Cloning Of Transcriptional Factors MxFIT And MxIRO₂and Their Relationship With Promoters In Malus Xiaojinensis

Iron deficiency is one of the important factors that affect the yield and quality in fruit trees, especially for apple tree. People have conducted a lot of researches about the mechanism of resistance to iron deficiency for years. However, the molecular mechanism of resistance to iron deficiency in apple tree is still not very clear. Malus xiaojinensis (Cheng et Jiang) is the first apple genotype identified as being resistant to iron deficiency by our research team. In this study, two iron-deficiency-induced transcription factors, MxFIT and MxIRO2, were cloned in M. xiaojinensis and the characteristics and functions were also analysed. MxIRTl and MxFRO2had been proved as two major genes related to iron uptake. The relationship of the promoters of MxIRTl and MxFR02with MxFIT and MxIR02transcription factors was studied in this paper. This work offers an important basis for further investigating the mechanism of fruit tree adaptation to iron-deficiency stress.According to the genome of Golden Delicious and the transcriptome sequencing of M. xiaojinensis, an iron deficiency response transcription factor gene, MxFIT, was cloned from M. xiaojinensis. MxFIT encoded a basic helix-loop-helix protein and contained a966bp open reading frame. MxFIT protein was targeted to the nucleus in onion epidermal cells and showed strong transcriptional activation in yeast cells. The antibody was obtained by injecting a New Zealand rabbit with the purified MxPIT-His fusion protein. Real-time PCR and Western blot analysis revealed that MxFIT was up-regulated in roots under iron deficiency at both mRNA and protein levels, while almost no expression was detected in leaves irrespective of iron supply. Ectopic expression of MxFIT resulted in enhanced iron deficiency responses in Arabidopsis under iron deficiency and stronger resistance to iron deficiency. Thus, MxFIT might be involved in iron uptake and plays an important role in iron deficiency response.In this study, another bHLH iron-related transcription factor IRO2, containing a762bp open reading frame and encoding253amino acids, had been cloned from M. xiaojinensis. The MxIRO2protein was targeted to nucleus in onion epidermal cells and had weaker transcription activation ability in yeast cells. The activation degree was restrained by1mM3-AT. The antibody was obtained by injecting a New Zealand rabbit with the purified MxIR02-His fusion protein. The spatiotemporal expression of MxIR02under iron deficiency was then assayed. The results indicated that MxIR02was induced in both roots and leaves when subject to iron deficiency. The expression model in transcription and translation level was increased at first and then decreased with iron deficiency stress.Yeast two-hybrid analysis proved that MxFIT could interact with MxIRO2. The promoters of MxIRTl and MxFRO2were activated in the tobacco leaves by coinfiltrated with agrobacterium containing MxFIT and MxIRO2. The binding sequences with MxFIT and MxIRO2transcription factor might locate-485～-236bp in MxIRT1promoter and-813～-564bp in MxFR02promoter. There might be reinforcing elements in-764～-485bp of MxIRT1promoter and-1704～-1228bp and-564～-329bp of MxFOR2promoter. Therefore, based on the relationship between transcription factors and functional gene promoters, we proved that MxFIT and MxIRO2could regulate MxIRTl and MxFRO2after interactions. Meanwhile, we found IRT1and FRO2were regulated by FIT in transgenic Arabidopsis. MxFTT and MxDRO2were two important transcription factors in regulation of iron absorption in Malus xiaojinensis.