Screening Resistance Related Genes To Powdery Mildew From TAC Library Of A Triticum Aestivum-Haynaldia Villosa Translocation Line 6VS/6AL

Powdery mildew,caused by Erysiphe graminis DC.f.sp.tritici Marchal,is one of the major diseases of common wheat worldwide.Development and utilization of wheat cultivars with resistance gene is an efficient,economic and environmentally safe strategy to control powdery mildew.Pm21,originally introduced into wheat from H.villosa through chromosome translocation of 6VS/6AL,is an effective powdery mildew resistance gene with broad spectrum in the world and has a good promise in wheat breeding.So cloning of Pm21 gene and powdery mildew resistance-related genes will play an important role in future disease resistance breeding.Hv-S/TPK gene,a resistantance related gene to powdery mildew,was cloned by using genechip,and located on the short arm of the 6V chromosome.A pair of primers (CINAU15)designed based on the sequence of Hv-S/TPK could be used as a co-dominant marker for selecting Pm21 located on 6VS.CINAU15 was used to screen a genomic TAC library of Triticum aestivum-Haynaldia vilossa 6VS/6AL translocation line consisting of 2x10~6 clones.The library was stored in twenty-two 96-well plates,each well containing approximately 1000 TAC colonies.By using a pooled PCR screening procedure,a positive TAC clone was screened.A 5-kb EcoR I fragment containing Hv-S/TPK was subcloned and identified.This fragment having 5160bp was determined by specific primer walking.The analysis of Hv-S/TPK genomic sequence showed one intron in 5'UTR,three introns and four extrons between start codes and stop codes.There was no difference comaparing four extrons with Hv-S/TPK cDNA sequence cloned from Haynaldia villosa.There was a TATA box in the promoter of Hv-S/TPK.The upstream of TATA box had many resistantance related elements,including two W-box,three OCS-like elements and two PAL box.We also forecasted more twenty cis-elements in this promoter,there were some tissue specific and hormone inducible cis-elements.Bombardment-mediated transient expression analysis of a pHv-S/TPK-GFP construct showed bright green fluorescence in immature wheat leaves inoculated by Erysiphe graminis DC.Another resistantance related gene to powdery mildew,Ta-LRR2,was cloned in our laboratory using SSH and silico cloning.A pair of primers(CINAU16)designed based on the sequence of Ta-LRR2 could be used as a co-dominant marker for selecting Pm21 located on 6VS.By using CINAU16 screening the genomic TAC library of 6VS/6AL,a positive TAC clone containing Ta-LRR2 was obtained.