Differential Expression Of Wheat Leaf Rust Resistance Gene Lr41

Wheat leaf rust caused by Puccinia triticina is one of very serious and the most widely distributed diseases of wheat. Using resistant cultivars is an economic and environmental-friendly way for minimizing the losses caused by the disease. However, new more virulent race of Puccinia triticina often make the resistant cultivars lose its resistance to the disease, Therefore, investigation on the interaction between wheat and its rust fungi may reveal the resistant mechanisms of the host plant and the pathogenic mechanism of the rust fungi, and also provide further information for selection and reasonable use of resistant wheat cultivars. In the present study, suppression subtractive hybridization (SSH) was adapted to construct a wheat leaf cDNA library induced by Puccinia triticina race 05-12-90 and a wheat leaf cDNA library between TcLr41 and Thatcher. In order to elucidate the wheat resistant mechanism at the molecular level, the cDNA libraries were screened by cDNA microarray. The differential genes expression were studied and resistance related genes were cloned. Several aspects included in the research were as followed:1 With the use of fluorescent microscopy, the histological features of Puccinia triticina on a susceptible host Thatcher and its near isogenic line TcLr41 resistant to wheat leaf rust were studied. By comparison, no significant differences of wheat leaf rust on different lines were found through histological studies before 24 hours post inoculation (HPI), but the hypersensitive reaction only appeared in TcLr41 after 36 HPI. The hypersensitive reaction produced speedily at 48 HPI. Therefore, we speculated that 48 h was the key timepoint for TcLr41 to defense leaf rust pathogen.2 A near isogenic line resistant to wheat leaf rust, TcLr41 was used to construct a suppression subtractive hybridization (SSH) library from wheat leaves induced by Puccinia triticina. A high quality SSH cDNA library was constructed successfully and 3,456 positive clones were obtained in the SSH library. With the group of the TcLr41 seedling cDNA as the tester and the corresponding group of the Thatcher seedling cDNA as the driver, the suppression subtractive hybrization (SSH) containing 2,544 clones was performed in this research, at the same time.3 The cDNA libraries were screened by cDNA microarray. Two hundred and forty differential expression sequence tags were obtained and 137 qualified ESTs showed high homology with the function known genes or ESTs. Through analysing the genes with known function, glutathione-S-transferase, mitogen-activated protein kinase, W1R1 protein, serine/threonine protein kinase, lipid transfer protein, thaumatin-like protein, GTP-binding protein, chitinase, and calreticulin, etc., were supposed to involve in the process of the incompatible interaction between wheat TcLr41 and the Puccinia triticina race.4 Real time quantitive PCR was used to detect gene expression of six genes. Based on the results of cDNA microarray, real time quantitive PCR was adapted for further accurate quantification. Six ESTs took different trends, but showed higher expression at 48 h infected by Puccinia triticina in TcLr41 than in Thatcher. By comparison, the results of two detection methods were identically.5 Rapid amplification of cDNA ends (RACE) were used to extend three sequences, 1,636 bp BJ1.1A3 sequence was obtained by RACE, which had higher similarity with rust resistance gene in barley. A full length sequence of 593 bp JD4.4E3 was obtained by RACE, which had the highest similarity to WIR1 either at the level. A 1,699 bp JD6.2D12 sequence encoded casein kinase II alpha was obtained by RACE in this research.