Bioinformatics Analysis and Functional Study of DREB Genes from Morus Spp

Plants often encounter in a variety of adverse conditions, including drought, high temperature, low temperature and high salt stress between their growth and decelopment in natural conditions. All of the abiotic stress is the major factor for the agriculturral production efficiency which seriously affect and limite the growth and development of plants, and even lead to death of the plant. Therefore,the research on abiotic stress related genes has important theoretical and practical significance.Transcription factor can active or inhibite the expression of other genes by binding to specific DNA sequences in promoter. As upstream regulatory factors, transcription factors play an important role in the study of plant resistance, which can activate a variety of stress-related and functional gene expression. Transcription factor is more significant than a single functional genes in the aspect of improving plant resistance. At present, the main transcription factors, which are more reserched in plant resistance, include MYB, WRKY, bZIP, AP2/ERF and NAC. As one of the main transcription factors in plant resistance, AP2/ERF can be divided into AP2, ERF, DREB, RAV and OTHERS by their minor difference in AP2domain. DREB(Dehydration responsive element binding) are one class transcription factors related to drought, high and low temperature and other abiotic stress, can specificly bind to DRE(Dehydration respossive element) and regulate the expression of genes which contain DRE element in their promoters,and improve the plant resistance. That is why more and more researchers pay their attention on DREB transcription factors. But the research on DREB are major focused on herbaceous plants, and little in woody plants.Mullberry is one perennial dicotyledonous woody plants, they can survival from latitude10°to north latitude50°. Our country is the main origin of mulberry and own the most germplasm resources of mulberry in the world.. Mulberry can resist waterlogging, drought and the infertile soil, and their ability for growth and taking root are strong. Mulberry as an excellent ecological and economic tree, its leaves have a strong tolerance and absorption purification ability to sulfur dioxide, hydrogen fluoride chlorine and other contaminants. However, the research on mulberry resistance is little, and the research of DREB transcription factors in mulberry is not been reported. Therefore,the research on DREB in mulberry will help to supply the regulation mechanism of DREB in woody plants. In this research we get30DREB protion sequences through informatic analysis, compared the expression levels of20genes in leaves under40℃,4℃, drought and high salt, cloned MnDREB4G and transformed them to Arabidopsis. The results are as follows:1Bioinformatic analysis of DREB genes in M.notabilisThrough bioinformatic analysis we got110protein sequences which contain AP2domain from protein database of mulberry, included seventeen AP2, three RAV, fifty-four ERF, thirty DREB and six others. The DREB proteins can divided into six subfamilies A1-A6, according the AP2domain. The number of every subfamily is2,5,2,8,8,5,similar to Arabidopsis. The protein molecular weight of DREBs are between20KD and30KD, and the isoelectric point in the range of4.78to8.93. And the most gene length of DREB are less than1000bp, and the27genes are single exon in addition to MnDREB2B, MnDREB2C, MnDREB2E. We conjecture that there are alternative splicing mechanism in these three genes. There are many of stress-related elements in mulberry promoters. Twenty-eight of the thirty genes exist transcriptome data in mulberry database, and the expression patterns of each subfamily are different, such as the transcriptome data of MnDREB6B is high in various organizations and the MnDREB6E is very low.2Stress induced expression patterns of DREBIn this study, we design20pairs primers available based on mulberry DREB sequences. The different gene show the different expression patterns under drough, high salt, high and low temperature, and the response to different stress is not inevitable with the elements in their promoter. Presumably the abiotic stress mechanism mediated by DREB in mulberry is more complex and require multiple genes’interaction. But it may be the promoter sequences in Morus notabilis C. K. Schneid and Morus alba var. multicaulis are different, and the response mechanism to abiotic stress are different.3The functional reseach of MnDREB4GIn order to study the function of MnDREB4G in mulberry, we cloned them and build the overexpression vector pLGNL-35S-MnDREB4G, and transformated them into Arabidopsis, successfully builded transgenic Arabidopsis strains. Analysis the stress-related downstream gene’s expression of rd17, LEA14and rd29A found that the expression of rd17and rd29A raised in transgenic Arabidopsis lines14,29and32, and similar to the target gene MnDREB4G. But the expression of LEA14showed a slight downward adjustment, and there is no effect with the target gene MnDREB4G. The resistance of transgenic Arabidopsis to abiotic stress were increased under low temperature and high salt, suggesting that the MnDREB4G gene may be involved in low temperature and high salt stress response in plant.