Molecular Cloning And Functional Analysis Of Transcription Factors Of CpICE1a, CpICE1b And CpDREB1 About Cold Resistance From Chimonanthus Praecox L.

Wintersweet(Chimonanthus praecox L.)belongs to Calycanthaceae,which is known as " cold tolerance,drought and shear resistance ",Its flowering period is from November to February of next year,It even blossoms in snow.In theory,the flower is the weakest part of the plant against the outside of the stress,but the flower of wintersweet can open in the winter with a low temperature,it is speculated that there are some special factors to regulate the tolerance of cold stress.At present,the research of the CBF signal transduction pathway is clearer than others.The genes of CBF/DREB1 are located in the crucial position in the CBF pathway.Their expression is affected by various transcription factors.ICE1 is the upstream transcription factor in the CBF signaling pathway.It can regulate the expression of downstream genes by binding to the CBF promoter region.At present,the research on the cold resistance of wintersweet is mainly focused on the functional genes.The study of these two types of transcription factors is important to find out the mechanism of cold resistance in wintersweet based on the pivotal role of ICE1 and CBF / DREB1 genes in the CBF signaling pathway.Based on the previous detection of two ICE transcription factors: CpICE1 a and CpICE1 b.First,the CpICE1 a and CpICE1 b gene and one DREB1 gene: CpDREB1 which have complete domain are screened out and cloned.Then,the expression of CpICE1 a,CpICE1b and CpDREB1 were detected by using the real-time PCR technique in different organs,flowering periods and low temperature stress.Furthermore the genes were inserted into pCAMBIA1302 to construct the plant binary expression vector,and the construct vector was transformed into the model plant tabacco mediated by Agrobacterium the transgenic plants were obtained.Finally,the molecular mechanism of CpICE1 a,CpICE1b and CpDREB1 overexpression increased freezing stress tolerance in transgenic tobacco were studied and analyzed.The main results are as follows.(1)The ORF and full length sequence of CpICE1 a,CpICE1b and CpDREB1 wer cloned.By using bioinformatics analysis method,We found that: CpDREB1 gene has no intron.The coding regions of CpICE1 a and CpICE1 b genes included two introns and three exons,the structures of CpICE1 a and CpICE1 b genes were different from the three introns and four exons of the ICE1 genes in other species included Arabidopsis thaliana,Malus domestica,Prunus mumes,and Vitis amurensis.They had the specificity of species.(2)431bp promoter fragment before the initial codon is attained in CpDREB1.there was a MYC binding site(CATTTG)and some Hormone induction regulatorysites such as methyl jasmonate(MeJA),gibberellin(GA)and salicylic acid(SA).(3)Real-time PCR technology was applied to detect CpICE1 a,CpICE1b and CpDREB1 gene expression in different organs,flowering periods and low temperature stress of H29.The results showed that CpICE1 a,CpICE1b and CpDREB1 were expressed in different stages of stem,leaf,flower,fruit and flower development of wintersweet.The three genes also could respond to low temperature stress,but the expression patterns of two types of genes are different.It is speculated that the wintersweet may have feedback mechanism in order to adapt to the low temperature environment.(4)Construction of CpICE1 a,CpICE1b and CpDREB1 expression vector and vectors of pCAMBIA1302 were transferred into tobacco.The number of transgenic tobacco which have the CpICE1 a,CpICE1b,CpDREB1 and pCAMBIA1302 are 47,47,44,36 plants.The transgenic plants and wild type have no difference from the phenotype,In the discovery of function of genes,First,the wild type and transgenic plants whose growth state almost were treated with 4 degrees for 24 h,the wild type of loss water was more serious than that of transgenic plants,we choosed the samples of transgenic plants and wild type which are treated for 3 hours with 4 degrees to measure the contents of Malondialdehyde and analysis of changes of related genes.The results showed that the content of MDA in some transgenic plants was smaller than that in wild type and the cold resistance of some transgenic plants was improved,Combined with physiological indicators and gene expression,we found that the MDA content of content is smaller,the expression level of target genes is higer,The results of these two analytical methods are relatively consistent.By analyzing the expression of target genes and the genes of related cold stress: NtLEA5 and NtERD10 D in wild-type and transgenic tobacco which before and after treatment,The main results are as follows: Under normal growth conditions,the expression of NtLEA5 was significantly increased in transgenic plants of CpICE1 a and CpICE1 b,The expression of NtERD10 D has no change.In the transgenic plants of CpDREB1,Even if the expression of CpDREB1 was higher,the expression of these two genes has no obvious change.So,we indicat that the introduction of CpICE1 a and CpICE1 b can increase the resistance to stress in tobacco.The CpDREB1 gene may have no effect to the expression of NtLEA5 and NtERD10 D in the normal growth conditions In low temperature,the expression of target gene and NtERD10 D increased in the transgenic plants of CpICE1 a,the expression of these two genes in the transgenic plant of CpDREB1 is still same with the before treatment.The low temperature improved the expression of NtLEA5 both in wild and transgenic.Because the expression of NtLEA5 is too high,we can not see the range of change by using the method of semi-quantitative and know the function of CpDREB1 in the tolerance to cold stress.It needs more experiment to test.