NDLI: Design and application of specific 16S rDNA-targeted primers for assessing endophytic diversity in Dendrobium officinale using nested PCR-DGGE

Content Provider	Springer Nature Link
Author	Yu, Jie Zhou, Xiao Feng Yang, Sui Juan Liu, Wen Hong Hu, Xiu Fang
Copyright Year	2013
Abstract	Novel specific 16S rDNA-targeted primers were successfully designed and applied to the characterization of endophytic diversity in Dendrobium officinale. Using the popular universal bacterial primers 27f/1492r, the fragments of chloroplast and mitochondrion 16S/18S rDNA were amplified from D. officinale. They shared high nucleotide identity with the chloroplast 16S rDNAs (99–100 %) and with the mitochondrion 18S rDNAs (93–100 %) from various plants, respectively, and both shared 73–86 % identities with the bacterial 16S rDNA sequences in GenBank. The current bacterial universal primers, including 27f/1492r, match well with the chloroplast and mitochondrion 16S/18S rDNAs, which accordingly renders these primers not useful for endophytic diversity analysis. Novel 16S rDNA-targeted primers fM1 (5′-CCGCGTGNRBGAHGAAGGYYYT-3′) and rC5 (5′-TAATCCTGTTTGCTCC CCAC-3′) were designed, which show good specificity compared to the 16S/18S rDNAs of D. officinale, and perfect universality within bacteria except for Cyanobacteria. The primers fM1/rC5, together with 515f-GC/rC5, which overlaps the whole V4 region of 16S rDNA, were subjected to nested polymerase chain reaction denaturing gradient gel electrophoresis (PCR-DGGE) to analyze the diversity of endophytic bacteria in D. officinale from three different sources in China. The results showed diversities in roots and stems of the plants from all three locations. Altogether, 29 bands were identified as bacteria, with the dominant group being Proteobacteria and the dominant genus being Burkholderia, some of which commonly has the function of nitrogen fixation and thus may play potentially important roles in D. officinale. Therefore, the nested PCR-DGGE method based on the novel primers provides a good alternative for investigating the communities and roles of endophytes in D. officinale.
Starting Page	9825
Ending Page	9836
Page Count	12
File Format	PDF
ISSN	01757598
Journal	Applied Microbiology and Biotechnology
Volume Number	97
Issue Number	22
e-ISSN	14320614
Language	English
Publisher	Springer Berlin Heidelberg
Publisher Date	2013-10-15
Publisher Place	Berlin, Heidelberg
Access Restriction	Subscribed
Subject Keyword	Dendrobium officinale Chloroplast 16S rDNA Mitochondrion 18S rDNA Novel primers Endophytic bacteria Nested-PCR-DGGE Microbiology Microbial Genetics and Genomics Biotechnology
Content Type	Text
Resource Type	Article
Subject	Medicine Applied Microbiology and Biotechnology Biotechnology

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