杨梅根瘤内生菌超微结构及其固氮酶结构基因的研究

Study on the Ultrastructure and nif HDK Gene of Nodular Endophytes of Myrica rubra

用透射电镜首次观察到杨梅根瘤内生菌泡囊结构存在两种类型,有隔或无隔,椭圆形,直径1.3～3.0μm。并首次从杨梅根瘤共生固氮的内生菌中通过PCR反应克隆了2675bp的固氮酶结构基因,包括nifH和nifD全长基因及nifK基因片段,对其nifH和nifD基因的核苷酸序列分析结果表明,其核苷酸与已报道的Frankia菌株同类基因的核苷酸的同源性高,达89%以上。861bp的nifH基因开放阅读框架和1458bp的nifD基因开放阅读框架的氨基酸序列与已报道的其他Frankia菌株同类基因的氨基酸序列的同源性分别为92%以上和89%以上,特别是起重要作用的氨基酸残基非常保守。nifH、nifD及nifK基因是连锁在一起的,nifH和nifD及nifD和nifK之间的间隔序列分别为49bp和205bp,与已报道的Frankia菌株同类基因之间的间隔序列在长度和同源性上差异较大,因而杨梅根瘤共生固氮的内生菌与其它属植物根瘤共生固氮内生菌及分离株存在菌株之间的差异。

Two kinds of vesicles with septum or without septum,oval in shape,about 1.3～3.0μm in diameter were discov-ered firstly in nodule of Myrica rubra by TEM.A2675bp sequence,including complete nucleotide sequences of nifHD gene and partial nucleotide sequence of nifK from nodular endophyte of Myrica rubra were determined and analyzed by PCR,cloning and sequencing.Sequence analysis of2675bp revealed two open reading frames and beginning of a third in the same orientation,each of them showed high degree of sequence homology with nifH,nifD and nifK of other Frankia strains re-spectively.The deduced amino acid sequence of the nifH ORF,consisting of861bp,showed sequence similarity of above92%with those of other Frankia strains,whereas that of nifD ORF,consisting of1458bp,showed more than89%similari-ty.The amino acid residues,playing important functional roles are very well conserved.Intergenic sequences(IGS)between nifH and nifD and between nifD and nifK were49bp and205bp respectively.The IGS between nifH and nifD or between nifD and nifK has big differences in length or homology with those of other Frankia strains.The result showed that Frankia strains from nodular endophytes of Myrica rubra were different from those isolated from nodules of other actinorhizal plants.