| China has a long history of apple cultivation,and its planting area and output account for more than 50%of the world.The Loess Plateau is one of the best eugenic areas for apple planting in China,but the sustainable development of the apple industry in this area has been significantly affected by drought.b HLH proteins are the second largest transcription factor family in plants.These proteins not only regulate plant growth and development,but also play an important role in abiotic stress response and resistance regulation of plants.However,there are few studies on the function of b HLH transcription factors in apple in regulating drought resistance.In this study,the drought stress responsive gene MdbHLH2 was cloned from apple,the expression patterns of MdbHLH2 in different tissues and under drought stress conditions were analyzed,and the drought resistance regulation function of MdbHLH2 was identified in detail in transgenic Arabidopsis,tomato and apple calli.The main results are as follows:1.The full-length coding sequence（CDS）of MdbHLH2 gene is 1068bp,and the length of the encoded protein is 355aa.MdbHLH2 contains a typical b HLH conserved domain.Promoter sequence analysis showed that the MdbHLH2 promoter contains multiple cis-acting elements,such as ERE,P-box,Box4,etc.,which are related to plant hormone or stress response.Subcellular localization assay showed that MdbHLH2 protein was localized in the nucleus.Expression analysis showed that the expression of MdbHLH2was induced by drought,salt,ABA and low temperature stress,suggesting that MdbHLH2may be involved in plant responses to abiotic stress.Transcriptional activation analysis showed that MdbHLH2 protein had significant transcriptional activation activity,which was in line with its transcription factor properties.Yeast two-hybrid（Y2H）experiments showed that the MdbHLH2 protein could interact with itself and with the drought resistance-related proteins Md FMA1 and Md BT2.2.It was found that MdbHLH2 negatively regulated the drought stress resistance of apples by osmotic stress treatment of transgenic apple callus with overexpression and interference expression of MdbHLH2.Under 100 m M mannitol treatment,the fresh weight of apple callus overexpressing MdbHLH2 was significantly decreased,and the MDA content was significantly increased,indicating that overexpression of MdbHLH2reduced the tolerance of apple callus to osmotic stress.On the contrary,the fresh weight of apple callus with MdbHLH2 expression interferenced was significantly higher,and the MDA content was significantly lower than that of the control,suggesting a stronger resistance to osmotic stress.In conclusion,MdbHLH2 negatively regulates the response of apple to drought stress.3.Through drought treatment of transgenic Arabidopsis thaliana overexpressing MdbHLH2,it was found that MdbHLH2 negatively regulates drought resistance.Under drought treatment,overexpression of MdbHLH2 increased the water loss rate,relative conductivity,and MDA,H2O2and O2-content,and decreased the activities of antioxidant enzymes such as SOD,POD,and CAT in transgenic Arabidopsis.Transcription levels of the drought resistance-related genes were also significantly lower in transgenic plants than of the control.The above results indicated that overexpression of MdbHLH2 reduced the drought resistance of transgenic Arabidopsis.4.Through drought treatment of transgenic tomatoes overexpressing MdbHLH2,it was further clarified that overexpression of MdbHLH2 reduces drought resistance.Under drought treatment,overexpression of MdbHLH2 increased the relative conductivity,and MDA,H2O2and O2-content of transgenic tomato leaves.The transgenic plants also showed lower PRO content,antioxidant enzyme activity,photosynthetic pigment content and Fv/Fm.Analysis of stomatal characteristics showed that the leaf stomatal aperture of transgenic plants was significantly larger than that of the wild type.These results indicated that overexpression of MdbHLH2 reduced the tolerance of transgenic tomato plants to drought stress. |
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