Cloning And Analysis Of Two Pathogenesis-related Protein Genes From TcLr19 In The Defense Responses To Puccinia Triticina

Plant pathogenesis-related proteins (PRs) are the proteins induced by the pathogen or environment condition of pathology which have been reported about many plants. The objective of this research was to deeply understand the mechanism of wheat resistance to leaf rust pathogen. RT-PCR and RACE was combined to isolate the full length cDNA sequence of TcLr19PR1 and TcLr19Glu from the near-isogenic line TcLr19 induced by Puccinia triticina. Quantitative real-time PCR (qRT-PCR) was used to detect the expression profiles of TcLr19PR1 and TcLr19Glu in the compatible and incompatible interactions between wheat and leaf rust pathogen. In the meantime, the recombinant plasmids pAHC-TcLr19PR1 and pAHC-TcLr19Glu were constructed and transformed into detached leaves and embryogenic callus of susceptible material by particle bombardment respectively. The main results are as follows:1. Wheat near-isogenic line TcLr19 and leaf rust strain 07-10-421-3 were used as the initial material. Total RNA of wheat leaves at different hours post inoculation (hpi) were mixed and used to synthesize first strand cDNA, the full length cDNA sequence of TcLr19PR1 and TcLr19Glu was obtained by RT-PCR and RACE, respectively.2. TcLr19 and Thatcher inoculated with leaf rust strain were used as the initial materials, temporal expression profile of TcLr19PR1 and TcLr19Glu in compatible and incompatible interactions at different hpi was detected by semi-quantitative RT-PCR and qRT-PCR. The results showed that TcLr19PR1 transcripts reached the maximum at 12 hpi in the incompatible interaction, and then declined, and more transcripts were accumulated in incompatible interaction than compatible interaction. TcLr19Glu transcripts were detected at as early as 12 hpi in the incompatible interaction, and reached the maximum at 36 hpi, and then declined, and more transcripts were accumulated in incompatible interaction than control and compatible interaction.3. TcLr19PR1 sequence in DNA was obtained by PCR using the genomic DNA of TcLr19 as templates. TcLr19PR1 gene had low copies in the wheat genome demonstrated by Southern blot. In the meantime, TcLr19PR1 gene was located on chromosome 7D by nulli-tetrasomic lines of“Chinese Spring”. 4. In order to understand the function ofβ-1,3-glucanase in the defense response to Puccinia triticina, the activity ofβ-1,3-glucanase in TcLr19 and Thatcher induced by leaf rust strain was determined. The results showed that the activity in Thatcher be increasing slowly from 0 to 144 hpi, and the activity level reached maximum at 144 hpi; however, the activity in TcLr19 increased significantly from 0 to 48 hpi, and the activity level reached maximum at 48 hpi. The activity level in TcLr19 was higher than that in Thatcher on the whole.5. The transgene vectors of pAHC-TcLr19PR1 and pAHC-TcLr19Glu were successfully constructed after inserting TcLr19PR1 and TcLr19Glu open reading frame into plant expression vector pAHC25 respectively. The molecular mechanism of the TcLr19PR1 and TcLr19Glu in wheat to the Puccinia triticina was analyzed by a transient expression system. The results showed that the hyphae development of leaf rust fungus was inhibited to the extent.6. pAHC-TcLr19PR1 and pAHC-TcLr19Glu were transformed into the embryogenic callus of the susceptible material Zhengzhou5389 by particle bombardment, and the regenerate wheat seedlings were obtained after series of selective cultivation.