Cloning And Function Analysis Of Tamlo3 Gene

Wheat powdery mildew, caused by Blumeria graminis, is one of the major fungal disease that make drop in the production and quality of wheat, especially in all the north of china. Because there is highly diversified differentiation in the pathogen and more than 30 physiological races has been identified, the pathogen mass changes at a rapid speed and the variety resistance is easily lost. However, mlo, first discovered in barley, is a kind of plant genes which related to the broad-spectrum resistance to all the known races of the powdery mildew. The MLO protein, as CaM binding protein, had been presumed as a resistance-suppressing factor in the interaction between barley and its powdery mildew pathogen. Research on the Mlo homologues in wheat will open a new field for wheat molecular breeding.According to the conservative sequence of the Mlo genes in barley and Arabidopsis, several pairs of primers for PCR were synthesized; and a cDNA fragment of 513 bp from wheat, shared highly similarity with barley Mlo gene and nominated TaMlo3 (GenBank accession number: AF336105), was isolated. Then the RACE technology was used for the 3'-fragment cloning. A 1322 bp fragment of wheat TaMo3 including the major functioning regions was gained. This fragment encodes 440 amino acids, which contains five transmembrane regions, one CaM binding domain, one nuclear locatization sequence (KKKVR), and several kinase sites.The Mlo gene expression in different varieties and in various interaction stages was analyzed with Northern blot, using the TaMloS 3'-fragment (SOObp) as probe. It was found that the expression dose in resistant varieties was much lower than in the susceptible ones. In susceptible varieties, the expression level was at the minimum 6 hours after inoculation. The results indicated that the Mlo expression was negatively related to the resistance in wheat.On the other hand, the cytochemical localization of CaM, which is recently confirmed as an upstream binding protein of MLO protein, was also carried out here by means of immune-gold labeling. The results showed that the CaM was mainly located in the vacuoles in the health cells. The gold particles were labeled much more in the successfully infected cells than in the unsuccessfully infected ones, both in resistant and susceptible plants. And in the later stages of the infection, CaM was entered and accumulated in the extra-haustorial matrix, implying that the protoplasmic membrane was destroyed. These suggested that CaM in the epidermal cells was a critical factor to keep low concentration of Ca2+ in the cell.