联合固氮酶活性测定中酶促反应时间的控制方法研究  

Studies on the method of controlling enzymatic reaction time of acetylene reduction in determining associative nitrogenase activity of associative system

作　　者：季天委[1] 方萍[1] 朱日清[1] 周钗美[1] 

机构地区：[1]浙江大学资源科学系,浙江杭州310029

出　　处：《浙江大学学报（农业与生命科学版）》2003年第4期382-386,共5页Journal of Zhejiang University:Agriculture and Life Sciences

基　　金：国家自然科学基金资助项目(30070443).

摘　　要：乙炔还原法是国际上普遍公认的测定生物固氮活性的方法,该法具有简便、快速、灵敏度高等优点.乙炔还原反应的温育时间不同,直接影响乙炔还原法测定固氮酶活性结果的可比性.为确保大批量的联合固氮酶活性测定条件的一致性,有必要选择一种固氮酶活性阻断剂,使联合固氮体系中的乙炔还原反应维持在特定时间后予以阻断.本文比较了3种灭菌剂对2株联合固氮菌的抑菌效果及其对水稻根际联合固氮酶活性阻断效果,结果表明,3.5%甲醛水溶液是一种理想的联合固氮酶活性阻断剂.Acetylene reduction assay (ARA) is a world wide used method for measurement of the biological nitrogenfixing activity, which is featured by its rapidity, convenience, and high sensitivity. Because of the varying culture time, the results of acetylene reduction reaction would be different and, hence, unable to compare with each other. To ensure the veracity of ARA in detecting numerous specimens, it is necessary to find a reagent capable of interdicting the activity of nitrogenase that catalyzes acetylene reduction reaction in an associative nitrogenfixing system at certain periods. Two experiments were conducted to compare the effects of three bactericides in restricting the growth of 2 associative nitrogenfixing bacteria strains W12 and FY and in interdicting their nitrogenfixing activity in rice rhizosphere. Firstly, bacterial inhibitory ring method was used to determine the effects of three kinds of bactericides each with two concentrations on the growth of W12 and FY. It was repeated twice for treatment and cultured at 30 ℃ for three days. The diameter of bacterial inhibitory ring was determined by crisscross method. It was found that 3.5% formaldehyde, 2.6% sodium hypochlorite and 0.1% mercuric chloride all showed high restriction effect on the growth of W12 and FY. Then these selected bactericides in the prior experiment were used to investigate their interdictory effect on the nitrogenase activity of W12 and FY associated with two rice varieties Minghui 63 and Zhenshan 97 by ARA. After 10% of the atmosphere in each tube had been replaced by C2H2, the tubes were cultured at 30 ℃ for further 72 h. Then 1mL bactericide solution was injected into each tube. 1　mL gas samples were assayed for C2H4 by GC14B of SHIMADZU CORPORATION and the amount of C2H4 in each tube was subject to detection at 10min, 8　h, 24　h, 48　h and 120　h after bactericide injection. Each bactericide treatment was repeated five times during the determination period. Results illustrated in Fi

关 键 词：联合固氮酶 酶活性 测定 酶促反应时间 控制方法 乙炔还原法 甲醛水溶液 酶活性阻断剂 

分 类 号：Q946.549[生物学—植物学] S144.5[农业科学—肥料学]