小麦内生固氮菌分离及其ACC脱氨酶测定  

Isolation of Wheat Endophytic Diazotrophs and Determination of 1-Aminocyclopropane-1-Carboxylate Deaminase

作　　者：秦宝军[1] 罗琼[1] 高淼[1] 胡海燕[1] 徐晶[1] 周义清[1] 孙建光[1] 

机构地区：[1]中国农业科学院农业资源与农业区划研究所/农业部作物营养与施肥重点实验室,北京100081

出　　处：《中国农业科学》2012年第6期1066-1073,共8页Scientia Agricultura Sinica

基　　金：国家公益性行业(农业)科研专项(201203045)

摘　　要：【目的】了解小麦内生固氮菌数量,筛选具有ACC(1-aminocyclopropane-1-carboxylate,1-氨基环丙烷-1-羧酸)脱氨酶活性的小麦内生固氮菌,确定筛选菌株的系统发育地位与分类地位,为微生物肥料生产收集菌种资源。【方法】样品表面灭菌后采用无氮培养法筛选内生固氮菌,乙炔还原法测定菌株固氮酶活性;采用ACC唯一氮源法筛选ACC脱氨酶阳性菌,比色法定量测定ACC脱氨酶活性;PCR扩增得到菌株16S rDNA,通过序列测定和相似性分析研究菌株的系统发育;通过形态、生理生化特征和16S rDNA序列比对鉴定菌种。【结果】小麦体内固氮菌数量为(0.2—17.8)×105cfu.g-1鲜重;分离到小麦内生固氮菌60株,固氮酶活性在1—36 nmol C2H4/h.mg蛋白,其中9株具有ACC脱氨酶活性,活性在0.87—9.32μmolα-丁酮酸/h.mg蛋白;新分离菌株9136固氮酶活性为1.82 nmol C2H4/h.mg蛋白,ACC脱氨酶活性为9.32μmolα-丁酮酸/h.mg蛋白,初步鉴定为假单胞菌Pseudomonassp.。【结论】田间自然生长的小麦体内有大量固氮菌,数量在105cfu.g-1鲜重,其中部分菌株具有ACC脱氨酶活性,个别菌株具有较高的ACC脱氨酶活性,可能对作物抵御不良环境具有作用。【Objective】 The objective of this study is to determine the ammount of wheat endophytic diazotrophs and screen for ACC（1-aminocyclopropane-1-carboxylate） deaminase activity from the diazotrophs,determine the phylogenetic and classific position of selected strains and prepare strains for microbial fertilizer production.【Method】 Surface sterilization and nitrogen-free medium were used to isolate diazotroph and ACC was used as sole nitrogen source to screen strains with ACC deaminase activity.Nitrogenase activity was determined with acetylene reduction assay.16S rDNA was amplified with PCR and analysed with MEGA software.Strain identification was carried out based on the morphology,physiology,biochemical test results and 16S rDNA analysis.【Result】 The ammount of endophytic diazotrophs at jointing stage of wheat was（0.2-17.8）×105 cfu？g-1 fresh weight.Sixty endophytic diazotrophs with nitrogenase activity ranging 1-36 nmol C2H4/h？mg protein were isolated from wheat.Nine of the 60 endophytic diazotrophs were ACC deaminase positive,the range of enzyme activity is 0.87-9.32 μmol α-ketobutryric acid/h？mg protein.New isolate 9136 with nitrogenase activity 1.82 nmol C2H4/h？mg protein and ACC deaminase activity 9.32 μmol α-ketobutryric acid/h？mg protein was identified as Pseudomonas sp..【Conclusion】 About 105 cfu？g-1（fresh weight） endophytic diazotrophs naturally colonized field grown wheat,some of these endophytic diazotrophs could produce ACC deaminase.A few strains showed relatively high ACC deaminase activity,and they might play a role in crop resistance to enviromental stress.

关 键 词：小麦 内生固氮菌 ACC脱氨酶 根际促生细菌 

分 类 号：S512.1[农业科学—作物学]