Cdna-aflp Analysis And Bacterial Blight Interaction In The Rice Gene Expression Profiles

cDNA-AFLP was employed to analyze the expression profiles of rice during compatible interaction between rice and Xanthomonas oryzae pv. oryzae, a causal agent of the bacterial blight disease of rice.Rice (Oryza sativa japonica L. cv. Nipponbare) callus was induced with the mature embryo, and then the suspension culture cells (Scc) were established in the liquid MS medium. The Scc interacted with the compatible pathogen of Xanthomonas oryzae pv. oryzae (JxoI strain) was harvested and ground to a fine powder under liquid nitrogen. Total RNA of the rice cells was isolated using LiCl differentially precipitation method, and the mRNA was separated and purified from the total RNA with the streptavidin magnesphere particles-oligo-dT. Double stranded complementary-DNA (cDNA) were synthesized from the mRNA with the primer of oligo (dT)₂₁.After digested using restriction endonuclease VspI and TaqI, and ligated with the relevant anchor, the cDNA was used as primary template for pre-amplification. The product of pre-amplification was diluted to the concentration of 1ng/μl, and used as the template for the selective amplification. The selective primer complementary to the TaqI-anchor was labeled using [γ-³²P]ATP. The resulting products were size-fractionated on sequencing type PAGE. After autoradiography and fingerprint analysis, the differential displayed rice transcripts were identified, isolated, sequenced and analyzed. In this study, 64 primer pairs were used and 384 differential expression transcript-derived fragments (TDFs) were obtained, of which 321 TDFs were up-regulated and the others were down-regulated. Over 100 TDFs were sequenced and determined using database homology searches. The results revealed that 23 TDFs were putative function genes and 41 TDFs were unknown function genes.Real-time PCR analysis was performed to confirm the cDNA-AFLP differential expression TDFs.'With the analysis information of TDFs, the complete sequence gene encoding basic transcription factor 3 was amplified and cloned with the T-vector (pMD 18-T).