Cloning Of The Transcription Factors MhERF017 And MhMYB114-like From Malus Halliana And Its Function In Response To Iron Deficiency

Iron(Fe)is one of the essential minerals during the growth and development of plants.In addition,Fe also participates in the composition of some enzymes.Fe deficiency leads to the inhibition of chlorophyll synthesis,and leaves appear yellow phenomenon,which seriously affects the quality and yield of apple fruit.In order to better adapt to Fe deficiency environment,plants have evolved two different Fe absorption mechanisms to balance the Fe content in plants.Among them,members of AP2 and MYB families play an important role in Fe deficiency.When plants are subjected to Fe deficiency,it will stimulate the regulation of related specific transcription factors,causing a series of physiological changes in the plant,thereby improving the tolerance.At present,the functions of some AP2 and MYB transcription factors have been mainly studied in model plants such as Arabidopsis and rice,however,comparatively few studies have been conducted on iron deficiency in apples.Therefore,it is practical importence to explore the regulation response to Fe deficiency in fruit trees.In this study,we used 'Malus halliana' as the material.The expression levels of transcription factors under Fe deficiency stress were determined by quantitative real-time PCR.We screened differentially expressed genes ERF017 and MYB114-like,and isolated genes MhERF017 and MhMYB114-like from Malus halliana.Then,we perform bioinformatics analysis of the genes.Finally,the role of the obtained MhERF017 and MhMYB114-like plants in response to Fe deficiency was preliminary studied.The major results are as follows:1.Real-time fluorescent quantitative PCR analysis showed that under Fe deficiency,ERF017 and MYB114-like were found to be expressed in the roots and leaves of 'Malus halliana',and the expression levels were significantly higher than other genes.2.We cloned genes MhERF017 and MhMYB114-like from Malus halliana.The fragments were 714 bp and 602 bp in size,encoding 227 and 200 amino acids,respectively.We predict that the molecular weight of ERF017 protein is 25359.96 KD and the isoelectric point is 4.86,while the molecular weight of MYB114-like protein is 23414.66 KD and the isoelectric point is 9.38.3.Amino acid sequence alignment showed that there was an AP2 domain at the N-terminus(between the 24 th and 87 th amino acids)of ERF017 protein in apple ERF017 and other species,and the amino acid sequence comparison of MYB114-like protein revealed that MYB114-like and other species of MYB114-like protein are relatively conserved at the N-terminus and show more specificity at the C-terminus.4.Phylogenetic tree results show that apple ERF017 had a closer relationship with jujube and rose,and the closest with rose is the highest homology,while apple MYB114-like has a closer relationship with wild strawberry.5.Construction of binary overexpression vectors for MhERF017 and MhMYB114-like genes,application of agrobacterium-mediated transformation of MhERF017 genes to apple callus and tobacco,and transformation of MhMYB114-like genes to apple callus and Arabidopsis,successfully obtained overexpressed transgenic plants.6.Overexpression of MhERF017 and MhMYB114-like genes significantly increased the tolerance of transgenic apple callus to Fe deficiency.Ectopic expression of MhERF017 gene in transgenic tobacco would also increase plant resistance to Fe deficiency stress.The ectopic expression of MhMYB114-like gene in transgenic Arabidopsis increased the chlorophyll content and increased the transgenic plant's tolerance to Fe deficiency stress.