L-丙氨酸对海南粗榧悬浮细胞合成三尖杉酯类碱的影响

摘要

该研究在海南粗榧悬浮细胞培养的不同阶段(5、10、15、20 d),分别添加不同剂量的L-丙氨酸(10、30、50、100 mg·L-1),测定细胞生长、细胞活力及产物含量,确定L-丙氨酸最佳的添加时间及添加剂量.结果表明:添加L-丙氨酸对细胞生长和细胞活力均有抑制作用;在海南粗榧悬浮培养第15天、添加30 mg·L-1 L-丙氨酸时,产物含量最高(4.853 6 mg·L-1),是对照(2.853 8 mg·L-1)的1.7倍.同时,为了探讨添加L-丙氨酸对海南粗榧悬浮细胞糖代谢的影响,对培养基糖耗程度、细胞内糖酵解途径(glycolytic pathway,EMP途径)关键酶丙酮酸激酶(Pyruvate kinase,PK)活力、磷酸戊糖途径(hexose monophosphate pathway,HMP途径)关键酶6-磷酸葡萄糖脱氢酶(glucose 6-phosphate dehydrogenase, G6PDH)活力进行了测定,结果显示添加L-丙氨酸后,植物细胞培养液中总耗糖速度与对照相比无明显差异,丙酮酸激酶(PK)活力与对照(25.37 U·g-1)相比下降了29.10%,G6DPH活力是对照组(53.49 U·g-1)的1.33倍.以上结果说明,糖代谢途径中碳通量在一定程度上由EMP途径转向了HMP途径,三尖杉酯类碱合成的前体物PEP积累,E4P合成量增加,均有利于产物三尖杉酯类碱含量的增加.%Cephalotaxus mannii is an indigenous plant to Hainan, China, which is the main material of cephalotaxuin eloids.The cephalotaxus alkaloids are anti-cancer drugs which have been used widely in clinic.But C.mannii is rare.The plant suspension cell culture can increase the yield of secondary metabolites steadily.And some adopted strategies have been developed to increase the yield in plant cell culture, such as add metabolic inhibitor.L-alanine is an effective inhibitor of pyruvate kinase (PK), which is an important limited enzyme in the glycolytic pathway (EMP).To investigate the effects of L-alanine on suspension cell growth and the cephalotaxine production of C.mannii, different doses of L-alanine (10, 30, 50 and 100 mg·L-1) were added into the C.mannii cells at different stages (10, 15, 20 and 25 d) of culture time.Then the cell growth, the cell activity and the cephalotaxine production were determined.The results showed that the cell growth and cell activity of C.mannii were inhibited after adding L-alanine, the cultures treated with 30 mg·L-1) L-alanine at day 15 showed the highest cephalotaxine yield (4.853 6 mg·L-1), which was 1.7 times that of the control cultures (2.853 8 mg·L-1).Then, the residue sucrose contents of the culture, the activity of pyruvate kinase (PK) which is the key enzyme of EMP pathway, the activity of glucose 6-phosphate dehydrogenase (G6PDH) which is the key enzyme of hexose monophosphate pathway (HMP pathway) were all been determined to investigate the effects of L-alanine on cell glucose metabolism of C.mannii.The results showed that after adding L-alanine, the sucrose consumption rate of treated cells had no significant difference compared with the control cells, the PK activity of treated cells was decreased by 29.10% compared with the control cells (25.37 U·g-1), the G6PDH activity of treated cells was 1.33 times of that in the control cells (53.49 U·g-1).The results suggest that the carbon metabolic flux flow to HMP pathway from EMP pathway in some degree, so it can be presumed that the precursor PEP accumulated and the other precursor E4P also increased, which all benefit to the synthesis of cephalotaxine production.