CDNA Library Construction And EST Analysis Of TcLr19 Induced By Puccinia Triticina

Wheat leaf rust caused by Puccinia triticina is a serious and widely distributeddisease of wheat. Resistant cultivars are the most economic, safety and environmentalfriendly way for minimizing the losses caused by the disease. As an important leaf rustresistance gene, Lr19 was firstly transferred into the wheat genome from Thinopyrum sp.in the form of the substitution line 'Agrus' in 1966. At present, Lr19 was a rsistance genewith great potential to be used for wheat production, cDNA library of TcLr19 induced byPuccinia triticina was constructed by SMART technique. Bioinformatics method andcDNA-AFLP technique were used to analyze the sequence characteristics besidessequencing strategy. A primary gene expression profiling of the disease resistance in wheatwas obtained with the material infected by Puccinia triticina at the whole stage.The leaves of NIL Lr19 were harvested at 6h, 12h, 18h, 24h, 36h, 48h, 60h, 72h and96h, respectively after inoculation with THTS biotype of Puccinia triticina. A cDNAlibrary was constructed using these leaves. The titer of library was 4.1×10~6pfu/mL. Theclones of the library had an average insert size of 1.0Kb, which ranged form 0.5Kb to 2.0Kb.A STS marker, co-segregated with Lr19, was used to screen the 21120 clones thatwere randomly selected from cDNA library, and no target fragment was found. Thefollowing study was conducted to detect the differential expression of genes in TcLr19inoculated with Puccinia triticina by cDNA-AFLP methods. Two hundred twenty fourpairs of primers were screened for cDNA-AFLP analysis. Fifty eight pairs of these primerscould generate polymorphic bands between TcLr19 and Thatcher, with 25.9% ofpolymorphism. Nine of the 58 primer combinations that were P-AA/M-GGG,P-AG/M-GAA, P-TA/M-GGC, P-GC/M-CGC, P-GC/M-GAG, P-TC/M-CAA,P-TA/M-GAA, P-CA/M-CGA, and P-AC/M-CAC, respectively amplified specific bandsSix specific bands were collected and sequenced. Blastn analysis showed that all of thebands had the homology sequences to some paints in the GenBank database. Thenucleotide similarity to known sequences was up from 90% to 100%. Two of the six bandswere the genes with known function, which code the signal transduction proteins,calmodulin-binding protein and serine/threonine kinase protein, respectively.The sequence 7719-Ca which was amplified by primer P-CA/M-CGA was obtainedvia silicon cloning and RT-PCR technique. The size of 7719-Ca was 1209bp. The sequencewas extended 137bp to left direction and 891bp to right. The sequence of 7719-Ca was sentto GenBank for homologue search using Blast programs and was found 87% homologuewith Calmodulin-binding protein 60-B of Oryza sativa. This protein encoded by 7719-Ca included one conserved domain for Calmodulin-binding.One hundred eight seven sequences with high quality were obtained from cDNAlibrary. The sequences were sent to GenBank for homologne search using Blastn andBlastx programs and further classified based on their functions. The results were as follows:one hundred thirty sequences showed high homologue to 11 classes of genes with knownfunction; forty two sequences were matched to the genes whose protein functions wereunclassified; twelve sequences showed homologue to ESTs; and two sequences did not hitany sequence in GenBank. Thirty four of 130 sequences that hit the genes with knownfunction, showed homologue to putative genes related to disease resistance or defensesreaction. These genes included GTP-binding protein like gene, ion channel component,participating in signal transduction gene, protein involved in defense response, and proteinrelated to cell self protection in disease-resistance response.Through reverse northern blotting test, the clones 205, 586, 345, 387, 130, and 183were always found in the results of the hybridization with the probes made by cDNA ofTcLr19. It showed the genes would be involved in Lr19 disease-resistance response. Threeclones, 658, 360, 581, were found in the hybridization results with inoculated TcLr19cDNA and Thatcher cDNA probes, it showed that the clones, 658, 360, and 581 wereinvolved in Puccinia triticina and Thatcher susceptible response, but in Puccinia triticinaand Lr19 resistance response those clones may be restrained.