| Arabidopsis broad-spectrum disease resistance gene RPW8 (resistance to POWDERY MILDEW locus, RPW8) is isolated from Arabidopsis thaliana MS-0, which contains two tightly linked genes:RPW8.1 and RPW8.2. A previous study showed that expression of the RPW8.2 is induced by powdery mildew infection, via the intracellular vesicular transport specific anchor to the extrahaustorial membrane (Extra-Haustorial membrane, EHM) of Powdery Mildew to confer broad-spectrum disease resistance. The study of RPW8.1 showed that it had a certain degree of similarity in the amino acid sequence to RPW8.2, and it also had broad-spectrum resistance to powdery mildew. However, the localization and expression of RPW8.1 protein in the cell is largely different from that of RPW8.2, which caused the concern and discussion of the disease resistance mechanism of RPW8.1. RPW8.1 is located surrounding chloroplasts in mesophyll cells and how dose it resist the powdery mildew in epidermal cell? To answer this question, we choose a transgenic line R1Y4 that stably expressing RPW8.1-YFP with resistance to powdery mildew as a base material in EMS-mediated mutagenesis. From the M1 Generation Plants we screened more than 100 mutants different from R1Y4 in leaf shape, leaf color and leaf cell death. By observation and comparison, among the mutants selected, three mutants altered the "pitted-leaf" phenotype of R1Y4 into lotus-like leaves that had shortened petioles with the most severe phenotype in b9-1 and accompanied by cell death, weakest in b6-3 and intermediate in b10-6. In addition, all the three mutants had leaves smaller than that of R1Y4 and were in smaller plant statue. Three mutants did not express R1 Y4 unique "pitted-leaf" phenotype. During the same period planting mutants and R1Y4, mutant plants bolting ahead of time and rachis and stem ratio significantly greater than R1Y4.Mutant b9-1 was selected for map-based cloning. An F2 segregating population was derived from a cross between b10-6 and Ler. In F2 population, the mutant phenotype plants accounted for one fourth, thus the mutant is controlled by single recessive gene. Selected 20 strains from F2 mutant phenotype plants for positional cloning, Through choosing 20 simple sequence length polymorphism (SSLP) markers in a linkage analysis, the mutant gene of b9-1 was preliminarily mapped to Chromosome 2, and in the outer end of the chromosome 2 SSLP markers T8O18, co-segregation with F3G5. Then according to the Col-gl and ler polymorphisms in F3G5, choosing 15 SSLP markers (T1B8, T20F21, F10I1, T1J8, T2N18, F16M14-1, F16M14-2, T6A23, T7F6, F12L6, T28M21, F27I1, T3K9, F4I1 and F11C10), in addition to the T20F21, T28M21, T3K9 polymorphism are poor or amplified no banding problems, other molecular markers between the mutant and ler has polymorphism; at the same time, we also screened with a mutant phenotype of plant a total of 133 strains from the F2 population, thus the mutant gene in b9-1 was located between the T2N18 and F16M14 (physical distance 0.4Mb). According to the reported genes between the two markers, combined with phenotypes, two genes were chosen for sequencing. The results showed that the At2g37630 (encoding asymmetric leaves1 and AS1) sites in the mutant b9-1 occurs a base substitutions, causing the AS1 gene At2g37630 with a G to A single nucleotide mutation, and thus resulting in a stop codon. The other two mutants have mutations in the same gene:a stop code in the b10-6, and in the b6-3 caused an amino acid replacement.Three mutants mutations located in different areas of the AS1 gene, and in different plants are conservative, understanding and awareness of the mutations in the three regions more in-depth will benefit for the prediction of the model of AS1 protein more scientific and reasonable. Previous reports showed that the AS1 gene can encode a myb domain transcription factor, inhibition expression of KNOX gene in leaf primordia, and KNOX genes is important to control apical meristem formation, it is not difficult to deduce that AS1 plays important role in Arabidopsis leaf development. Futher work will be carried on charactering the functions of AS1 gene, and its relationship with RPW8.1. |
