Cloning And Stress Resistance Functional Analysis Of DREB Transcription Factor Genes From Sugarcane

Sugarcane is the most important sugar crops in China.In our country,80%of the sugarcane planting areas are dry sloping fields,which are characterized by low rainfall,uneven rainfall and low soil fertility.They are prone to drought and high salt stress,resulting in the loss of total yield and quality of sugarcane.Improving sugarcane resistance is always a continuous and important challenge in sugarcane breeding.Mining and identifying resistant genes are the basis of sugarcane transgene and marker assisted resistance breeding,and can provide potential practical utilization of gene resources.Transcription factor,being as a type of stress regulating factor which can regulate a variety of stress resistant genes,is more effective than a single functional protein gene in the improvement of plant stress resistance.DREB(dehydration responsive element bingding protein),as a subfamily of the AP2/ERF transcription factor,is closely related to abiotic stress.Considering the important role of DREB gene in plant resistance to stress,and the systematic study of sugarcane ScDREB family genes has not been done yet,whether its functional differentiation was similar to other plant DREB genes was not known.Therefore,this study mining 15 ScERF transcripts based on the sugarcane transcriptome database in our previous reseach.First of all,through the search of conserved domain of 15 ScERF genes prediction protein,the specific amino acid alignment AP2 conserved domain and phylogenetic tree clustering analysis,five ScDREB Unigene sequences were obtained.Secondly,three ScDREB(genes ScDREB2A、ScDREB2B-l and ScDREB2B-2)were actually cloned from sugarcane.The structure character of the gene encoding protein of this three ScDREB genes were analyzed.Their gene expression patterns in different sugarcane genotypes(ROC22 and Badila),which were treated by NaCl,PEG and ABA stresses were conducted by the real-time fluorescence quantitative(qRT-PCR).Thirdly,the subcellular localization,transcription activity analysis,yeast expression analysis and preliminary function verify after genetic transformation of ScDREB2A and ScDREB2B-1 were performed in Nicotiana bentamiana.The purpose of this study is to provide genetic resources with independent intellectual property rights and potential breeding application value to sugarcane breeding,which will improve the resistance of sugarcane varieties with molecular breeding methods and lay the foundation for the use of ScDREB family genes in the sugarcane stress resistance breeding.1.Mining of ScDREB genes in sugarcane and their bioinformatic analysisWe screened five sugarcane ScDREB gene transcripts through the homology comparison,specific amino acid search and phylogenetic tree cluster analysis were screened from 15 sugarcane ScERF genes,and named as ScDREB2A,ScDREB2B-1,ScDREB2B-2,ScDREB5A and ScDREB5B,respectively.Among them,the coding proteins of ScDREB2A,ScDREB2B-1 and ScDREB2B-2 belonged to DREB(A-2)subgroup,and were closely related to sorghum SbDREB.ScDREB5A and ScDREB5B belonged to DREB(A-5)subgroup.The number of amino acids and molecular weight of the five sugarcane ScDREB proteins were between 236 aa-330 aa and 25.97 KDa-35.52 KDa,respectively,moreover,one conserved AP2 domain in these five sugarcane ScDREB proteins was contained,and its 14th and 19th amino acids were valine and glutamic acid,which accorded with the specificity of AP2 domain in DREB protein that the 14th and 19th amino acids were conservative.2.Cloning and expression pattern analysis of ScDREB genes in sugarcaneThe full-length cDNA sequences of three ScDREB genes ScDREB2A,ScDREB2B-1 and ScDREB2B-2 were cloned from sugarcane variety ROC22.There was only one AP2 domain composed of YRG and RAYDAP2 conservative motifs in these three ScDREB genes encoded proteins.The C terminal of ScDREB2A,ScDREB2B-1 and ScDREB2B-2 proteins contained a complete GDDGFSLFAY motif of DREB(A-2)subgroup,but partial amino acid residues in the SLFAY were changed into the form of DVDEM in ScDREB2B-1 and ScDREB2B-2 proteins.Under PEG,NaCl and ABA treatments,ScDREB2A,ScDREB2B-1 and ScDREB2B-2 genes were basically induced and up-regulated in the roots and leaves of sugarcane ROC22 and Badila revealed by qRT-PCR.However,there were differences in the expression time and expression levels of ScDREB2A,ScDREB2B-1 and ScDREB2B-2 genes in different sugarcane genotypes and tissues after the induction of these three different stress factors.3.Preliminary biological function analysis of ScDREB genes in sugarcaneThe subcellular localization results of ScDREB2A and ScDREB2B-1 genes showed that both ScDREB2A and ScDREB2B-1 proteins were located on the nucleus and cell membrane.The yeast two hybrid selfactivation experiment suggested that ScDREB2A protein had no transcriptional activation activity,but did ScDREB2B-1 protein.Recombinant yeast carrying sugarcane ScDREB2A or ScDREB2B-1 gene was growed in YDPS culture medium containing PEG(5%,10%,15%)and NaCl(100 mM,150 mM,200 mM).The results showed that the ScDREB2A gene can enhance the tolerance to PEG and NaCl stresses in corresponding recombinant yeast cellsbut not ScDREB2B-1 gene.Furthermore,the ScDREB2A gene was constructed on the plant overexpression vector pEraleyGate 203(N-Myc)-CD3689 and transformed in N.benthamiana by Agrobacterium mediated leaf disc immersion.Eight T0 ScDREB2A transgenic plants with different oreign expression levels(including high,medium and low expression)were obtained.The T1 generation transgenic lines OE8,OE2,and OE7 with high,medium,and low expression level of ScDREB2A increased the resistance of N.benthamiana to NaCl and mannitol stresses.These results indicated that ScDREB2A may play a regulatory role in resisting high salt stress and mannitol drought stress in sugarcane.This study provides the objective gene elements and plant experiment materials for the further investigation of the functions of ScDREB2B-1 and ScDREB2A.