Cloning And Functional Analysis Of SbDREB2Gene In Sorghum (Sorghum Bicolor)

Drought, salinity, low-temperature and other abiotic adverse stress, impactingon plant growth development significantly, under extreme conditions even leading toplant death, are important factors in reduction of crop production. Higher plants formmany complex defense mechanism in the long-term evolutionary process in order toresist these stresses. The DREB protein is a part of plant-specific family oftranscription factors binding specifically with the dehydration responsive element.DREBs play important roles in regulating the expression of downstream genes inresponse to a variety of abiotic stresses and can improve the content of proling andsucrose. Therefore, in plant genetics and breeding, the DREB kinds transcriptionalfactor to be able to synthesize the improvement plant the resistance character, is untilnow the more ideal resistance gene.In this study, sorghum dealt with the salinity was selected as experimentmaterial. Using silico cloning and RT-PCR technology, we obtained a cDNAsequence of Sorghum bicolor DREB transcription factor. The expression patternanalysis under different adversity was carried out by quantitative real-time PCR.Prokaryotic Expression was carried out and plant expression vector was constructedsuccessfully. The results are as follows:1. According to published EST sequences in GenBank of Sorghum bicolor DREBtranscription factor, primers OF and OR were designed and a cDNA sequence with1152bp in length is amplified by RT-PCR. The genome DNA was amplified with thesame primers. It was found that there is a740bp intron in the DNA sequence of thegene. SbDREB2contains a complete open reading frame (ORF)789bp whichencodes a peptide of262amino acids. The amino acids analysis indicated that thepredicted protein sequence contained a typical AP2DNA-binding domain in the82~145region. Multiple sequences alignment revealed that the SbDREB2encodingproduct showed84%and69%sequence similarities with Zea mays DREB2A andOryza sativa DREB1, respectively.2. Using quantitative real-time PCR analysis of expression modes showed thatSbDREB2gene was constitutive expression. We found that the expression of SbDREB2was in roots, stems, leaves and expression level in roots was about2.5times than in stems. The gene was strongly up-regulated by treatments with highsalinity, dehydration and exogenous application of abscisic acid (ABA). However,the expression of SbDREB2was not affected by low temperature.3.The specific primer DF and DR, containing EcoR I and Xho I restriction sites,were designed. Plasmid containing SbDREB2gene and pET28a vector were digestedwith EcoR I and XhoI, respectively. The T4DNA Ligase was used to be ligated.Prokaryotic expression vector pET28a-SbDREB2was established and transformedinto E. coli competent cell. The positive clone was selected and sequenced. AfterIPTG induction, the fusion protein relative molecular weight was32.5kD, whichwas consistent with theoretical value.4.The specific primer PF and PR，containing BglⅡand Nco I restriction sites, weredesigned. The target DNA containing SbDREB2and pCMABIA3301vector weredigested with BglⅡand Nco I, respectively. The T4DNA Ligase was used to beligated. Plant expression vector pCAMBIA3301-SbDREB2was established.5.The plant expression vector pCAMBIA3301-SbDREB2was transformed intoAgrobacterium EHA105using freeze-thaw method. The PCR amplification specificprimers was carried out in transformed bacteria. Expression vector was confirmed totransfer into the Agrobacterium.Then, the plant expression vector was transformedinto tobacco and obtained transgenic palnt.