Gene Cloning And Functional Identification Of MdHEMA1 Encoding The Key Enzyme For Chlorophyll Biosynthesis In Apple

Two apple cultivars ‘Golden Delicious and Gala’ and Ailsa Craig tomato seedlings were used as plant materials in this study. Golden Delicious fruits were bagged with 4 different color bags, then the light transmittance rates of bags, fruit coloration, chlorophyll content and the expression of genes involving in chlorophyll biosynthesis, the content of soluble solids, titratable acidity and Vc were measured, based on these data, the effects of light transmittance rates on fruit coloration and internal quality were analyzed. MdHEMA1 encoding the key enzyme in chlorophyll biosynthesis pathway in apple was cloned and inserted into plant expression vector. Transgenic tomato over-expressing MdHEMA1 were obtained with agrobacterium-mediated genetic transformation and the relative expression of genes involving in chlorophyll biosynthesis of transgenic tomato were measured. This study not only had important theoretical significance for the regulation of chlorophyll synthesis, but also had potential applications in enhancing photosynthetic efficiency, improving fruit quality and other aspects in apple. The main results are showed as follows:1. Bagging with different color bags had effects on both fruit coloration and internal quality. Blue and red bags with high light transmittance promoted the accumulation of chlorophyll and enhanced yellow or green saturation on fruit surface. The contents of total soluble solids, titratable acid and Vc were higher than those under other treatments. So we believed that bagging with blue or red bags was beneficial to the formation of internal quality of Golden Delicious.2. The relative expression of Md HEMA1 and its downstream genes were positively correlated with the level of chlorophyll accumulation.3. Md HEMA1 is located on chromosome 8 of apple. The amplified sequence of this gene contains a1638 bp coding sequence, which encodes a protein of 545 amino acid residues.Sequence analysis showed that MdHEMA1 protein contains 3 conserved domains. The phylogenetic relationship of this gene is closer with Pyrus×bretschneideri. Promoter analysis revealed that there are multiple putative cis-acting elements that are involved in light, plant hormone, low-temperature and drought responsiveness.4. qRT-PCR results showed that the MdHEMA1 gene was expressed in all tested tissues tested.However, photosynthetic tissues had a higher expression level. The expression level of MdHEMA1 varied at different developmental stages and decreased dramatically with the reducing of chlorophyll content. The expression of MdHEMA1 could also be up-regulated by drought and other stresses, which was consistent with the analysis of cis-acting elements on the promoter of MdHEMA1. These results suggested that MdHEMA1 may also involved in the photosynthesis and stress response in apple.5. MdHEMA1 was inserted into the plant expression vector pRI101. Transgenic tomato was obtained through the agrobacterium-mediated genetic transformation. qRT-PCR results showed that the expressions of MdHEMA1 in transgenic tomato were higher than that in wild line. Meanwhile, the expressions of downstream genes involved in the chlorophyll biosynthesis pathway were alsoup-regulated in the transgenic tomato. These results suggested that Md HEMA1 could regulate the chlorophyll biosynthesis in tomato.