Characterization And Functional Analysis Of APX And Mlo Genes In Muskmelon

Melon (Cucumis melon L.) is a valuable cash crop grown throughout the word. It is a member of the genus Cucumis, in the family Cucurbitaceae. Powdery mildew is one of the most important limiting factors for cucurbits production. Powdery mildew is a severe disease which causes the reduction of melon production in worldwide. As the most characteristic visual symptom the disease induces development of a whitish, talcum-like powdery growth on stems, petioles and leaf surfaces. Infected leaves usually wither and die, and plants senesce prematurely. It frequently causes significant yield losses and reduction of fruit quality in agricultural settings, including field farming and greenhouse. It has been suggested that recombination may play an important role in the virulence variation of P. xanthii in cucurbits. So it is difficulty to develop a normal breeding method to obtain melon cultivars resistance to different fungal races. Therefore, through genetic engineering to obtain the durable, broad-spectrum resistance variant of melon is very important.We successfully cloned the APX and Mlo family genes form muskmelon. The analysis showed that these genes maybe related with occurrence of the powdery mildew. The result of RT -PCR and Real-time PCR indicate that the CmMlo2 functions as a negative regulator that suppressed plant defenses in uninfected tissues. Loss of MLO reduces the threshold at which cells respond defensively to powdery mildew infections. We obtained the translated melon plant after constructed RNAi vector.1. The RT PCR and RACE methods were used to obtain the cDNA sequence of an ascorbate peroxidase (APX) gene after the leaf was induced with powdery mildew. A pair of primers was designed for obtaining the cDNA and genomic DNA sequence. The cDNA length of APX gene is 1047bp with a 750bp ORF encoded a 249 amino acid and the molecular weight of APX protein is 27.3 kDa. The analysis showed that the CmAPX genomic DNA contained 10 extrons and 9 introns .The identity of the amino acid sequence deduced from the cDNA with the APX family of other homologous members was about 74-97%.2. A Full-length of ORF was sub-cloned into prokaryotic expression vector pET24a. The recombinant proteins had high expression level in E.coli. This is the first report on cloning and sequencing the APX gene from melon.3. For determining the function of CmAPX we inoculated the leaves with powdery mildew, moderate accumulation of CmAPX mRNA was observed in initial leaves, and then increased during infection process. In the melon leaves the mRNA level sharply increased after 3d,and remained high to 5d, after 7d the mRNA level reached to normal level. We also detected the APX activity of the melon leaves. The results showed that the APX activity was increased during the leaves was induced with powdery mildew .The activity reached a maximum level after the leaves induced within 5d. These results suggested that CmAPX involved in the infection process of powdery mildew.4. The RT-PCR and RACE methods were used to obtain the cDNA sequence of three Mlo genes of muskmelon after the leaves were induced with powdery mildew. The three Mlo genes of melon were designated CmMlo1 (GeneBank Acc: FJ713541), CmMlo2 (GeneBank Acc: FJ713542) and CmMlo3 (GeneBank Acc: FJ713543). CmMlo1 was 1551 bp in length and contained a putative ORF encoding 516 amino acid residues, while CmMlo2 cDNA was 1713 bp in length and contained a putative ORF encoding 570 amino acid residues. CmMlo3 was 1464 bp in length and contained a putative ORF encoding 487 amino acid residues. To detect similarities and differences in individual amino acid sequence positions, we aligned the deduced amino acid sequence of CmMlo with the sequence of other plant Mlo in the database of NCBI Blast search. The comparison of identities among the homologues isolated in this study indicated that CmMlo1, CmMlo2 and CmMlo3 encode polypeptides that are highly sequence related to barley Mlo, Arabidopsis AtMLO2, AtMLO6, and AtMLO12. Semi-quantitative RT-PCR and Real-time PCR indicated Cmmlo2 may play a important role in occurrence of powdery mildew.5. Predicted topology of Cmmlo2 and assays of pROK-Cmmlo2-GFP fusion protein in onion showed Cmmlo2 is an integral plasma membrane-localized protein.6. After The efficient of dsRNA constructs were examined via Cmmlo2-GFP fusion protein and pFGC1008-Cmmlo2 were Co-transformation in Nicotiana benthamiana the dsRNA constructs were introduced into Agrobacterium tumefaciens and transformed into melon wild type with the methods of Agrobacterium-mediated leaf disc transformation. The material of broad-spectrum powdery mildew resistance against P. xanthii in muskmelon was obtained by ihpRNAi-mediated knock-down of Cmmlo2.