Apple bHLH Transcription Factor MdTTL1 Promotes Anthyocyanin Biosynthesis and Fruit Coloration Via Multiple Pathways at Low Temperature
|School||Shandong Agricultural University|
|Keywords||Apple Coloration Anthocyanin bHLH Hypothermia|
Fruit color is an important agronomic traits of fruit trees, apple and other fruit trees decided edible and market value. Apple peel color is determined by the anthocyanin. Anthocyanin synthesis by light, temperature and nutrition and other environmental factors. In the past century, global temperatures continue to rise. This climate change is already affecting Apple's coloring. So, to clarify inhibition induced by low temperature or high molecular mechanism of apple fruit coloration is essential for the production of apples. In this paper, the new Red Star apple (Malus Domestica Borkh cv. Starkrimson) fruit as test materials, the use of differential screening method, cloned into a low-temperature responsive bHLH transcription factor, named MdTTL1 (GeneBank accession number JF920725). cDNA was 2687bp, encoding 710 amino acids, conserved domain analysis showed that, MdTTL1 protein contains bHLH (basic Helix-Loop-Helix) functional domains. All the protein and bHLH proteins in Arabidopsis phylogenetic tree analysis showed MdTTL1 subgroup with Ⅲ f flavonoids and regulation of epidermal cell fate proteins clustered into one class, and the highest homology with AtTT8, so named for the MdTTL1 (TT8 like1). Further analysis showed that, MdTTL1 with Apple MdbHLH3, grapes VvMYC1, petunias and other plants PhAN1 anthocyanin metabolic regulation bHLH protein homology. RT-PCR analysis showed that, MdTTL1 in apple fruit, leaves and stems are expressed, but in the colored peel the highest expression; and induced by low temperature. According epitope prediction software analysis and biological sequence alignment results, select a coded protein, synthetic peptides as antigen antibody production. Western blot analysis showed that, MdTTL1 treatments in UV17 ℃ protein expression increased and UV27 ℃ showed lower protein degradation trends. Yeast two-hybrid and bimolecular fluorescence complementation analysis showed MdTTL1 capable MdMYB1 interaction, shear test found that the N-terminal 1-228 amino acids are necessary for the interaction with MdMYB1 area. In addition, EMSA and ChIP vitro and in vivo are two ways to prove MdTTL1 with MdUFGT and MdDFR promoter binding, further promoter GUS transient expression and stable expression analysis showed that, MdTTL1 protein is regulated MdUFGT promoter and promoted at low temperatures stronger. To further determine the function of the Apple MdTTL1, we constructed a pBI121-MdTTL1 and pBIN-MdTTL1-GFP overexpression vector was transformed into Agrobacterium infection Gala callus tissue culture and Wang Lin. Low temperature and the treatment of transgenic callus UVB analysis of transgenic callus anthocyanin MdTTL1 trends and protein expression level, indicating that the UVB MdTTL1 transgenic calli can be accumulated in the presence of anthocyanin, and in the low temperature cyanine glucosinolate content increased expression. Little change in protein levels, it can be inferred MdTTL1 exist post-translational modification. Western blot results demonstrate MdTTL1 occurs at low temperatures protein phosphorylation, and this cold-induced phosphorylation may be out Staurosporine (10μm stau) are inhibited. Turn Jiyingala significantly increased the roots of anthocyanin and anthocyanin at low temperatures even higher. In addition, there are transgenic plantlets dwarf phenotype, ChIP experiments show, MdTTL1 MdCBF2 and MdCBF3 binding to the promoter. To prove MdTTL1 apple fruit coloring process in the feature vector using transient expression IL-60-BS to its overexpression Starkrimson peel around the hole to facilitate the injection process, coloration, suppresses the expression of inhibition around the hole injection coloration, It showed MdTTL1 apple fruit coloring process plays a positive regulatory role.