The Construction Of Apple MdBAK1s Genes Expression Vector,Callus Transformation And Apple Ring Rot Disease Resistance Characterization

BAK1 is one of the important types of kinase receptors in plants.In Arabidopsis thaliana,BAK1 mediates the signal transduction of brassinolide,innate immune response and toxicity of pathogenic bacteria protein to the plant.MdBAK1 s in apple has a high similarity with BAK1 in Arabidopsis thaliana.But until now,there are few studies on the role of BAK1 in resistance to various diseases and in apple development at home and abroad.Therefore,in this study,we cloned 2 homologous genes of BAK1(named as: MdBAK1-1 and MdBAK1-2)in apple and analysed their amino acid sequence.Then,we studied the change of the growth of overexpression transgenic apple callus,the impact on BR processing,the disease resistance and the signal response to MAPK after inhibiting expression.These studies will lay the foundations for further research on the role of MdBAK1 s in apple growth and disease resistance.The main results are as follows:1.Two homologous genes of BAK1 were cloned from the tissue culture seedlings of the ‘gala',named as: MdBAK1-1 and MdBAK1-2.Amino acid sequence analysis showed that: the sequences of MdBAK1-1,MdBAK1-2 highly conserved with Arabidopsis BAK1,the similarity was 83% and 84% respectively.Thereinto,the conserved region of intracellular kinase is more conservative.And they all have a signal peptide region,leucine zipper region,5 leucine rich region,proline rich region,transmembrane region and intracellular kinase region.2.Transgenic apple callus and transgenic apple plants with stable over expression of MdBAK1 s were obtained.By preliminary observation of the phenotypic characteristics,we found that the transgenic apple plants growing weaker,some leaves gradually turn yellow with the growth.3.The expression of MdBAK1 s gene was knocked out by the method of RNA interference and transgenic apple callus with inhibited expression of MdBAK1 s was obtained.By preliminary observation of the phenotypic characteristics,we found that the transgenic apple callus with inhibited expression was relatively light in color comparing with control callus.4.The difference of the growth of the transgenic apple callus with overexpression of MdBAK1 s and the control callus in different periods was studied.The results showed that the growth of 15 days of transgenic apple callus with overexpression of MdBAK1-1 was significantly higher than that of the control callus.The growth of transgenic apple callus is 2.3 times of the control callus.5.The difference of the growth of the transgenic apple callus with overexpression of MdBAK1 s and the control callus in different concentration of BR treatment was studied.The results showed that when the concentration of BR was 1.0 nmol/L,the growth of apple callus could be promoted.With the increase of BR concentration,the growth of apple callus could be inhibited.But when the concentration of BR was 1000 nmol/L,the growth of the transgenic apple callus with overexpression of MdBAK1 s was significantly lower than that of the ordinary callus.6.The resistance of the transgenic apple callus with overexpression of MdBAK1 s and the control callus to the apple rot was studied.The result showed that transgenic apple callus with overexpression of MdBAK1 s was more susceptible to disease.7.The expression difference of the MAPK6 protein in response to the flg22 signal indicative between the transgenic apple callus with overexpression of MdBAK1 s and the control callus under flg22 treatment was studied.The result showed that the expression of MdBAK1 s was inhibited,and the expression of MAPK6 of background level was weaker than that of the control.