Analysis Of Incompatible Expressed Sequence Tags From Wheat Leaves Induced By Puccinia Triticina And CDNA Cloning And Expression Analysis Of Gene TaRLP19 And TaSTKC8

Leaf rust, caused by Puccinia triticina, is one of the most destructive diseases of wheat worldwide. Its epidemics sometimes cause significant yield losses. Resistant cultivars are the most economic, safely and environmental friendly way for minimizing the losses caused by the disease. As one of the few widely effective genes conferring resistance to leaf rust in wheat, Lr19 was transferred into the wheat genome from Agropyron elongatum. To date, it has great potential to be used for wheat production. TcLr19 cDNA library induced by P. triticina was analyzed to develop an EST database of wheat infected by avirulent P. triticina isolate and provided a base for understanding the wheat resistant mechanism to the pathogen at the molecular level. Also we used RACE (rapid amplification cDNA ends) technique to obtain full length sequence of the specific expression gene in the near isogenic lines TcLr19, then we detected the gene expressions of two sequences in the compatible and incompatible interactions of wheat and P. triticina by real-time quantitative PCR technique. The main results were as follows:1. A total of 1516 ESTs were randomly picked and tested using PCR with universal primers M13 derived from the sequence flanking the vector. The positive clones from the library were combined and assembled into 948 unigenes which consisted of 241 contigs and 734 singlets for further analysis. Results of function annotation and clusters of orthologous groups of proteins, 651 unisequences accounted for 68.67% of all the unisequences showed high homology with the function known genes or ESTs, 213 unisequences accounted for 22.49% of all the unisequences showed high homology with the hypothetical protein, 10 unisequences accounted for 1.05% of all the unisequences showed high homology with the P. triticina, and 74 unisequences have no significant homology to any sequence in the database. The incompatible cDNA library included energy production and conversion genes which contained amino acid, carbohydrate, coenzyme, inorganic ion, lipid transport and metabolism and photosynthesis related genes, disease defence, information storage processing, signal transduction, transcription and translation, cytoskeleton genes and so on. 2. Within the function known ESTs, ribulose bisphosphate carboxylase/oxygenase activaseA, chlorophyll a/b binding protein, zinc finger protein, ascorbate peroxidase, SGT1 protein, RPP-8 like protein, HSP70/90, PR-10 protein, F-box containing protein, WIR1A protein, biotic or nonbiotic stress-related genes, the ABC transporter, H-ATP enzymes, GTP protein, serine/threonine protein kinase, receptor-like protein kinase, etc., were supposed to involve in the process of the incompatible interaction between wheat TcLr19 and the P. triticina race. The cDNA library is useful for identifying the functional genes involved in the wheat-leaf rust incompatible interaction, and thus laying a foundation for establishment of a new database for studying leaf rust pathogenesis genes and wheat defense genes.3. One cDNA sequence of the aimed gene was cloned from TcLr19 carrying the Lr19 gene by using RACE technique based on the target fragment 19-9 amplified by cDNA-AFLP. It was verified further in TcLr19 leaves induced by P. triticina with RT-PCR.The sequence named TaRLP19 had a length of 2545 bp, including a 2136 bp complete open reading frame encoding 711 amino acid, a 180 bp 5′untranslated terminal region (5′UTR), a 243 bp 3′untranslated terminal region (3′UTR) and 31bp polyA tails. The deduced amino acids of TaRLP19 protein consisted of an extracellular domain, a single-pass transmembrane domain and a small cytoplasmic domain which not contained serine/threonine protein kinase domain; the extracellular domain contained six leucine-rich repeats (LRR) domains which had 23-24 amnio acid. It suggested that the TaRLP19 gene should be a member of a class LRR-TM type resistance gene. The gene has been submitted to the GenBank database, with the accession number of JN872563.4. Another cDNA sequence of the aimed gene was cloned from TcLr19 by using RACE technique based on the target fragment Sr-19 amplified by cDNA-AFLP. It was verified further in TcLr19 leaves induced by P. triticina with RT-PCR. The sequence named TaSTKC8 had a length of 2062 bp, including a 1363bp complete open reading frame encoding 450 amino acid, a 275 bp 5′untranslated terminal region (5′UTR), a 434 bp 3′untranslated terminal region (3′UTR) and 27bp polyA tails. The deduced amino acids of TaSTKC8 protein contained serine/threonine protein kinase domain and major sperm protein domain.5. TcLr19 and Thatcher, which were uninoculated and inoculated at different times (0h, 6h, 12h, 18h, 24h, 36h, 48h, 60h, 72h, 96h, 120h) by using real-time PCR, was analyzed. The results showed that the TaRLP19 and TaSTKC8 genes were constitutive genes. TaRLP19 was expressed earlier and expression down-regulated comparing inoculated TcLr19 with non-inoculated. The gene TaRLP19 was expressed earlier and expression down-regulated in inoculated TcLr19 than non-inoculated TcLr19. TaSTKC8 was expressed earlier and expression up-regulated in inoculated TcLr19 than non-inoculated TcLr19. The gene TaSTKC8 may play a positive regulatory role in the process of leaf rust infection and some specific genes or HR/SAR response protein could be induced and worked during the reaction.