摘要
为了解烟草内生细菌的多样性,采用改良的NA培养基和稀释平板法从不同生长期的烟草的根、茎、叶中分离得到127株内生细菌。利用细菌16S rDNA特异引物扩增得到16S rDNA的部分片段,长约700 bp,分别用内切酶MspⅠ、HaeⅢ、AfaⅠ、HinfⅠ对扩增产物进行限制性酶切。16S rDNA PCR-RFLP分析中,在100%相似性水平处,除了cj23、ty16、cg23、wy6、cj25、wy13、my23、ty7、cj14、cg25、cg2、cj13、my11,my27单独成群外,其余菌株分为15个分类操作单元,其中Otu1、Otu2,Otu27类群最大,分别包括了27、18,26个菌株。对29个代表菌株16S rDNA序列系统发育分析表明,这些菌株主要分布于芽孢杆菌(Bacillus)分支,其余菌株分布于葡萄球菌属(Staphylococcus)、微杆菌属(Microbacterium)、单胞菌属(Sphingomonas)、中华根瘤菌属(Sinorhizobium)、土壤杆菌属(Agrobac-terium)、根瘤菌属(Rhizobium)、纳西杆菌属(Naxibacter)、贪铜菌属(Cupriavidus)、寡养食单胞菌属(Stenotrophomonas)、不动杆菌属(Acinetobacter)、假单胞菌属(Pseudomonas)、肠杆菌属(Enterobacter)、泛菌属(Pantoea)、克雷伯菌属(Klebsiella)、Bifissio属系统发育分支。通过Shannon多样性指数对不同生长期分离到的菌株进行多样性分析和比较,结果表明团棵期叶片中分离到的内生细菌多样性最大,随后是旺长期,苗期次之,成熟期最小。
127 endophytic bacteria strains were isolated from roots, stems and leaves at various growing stages by dilution and spread plate technique with improved NA medium to study tobacco endophytic bacteria diversity. 16S rDNA genes were amplified with peculiar primers 799f and 1492r to produce a single band of approximately 700 bp. PCR products were digested with restriction endoenzyme MspMsp Ⅰ ,HaeⅢ ,Afa Ⅰ ,Hinf Ⅰ Msprespectively. Results of 16S rDNA PCR- RFLP analysis showed that most strains could be clustered into 15 Outs except the unique strains of cj23, tyl6, cg23, wy6, cj25, wyl3, my23, tyT, cjl4, cg25, eg2, cj13, myll and my27. The largest groups were Otul, Otu2 and Otu27 which consisted of 27, 18 and 26 strains respectively. 16S rDNA gene sequences of 29 representative strains were determined and a phylogenic tree was constructed. It showed that these bacteria were mainly distributed in the phyloge- netic branches of Bacillus, whereas the remaining strains belonged to genus Staphylococcus, Microbacterium, Sphin-gomonas, Sinorhizobium, Agrobacterium, Rhizobium, Naxibacter, Cupriavidus, Stenotrophomonas, Acinetobacter, Pseud- omonas, Enterobacter, Pantoea, Klebsiella and Bifissio. In addition, Shannon diversity index used for analyzing strains＇ diversity indicated that the order of diversity in leaves of tobacco plants at different growing stages is that resettling stage followed by growth peak period, seedling stage and mature period.
出处
《中国烟草学报》
EI
CAS
CSCD
北大核心
2012年第1期92-100,105,共10页
Acta Tabacaria Sinica
基金
国家自然科学基金资助项目(No.30860017)
关键词
烟草
可培养
内生细菌
RFLP
tobacco
cuhivability
endophytic bacteria
restriction fragment length polymorphism
