Characterization Of A Wheat Stripe Rust Hsp70Gene And Its Expression In Response To Heat Shock Process

Stripe (yellow) rust, caused by Puccinia striiformis f. sp. tritici (Pst), is a serious disease of wheatoccurring in most wheat areas with cool and moist conditions during the growing season. In the past, itwas believed the pathogen could only over summer in the areas where the hottest summer averageten-days temperature was below23℃. However, in recent years, the lowest elevation of theover-summering areas is reducing, the pathogen can over summer in the areas where the hottest summeraverage ten-days temperature was above23℃. It could be concluded that the Pst populations mighthave developed adaptation to higher temperature. In this study, a stripe rust hsp70gene was clonedusing RACE technology and the differences of its expression level in response to heat schock processwas analyzed using Quantitative Reverse Transcription Polymerase Chain Reaction (qRT-PCR). Still,screening polymorphic sites relative of temperature-sensitive traits was tried using Sequence RelatedAmplified Polymorphism (SRAP). The results would provide a base for clarifying the high temperatureendurance change mechanism of Pst on molecular level. The major results were summarized as follows:1. The gDNA and cDNA sequence of Pst hsp70gene was cloned via RACE and PCR. The fullDNA length was2817bp, including7introns and the open reading frame (ORF) was1917bp, encoding639amino acids. Blasted in NCBI, the sequence showed a highest homology with the wheat stem rustheat shock protein HSS1, and the homology proportion of cDNA sequence was85%while thehomology proportion of amino acids sequence was92%. Searching the sites of protein functional usingExPasyProsite, we could find the amino acid sequence contained the signature domains of HSP70family. Meanwhile, there was regulatory motif EEVD at C-terminal, so the protein was cytoplasmHSP70.2. Using the technology of qRT-PCR, the hsp70relative transcript levels were distinctly sensitiveto temperatures were researched, and the actin gene acted as the reference gene. The results showed thatthe hsp70relative transcript level of the low temperature-sensitive isolate11-6-4-1maintained at a lowlevel during the first2hours after heat shock. At2hours, the level increased rapidly, it was17.4-foldhigher than the untreated isolate, and4.9-fold higher than the high temperature-sensitive isolate10-9-1-9after2hours heat shock. For different temperature-sensitive isolates, the hsp70relativetranscript levels had a striking difference (P=0.05). The level of the low temperature-sensitive isolatewas higher than the high temperature-sensitive isolate, and its mean level was2.5-fold higher than thehigh temperature-sensitive isolate. It tuned out that hsp70gene showed obvious differences betweentwo types of Puccinia striiformis f. sp. tritici and it played a significant role in the process of resistinghigh temperature.3. Twenty Puccinia striiformis f. sp. tritici were filtered to establish two classes of differentsensitivity to temperature. Utilizing the technology of SRAP, we tried to find polymorphic sites whichhad a relation with the sensitivity to temperature of wheat stripe rust. There were9couples of primerswhich had obvious difference among180couples of primers carried on. Analyzing the data andbuilding a tree of phylogenetic. The consequence showed that the similarity coefficient of the20strains was among0.63to0.93. Six strains from Yunnan which were differently sensitive to temperature weredivided at0.85. Three high temperature-sensitive strains gathered as same as three lowtemperature-sensitive strains, illustrating there was a higher polymorphism of wheat stripe rust in Yunanand we could gather parts of same sensitivity to temperature strains through the skill. But we could notcompletely seprate all the strains which had the same sensitivity to temperature, so it needed a furtherexploratory to improve the skill to find polymorphic sites which had a relation with the sensitivity totemperature of wheat stripe rust.