Molecular Cloning And Expression Analysis Of Transcriptional Activator Factor ScCBF1 Gene From Sugarcane

Abiotic stress,such as low temperature,drought and high salt,is one of the important limiting factors affecting plant growth and development.It can cause plant death and do great harm to agricultural production in severe cases.The low temperature may cause the mechanical damage on plant cells,and cause lipid phase transition of the cell membrane.Drought and salt stress belong to osmotic stress.Draught can led to cell water loss,even cause death when serious.Salt injury can suppression plant growth,decrease photosynthesis and accumulate active oxygen and other adverse effects.Studying the response mechanism of plant to abiotic stress and improving the resistance to stress always the research hotspot in plant science.A low temperature and dehydration responsed pathway,which dominated by CBF combination factor(C-repeat/dehydration-responsive element binding factor),exists in plant.This pathway the core of the genetic regulatory net,work in plant responsing to low temperature.CBFs transcription factor is a kind of regulating factor,which specificly exist in plants,and play an important role in abiotic stress such as drought or low temperature.The CBFs can specifically identify the DRE/CRT(dehydration responsive element/C-repeat)cis elements and combine with DRE/CRT cis element,thus to activating the expression of a series of downstream genes involving in the response to drought,high salt or low temperature and so on.Therefore,transcription factors play an important role in plant growth,development,signal transduction and gene expression regulation.Sugarcane(Saccharum ofcinarum)is the primary sugar and energy crops in the world.Therefore,the study of sugarcane variety of abiotic stress response mechanism to improve the resistance of sugarcane for screening foster resilience sugarcane varieties and materials have important theoretical significance and application value.In China,85%sugarcane planting area surfers from drought.Drought is one of the key factors limiting the sugarcane production in China.Diseases and insect also threaten the sugarcane production because of culture without rotation.In recent years,disastrous weather,such as low temperature,happened frequently and threaten the sugarcane industry in China.Therefore,investigation in the mechanism of sugarcane plant responding to the biotic and abiotic stresses will benefit the selection or breeding of resistant sugarcane germplasm or cultivars.It is theoretically significance and has potential application value.In this study,we cloned the whole cDNA sequence of the ScCBF1 gene from sugarcane base on the homologous cloning and predicted the structure and function of the ScCBF1 protein through the bioinformatics.Meanwhile,the expression pattern of the ScCBF1 gene in different tissues and under different stress treatments was studied by quantificational real-time PCR.Then we transient expressed the ScCBF1 gene in Nicotiana benthamiana by agrobacterium infection to identify the function of ScCBF11 gene.This study will provide important information about the biological function and role of CBF1 gene under stresses.Main results and conclusions are as follows:1.cDNA cloning and sequence analysis of CBF1 gene from sugarcaneIn this paper,a new CBF-like gene,designed as ScCBF1,was cloned by bioinformatics and RT-PCR method from sugarcane.Sequence analysis showed that ScCBF1 contained a 603 bp open reading frame(ORF)and encoded a deduced protein of 200 amino acids.Its molecular weight and isoelectric point were predicted as 22.80 kD and 10.31,respectively.The amino acid sequence alignment results showed that ScCBF1 shared the similarity of 96%and 94%with the CBF1 protein from Sorghum bicolor and Zea mays,respectively.Phylogenetic analysis revealed that ScCBF1 had closer relationship with that of Sorghum bicolor.2.The Expression patterns of ScCBFl in sugarcaneqRT-PCR showed that ScCBF1 expressed in root,stem and leaf in sugarcane with the highest expression level in roots.ScCBF1 gene was induced by low temperature,drought or abscisic acid(ABA),but was down regulated under high salinity.3.The expression of ScCBF1 in prokaryotic expression systemThe prokaryotic expression vector of pGEX-6P-1-ScCBFl was constructed successfully and the recombinant protein 44.80 kDa was expressed in E.coli Rosetta(DE3)cells under IPTG induction.The growth curves of E.coli containing the recombinant plasmids pGEX-6P-1-SCCBF1 and pGEX-6P-1,respectively,showed not significantly different.4.The transient espression of ScCBF1 into Nicotiana benthamianaThe over expression vector of pCAMBIA-1301-ScCBF1 was constructed successfully.And then,this plasmid carrying ScCBFl gene were transformed into Nicotiana benthamiana by Agrobacterium-mediated method.The phenotype of TO generation tobacco plants were investigated.It provides the basis for the further functional identification of the target genes in plants.In conclusion,this study gave the preliminary identification of the function and expression patterns of the ScCBF1 gene and may benefit the selection or breeding of sugarcane germplasm or cultivar charactered as stress tolerance or resistance.