The Correlation And Expression Characteristic Of Six Stress-induced Transcription Factors And Alcohol Acyltransferase Gene In Apple

Alcohol acyltransferase (AAT), as the key enzyme, plays important roles in ester biogenesis. In our previous reports, the expression of apple AAT gene (MdAAT2) was shown to be induced on different levels by salicylic acid (SA), Ethephon (ETH), and methyl jasmonate (MeJA). This indicated that MdAAT2 gene may be involved in defense signaling pathways. At present, the research of AAT genes are mostly foucs on fruit and flower aroma synthesis. But the mechanism of transcription factors on regulation of MdAAT2 promoter has not been well established. Using bioinformatics technology, we isolated and investigated six candidate stress-induced transcription factors, i.e., MdMYB1, MdMYB2, MdMYB6, MdERF1, MdERF2 and WRKY. In order to investagate the transcript correlation between these TFs and MdAAT2 gene during fruit development series and different hormone treatments, reverse transcription-polymerase chain reaction (RT-PCR) and statistical analysis were performed. The main results are as follows:1.To identify potential cis-acting regulatory elements in MdAAT2 gene romoter, Plant CARE and other available databases were performed. Sequence analysis showed that the transcription factor binding sites of two salicylic responsive element W-box and a methyl jasmonate (MeJA) responsive element TGACG-motif were found in MdAAT2 promoter region, located at -661bp, -1130bp and -1129bp,respectively. Based on the results presented here and homologous analyze, we isolated six candidate transcription factors MdMYB1 (GU013684), MdMYB2 (GU013681), MdMYB6 (GU013682), MdERF1 (AB288347), MdERF2 (AB288348) and WRKY (GU013683) from "Golden delicious"apple.2.The transcript pattern of these TFs and MdAAT2 gene was analyzed by RT-PCR in apple fruit and flower tissues. The results indicated that the transcript expression of six TF genes was all detected in stigma.The transcript expression of MdERF1 and MdERF2 were also detected in fruit and flower tissues. Sequence analysis showed the MdAAT2 promoter contained two pollen-relative elements POLLEN1LELAT52 which located at the -82 and -882. Accordingly, in apple fruit and flower tissues, these TF genes may be involved in the regulation of functional genes or interacted with the MdAAT2 gene during volatile ester biosynthetic pathway.3.The transcript pattern of these TFs and MdAAT2 gene during fruit ontogeny was analyzed by RT-PCR. There were comparatively low correlation of the transcript levels between two transcription factors WRKY, MdMYB6 and MdAAT2 gene by correlation analysis. But as compared with the MdAAT2 gene expression, the transcript levels of MdMYB1, MdMYB2, MdERF1 and MdERF2 showed a significant correlation. Therefore, MdMYB6 (GU013682) and WRKY (GU013683) may not affect in fruit development. On the contrary, MdMYB1, MdMYB2 and MdERF1 may be responsible for the regulating the expression of MdAAT2 gene which results in the volatile esters metabolizes.4.Transcript pattern of MdERF1 was almost consistent with MdAAT2 and the correlation analysis also showed a high degree after application of 2.0 mM SA treatment. And there was a well correlation between the gene of MdMYB6 expression and MdAAT2 gene expression. These results indicate that MdERF1 and MdMYB6 probably play an important role in regulation of MdAAT2 gene expression during the SA defense signaling pathway.5.Transcript pattern of two TF genes MdMYB1 and MdMYB6 showed a significant correlation with MdAAT2 gene expression after 2.0 mM ETH treatment. Therefore, MdMYB1 and MdMYB6 might serve as a candidate activator of MdAAT2 gene expression in the ethylene signaling pathway. 6.Under 2.0mM MeJA treatment, the transcript levels of MdMYB2 and MdERF2 were observed to be a significant correlative with MdAAT2 expression. Thus, MdMYB2 and MdERF2 probably regulated the expression of MdAAT2 gene after MeJA inducement.7.Sequence analysis indicated CAAT,CCAAT elements and several MYB transcription factor binding sites exist in MdAAT2 promoter region. And the MdMYB1 and MdMYB6 sequences in"Golden Delicious"revealed similarities to CAAT or CCAAT consensus sequence of two protein complexes. Thus, the transcription factors MdMYB1 and MdMYB6 might serve as activators of MdAAT2 gene involved in stress signaling pathways.