Gene Cloning And Functional Identification Of MdMYB2 In Apple

Diverse environmental stresses,such as high salinity,drought,extreme temperature,heavy metal pollution,and nutrient deficiency,affect plant growth and development.In order to adapt to the diversely environmental stresses,plants have evolved the sophisticated adaptive mechanisms that enable them to recognize and respond to stress stimulus.When plants suffer from environmental stresses,a variety of stress-responsive genes are up-regulated and lots of changes in plant physiology occurred,thus protecting plants from the stress damage.Moreover,the expression of these genes is mainly regulated by specific transcription factors.Among these transcription factors,members of MYB,bHLH,WRKY and NAC families play vital roles in response to abiotic stress in plants.Previous studies have shown that MYB transcription factors are responsive to a variety of abiotic stress,such as drought,high salinity,and cold stress.Overexpression or suppression of MYB genes affects plant stress tolerance.Up to now,most researches on MYB transcription factors are restricted in the model plants,such as in Arabidopsis and rice.The reports that concern the function of MYB transcription factors on abiotic stress response in woody plants,especially in apple,are still unclear.Apple is an important economic crop.China's apple production and export volume rank top in the world.Environmental stress seriously affects apple production and quality.Therefore,it is of great practical significance to study the effects of various stresses on apple production and the response mechanism of fruit trees to stress to improve apple yield and fruit quality.In this study,a MYB transcription factor named MdMYB2(GenBank accession number: MDP0000823458)was isolated from Malus × domestica ‘Royal Gala' by homologous cloning and PCR method.Bioinformatics analysis showed that MdMYB2 contained a conservative R2R3-MYB domain and had the highest homology with PbMYB2 from pear.The expression patterns were examined using qRT-PCR,and the results showed that MdMYB2 was existed in all tissues and was responsive to various hormones and abiotic stresses.In order to test the function of Md MYB2,the overexpression(MdMYB2-OE)and suppression(MdMYB2-Anti)of MdMYB2 transgenic apple calli,and ectopic expression of MdMYB2 transgenic Arabidopsis by Agrobacterium-mediated genetic transformation were obtained.Then MdMYB2 transgenic apple calli and Arabidopsis were used to test the role of MdMYB2 in abiotic stress response.The main work and results were shown as follows:1.The open reading frame(ORF)of MdMYB2 contained 873 bp,and encoded 290 amino acid.The predicted MdMYB2 protein had a molecular mass of 32.92 kD and a pI value of 4.91.The result of amino acid sequence alignment indicated that MdMYB2 contained a R2R3-MYB domain.Phylogenetic analysis demonstrated that MdMYB2 had the nearest relationship to PbMYB2 from pear.2.The expression analysis showed that MdMYB2 gene was universally expressed in different apple tissues,and its expression level was higher in young fruit and stem than in others.The expression of Md MYB2 gene was noticeably induced by salt,mannitol,PEG,ABA,high and low low temperature stresses.3.The tolerance assays showed that overexpression of MdMYB2 in apple calli remarkably enhanced the tolerance to high salinity,mannitol,and the ABA sensibility.Ectopic expressing MdMYB2 in Arabidopsis also positively regulated the tolerance to salt,mannitol,drought stresses,and the ABA sensibility.These results indicated that MdMYB2 plays an important role in plant abiotic stress response.