| Ten different stages callus of the Nicotiana benthamiana (tobacco) leaves were cultured in theMS medium. Observation from the morphologic varieties of nucleolus was performed by the Leicafluorescent microscope, the irregular nucleus dyed with Hoechst 33258 were found in every stagescallus, which was the type character nuclear change in the process of Programmed cell death (PCD);DNA ladder was tested during ten different stages of the induced callus tissue of tobacco leaves,showed different extents of DNA ladder during ten different stages, and got most fragments at stagesix, leaf Segments cultured at 13 days, PCD reach to peak.The content of ABA at the beginning of callus inducement was almost zero, at the stage five, leafsegments cultured 11 days appeared a great deal of ABA, the ABA content at stage six, leafsegments cultured 13 days was the highest, more than 3 times of stage five, the same with DNAladder. It was inferred that the auxins added in culture medium is the crucial factor to triggered PCDin the process of callus inducement.We amplified Beclin-1 gene by RT- PCR from ten different stages callus and detected by 1.2%agarose electrophoresis showed that nine different stages, leaves segments cultured at 3, 5, 7, 9, 11,13, 15, 17, 19 days had Beclin-1 gene expression.The results of cloning and sequencing showed thatthe homology of the nucleotide between the cloned Beclinl-like gene and tobacco leaf Beclin-1(AY701316) gene was 95%. Transcribed Beclin-1 gene as a RN A probe, Northern blotting resultsshowed that the hybridization signal of Beclin-1 gene in the stage six, the leaf segments cultured 13days was the highest in the other nine stages, which indicated the expression of Beclin-l gene in the13th day of callus inducement was higher than those in the 19 days.It inferred that the endogenous hormones ABA might influence the expression of Beclin-1 geneand Beclin-1 gene might promote the signal of PCD inside the cell.
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