为研究银杏细胞的耐盐能力和提高细胞培养生产黄酮的产量,通过添加0、50、100、150 mmol/L的NaCl到液体培养基中进行悬浮细胞振荡培养,对盐处理下银杏悬浮细胞的生长、耐盐能力和黄酮积累的动态变化进行了研究。结果表明:在盐处理早期(4～8 d),各处理的细胞生长量随着盐处理浓度的增加而增加,而在后期(12～20 d)则随着盐处理浓度的增加而下降。中、高浓度盐处理(100～150 mmol/L)的细胞在培养早期表现出对盐刺激有一定的耐受性; 8～16 d时盐胁迫加强,细胞通过提高脯氨酸含量增强渗透调节的能力,同时通过促进细胞内黄酮的积累增强抗氧化能力,减少盐胁迫对细胞的伤害; 而在20 d时盐胁迫强度超过细胞的自我调节能力,细胞受到严重的伤害而出现褐化。50 mmol/L NaCl处理的悬浮细胞生长受抑制较轻,PAL活性在第8天最高,高的黄酮含量和产量(129.0 mg/L)出现在第12天,在整个处理期内能够维持生长。100 mmol/L盐处理的细胞在第16天时黄酮含量比对照增加了29.0%,但总黄酮产量(98.4 mg/L)只比对照增加了10.9%。150 mmol/L盐处理的细胞在第8天和第16天都维持较高水平的黄酮含量,第8天时比对照增加了66.3%,总黄酮产量最高达104.0 mg /L。因此50 mmol/L和150 mmol/L NaCl处理下,悬浮培养的银杏细胞分别在第12天 和第8天时收获较好。
Abstract
The salt tolerance ability, cell growth and flavonoids accumulation in Ginkgo biloba L. suspension cells were investigated by adding 0, 50, 100, 150 mmol/L NaCl into the MS liquid culture medium. The results showed that the cell growth increased with the increasing of both salt treatment time and concentration in the early treatment(4-8 days), while decreased during 12-20 days under NaCl treatments. Moderate and high dose of salinity(100-150 mmol/L)had a certain tolerance to salt stimulation by increasing the soluble protein content at early NaCl treatment(4-8 days)compared to the control. The growth biomass was reduced by the increase of salinity dose during 8-16 days, whereas the high level of proline and flavonoids was found and it suggested that the Ginkgo biloba cells could enhance the osmotic adjustment capacity and oxidation resistance ability by stimulating the biosynthesis of proline and flavonoids at this period. Furthermore, the intensity of salt stress was higher than the cell self-regulation capacity, and serious damage and brown appeared in cells at the 20th day. The growth of suspension cells was not obviously restrained under 50 mmol/L NaCl treatment compared to the control, due to the low level of relative electric conductivity(REC)and MDA content in cell observed. Furthermore, the PAL activity increased significantly at the 8th day and the highest flavonoid accumulation and total flavonoids yields(129.0 mg/L)were found at 12th day. The flavonoids content at 100 mmol/L NaCl treatment increased by 29.0% than the control, while the total flavonoids yield(98.4 mg/ L)was only 10.9% higher than the control. However, the flavonoids content at 150 mmol/L NaCl could maintain the high level at 8th day and it was 66.3% times higher than control, meanwhile the total flavonoids yield was(104.0 mg /L)attained at 8th day, too. Therefore, the cell sampled time at 12th day and at 8th day was beneficial to flavonoids production under 50 mmol/L and 150 mmol/L NaCl treatment, respectively.
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基金
收稿日期:2015-06-13 修回日期:2015-09-02
基金项目:“十二五”国家科技支撑计划(2011BAD38B01); 江苏省自然科学基金项目(BK2012818); 江苏高校优势学科建设工程资助项目(PAPD)
第一作者:陈颖,副教授,博士。E-mail: chynjfu@163.com。
引文格式:陈颖,罗永亚,邱娜菲,等. NaCl处理对银杏悬浮培养细胞生长、耐盐性和黄酮积累的影响[J]. 南京林业大学学报:自然科学版,2015,39(6):45-50.
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