Isolation And Functional Analysis Of Malus Baccata (L.) Borkh ERF Transcription Factors MbERF11 And MbERF12

In this study,Malus baccata(L.)Borkh was used as a material to clone two novel ethylene response factors(ERFs).The ethylene response factor MdERF011-like gene sequence of the map was used to design the primers,extraction the RNA of the Malus baccata and reverse transcriptase was used to reverse transcribe and synthesize the single-stranded cDNA.The full length of the ERF gene of the Malus baccata was obtained by PCR amplification.The two genes were found to have high homology of 98.38% in their amino acid sequences by DNAMAN 5.2 software.These two genes were named MbERF11 and MbERF12.Sequencing results showed that the open reading frames of MbERF11 and MbERF12 were 483 bp in length.The nucleotide sequence of the MbERF11 and MbERF12 genes was translated into amino acid sequences by DNAMAN5.2 software.It was speculated that the MbERF11 and MbERF12 genes were responsible for encoding proteins consisting of 160 amino acids.The predicted MbERF11 protein has a theoretical molecular mass of MW=17.967 kDa,a theoretical isoelectric point pI=9.27,an average hydrophilicity coefficient of-0.959.The theoretical molecular mass of MbERF12 protein was predicted to be MW=17.918 kDa,the theoretical isoelectric point pI=9.3,and the average hydrophilicity coefficient was-0.972.Subcellular localization of MbERF11 and MbERF12 proteins showed that they are localized in the nucleus.The amino acid and nucleotide similarity analysis of the ERF genes of the MbERF11 and MbERF12 genes and other species showing that both them are related to the apple(Malus domestica)ERF011-like The highest homology.Semi-quantitative PCR results showed that MbERF11 and MbERF12 genes have organ-specific expression in different parts of the Malus baccata.In the normal Hoagland nutrient solution culture(0 h),MbERF11 and MbERF12 were expressed in all organs tested,and the abundance in the new roots and stems was higher.At low temperature,the expression levels of MbERF11 and MbERF12 genes began from the 3 rd hour of treatment to the 24 th hour.The expression of these genes in new leaves was significantly and continuously enhanced,and expression in new roots began from the 3 rd hour.The increase reached the highest point at the 12 th hour.Similarly,the expression of the them in the new leaves and new roots was increased first and then decreased in the high salt treatment.In the new leaves,the expression reached the highest at 12 h after treatment,and reached the highest in the 6 h in the new root.Real-time fluorescence quantitative PCR analysis showed that the expression of MbERF11 and MbERF12 genes in different parts of the normal Hoagland nutrient solution(0 h)was: the expression of new roots was the highest,followed by the stem,new leaves,and mature leaves.Under salt stress,low temperature,and ethephon treatment,the expression levels of them in the new leaves of Malus baccata were first increased and then decreased with the prolonged treatment time.In the salt stress treatment,the maximum value was reached at 12 h.It reached a maximum value at 24 h after low temperature,while it reached the maximum at 4 h after ethephon treatment.In the new roots of Malus baccata,the expression levels of MbERF11 and MbERF12 also increased first with increasing treatment time under salt stress,low temperature and ethephon treatments.After reducing the trend.The maximum value was reached at 8h after salt stress treatment.The maximum value was reached at the low temperature treatment for 12 h,while it reached the maximum at 2 h after the ethephon treatment.The expression levels of MbERF11 and MbERF12 genes in the new roots were increased faster than those in the new leaves,which were consistent with semi-quantitative PCR results.In this study,the Malus baccata MbERF11 and MbERF12 genes were overexpressed in Arabidopsis thaliana by Agrobacterium-mediated method.Three transgenic lines were obtained and treated with NaCI for 7 days.Compared to before treatment,the wild type showed a clear yellowing phenomenon after salt stress treatment,but the transgenic Arabidopsis performed relatively well and showed no yellowing.The resistance of Arabidopsis overexpressing MbERF11 or MbERF12 genes was identified through the identification of cold resistance.The freezing capacity was significantly higher than that of the wild type.The physiological indices related to plant resistance tested by us showed that the contents of chlorophyll and proline and the activities of SOD,POD and CAT in the transgenic lines of MbERF11 and MbERF12 were higher than those in the untreated group.Compared with control group,there was no difference of MDA content.The results showed that the plants transformed to MbERF11 and MbERF12 got less damaged after low temperature and salt treatment,and the resistance to low temperature and salt stress was significantly enhanced.In summary,over-expression of MbERF11 and MbERF12 genes in Arabidopsis thaliana can improve the tolerance of transgenic Arabidopsis thaliana to low temperature and salt stress.