小麦叶绿体果糖-1,6-二磷酸醛缩酶活性的测定方法及应用

The Methods for Measuring the Activity of Chloroplast Fructose-1,6-bisphosphate Aldolase in Common Wheat and Its Application

高等植物中的果糖1,6-二磷酸醛缩酶(FBA)在叶肉细胞的叶绿体中高效表达,可逆催化卡尔文循环中果糖-1,6-二磷酸(FBP)和景天庚酮糖-1,7-二磷酸(SuBP)的合成反应,在各种非生物胁迫的逆境条件下,FBA会表现出不同的响应。为了测定小麦叶绿体FBA(CpFBA)的活性,进一步研究小麦中CpFBA的功能,本研究比较了两种酶活性测定方法:一种方法是根据FBA催化FBP分解的过程中NADH转化为NAD+的量来确定FBA的活性;另一种是利用谢利万诺夫试剂(Sediwanoff reagent)与酮糖FBP作用生成鲜红色复合物,根据OD520的变化测定FBA的活性。在此基础上,测定了两个小麦材料在4℃低温处理过程中CpFBA活性的变化。结果表明,谢利万诺夫试剂法对于测定小麦叶绿体FBA活性较为可行。CpFBA酶活性受到温度的影响,随着处理时间的延长,在35d内两个材料的FBA酶活都呈现升高-降低-升高的交替变化,相比较而言,对低温不敏感的小麦矮变1号相对稳定,对低温敏感的返白系小麦出现先升高后又逐渐降低的趋势。

In higher plants,plastidic fructose-1,6-bisphosphate aldolase（FBA）,participate in two reversible reactions of the formation of fructose-1,6-bisphosphate and sedoheptulose-1,7-bisphosphate in Calvin cycle,which express specially in chloroplast of mesophyll cell.FBA have been shown to be related to the plant response in all kinds of abiotic stress.In this study,two methods of measuring FBA enzyme activity were compared for investigating the function of chloroplast FBA（CpFBA） in wheat.One is testing the quantity of NADH transferred to NAD＋ in the process of FBP degradate,the other is according to the change of OD520 of resorcinol bright red compounds produced by the reaction of HMF formed by ketose with Sediwanoff reagent.Then,the changes of CpFBA activity of two common wheat materials during cold stress at 4℃ were determined.The results showed the method with Sediwanoff reagent is more effective,and the CpFBA activity of wheat were influenced obviously by low temperature.During the 35 days of 4℃ cold stress,the CpFBA enzyme activities of both wheat cultivars were found to change as fall-raise-fall alternately.By comparison,the enzyme activity of Aibian 1 was relative stable,but of albinism line,the cold sensitive wheat,was first soar up and then slump.This may provide a basis to investigate the biologic function of CpFBA in low temperature stress.