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

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

[1]  P. Lim,et al.  Identification of Differentially Expressed Genes in Flower Buds of Calanthe discolor and C. sieboldii , 2010, Journal of Plant Biology.

[2]  D. Scheel,et al.  A cell wall extract from the endophytic fungus Piriformospora indica promotes growth of Arabidopsis seedlings and induces intracellular calcium elevation in roots. , 2009, The Plant journal : for cell and molecular biology.

[3]  P. Venkatachalam,et al.  Molecular Identification and Characterization of a Gene Associated with the Onset of Tapping Panel Dryness (TPD) Syndrome in Rubber Tree (Hevea brasiliensis Muell.) by mRNA Differential Display , 2009, Molecular biotechnology.

[4]  A. Shete,et al.  Molecular characterization of an expressed sequence tag representing the cuticle-degrading serine protease gene (PII) from the nematophagous fungus Arthrobotrys oviformis by differential display technology. , 2008, Genetics and molecular research : GMR.

[5]  X. Yao,et al.  Copacamphane, picrotoxane and cyclocopacamphane sesquiterpenes from Dendrobium nobile. , 2008, Chemical & pharmaceutical bulletin.

[6]  M. Akkaya,et al.  Identification of differentially expressed transcripts from leaves of the boron tolerant plant Gypsophila perfoliata L. , 2008, Plant Cell Reports.

[7]  Jichen Xu,et al.  Identification and characterization of an expansin gene AsEXP1 associated with heat tolerance in C3 Agrostis grass species. , 2007, Journal of experimental botany.

[8]  A. Bago,et al.  A gene from the arbuscular mycorrhizal fungus Glomus intraradices encoding a binding protein is up-regulated by drought stress in some mycorrhizal plants , 2007 .

[9]  I. Baldwin,et al.  Sebacina vermifera Promotes the Growth and Fitness of Nicotiana attenuata by Inhibiting Ethylene Signaling1[W] , 2007, Plant Physiology.

[10]  J. Cairney,et al.  A simple and efficient method for isolating RNA from pine trees , 1993, Plant Molecular Biology Reporter.

[11]  C. Town,et al.  Analysis of indole-3-butyric acid-induced adventitious root formation on Arabidopsis stem segments. , 2005, Journal of experimental botany.

[12]  L. Altschmied,et al.  The Endophytic Fungus Piriformospora indica Stimulates the Expression of Nitrate Reductase and the Starch-degrading Enzyme Glucan-water Dikinase in Tobacco and Arabidopsis Roots through a Homeodomain Transcription Factor That Binds to a Conserved Motif in Their Promoters* , 2005, Journal of Biological Chemistry.

[13]  P. Karlovsky,et al.  Conversion of cDNA differential display results (DDRT-PCR) into quantitative transcription profiles , 2005, BMC Genomics.

[14]  Song Jy,et al.  Effects of fungus on the growth of Dendrobium candidum and D. nobile in vitro culture , 2001 .

[15]  S. Guo,et al.  [Effects of fungus on the growth of Dendrobium candidum and D. nobile in vitro culture]. , 2001, Zhongguo yi xue ke xue yuan xue bao. Acta Academiae Medicinae Sinicae.

[16]  L. Zettler,et al.  Propagation of the little club-spur orchid (Platanthera clavellata) by symbiotic seed germination and its ecological implications , 1998 .

[17]  P. Liang,et al.  Differential screening of differential display cDNA products by reverse northern. , 1997, Methods in molecular biology.

[18]  J. Antoniw,et al.  An analysis of differential display shows a strong bias towards high copy number mRNAs. , 1995, Nucleic acids research.

[19]  J. Reich,et al.  Identification of differentially expressed mRNA species by an improved display technique (DDRT-PCR). , 1993, Nucleic acids research.

[20]  C. N. Stewart,et al.  A rapid CTAB DNA isolation technique useful for RAPD fingerprinting and other PCR applications. , 1993, BioTechniques.

[21]  A. Riggs,et al.  Genomic Sequencing , 2010 .

[22]  A. Pardee,et al.  Differential display of eukaryotic messenger RNA by means of the polymerase chain reaction. , 1992, Science.