Cloning And Functional Analysis Of Transcription Factor Involved In Abiotic Stress In Malus Robusta Rehd And Oryza Sativa L.

Plants are constantly challenged by various biotic and abiotic stresses such as drought,high salinity,low temperature and disease in their natural environment,which cause adverse effects on the growth of plants and serious reductions in the quantity and quality of agriculturally important crops.Encountering environmental stress,many signal transduction pathways are activated and plenty of genes involved in abiotic stress response are induced to express in plants.In the process of environmental stress response,transcription factors play important role.Till now,a number of transcription factor families have been found involving in plant stress responses,and each contain a different type of DNA-binding domain,including AP2/EREBP,Myb,WRKY,and bZIP and so on.In this study,we isolated five novel transcription factors,MrDBP1 form Malus robusta Rehd.,RCBF2,OsHSF6,OsHSF7,OsHSF12 from Oryza Sativa L, which belonged to AP2/EREBP and HSF,respectively.In this study,a novel AP2/EREBP type transcription factor,MrDBP1,was isolated using rapid amplification of cDNA ends(RACE) method.The full-length cDNA of MrDBP1 has a 483bp open reading frame(ORF) and encode a DREB subgroup transcription factor consisting in 160 amino acids.The result of Southern blot showed MrDBP1gene might be has several copies or high level of homologous genes in Malus robusta Rehd.genome.Subcellular location of MrDBP1 showed that the transcription factor MrDBP1 had obvious ability of nucleus location.The result of RT-PCR indicated that the expression of MrDBP1gene was induced by drought stress,but not low-temperature and high salinity and the transcript level of MrDBP1gene was the highest in Malus robusta Rehd.seeds.In our study,a transcription factor RCBF2 which interacts with C-repeat/DRE was isolated from Oryza sativa L by yeast one-hybrid method.Analysis of the deduced RCBF2 amino acid sequence revealed that RCBF2 contained a conserved EREBP/AP2 domain of 59aa and a potential nuclear localization sequence.The result of rice semi-quantitative RT-PCR(s-Q RT-PCR) indicated the expression of RCBF2 gene was induced by cold,dehydration and high-salinity,but not by ABA,and the transcription of RCBF2 gene accumulated primarily in rice immature seeds,growing point and shoots. The expression of OsHSF6 and OsHSF7 gene was induced immediately and largely by high temperature,and the mRNA transcript of OsHSF7 was found to be most abundant in leaf.This indicated that OsHSF7 might involve in heat stress reception and response. Over-expression of OsHSF7 in transgenic Arabidopsis could not induced over-expression of most target heat stress-inducible genes of HSFs.However,after two hours heat stress treatment,the transcript of some HSF target genes containing HSE in their promoter regions were more abundant in transgenic plants than in wild type, meanwhile,those transgenic plants also revealed higher tolerance to heat stress. Collectively,our results indicated that OsHSF7 might play an important role in responding high temperature and be potentially useful for producing transgenic monocots,which were induced to over-express some protective genes,such as HSP, GolS,under high temperature stress,and resulted in the tolerance to heat.In addition,the function of homologous genes of DREB1B/CBF1 was analyzed and compared in Malus robusta Rehd.,rice and Arabidopsis.The results suggested the function of three genes was similar.However,there were some differences the function of three genes in phenotype and expression of downstream genes.Three types of transgenic plants all presented retardant phenomenon at different extent,respectively. Under long light condition,D35S::MrCBF1 plants had still not flowered after six months development,D35S::OsDREB1A plants started to flower after four months development,while D35S::AtCBF1 plants and wild type plants had finished a life cycle in four or three months,respectively.Furthermore,as the ability about induction of expression of down stream genes as concerned,the level of expression of COR15A, RD29A and KINI genes in D35S::MdCBF plants was higher than that in D35S::AtCBF1 and D35S::OsDREB1A plants.