Molecular Cloning And Primary Functional Analysis On AlDREB2 And AlTINY From Aloe Vera L. Var. Chinensis (Haw.) Berger

Aloe vera L.var.chinensis(Haw.) Berger,one of perennial succulent plant of Liliaceae and the genus Aloe,considered as a variant of Aloe Vera L.,origins from the stone desert and desert of African continent,and belongs to Crassulaceae,Most of the volumn of mature leave are storage parenchyma,and exremely water,heat,drought resistance characteristics.Because of containing the rich bioactive substances in its leave,Aloe has been used on the industry of pharmaceutical and chemical widely,research mainly concentrated in this area and reached a higher level.But the studies of Aloe resistance focused on the aloe leaf morphology,and were less from molecular level.On the other hand,Aloe is classical xerophyteo.Previous researches have also showed that Aloe have ability of resistance to different stresses,such as high salt,pests and diseases.Plant resistance research are always hot point,but mainly focus on the important crops and model plants.There are a lot of plants that have strong resistance in the nature,which made them accumulate rich resistance genes on the long process of evolution.Aloe can resist to various stresses,and have been studied less on the resistance.The resistance research of Aloe have been from the physical level,but hardly from the molecular level.In this paper,it was reported that two stress-related genes had been cloned from Aloe,named as AlDREB2 and AlTINY,which belongs to DREB transcription factor family.From the various plants the many DREB-like genes had been isolated,and verified that they played an important role in the resistance to environmental stress.The expression of AlDREB2 and AlTINY was analyzed through the different treatment with Aloe plants.1.The cloning of AlDREB2 and AlTINYUsing the 3'RACE method,the genes expressed were obtained by cold and dehydration treatment. Lastly,the two sequences had been isolated through analysis,the 5'end of which was gained using the PCR-walking.After splicing,two genes that had complete coding sequence were got.Two same fragments were gained by amplifying the template of DNA and cDNA,which indicated that AlDREB2 and AlTINY gene have no intron.2.Bioinformatic analysis of AlDREB2 and AlTINYAlDREB2,636bp ORF frame,encoded 211 amino acids.AlTINY,552bp ORF frame,encoded 183 amino acids.The BLAST alignment in the NCBI showed that AlDREB2 had the highest identity to Aloe.L DREB1,AlTINY had the highest identity to Arabidopsis thaliana TINY2.Typical AP2-binding domain and conversed amino sites binding DRE-cis element of DREB family,were observed.Phylogenetic tree analysis showed that AlDREB2 belonged to the 6^th subgroup and AlTINY belonged to the 4^th subgroup, both of them had no signal peptied.Subcellular localization predicted that AlDREB2 most likely located in the mitochondrial matrix and AlTINY most likely located in the nucleus.Three dimensional structure prediction illustrated that AlDREB2 and AlTINY can form typical DREB-like transcription factor structure.3.Expression analysis of AlDREB2 and AlTINY geneAfter the treatment with ABA,dehydration,cold,high-salt and ethephon,in Aloe the expression of AlDREB2 and AlTINY gene were analyzed.The results showed that although two genes belonged to the same gene family,the expression changes were different.In addition,it was found that they were induced by ethephon and had high-level expression,which was different from the other reported genes of DREB family.This may be able to explain that Aloe had not only strong resistance to environmental stress,but also be able to resist pests and diseases,such as abiotic stress.