| Wheat is one of the major crops in the word, which provides main food for the35%-40%of the population. But, wheat production is seriously affected by powdery mildew (PM) caused by Erysiphe graminis f. sp. tritici. Breeding resistant cultivars to the pathogen is an effective method to control this disease. The Triticeae genetic resources have potential value to the improvement of wheat and other Triticeae crops by introducing some useful alien genes through genetic manipulation. Haynaldia villosa is a wild relative of wheat, which harbors many useful genes including disease-resistant and drought-tolerant ones, especially, it appears excellent resistance to PM. However, the genomics of Haynaldia villosa has not been studied in-depth. In addition, different Haynaldia villosa landraces derived from different regions might carry different resistant genes to biotic or abiotic stresses. Therefore, it is necessary to further investigate the PM resistant genes in Haynaldia villosa with different originations.To find out the similarities and differences of the gene sequences resistant to PM in different Haynaldia villosa and clarify their resistant mechanism to the pathogen, two different originated Haynaldia villosa accessions and their derived wheat-Hynaldia villosa translocation lines of Pm97033, Pm97034and92R137with good resistance to powdery mildew were used as materials in this study. Based on the sequence of serine/threonine protein kinase gene Stpk-V (GenBank accession number:HQ864471.1), we cloned and analyzed its homologous genes from Haynaldia villosa (No.1026, from the former Soviet Union), and compared the sequences with the Stpk-V genes from the other Haynaldia villosa derivative and wheat genome. At the same time, their bioinformatics, expression patterns, copy numbers, molecular markers, and function evaluation through transgenic approach were explored. Some main results were achieved as follows:(1) Two serine/threonine protein kinase genes (Stpk-V2, Stpk-V3) were cloned from No.1026Haynaldia villosa, which are highly homologous compared with the original Stpk-V. A22bp insertion and a78bp insertion were found in the fourth intron of the new sequences, respectively, comparing with Stpk-V. The coding sequence of Stpk-V3is consistent with that of Stpk-V.(2) Stpk-V2is located on wheat chromosomes other than the6VS of Haynaldia villosa by using allele-specific PCR according to the SNP between Stpk-V2and Stpk-V3.(3) Two functional markers were developed based on the sequences of Stpk-V and Stpk-V3, which can be used to identify the origination of the resistant resouces or varieties to PM related to Haynaldia villosa. The resistant line of CB037with PM resistance was identified with the2functional markers, and the result is consistent with that using other molecular markers.(4) Bioinformatic analysis of Stpk-V2revealed that it encodes a non-secretory protein without signal peptide, containing a transmembrane region at the N-terminal, a ATP binding site between the fifty-second and seventy-fourth amino acid, a kinase active site between the one hundred sixty-eighth and one hundred-eightieth amino acid, and five glycosylation sites. Prokaryotic expression analysis was conducted by constructing a prokaryotic expression vector based on the conserved region of its coding sequence, and the target protein of33kD around in size was successfully expressed in E.coli as expected.(5) Putative promoter of Stpk-V2was cloned and the partial promoters of serine/threonine protein kinase gene was amplificated by using tetraploid substitution wheat lines in which the chromosomes in A and B genome were substituted by the chromosome in D genome one by one, respectively, and Chinese Spring nulli-tetrasomic lines in group6. The results suggested that there was at least one copy of serine/threonine protein kinase gene existing on6A,6B,6D chromosome, respectively, and a78bp deletion in the corresponding promoters region on6B and6D chromosome compared with6A chromosome.(6) Expression analysis of Stpk-V (Stpk-V3) in the leaves of92R137and Pm97034induced by Blumeria graminis f. sp. tritici E09was conducted with semi-PCR and real-time quantitative PCR. The results showed that the gene response patterns were slightly different among the two translocation lines. Nevertheless, the reason causing the difference needs to be studied further. (7) Two RNAi expression voctors for Stpk-V3gene silence were constructed. Genetic transformation mediated by Agrobacterium and biolistic particle were conducted using the mature embryos and immature embryo of Pm97034as receptor tissues. One positive plant containing the target gene was obtained by PCR test. Unfortunately,6VS was found to be lost in this plant according to the moleaular marker. Function analysis for the resistant gene in Pm97034need to be researched further by crossing the positive plant with Pm97034for the pyramiding of the target gene and6VS.In conclusion, there are differences in gene structure and expression of serine/threonine protein kinase genes (Stpk-V, Stpk-V3) of Haynaldia villosa No.1026and H.v#2... |
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