Identification Of Wheat Germplasm Resistance To Stripe Rust (Puccinia Striiformis) And Expression And Cloning Of Differential Gene

Common wheat(Triticum aestivum L.)is the most widely grown food crop. Its production directly affects food security in the world. Stripe rust, caused by the obligate biotroph fungus Puccinia striiformis f. sp. Tritici Eriks (Pst), is one of the most destructive disease in common wheat production worldwide. Therefore, it is urgent to identify new stripe rust resistance germplasm and genes, broaden the spectrum of disease-resistant varieties, and study gene expression profiling and pattern for dissecting the regulatory mechanism and transcriptional networks that underlie phenotypic responses.T. aestivum ssp. tibetanum Shao, Tibetan wheat landraces (T. aestivum L.) and common wheat-Austrila rye (Secale cereale L.) derivatives as materials, their stripe rust resistance and germplsm genetic diversity were analyzed. Molecular cytogenetic identification of screened germplasm wheat- S. cereale derivative NR1121 immune to stripe rust was done. A SSH(Suppression subtractive hybridization)-cDNA library was constructed with the seedling leaves from wheat germplasm NR1121 infected by Chinese Pst race CY32. Resistance-related ESTs from SSH-cDNA library were anlyzed by bioinformatics methods, and resistance-related gene expression profiles was obtained, and then resistance-related novel genes were screened. The expression pattern of novel genes were analyzed by the semi-quantitative RT-PCR and real-time PCR, and the novel genes were cloned. The function of novel genes was verified by VIGS (Virus induced gene silencing) system constructed with BSMV (Barley stripe mosaic virus). The main results are as follows:1. Based on the identification results resistant to Pst isolation CY32 at seedling and adult, two T. Tibetanum Shao accessions(Longzizheda7and Longzizheda10) highly resistant to Pst and wheat- S. cereale derivative NR1121 immune to Pst were screened from 136 T. Tibetanum Shao accessions, 119 Tibetan wheat landraces and 70 wheat- S. cereale derivatives. The genetic diversities of 136 T. Tibetanum Shao accessions and 119 Tibetan wheat landraces were detected by using PCR method with ISJ (intron-splice junction) primers and long random primers. The results indicated 33(11%) primers or primer combinations out of 26 ISJ primers and 300 primer combinations could yield polymorphic bands. 333 good bands were produced from T. Tibetanum Shao accessions and 243(72.97%) bands were polymorphic. 316 good bands were produced from Tibetan wheat landraces and 197(72.97%) bands were polymorphic. The genetic diversities of T. Tibetanum Shao accessions were more than that of Tibetan wheat landraces. The genetic difference between T. Tibetanum Shao and Tibetan wheat landraces were identified by high polymorphic ISJ markers.2. S.cereal chromation in NR1121 was detected by chromosome karyotyping, C-banding, GISH (genomic in situ hybridization), SSR, SCAR, STS and A-PAGE. NR1121 and Austrian rye were immune to Pst isolate CY32 and Shaanmai 611 and six wheat cultivars (Lovrin 10,Lovrin 13,Qingmai No.9,Fengkang No.8,Shaan229 and Yanshi No. 9) with Yr9 were highly susceptible to CY32. NR1121 was stable in cytology with chromosome numbers 2n=42=21Ⅱ. Giemsa C-banding result showed that one pair of rye 1R chromosomes was detected in NR1121. GISH results with Austrian rye genomic DNA as a probe showed that two chromosomes of NR1121 was transferred from S.cereal. SCAR (primer combination AF1 and AF4, and primer SCM-9) and STS (primer combinationω-sec-p3 andω-sec-p4) markers indicated that NR1121 possessed chromatin of rye 1R chromosome. A-PAGE results indicated that Gli-B1 inωregion of NR1121 had the specific band of Austrian rye 1R short chromosome arm. All the results demonstrated that NR1121 was a wheat-S. cereale 1R disomic substitution line and immune to CY32. The resistant gene in NR1121 was derived from Austrian rye and probably had a new gene differing from Yr9.3. A SSH-cDNA library was constructed with the seedling leaves from NR1121 infected by Chinese Pst race CY32. From the forward library, 350 positive clones were randomly selected for plasmid extraction and sequencing, and 96 ESTs (expressed sequence tags) were obtained after removing repeated and redundancy sequences and submitted to GenBank. Accession numbers of GenBank were from GO254150 to GO254231, GR884577 to GR884584 and GT270714 to GT270718. BlastX alignment revealed that 78 of 96 ESTs predicted genes had more than 90% similarity to proteins and 50 ESTs with known function were involved in metabolism (12.8%), energy (9.0%), disease/defense (16.7%), signal transduction (6.4%), transportation (5.1%), protein synthesis, destination and storage (5.1%), transcription (5.1%), cell growth/division (2.6%), and the immune system (1.3%). The function of 35.9% ESTs were not known. ESTs with a high similarity to sequences of senescence-related proteins, UPL2 (Ubiquitin-protein ligase 2), maturase K, membrane transporter YKT61, SAMDC (S-adenosylmethionine decarboxylase), and S/PKSNT7 (Serine/threonine protein kinase SNT7) were discovered. There were 22 ESTs associated with stripe rust resistance, including 5 ESTs for signal transduction, 1 for HR (Hypersensitive necrosis reaction) system, and 16 for SAR (Systemic acquired resistance) system, respectively. 4. A total of 150 positive clones were randomly chosen from the inverse SSH-cDNA library and sequenced. 69 ESTs were obtained after removing repeated and redundancy sequences and submitted to GenBank. Accession numbers of GenBank were from GR884585 to GR884652. BlastX alignment revealed that 57(82.6%) of 69 ESTs predicted genes had 90% similarity to known genes and ESTs with known function were involved in metabolism (12.3%), energy (14.0%), disease/defense (1.8%), signal transduction (7.0%), transportation (5.3%), protein synthesis, destination and storage (5.17%), cell growth/division (3.8%), and the immune system (1.9%). The function of 48.2% ESTs was not known. There were 9 ESTs associated with stripe rust resistance, including 4 ESTs for signal transduction, 1 for HR system, and 4 for SAR system, respectively.5. The expression pattern of eight genes, which may participate in energy metabolism, disease resistance and defense, transportation, and signal transduction, were analyzed in Shaanmai 611 (Compatible interaction) and NR1121 (Incompatible interaction) by the semi-quantitative RT-PCR and real-time PCR. These genes were AcsA (Acyl-coenzyme A synthetase), GST ( Glutathione-S-transferase ) , LTP2 (Lipid transfer protein), CP450 (Cytochrome P450), UPL2, S/PKSNT7, SHMT (Serine hydroxy methyl transferas) and SAMDC. The results from semi-quantitative RT-PCR and real-time PCR of eight predicated genes were consistent with each other. The expression time and level of these genes were obviously different between Shaanmai 611 and NR1121. The genes AcsA was up-regulated to their highest expression level at 24 hpi(hours post inoculation) after Pst infection in Shaanmai 611 and NR1121, but the expression level was different. The expression level and time of the genes GST, LTP2, CP450, UPL2, S/PKSNT7, SHMT and SAMDC were different in between Shaanmai 611 and NR1121. These six genes were up-regulated to their highest expression level at 24 hpi or 48 hpi after Pst infection in NR1121, but the gene UPL2 and SAMDC were down- regulated and the other five genes up-regulated to their highest level at 72 hpi or later in Shaanmai 611. The results demonstrated that GST, LTP2, CP450, UPL2, S/PKSNT7, SHMT and SAMDC were highly induced at 24 hpi or 48 hpi after Pst infection in NR1121, suggesting they are transcriptionally activated for the host defense response. The key expression stage of resistance-related genes were from 24 hpi to 48 hpi in NR1121 seedling (Incompatible interaction) after Pst infection, which could be the optimal stage constructed seedling SSH-cDNA library of wheat resistant (Race specific) to CY32.6. Two new wheat genes, which were SAMDC gene designated as TaSAMDC2(GenBank accession number is GU016570) and S/PKSNT7 gene designated as TaSNT7(GenBank accession number is GU574209), were isolated from NR1121with in silico cloning,RT-PCR and RACE techniques. The cDNA complete sequence of TaSAMDC2 was 2003 bp in length and its 5' untranslated region (5'-UTR) and 3' untranslated region (3'-UTR) with Poly(A) were 553 and 283 bp , rsspectively. Open reading frame (ORF) of TaSAMDC2 gene was 1167 bp and encoded 388 amino acids containing conserved sequence with the proenzyme cleavage site and one typical conserved PEST domain of pathogenesis related protein SAMDC family. The genome DNA complete sequence of TaSAMDC2 was 2539 bp in length and its 5'-UTR contained 526bp intron with the splicing sites of GT-AT bi-nucleotidesequence. Homologous analysis found that TaSAMDC2 were 95.0%, 85.0%, 80.0% and 80.0% of sequence similarity with SAMDC proteins from barley(Hordeum vulgare L.), rice (Oryza sativa L.), maize (Zea mays L.) and wheat (T. monococcum L.), respectively. The cDNA and genome DNA complete sequence of TaSNT7 was 1555 bp in length. Open reading frame (ORF) of TaSNT7 gene was 1035 bp and encoded 388 amino acids. Homologous analysis found that TaSNT7 were 84.0%, 84.0%, 25.0% and 25.0% of sequence similarity with S/PKSNT7 proteins from rice (Oryza sativa L.), maize (Zea mays L.), solanum (Solanum lycopersicum) and tobacco (Nicotiana tabacum) , respectively. TaSNT7 was located on 1D chromosome by Chinese spring nullisomic-tetrasomic lines.7. With the established VIGS system, function analysis of two genes, TaSAMDC2 and TaSNT, were carried out in order to clarify their role during wheat in response to Pst attack. RNA was reversely transcripted into cDNA and BSMV recombination system was constructed to inoculate wheat. Real-time PCR results indicated that the low transcription products were detected in wheat inoculated by BSMV-TaSNT7 and BSMV-TaSAMDC2, which suggesting that TaSNT7 and TaSAMDC2 genes were silenced or partially silenced. Phenotype results indicated that the gene TaSNT7 played a role in stripe rust resistance pathway in wheat.