摘要
为探讨果胶酶基因与内生芽孢杆菌在植物体内定殖的相关性,PCR克隆了2株内生芽孢杆菌的果胶酶全长基因,并将全基因序列插入穿梭载体pGFP4412,构建了果胶酶基因过表达载体,转化内生细菌实现了果胶酶过表达.平板果胶酶酶活检测表明野生菌的水解圈直径仅为过表达突变体水解圈直径的50%.通过实时荧光定量PCR分析突变体和对照菌株(BS2-gfp和TB2-gfp)中果胶酶基因的表达情况,发现突变体的表达量分别是对照菌株表达量的82和85倍.统计了突变体与对照菌株28 d内在小白菜各组织中的定殖数量,结果表明:接种后1-3 d,突变体在根茎叶中的定殖数量与对照菌株比较,差异性显著(P<0.05).接种后7-28 d,除第7天(d 7)过表达菌株与对照菌株在根中的定殖菌量有显著性差异(P<0.05)外,其余统计结果均无显著性差异(P>0.05).在为期28 d的分离过程中,突变体的定殖数量经历了一个由显著高于对照菌株,到略高或逐渐与对照菌株的菌量趋于一致的过程.本研究初步证明了果胶酶基因在内生芽孢杆菌的定殖初期起到了一定的作用.
In order to investigate the correlation between pectinase gene of endogenous Bacillus and colonization in plants, the full length genes of pectinase from two endophytic Bacillus strains were cloned; the overexpressing vectors of pectinase genes were constructed by inserting the full length genes of pectinase into shuttle vector pGFP4412, then transformed into the endophytic Bacillus strains and overexpressed. Pectinase enzymatic assays showed that the diameters of hydrolyzed circles caused by the wild type strains were only 50% of the overexpressing mutants on LB agar plates. The real-time quantitative PCR results showed that the relative mRNA transcription levels of pectinase gene in mutant bacteria were 82 and 85 times as much as the level in the control strains （BS2-gfp and TB2-gfp）, respectively. The colonizing bacteria amounts of pectinase mutants and the control strains in the tissues of cabbage within 28 d indicated a significant difference between mutants and control strains in roots, stems and leaves in the first 1-3 d after inoculation （P 〈 0.05）. The amount of mutant bacteria in roots was notably higher than that of control strains in the 7th day （P 〈 0.05）. But for the other statistic data, no significant difference was detected. The results of continuous isolations during 28 d showed a remarkably higher population density in mutants than the control strains at the beginning, and a gradually decreasing difference between the two. This study presented preliminary evidence that pectinase gene plays a role in endophytic Bacillus bacteria colonization in plant at the initial stage. Fig 7, Tab 2, Ref 23
出处
《应用与环境生物学报》
CAS
CSCD
北大核心
2013年第5期805-810,共6页
Chinese Journal of Applied and Environmental Biology
基金
福建省科技厅自然科学基金项目(2010J05049)资助~~
