SSH CDNA Library Construction Of Wheat With New Rust-resistant Gene And Preliminary Screening

Wheat stripe rust, caused by Puccinia striiformis f.sp.tritic, is the major disease in China. Especially in west regions of our country and due to suitable conditions, the disease recently turns to be most serious there. The reason can be concluded as fast variation of pathogen, lack of resistance genes and single resistant inheritance basis of wheat variety. Another important reason is complicated resistant mechanism between pathogen and plant interaction. The molecular mechanism is still unclear so as to restrict the breeding of new resistant variety. Thereby, while constantly finding out new resistant genes and fast breeding of new wheat variety with durable resistance researches should be focused on molecular mechanism of pathogen-host recognition and plant resistance and cloning resistance genes and resistant-related genes, all of which are crucial for fundamentally solving threats from wheat stripe rust.In this research, rust-resistant variety named CN19 involved in a new rust-resistant gene Yr41 were taken as materials and we employed suppression subtractive hybridization technique characterized by high specificity and output rate to construct rust-induced SSH cDNA library and obtain positive clones through differentially screening. And then, resistant gene expression analyses on correlative ESTs were made. The results could be the basis for making resistant mechanism fatherly understood, discovering resistance-related genes, mapping the new resistance gene Yr41 and molecular marker assisted breeding. Meanwhile, there are important theoretical and practical significances in solving problems in wheat production threatened by rust disease and breeding of new resistant variety. Main results could be summarized as follows:1. Rust pathogen-induced SSH cDNA library was successfully constructed and 6972 recombinant clones were preserved. Key points in the course of construction, including qualities of total RNA and starting mRNA, adaptor-ligated efficiency (about 70%) and hybridization efficiency, were controlled for the better library quality. Inserted cDNA fragments were distributed between 200 and 700bp and accorded with expectation. All of the above were the basis of screening differentially expressed genes with high efficiency.2. Dot-blotting was performed in 4000 clones screening and 216 positive clones were acquired. After sequencing with ramdon 60 positive clones 56 effective ESTs were obtained. BLAST analyses in GenBank indicated that 14 ESTs might be relatd to disease-resistant responses, including one resistance regulation gene, one pathogen-resisted protein, one enzyme to important secondary metabolism, one hydrolase and two reactive oxygen species scavengers. In addition, 34 ESTs were involved in photosynthesis, energy metabolism and other pathways. Four ESTs were found similarities in GenBank, but functions are unclear. Four ESTs were found no significant similarities and they may represent new genes or high variant non-coding regions of cDNAs.