MRNA Expression Difference Of Six Near-isogenic Lines For Wheat Leaf Rust Resistance Genes

Wheat leaf rust caused by Puccinia triticina is one of important diseases of wheat, which seriously reduced the yield of wheat.Resistant cultivars are the most economic, safety and effective ways for minimizing the losses caused by the disease.Wheat leaf rust resistance genes Lr9,Lr15,Lr19,Lr25,Lr38 and Lr45 confer strong resistance to Puccinia triticina in China till now.TcLr9,TcLrl9,TcLr38 and TcLr45 are resistant to Puccinia triticina race No. 05-21-116③,and TcLrl 5,TcLr25 are susceptible.The infection types of No.05-21-116③to TcLr9,TcLrl9,TcLr38,and TcLr45 are 0,;,1,1 respectively,and the infection type of No.05-21-116③to both of TcLrl5,TcLr25 is 4.The leaves of uninoculated and inoculated NILs TcLr9,TcLrl5,TcLrl9,TcLr25,TcLr38,TcLr45 and Thatcher were harvested at 0 h,6 h,12 h,18 h,24 h,36 h,48 h,60 h,72 h and 84 h respectively after inoculation with Puccinia triticina race No.05-21-116③.Then genes expression difference were analysed by cDNA-AFLP among TcLr9,TcLrl 5,TcLrl 9,TcLr25,TcLr38, TcLr45 and Thatcher.The main results were as follows:1.Two hundreds and twenty six pairs of primers were used to screen for differential expression analysis.As a result,7 554 bands were detected totally,and 33 bands were amplified by each primer pair on average.2.68 primer pairs could amplify differential expression bands among TcLr9,TcLrl 5, TcLrl9,TcLr25,TcLr38,TcLr45 and Thatcher,and the detection rate of differential expression bands was at 30.1%.84 differential expression bands amplified by the 68 primer pairs were classed into 9 types of differential expression(TypeⅠ～Ⅸ).TypesⅠ,ⅤandⅦwere constitutive expression,in all 49 bands;TypesⅢ,Ⅳ,ⅥandⅧwere up-regulated,in all 21 bands;TypesⅢandⅨwere down-regulated,in all 14 bands.Fives of nine types(TypeⅣ,Ⅴ,Ⅵ,Ⅶ,Ⅷ)were probably linked with disease resistance,and the five types were classed into two subtypes again.The first subtype was induced expression(TypesⅣ,Ⅵ,Ⅷ),the second was constitutive expression(TypesⅤ,Ⅶ).3.Eight differential expression bands were cloned and sequenced.Blastn analysis showed that seven of them had highly homology(81%～100%)with EST sequences of Triticum aestivum and Hordeum vulgare.Six of the seven sequences came from Triticum aestivum,and one sequence came from Hordeum vulgare.Blastx analysis showed that the seven sequences all had the homology sequences,and the amino acids showed similarity with the known sequences from 59%～96%.Cytochrome P450 enzyme family, GDA1/CD39 family protein,abscisic stress ripening protein 2 probably were linked with disease resistance.Proteins deduced by No.TcLr45-4,No.TcLr38-15 and No.TcLr45-16 had homology(68%,72%and 85%respectively)with Cytochrome P450 enzyme family, GDA1/CD39 family protein,abscisic stress ripening protein 2,respectively.The gene expression differences among TcLr9,TcLrl5,TcLrl9,TcLr25,TcLr38, TcLr45 and Thatcher by complementary DNA amplified fragments length polymorphism (cDNA-AFLP)were analysed,in order to screen differential expression fragments that are in correlation with disease resistance and establish the foundation for finding out resistance mechanism of leat rust resistance genes,screening and cloning resistance genes.