavones in soybean callus, and the PAL is a key enzyme for it.
4. The effect of culture conditions on growth of callus and accumulation of isoflavones:
(1) The illumination has great influence on the growth of callus and the accumulation of isoflavones. Comparede to illumination (24h) and darkness (24h), the contents and yield of isoflavones with the combination of illumination and darkness (16/8h) increased up to 11.09 (mg/g) and 12.04 (mg/Flask).
(2) It was showed that improving sucrose concentration enhanced the growth of callus and stimulated accumulation of isoflavones. In comparison with 2% sucrose, the contents and yield of isoflavones with 4% sucrose in the medium increased by 30 % and 61%.
(3) There was concentration effect on the growth of callus and the accumulation of isoflavones for 2,4-D,NAA and 6-BA. In comparison with control, 1.5mg/L 2,4-D was the best concentration, it had made the biomass of callus and the yield of isoflavones improve synchronistically. Among the 6-BA concentrations tested, 0.4 mg/L 6-BA enhanced the growth of callus and stimulated accumulation of isoflavones markedly.
(4) The phenylalanine stimulated the accumulation of isoflavones markedly; however, it restrained the growth of callus remarkably. The results showed that among the phenylalanine concentrations tested, 0.120 mmol/L phenylalanine improved the accumulation of isoflavones, and the yield of isoflavones reached 12.36mg/Flask, which has increased by 7 % compared to the control.
(5) By the experiments in this paper, SA which was chosen as an effective elicitor for isoflavones synthesis enhanced the growth of callus and stimulated accumulation of isoflavones remarkably. In comparison with control, the contents and yield of isoflavones with 120μmol/L SA in the medium increased by 35 % and 58 %.
本实验与HPLC相比较,通过优化电泳条件和采用內标分析,改进了测定大豆异黄酮含量的高效毛细管电泳方法;探讨了大豆种质异黄酮含量差异与其愈伤组织中异黄酮积累的相互关系,明确了组织培养过程中异黄酮的积累时期和关键酶PAL的动态变化,并在此基础上探索了影响愈伤组织积累异黄酮的培养条件。主要实验结果如下:
1.通过优化电泳条件和采用內标分析,改进了测定大豆异黄酮含量的高效毛细管电泳方法(HPCE)。最终电泳条件为:含有5%甲醇的40mmol/L硼砂溶液(pH10.0)为电泳缓冲液,压力进样20s,柱温25℃、分离电压16.5kV,检测波长为254nm,以鹰嘴豆芽素A作为內标物。在此条件下,6种异黄酮组分的峰面积和迁移时间的相对标准偏差(RSD)分别在0.5%～2.4%之间和1.8%～3.8%之间,加样回收率在97.2%～103.3%之间,RSD值均小于4.0%,6种异黄酮组分可在21min內完全分离并具有良好的线性关系。HPCE与HPLC对大豆异黄酮分离均较好,测定结果无明显差异。HPCE是一种检测异黄酮成分较为理想的方法。
2.不同大豆品种诱导形成的愈伤组织,其生物量、异黄酮含量和产量的差异均达极显著水平;大豆品种与外植体的互作对愈伤组织生物量、异黄酮含量和产量的影响均达极显著水平;不同外植体诱导的愈伤组织之间,其异黄酮含量和产量的差异极显著,但生物量的差异不显著。相关分析表明,不同大豆品种(整粒、胚轴和子叶)异黄酮含量与其愈伤组织(分别由胚轴和子叶诱导)的异黄酮含量和产量均呈显著正相关,由此可见大豆品种异黄酮含量与其愈伤组织的异黄酮含量和产量密切相关,选育异黄酮组培专用型品种具有种质遗传基础。实验结果还表明,大豆品种来源、播期类型和异黄酮含量高低对其愈伤组织生长和异黄酮积累有重要影响。由建阳秋大豆胚轴诱导的愈伤组织,其生物量和异黄酮产量均较高,分别为1.24g/Flask(瓶)和13.16mg/Flask(瓶),适合作为诱导愈伤组织积累大豆异黄酮的材料。
3.由愈伤组织生长和大豆异黄酮积累的动态变化可知,愈伤组织生物量和大豆异黄酮产量的增长趋势基本上一致,呈现“S”形曲线特征,但子叶和胚轴诱导产生的愈伤组织间存在差异。在整个愈伤组织培养过程中,PAL活性呈双峰型变化,大豆异黄酮积累之前先有一个酶活性增加过程,酶活化与最终产物的出现存在短暂的滞后时间,PAL酶活性对愈伤组织异黄酮合成存在重要影响。由此可见,在培养过程中愈伤组织大豆异黄酮的积累与初级代谢有密切联系,PAL是组织培养合成大豆异黄酮的一个主要的限速酶。
4.培养条件对大豆愈伤组织生物量、异黄酮含量和产量的影响:
(1)光照时间变化对大豆愈伤组织异黄酮的合成具有显著影响。在黑暗和光照交替处理下(8H/16h),愈伤组织的异黄酮含量最高为11.09(mg/g),同时异黄酮产量也最高,为12.04(mg/Flask)。结果表明,光照和黑暗处理相结合可有效促进愈伤组织生长和异黄酮的积累。
(2)蔗糖浓度变化显著影响愈伤组织的生长和异黄酮积累,增大蔗糖浓度可以促进大豆愈伤组织中异黄酮积累,其中4%蔗糖浓度处理效果较好,异黄酮含量和产量分别为2%处理的1.30和1.61倍;
(3)大豆愈伤组织对2,4-D、NAA和6-BA处理存在明显的浓度效应,2,4-D浓度为1.5mg/L时效果较好,能够实现愈伤组织生物量和异黄酮含量的同步增长;6-BA浓度为0.4mg/L时能显著促进愈伤组织生长和异黄酮积累;
(4)苯丙氨酸添加浓度对愈伤组织生长和异黄酮积累有显著影响。浓度升高有利于愈伤组织异黄酮的积累,但对愈伤组织生物量有较大抑制作用。苯丙氨酸添加的较适浓度在0.120mmol/L左右,在该浓度下愈伤组织异黄酮产量最高,为12.36mg/瓶,是对照的1.07倍。
(5)水杨酸(SA)作为诱导子对大豆愈伤组织异黄酮积累和愈伤组织生长诱导作用明显,当SA浓度为120μmol/L时,异黄酮含量和产量都达最高水平,分别是对照的1.35和1.58倍。
Soybean isoflavones are a large class of secondary metabolites found mostly in legumes. Recently, the isoflavones which have been associated with several health benefits, have been the one of the hottest topics in secondary metabolites. Isoflavones are usually extracted from soybean seeds and the waste water of the processing, so isoflavones resource is relatively limited. The use of plant cell cultures for large-scale production of active compounds is of interest from scientific and economical points of view. Many secondary metabolites have been reported to be produced by plant cell cultures. So soybean cell cultures can be used to provide high concentrations of bioactive isoflavones.
Compared with HPLC, HPCE was improved to detect the contents of isoflavones with the method of internal standard analyze. The effect of soybean varieties and the explants on the growth of callus and accumulation of isoflavnones has been investigated. Meantime, dynamic changes of the accumulation of isoflavnones and the activity of PAL (EC 4.3.1.5) have been studied. Then the culture conditions which influence the synthesis of isoflavones in soyben callus has been explored. The main results were as follows:
1. High performance capillary electrophoresis was improved to quickly analyze contents of isoflavones in soybean seed and callus. The biochanin was used as an internal standard. Electrophoresis conditions were as follows: a capillary tube with 40mmol/L sodium borate containing 5% methanol as the running buffer(pH10.0) and an applied voltage of 16.5kV, at 25℃, detected at a wavelength of 254 nm. Under the optimum conditions, 6 kinds of isoflavones were separated successfully within 21 minutes. The corrected peak areas of isoflavones increased linearly with components of isoflavones, and the recoveries were ranged from 97.2%～103.3%. The analytical results demonstrate the method is simple, quick and well reproducible, and can be used for the determination of isoflavones.
2. There were significant differences of the biomass, content and yield of isoflavones among soybean callus from difference varieties and the interplay between variety and soybean callus. There were significant differences of the content and yield of isoflavones, but no significant difference of the biomass among the calluses from different explants. It was found that there was very significant positive correlation between the content of isoflavones of different varieties and the content and the yield of isoflavones in soybean callus. In a word, the content of isoflavones in difference varieties has significant effect on accumulation of isoflavones in soybean callus. By the experiments in this paper, there always has high contents and yield of isoflanoves in soybean callus from Jianyangqiudadou hypocotyls. So it can be used as a potential material for producing isoflavones in the future.
3. The growth of soybean callus was approximately consistent with the accumulation of isoflavones by studying the dynamic changes of the accumulation of isoflavnones and the activity of PAL (EC 4.3.1.5). The shape of dynamic trends like a "S" curve, but there was little difference among the different explants. So there was close connection between accumulation of isoflavones and primary metabolism. During the entire course of culture of callus, the activity of PAL changed in a double-wave. The activity of PAL was activated before the accumulation of isoflavones. There was a short lag phase between activation of PAL and synthesis of isoflavones. Taken together, the activity of PAL has significant affect on the synthesis of isoflavones in soybean callus, and the PAL is a key enzyme for it.
4. The effect of culture conditions on growth of callus and accumulation of isoflavones:
(1) The illumination has great influence on the growth of callus and the accumulation of isoflavones. Comparede to illumination (24h) and darkness (24h), the contents and yield of isoflavones with the combination of illumination and darkness (16/8h) increased up to 11.09 (mg/g) and 12.04 (mg/Flask).
(2) It was showed that improving sucrose concentration enhanced the growth of callus and stimulated accumulation of isoflavones. In comparison with 2% sucrose, the contents and yield of isoflavones with 4% sucrose in the medium increased by 30 % and 61%.
(3) There was concentration effect on the growth of callus and the accumulation of isoflavones for 2,4-D,NAA and 6-BA. In comparison with control, 1.5mg/L 2,4-D was the best concentration, it had made the biomass of callus and the yield of isoflavones improve synchronistically. Among the 6-BA concentrations tested, 0.4 mg/L 6-BA enhanced the growth of callus and stimulated accumulation of isoflavones markedly.
(4) The phenylalanine stimulated the accumulation of isoflavones markedly; however, it restrained the growth of callus remarkably. The results showed that among the phenylalanine concentrations tested, 0.120 mmol/L phenylalanine improved the accumulation of isoflavones, and the yield of isoflavones reached 12.36mg/Flask, which has increased by 7 % compared to the control.
(5) By the experiments in this paper, SA which was chosen as an effective elicitor for isoflavones synthesis enhanced the growth of callus and stimulated accumulation of isoflavones remarkably. In comparison with control, the contents and yield of isoflavones with 120μmol/L SA in the medium increased by 35 % and 58 %.
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