Construction Of CDNA Libray From Wheat Leaves Challenged By Puccinia Striiformis And Analysis Of Expressed Sequence Tags

Stripe rust, caused by Puccinia striiformis f.sp. tritici, is one of the most destructive diseases of wheat in the world. Recently, there have been many researches on the compatible interaction between wheat and stripe rust from such aspects, like morphology, histology, cytology, and physiological biochemistry, and the compatible interaction mechanisms have been explained from different levels. However, studies on molecular mechanisms of stripe rust pathogenicity have attracted relatively attention seldom and little is known about the molecular mechanisms of interaction between stripe rust and its host. Studies on molecular interaction from molecular biology and genetics ways may elucidate bilateral relationship in genetic levels. Further more it will be of significant importance either in practices and theory.For this reason, compatible reaction has been established based on the cultivar Shuiyuan 11 and stripe rust CY31 interaction. Total RNA was extracted from inoculated wheat leaves at 72 h, 120 h, and 192 h respectively, equal amount of which then were mixed as reverse transcription template. Compatible cDNA library was constructed utilizing SMARTTM cDNA Library Construction Kit. 6.3×106 pfu/mL for primary library titer, 9.7×109 pfu/mL for amplified library titer, and 98% recombinants were obtained. The inserted cDNA lengths ranged from 0.5 kb to 2.2 kb, majority at about 1 kb.About 600 clones were randomly chosen for sequence. After removing the vector and repeat sequences, 594 ESTs were acquired. The ESTs lengths mainly distribute from 400～500bp. Fifty Contigs and 349 singletons were clustered by CAP3 application. And the ESTs similarity analysis based on BLASTx software was carried on by comparing sequences in non-redundant database of GenBank. 44% ESTs had the higher homology with plant, which was the largest category; 32% ESTs had the higher homology with fungi, and no-hits found ESTs were 18%. The results showed that PIG28 encoding protein, ABC transporter, metallothioneine, polyubiquitin 2, plasma membrane H+-ATPase, amino acid permease etc. were supposed to be involved in the process of compatible interaction. There have many no hits found and predicted proteins in the results compared with GenBank, and whether these ESTs were expressed by induction or not still need further verification.A full lengh cDNA library was constructed based on the compatible interaction induced by Puccinia striiformis f.sp. tritici. Our work laid a sound foundation for isolation of...