Isolation Of The Wheat Seedlings' CDNA Related With The Genes That Express Differentially In The Yr4 Near-isogenic Lines Inoculated With Puccina Striiformis F.sp.tritici

The wheat stripe rust resistant near-isogenic line (NIL) Yr4/6* Taichung29 was obtained by backcrossing to its susceptible parent Taichung29 six times. Its' genetic background was very similar to Taichung29 except for wheat stripe rust resistance. Isolation of the differential expressed genes between the two lines is very important to elucidate mechanism of the host-pathogen interaction.We chose the seedlings of the resistant near-isogenic line(NIL) Yr 4/6* Taichung29 and its susceptible recurrent parent Taichung29 inoculated with the race CY26 of Puccinia striifomis f. sp. tritici as the materials. With the group of the Yr4/6* Taichung29 seedlings collected at the 24h,48h,and 72h after inoculated as the tester and the corresponding group of the Taichung29 seedlings as the driver, Suppression Subtractive Hybridization(SSH) was performed. Then a subtractive library which contains approximately 1300 clones was constructed.Ninty randomly picked clones were analyzed with reverse Northern blot analysis. The process was as follows: three probes, prepared from total RNA of Yr4/6*Taichung29, inoculated Yr4/6*Taichung29 and inoculated Taichung29 respectively, were hybridized to three duplicated membranes prepared from PCR products of the SSH clones. As a result, four clones presented different.Whereas the different clones were very few, 20 randomly picked clones were sequenced for further analysis of the library. The result showed that all of the cDNA were from wheat. The reconstruction of Rsa 1 restriction enzyme site analysis also confirmed that the adapter ligation and the hybridization of SSH were no problem. Thus, we screened 400 clones from the library with three probes prepared from Yr4/6*Taichung29, inloculated Yr4/6*Taichung29 and Taichung29 respectively. Eleven clones presented different signals among the three probes.Four out of the total 15 clones were analyzed by Northern blot Two of them were confirmed expressed differentially. Sequencing of the two clones showed that the two genes encode thioredoxin and leucine-rich repeat protein respectively.