Screening and cloning of differentially expressed genes in Dendrobium nobile induced by orchid mycorrhizal fungus promoting the growth

Abstract

Appropriate mycorrhizal fungi could effectively promote plant growth and development. Our previous research results showed that the growth of Dendrobium nobile was obviously promoted under inoculating one orchid mycorrhizal fungi, Epulorhiza sp. AR-18. To understand the growth-promoting molecular mechanisms, differential displayed real time polymerase chain reaction (DDRT-PCR), reverse Northern blot and Southern blot were used to isolate and identify genes whose transcription were altered in cultured D. nobile plants that were treated with Epulorhiza sp. AR-18. Amplified by 8 primer combinations from one anchor primers and 8 random primers, a total of 14 complementary DNA (cDNA) fragments including 12 differentially expressed cDNA bands were isolated. Reverse northern blot analysis showed that only 2 genes were differentially displayed cDNA bands. One band was an especially expressed fragment, expressed in the treated group but not in the control; while another was a differentially expressed fragment, weak in the treated and strength in the control. Southern blot analysis demonstrated that the two gene fragments were from the plant and not from the fungus. Sequence analysis and database searches revealed no significant homology to any known sequences. The results suggested that the usefulness of messenger RAN (mRNA) differential display technique for the detection of differentially expressed genes in D. nobile whose growth could be promoted by mycorrhizal fungi.

Keywords: Dendrobium nobile, differential displayed real time polymerase chain reaction (DDRT-PCR), orchid mycorrhizal fungus, Epulorhiza sp., reverse northern blot