Cloning And Genetic Transformation Of 4CL,COMT,EDS1 And EDS2 Genes Related To The Resistance Of Apple Ring Resistance

China is a big country for apple cultivation,and its planting area and output rank first in the world.As the main cultivar of fushi in China is susceptible to apple wheel disease,the disease is widespread and serious in China,which restricts the development of apple industry in China.In this study,genes were selected from transcriptome analysis in response to the infection of apple in the laboratory,which were 4CL,COMT,EDS1 and EDS2,respectively.In this study,the leaves of the disease-resistant strain "1-1-46" and the susceptible strain "1-1-40" selected from the hybrid offspring of apple varieties "chicken comb" and "Fuji" were inoculated with Botryosphaeria dothidea.The heterologous genetic transformation of pCAMBIA 1300s-4cl was carried out in arabidopsis thaliana by agrobacterium-mediated method,in order to prepare for the later in-depth study on its role in the resistance to apple disease.The main research is as follows:1.In the whole period after indoor inoculation,the leaf spot area of disease-resistant strains "1-1-46" was significantly smaller than that of susceptible strains "1-1-40".The growth peak of disease spots occurred at 4 d-5 d after inoculation.According to the disease spot area at 6 d,the disease-resistant strain "1-1-46" was of moderate resistance,and the susceptible strain "1-1-40" was of high susceptibility.2.Relative gene expression levels of 4CL,COMT,EDS1 and EDS2 were measured,and it was found that 4CL and COMT genes were rapidly expressed in the resistant plants after inoculation,and the expression levels reached the maximum on day 1 after inoculation.However,the expression of EDSI and EDS2 genes reached the maximum at the 6th day after inoculation.3.The sequence length of apple 4CL was 1644 bp,the total number of atoms was 8526,and a total of 547 amino acids were encoded.The predicted relative protein molecular weight was 60.3 kDa,the isoelectric point was 5.57,and the encoded protein was transmembrane protein.It can be seen from the phylogenetic tree constructed that apple 4CL protein has the closest relationship with white pear and the highest homology.The sequence length of apple COMT was 1098 bp,encoding 365 amino acids,and the predicted relative protein molecular weight was 39.7 kDa,and the isoelectric point was 5.44.The protein encoded was not transmembrane protein.It can be seen from the phylogenetic tree constructed that apple COMT protein has the closest relationship with white pear and the highest homology.The sequence length of apple EDS1 was 1857 bp,encoding 618 amino acids,and the predicted relative protein molecular weight was 70.6 kDa,and the isoelectric point was 6.39.The protein encoded was not transmembrane protein.It can be seen from the phylogenetic tree constructed that it has the closest relationship with white pear and the highest homology.The sequence length of apple EDS2 was 1836 bp,encoding 611 amino acids,and the predicted relative protein molecular weight was 68.9 kDa,the isoelectric point was 5.67,and the protein encoded was transmembrane protein.It can be seen from the phylogenetic tree constructed that apple EDS2 has the closest relationship with white pear and the highest homology.4.pCAMBIA 1300s-4CL was obtained by homologous cloning method,and the 4CL gene was heterogenously introduced into arabidopsis plants by agrobacterium-mediated method,and the target genes of 4 transformed plants were detected,proving that all 4 strains were transformed strains.