| Gibberellins (GA) play important roles in several aspects of plant growth and development. SLENDER RICE 1 (SLR1) is a gene involved in regulating plant height. It was previously cloned from rice and homologues were identified from maize, sorghum and wheat. In previous studies, it was shown that SLR1 might be a transcriptional regulator that acts as a repressor of the gibberellin signaling pathway. SLR1 protein acts by participating in a large multiprotein complex that represses transcription of GA-inducible genes. Upon GA application, SLR1 is degraded by the proteasome, allowing the GA signaling pathway. In contrast, when this gene was overexpressed, it prevented the GA signaling pathway and induced a dwarf phenotype. In this study, we identified a functional SLR1 gene from switchgrass (Panicum virgatum), which is thought to contribute to the plant height of switchgrass. The PvSLR1 gene from switchgrass was successfully cloned based on sequence information from other well studied species including rice, maize, sorghum and wheat. A 1,540-bp SLR1 fragment was amplified by PCR from the switchgrass cDNA by LA Taq and was sequenced. Based on this sequence, a 2,458-bp SLR1 cDNA, containing 1,863-bp ORF was isolated by 5&feet;- RACE and 3&feet;- RACE. To investigate the role of PvSLR1 as a proposed negative regulator that represses GA response, the overexpression contruct of PvSLR1 was made. The PvSLR1 cDNA corresponding to full length was amplified by specific primers and cloned into the KpnI and SpeI site of the plant binary vector pTCK303. The pTCK303-PvSLR1 construct was transferred into Agrobacterium tumefaciens strain AGL1 and six independent transgenic plants were regenerated in the model grass Brachypodium distachyon genotype Bd21-3. Although the transgenic plants died before reaching to maturity and we are unable to conclude about the effect of PvSLR1 on plant height, we have successfully cloned the gene from switchgrass, made overexpression construct, and transformed it into Brachypodium distachyon using Agrobacterium-mediated transformation. In the process we have mastered the techniques of recombinant DNA construction and optimized the Brachypodium tissue culture. The construct is now available both in E.coli and in Agrobacterium, which the next student can use to transform Brachypodium and switchgrass to evaluate the effect of PvSLR1 and manipulate plant height for biomass feedstock improvement. It is anticipated that if PvSLR1 is a functional orthologue of rice SLR1 and indeed acts as negative regulator of GA response, overexpression of PvSLR1 in both Brachypodium and switchgrass will cause a dwarf phenotype and silencing of PvSLR1 in switchgrass will produce a tall plant, which would improve total biomass yield in switchgrass for lignocellulosic biofuel production. |
