Cloning Of Haynaldia Villosa And Aegilops Tauschii CMPG Gene Promoters And Preliminary Functional Analysis In Powdery Mildew Resistance

Powdery mildew, caused by Blumeria graminearum f.sp tritici (Bgt), is one of the most serious diseases threatening the production of common wheat. In recent years, the epidemic frequency and yield loss caused by powdery mildew have increased due to the changes of global climate and farming system. The identification of new resistance gene resources and the utilization of resistance varieies are regarded as the most efficient strategy for disease control. The rapid development of molecular biology has significantly accelerated the improvement the molecular breeding, which has become one of the most important ways in crop improvement for disease resistance.Pm21, a powdery mildew resistance gene introduced from wheat relative species, H. villosa, showed high and broad spectrum resistance. The gene are now widely used in wheat breeding program of China for powderymildew resistance, and more than 20 wheat varieties have been released. In order to clone genes related to the Pm21-mediated powdery mildew resitance the Pm21 gene, in previous research, microarray analysis using the Barley GeneChip was employed. A contig upregulated by the inoculation of Bgt was identified. The full length cDNA sequence was cloned from H. villosa. The gene encodes a 467 amino acid protein, containing four conserved amino acid residues Cys, Met, Pro and Gly. It has higher similarities with CMPGs from rice, Arabidopsis, tobacco and parsley, and was named as Hv-CMPG (Liu,2007). Wang (2010) cloned the genomic DNA sequence of the gene and found that it had no intron. In vitro ubiquitination activity assay proved that the protein had the E3 ligase activity。Overexpression of Hv-CMPG by stable transformation showed that its overexpression enhanced the powdery mildew resistance of wheat varieties Yangmail58.Fei (2011) found that the Hv-CMPG gene was rapidly upregulated in leaves of H villosa (45min post-inoculation of Bgt). The expression level of Hv-CMPG was also up-regulated by SA、ABA and H2O2. Overexpression of Hv-CMPG leads to the accumulation of active oxygen and the cell wall protein cross-linking after inoculation of Bgt.In order to reveal the molecular mechanism for the expression pattern of Hv-CMPG, we will clone the CMPG promoters from H.villosa and and two Aegilops tauschii accessions, which showed resistant and susceptible to powdery mildew, respectively.The structure characteristics and the promoter activities will be analyzed. Their potential use in genetic engineering for improvement of powdery mildew resistance will be evaluated. The main results obtained are as follows:1. CMPG promoter gene cloning and the analysis of sequence featuresAccording to the A genome of T.urartu (accession:G1812), D genome of Ae.tauschii (accession:AL8/78) and the genomic sequence of Hordeum vulgare, we obtained the 2.1kb sequences upstream of the start codon of CMPG gene. Sequence alignment found that similarity of three sequences was 45.34%. Their sequence consistencies at the 1-1.7kb and 1.7-2.1kb regions were 37.32% and 79.43%, respectively. Therefore, in this study, four specific primers, P-F1, P-F2, P-F3, P-F4, were designed based on the more conserved 1.7-2.lkb region. The promoter sequences of CMPG gene were cloned by homology cloning from the genomic DNA of H. villosa and Ae. tauschii. By PCR amplification, agarose gel recovery, T/A cloning, and sequencing, the CMPG coding region sequences were removed. Three CMPG promoter sequences from H.villosa, powdery mildew susceptible and resistant Ae. tauschii were obtained, their length were 382bp,381bp and 1635bp, respectively. The promoters were designated as pHv-CMPG, pAtR-CMPG and pAts-CMPG, respectively. Sequence analysis using the PlantCARE software showed that the three promoters contained various typical cis-elements identified in other plant species. They have their specific cis-acting elements, however, same cis-acting elements in H.villosa and Ae. tauschii were also identified, including pathogen responsive W-box and meristem activation A-box.2. Functional analysis of CMPG promoters in response to powdery mildew resistanceExpression vectors of the three promoters were constructed and used for bombardment the leaves of wheat variety Yangmai 158. The vectors p-HS-001 and p-HS-001-negative were used as positive and negative controls. The results showed that before and after Bgt inoculation, no significant change of GUS expression epidermal cell was observed when transforming p-HS-001, while significant increase of GUS expression cells were present when transforming pHv-CMPG. No GUS expression cell was observed when transforming pAfR-CMPG and pAtS-CMPG. These indicated that only the pHv-CMPG had the promoter activity, and pAtR-CMPG、pAtS-CMPG had no or extremely low promoter activity.