Screening Differential Profile Of Genes Expressed During Compatible Interactions Between Puccinia Striiformis And Wheat By Suppression Subtractive Hybridization

Stripe rust is one of the most serious diseases of wheat in China, which is caused by the fungus Puccinia striiformis. It can dramatically reduce crop yield during the epidemic, which threaten our national food safty significantly. Along with the development of ESTs technique, isolation of differentially expressed genes induced by Puccinia striiformis has become the first step to reveal the molecular mechanism of interaction between wheat and stripe rust fungus.Wheat cultivar Suwon 11 and Puccinia striiformis CY31 was used as the initial materials to construct a wheat SSH-cDNA library using mixed RNA samples isolated from wheat leaves 36 h, 72 h and 120 h after inoculation, respectively. In order to use the suppression subtractive hybridization technique effectively, it is necessary to fumble for a good RNA extraction method of wheat leaves. Through comparison the total RNA that was isolated from the wheat leaves by Biozol reagents and RNeasy Plant Mini Kit, we discovered that the purity and integrity of the total RNA extracted by Biozol reagent was as good as RNeasy Plant Mini Kit, while the output ratio of Biozol reagent was 1.47 times as much as RNeasy Plant Mini Kit. So Biozol reagents is very useful for the experiment which required more wheat leaves RNA, such as suppression subtractive hybridization technique and Northern bloting.A total of 1 983 clones were sequenced, which produced 1 707 high quality ESTs. 787 unigenes (332 contigs and 455 singlets) were obtained after clustering by CAP3 software. Functional annotation and category showed that about 50.4% unigenes were functionally unknown, including 17% matching unknown or hypothetical protein (unknown) and 33.4% without or with weak similarities to the proteins in the GenBank non-redundancy database (no hits). With respect to the annotated unigenes, the percentage of genes involving in primary metabolism, energy, transport, disease/defence, singal transduction were 9.3%, 6.5%, 4.6%, 4.2%, 4.1%, respectively. There were also 3.8%, 3.2%, 3.3%, 2.9% unigenes were thought to be concerned with protein destination/storage, protein synthesis, transcription, secondary metabolism, respectively. Only 3 unigenes, however, had higher similarity with pathogenic protein in this library. As previous researches described that plants hold defence reactions against pathogens even in compatible plant-pathogen interactions, many resistance-related genes were isolated, such as hypersensitive-induced reaction protein, Zinc-finger proteins and phenylalanine ammonialyases. Compared with the unigenes in the library constructed with wheat cultivar Suwon 11 and Puccinia striiformis CY23, more no hits were found in this library, some of the no hits may be with pathogen origin. Some genes which had higher similarity with catalase, elongation factor and cell death suppressor protein were supposed to have important roles during compatible interaction between Puccinia striiformis and wheat. In addition, eight ESTs were selected to analyze the expression patterns by RT-PCR, and six ESTs showed differential expression profile.