Isolation And Functional Analysis Of MbCBF1 And MbCBF2 In Malus Baccata(L.) Borkh

In this study,Malus baccata(L.)Borkh was used as a test material.The purpose was to clone two new CBF transcription factors and determine their functions.Agrobacterium transformation method was used to transform the target gene into Arabidopsis thaliana to further understand the functional characteristics of the CBF gene family;it laid a molecular foundation for studying the cold resistance mechanism of plants;in addition,it provided a molecular basis for the subsequent selection of apple rootstocks;At the same time provide new candidate genes for cold resistance breeding.Isolate the total RNA of the Malus baccata(L.)Borkh,reverse-transcribe it into singlestranded c DNA,and obtain the CBF gene of the Malus baccata(L.)Borkh by PCR amplification.The two genes were compared using DNAMAN 5.2 software and found that the two amino acid sequence homology was as high as 83.9%.These two genes were named MbCBF1 and MbCBF2,respectively.The sequencing results showed that the MbCBF1 open reading frame was 666 bp and the MbCBF2 was 663 bp.The obtained nucleotide sequence was translated using DNAMAN 5.2 software.MbCBF1 and MbCBF2 gene encode a protein consisting of 221 amino acids and 220 amino acids,respectively.The theoretical molecular weight(MW)of MbCBF1 protein was predicted to be 24.094 k Da.the theoretical isoelectric point(p I)is 5.21 and the average hydrophilic coefficient is-0.495.Among them,Ala(13.1%),Ser(10.0%),Arg(8.1%),Asp(7.7%),Pro(7.7%)accounted for a large proportion.The theoretical molecular weight(MW)of MbCBF2 protein was predicted to be 24.206 k Da.the theoretical isoelectric point(p I)is 5.18 and the average hydrophilic coefficient is-0.456,while Ala(14.5%),Ser(10.0%),Glu(7.7%),and Pro(7.3%)accounted for a large proportion in the protein sequence.Subcellular localization of MbCBF1 and MbCBF2 proteins indicated that both MbCBF1 and MbCBF2 proteins were localized in the nucleus.The phylogenetic tree of MbCBF1 and MbCBF2 genes homologous to other species was constructed using MEGA 7 software,showing that both MbCBF1 and MbCBF2 have the highest homology with Prunus dulcis Pd CBF4.The results of q-PCR showed that MbCBF1 and MbCBF2 genes had organ expression specificity in different parts of the Malus baccata(L.)Borkh.During normal Hoagland nutrient culture(0 h),MbCBF1 and MbCBF2 were expressed at all detection sites.MbCBF1 was highest expressed in new leaves;MbCBF2 was highest expressed in roots and both of MbCBF1 and MbCBF2 had lowest expression in old leaves.Under low temperature,high salt,drought and high temperature stresses,the expressions of MbCBF1 and MbCBF2 all increased first and then decreased.The expression levels of MbCBF1 in new leaves reached the peak value at 8 h of low temperature treatment,12 h of high salt treatment,5 h of drought treatment,and 5 h of high temperature treatment,respectively.In the roots,peaks were reached at 12 h under low temperature treatment,8 h at high salt treatment,2 h at drought,and 5 h at high temperature.In the new leaves,the expression levels of MbCBF2 reached the peak value at the 8h of the low temperature treatment,the 12 h of the high salt treatment,the 5 h of the drought and high temperature treatment.The expression level in roots reached the peak at 12 h of low temperature treatment,5 h of high salt treatment,2 h of drought,and 5 h of high temperature treatment.Among the four stresses,MbCBF1 and MbCBF2 are more sensitive to low temperature and high salt stress,that is,MbCBF1 and MbCBF2 are more susceptible to low temperature and high salt stress.MbCBF1 and MbCBF2 genes were overexpressed in Arabidopsis thaliana by Agrobacteriummediated method,and three transgenic lines were obtained.Wild-type Arabidopsis,unloaded Arabidopsis,and transgenic line Arabidopsis were treated with 200 m M Na Cl for 7 days,respectively.It was found that wild-type Arabidopsis and unloaded Arabidopsis thaliana showed a more significant yellowing phenomenon.The cold resistance of transgenic Arabidopsis with MbCBF1 and MbCBF2 genes was significantly higher than that of wild-type Arabidopsis(WT)and unloaded Arabidopsis(UL).Detection of physiological indicators related to plant resistance showed that during the low temperature and salt stress,the proline content,chlorophyll content and the activities of SOD,POD and CAT in all Arabidopsis transgenic lines of MbCBF1 and MbCBF2 genes were increased compared to the WT,while the MDA content reduced.These test results indicated that under low temperature and salt stress,the transgenic Arabidopsis plants with MbCBF1 and MbCBF2 genes suffered less damage indicated that their resistance to low temperature and salt stress was enhanced.In summary,both MbCBF1 and MbCBF2 genes are susceptible to low temperature and high salt stress.Overexpression of these genes can enhance the tolerance of transgenic Arabidopsis to low temperature and salt stress.