Construction Of CDNA Library Of Incompatible Interaction Between Wheat And Puccinia Striiformis And Analysis Of Expressed Sequence Tags

The disease——Stripe rust of wheat, caused by Puccinia striiformis f. sp. tritici, is one of the most important diseases of wheat worldwide. Growing resistant cultivars is the most effective and economical method of disease control. However, Cultivars with race-specific resistance often become susceptible soon after they are released because of the rapid evolution of new races, which is becoming the most important problem for the application of resistant cultivars. Presently, there are many researches on the interaction between wheat and stripe rust from morphology, histology, cytology, and genetics, while little is known about this interaction on molecular level. So studies at the molecular level on this interaction and revealing the molecular mechanism are significative on both practice and theory for reasonably utilization of resistant cultivars and sustainably control of stripe rust.In order to know more about the genes expression profile and the molecular mechanism of incompatible interaction between wheat and Puccinia striiformis, incompatible reaction has been established based on the cultivar Suwon 11 and stripe rust CY23 interaction. Total RNA was extracted from inoculated wheat leaves at16h, 24h, 36h and 48h respectively, equal amount of which then were mixed as reverse transcription template. Compatible cDNA library was constructed utilizing SMARTTM cDNA Library Construction Kit. 1.2×106 pfu/mL for primary library titer, 2.0×109 pfu/mL for amplified library titer, and 97% recombinants were obtained. The inserted cDNA lengths ranged from 0.4 kb to 3 kb.Above 5200 clones were randomly chosen to be sequenced from the 5' end of the cDNAs, which generated 1515 high quality ESTs after removing the vector and repeat sequences, containing 494 contigs and 1021 singlets after Cap3 analysis. Then the ESTs similarity analysis based on BLASTx software was carried on, result shows that nohit(46%) is the most largest one among all the unigenes. After function annotation of the ESTs, we concluded that the most largest one was relative to enery and metabolism(32%), in which ribulose-1,5-bisphosphate carboxylase/oxygenase, chloroplast precursor, chlorophyll a-b binding protein and photosystem protein et al. with high frequency. The second largest one was relative to unclear and unknown(22%), next was disease and defence, including induced resistant proteins, several PRs participating in the SAR pathway while pollen allergen Phl p 4 taking in part of HR pathway. The others had the high homology with signal transduction, protein synthesis and destination, transports. There were several ESTs encoding proteins from E coli. or other procaryotic organism. We chose some interested genes and prepared to clarify their functions in the interactions, expecting to find the new resistant genes.