Molecular Cloning, Structure Analysis, And Function Identification Of CBF Transcription Factor In Cold Acclimation From Fragaria×Ananassa

Strawberry production is the biggest planting area at present.The temperature is warm in south,the overwintering safeguard of strawberry is not severe and the low temperature reversion phenomena occurred so that strawberry would suffer little low temperature injury contingently during planting and great loss was peoduced.CBF(CRT/DRE binding factor, CBF) is a type of transcription factors related to abiotic stresses.These proteins are able to discern and bind the CRT/DRE element in the promoter of COR gene concerned with cold, resulting in enhancing the cold-resistance of plants.Therefore,it would be a good strategy to improve tolerance of various kinds of agriculturally important plants to abiotic stresses by CBF genes transfer.In this present studies,a pair of degenerate primers which was designed by comparing CBF genes from GenBank,and the conserved region with a length of 464 bp of the transcription factor CBF gene by touch down PCR from Fragaria×ananassa.Then,a full length of open reading frame of CBF gene was obtained by SON-PCR and RT-PCR.The cDNA was 794 bp in length and a real CBF gene of strawberry designed as Facbfl with an accession number FJ767754 in GenBank.The cloned cDNA contains an ORF of 636 bp, which encodes for a polypeptide of 211 amino acid residues with a molecular mass of 23.4 kDa and a pI of 6.53.FaCBF1 sequence contains a functional domain:AP2 binding domain, on the both side of which there exit two short peptides:PKK/RPAGRxKFxETRHP and DSAWRL.Semi-quantitative RT-PCR is used to analysis the expression of Facbfl gene under different time of treatment.The results showed that without a cold treatment there was no expression of Facbfl genes.But after the cold treatment,the level of Facbfl transcription was rapid increased in a short period of time and reached a peak at 12h and may last up to 24h and then declined.Protein structure was predicted with different methods.The results of primary structure analysis showed that FaCBF1 was soluble and hydrophobic and belonged to CBF family.Secondary structure analysis indicated that FaCBF1 contains conserved Alpha helix and random coils domain.3D structure of protein coding by the gene was predicted using the ESyPred3D software.It contains oneα-helices and threeβ-sheets structural domain and looks like the GCC-box binding protein.Function prediction was carried out in net server. The results showed that FaCBF1 was a member of CBF family,containing AP2-binding domain with some phyosphorylation and O-glycosylation sites,and it had no signal and trasmembrane domain.Electronic expression profiles revealed that FaCBF1 expressed in whole plants and during different development,and involved in different abiotic stresses. All of the results suggest that FaCBF1 played an important role in the growth a development of plants.Cold acclimation can enhance the ability of cold-resistance,but if the temperature is too low or too long it will cause the death of plants.Under different times of lowtemperature treatment,the target of a variety of physiological tests showed that the lowtemperature environment of strawberry seedlings must have the ability to adapt to the cold, that is,to some extent,improve the plant's cold-resistance,deal with one of 0℃can significantly improve plant adaptation to low temperature.Therefore,an appropriate lowtemperature treatment can be induced to enhance the cold-resistance of strawberry.This may be when the outside temperatures drop,plants feel low-temperature signals,and transmission of low-temperature signals to the nucleus to activate transcription factors, thereby regulating gene transcription or give rise to a series of physiological and biochemical reactions,such as antioxidant enzymes(SOD,CAT,APX and GR) activities and increased the contents of proline,soluble protein and soluble sugar,as well as chlorophyll and carotenoid content decreased,the adaptive changes of these substances laid the foundation of enhancing the cold-resistance of plants.It can be seen that lowtemperature induced the physiological and metabolic changes of plants in order to adapt to low temperature.This is an active physiological and biochemical process,which improve the cold-resistance of plants.These obtained results and conclusions provided important information not only for the physiology and molecular biological research of cold-resistance in horticulture plants but also for the genetic improvement of cold-resistance in agriculture and horticulture plants.