橡胶树丙酮酸脱氢酶激酶基因的克隆与表达分析

Molecular Cloning and Expression Analysis of HbPDK of Hevea brasiliensis

摘要: 丙酮酸脱氢酶激酶(PDK)通过磷酸化丙酮酸脱氢酶复合体(PDC)，负调控PDC的酶活性，抑制丙酮酸的氧化脱羧反应，在中间物代谢过程中起着重要的调控作用。橡胶树丙酮酸脱氢酶激酶基因，HbPDK，编码区长1110 bp，编码369个氨基酸。HbPDK主要在橡胶树叶片、胶乳和花组织中表达，在叶片不同发育时期，其表达量变化不明显。乙烯利处理只是轻微下调HbPDK的表达量，但割胶处理却能显著下调HbPDK在新开割树胶乳中的表达，HbPDK的表达量下降了近5倍。结果说明HbPDK与割胶促进胶乳产量的增加呈明显的负相关，可以通过调控乙酰辅酶A的生成而影响橡胶树产量。pET32a原核表达载体的Trx标签，促进了HbPDK在大肠杆菌中的可溶性表达，通过镍柱亲和层析，获得了纯度在90%以上的重组蛋白。

Abstract: Pyruvate dehydrogenase kinase (PDK) through phosphates pyruvate complex (PDC), negatively regulates the activity of PDC, PDK inhibits the oxidative decarboxylation of pruvate, plays an im-portant role in regulating intermediary metabolism. The pyruvate dehydrogenase kinase gene of Hevea brasiliensis, HbPDK, its coding region is 1110 bp and encodes 369 amino acids. HbPDK is mainly expressed in leaves, latex and flowers. The expression of HbPDK didn’t change much in dif-ferent leaf development. After Ethrel treatment, the expression of HbPDK in latex reduced slightly. However, HbPDK reduced significantly after tapping in virgin trees, it was down-regulated about 5 folds. HbPDK is negatively correlated with the increasing yield of latex due to tapping, it produces an effect on the latex production through regulating the generation of acetyl coenzyme A. The Trx tag of pET32a promoted the soluble expression of HbPDK in E. coli, the recombinant protein of HbPDK was purified by nickel-nitrilotriacetic acid chelate chromatography; the purification of pu-rified recombinant protein was over 90%.