Cloning And Functional Analysis Of Powdery Mildew Resistance Gene Mlo In Rose

Powdery mildew caused by Podosphera pannosa was the most common disease in cut rose production globally. It is also one of the main diseases of garden roses and pot roses. China is one of the distribution centers of wild roses in the world, and it is also one of the main cut rose producing areas in the world. However, the unclear resistance mechanism of wild roses to powdery mildew and the lack of related resistant genes have been limiting the utilization of wild roses in the genetic improvement of modern rose cultivars. Mildew resistance locus o（Mlo） genes are new type of disease resistance genes.In dicots such as arabidopsis, pea, and tomato, loss-of-function mutations in Mlo genes confer high levels of broad-spectrum resistance in powdery mildew.Rosa multiflora and its hybrids were used in this study to isolate four c DNA and genomic Mlo sequences based on homologous cloning technique. Related analysis software was used for structural analysis of the four candidate genes. We mapped all four genes in the diploid segregating population BC1 by genetic linkage mapping method. We analysed spatio-temporal expression patterns of the four Mlo genes based on real-time fluorescence quantitative PCR（FQ-PCR） technology. Finally, We constructed two plant expression vectors with sense and antisense Rh MLO1 gene, which were introduced into‘Baiyu’ respectively by Agrobacterium-meidiated transformation. We tested the powdery mildew resistance level of control plants and transgenic plants and verified Rh MLO1 function in rose by pros and cons of transgene method. Main results and conclusions in this study are listed as follows:1 Cloning and sequence analysis of four Mlo genes in roseR. multiflora and its hybrids were used to isolate four c DNA and genomic Mlo sequences based on RT-PCR and RACE technologies. Names of the four genes we got are Rh MLO1, Rh MLO2, Rh MLO3 and Rh MLO4, respectively. c DNA full-length of the four genes is 1779 bp, 1767 bp, 1692 bp and 1695 bp, with open-reading frame（ORF）encoding 592, 588, 563 and 564 amino acids, respectively. Amino acid sequence analysisrevealed that each gene has seven transmembrane domains and two conserved MLO motifs（Ca MBD and D/E-F-S/T-F） in the protein C-terminal. Protein molecular weight of the four genes is 67.79 k Da, 67.22 k Da, 64.55 k Da and 64.61 k Da, with theoretical isoelectic point 9.53, 8.96, 9.00 and 9.26, respectively. Genomic DNA length of the four genes is 6.1 kb, 3.7 kb, 9.3 kb and 3.5 kb, respectively. Each of them consisted with fifteen exons and fourteen introns. These results indicated that Rh MLO1-Rh MLO4 genes are new members of Mlo family, which provide the foundation for further study on structure and function of them.We conducted BLAST analyses of the four Rh MLO sequences with other MLO sequences from other species. The results showed that genetic relationship between Rh MLOs and MLOs from dicots such as arabidopsis, tomato, pea was more relative than that between Rh MLOs and MLOs from monocots such as barley, wheat, rice and corn,which was consistent with traditional classification system of botany. 2 Genetic linkage map construction of the four Mlo genes in roseWe constructed genetic linkage map of the four Mlo genes based on Single-Stranded Conformation Polymorphism（SSCP） PCR technology and Join Map2.0 software.Rh MLO1 is located on LG5 between markers NBS104-3₁ and CAg-ATg355-3₁.Rh MLO2 is located on LG3 near the double flower locus Blfo. Rh MLO3 and Rh MLO4 are closely linked to one another on LG1 near the blackspot resistance gene and a QTL for powdery mildew resistance. The results provide important information for our next practice work based on molecular marker assisted breeding（MAS）.3 Spatio-temporal expression analysis of the four Mlo genes in roseWe analysed spatio-temporal expression patterns of the four Mlo genes based on FQ-PCR technology. The results showed that expression patterns of Rh MLO1 and Rh MLO2 were similar in different tissues and stressed leaves. Transcriptional levels of Rh MLO1 and Rh MLO2 were up-regulated more than two-fold by external stimuli,especially by inoculation with powdery mildew fungus P. pannosa at different time points.The results indicated that Rh MLO1 and Rh MLO2 likely play important roles in rose-powdery mildew pathogen interactions.4 Functional verification of candidate gene Rh MLO1We constructed two plant expression vectors with sense and antisense Rh MLO1 gene based on digestion and connection methods, which were introduced into embryogenic callus of ‘Baiyu’ respectively by Agrobacterium-meidiated transformation. The transgenic lines were confirmed integration based on PCR and FQ-PCR. We compared the difference of powdery mildew resistance level between control plants and transgenic plants by isolated identification and microscope observation. The results showed that Rh MLO1-transgenic lines decreased resistance ability against the pathogen and anti-Rh MLO1-transgenic lines increased resistance against the pathogen. In conclusion,the results indicated that Rh MLO1 plays an important role in rose-powdery mildew pathogen interactions.