Isolation And Functional Analysis Of Malus Baccata(L.) Borkh NAC Transcription Factors MbNAC25 And MbNAC29

In this study,two new NAC stress response factors were successfully cloned from the Malus baccata?L.?Borkh.According to the homology of NAC genes with other plants,these two response factors were named MbNAC25 and MbNAC29,respectively.It was found by sequencing that the open reading frames of MbNAC25 and MbNAC29 had the same length of1,122 bp.After translating the gene sequences of MbNAC25 and MbNAC29,it was found that the MbNAC25 and MbNAC29 genes were responsible for encoding 373 amino acids,both of which contain a NAC domain.The theoretical molecular masses of proteins MbNAC25 and MbNAC29 were 41.488 k Da and 41.985 k Da;the theoretical isoelectric points p I were 8.70 and9.10;the average hydrophilic coefficients were-0.684 and-0.579,respectively.It shows that both proteins are hydrophilic proteins.The results of subcell localization showed that both MbNAC25 and MbNAC29 protein are located in the nucleus.The systematic evolution trees were built for the construction of MbNAC25 and MbNAC29 genes and homologous genes in other species by MEGA 5.0 software.It shows that both MbNAC25 and MbNAC29 have the highest homology with Malus domestica.The results of semi-quantitative RT-PCR showed that the expression of MbNAC25 and MbNAC29 genes was specific in different parts of the Malus baccata.MbNAC25 was expressed higher in new leaves and stems,and MbNAC29 was expressed higher in new and old leaves in the normal Hoagland nutrient solution culture?0 h?.In the stress of adversity,the expression of the MbNAC25 and MbNAC29 genes in the new leaves and roots showed a tendency of first enhancement and later weakening.At the treatment of low temperature,the expression of the two genes reached the highest at 3 h in the new leaves and at 12 h in the roots;In salt treatment,the MbNAC25 and MbNAC29 genes reached their highest levels at 12 h in new leaves and at 6 h in roots;Drought treatment of MbNAC25 and MbNAC29 peaked at 24 h in the new leaves and peaked at 12 h in the roots;In high-temperature treatment,the two genes reached the highest at12 h in the new leaf and at 24 h in the roots.The results of real-time PCR showed that the MbNAC25 gene had the highest expression in new leaves in normal Hoagland nutrient culture?0 h?,followed by stems,old leaves,and roots;And the expression of the MbNAC29 gene ranged from high to low,new leaves,roots,old leaves,and stems.In the treatment of low temperature?4??,salt stress(200 mmol/L^–1 Na Cl),drought and high temperature?38??,the expression of MbNAC25 and MbNAC29 genes in new leaves showed a trend of first enhancement and then weakening.In low temperature,high salt and drought treatment,the expression of MbNAC25 and MbNAC29 genes reached a maximum in 4 h,12 h and 24 h,respectively.In high-temperature treatment,the two genes peaked at 24 h and 12 h,respectively.In the roots,the expression of MbNAC25 and MbNAC29 genes also showed a tendency of first enhancement and then weakening.In low temperature,high salt,drought and high temperature treatments,the expression of MbNAC25 and MbNAC29 genes reached maximum at 12 h,6 h,12 h and 24 h,respectively.MbNAC25 and MbNAC29 gene superexpression vector were constructed,and the wild Arabidopsis was transformed through the agarum mediated method to obtain MbNAC25 and MbNAC29 transgenic plants.After low-temperature stress identification?-4??,there was a significant wilt phenomenon in wild Arabidopsis,while the MbNAC25 and MbNAC29 gene Arabidopsis was normal.Under salt stress,wild-type Arabidopsis showed obvious yellowing phenomena,while transgenic Arabidopsis showed no obvious yellowing phenomena.MbNAC25 and MbNAC29 genes improved the salt-alkali resistance of Arabidopsis thaliana.In addition to MDA,other physiological indicators such as chlorophyll content,proline content,SOD,POD and CAT activities of transgenic Arabidopsis plants with MbNAC25 and MbNAC29 genes under low temperature and salt stress were significantly higher than those of wild Arabidopsis thaliana.In conclusion,overexpression of MbNAC25 and MbNAC29 genes can significantly enhance the cold and salt tolerance of Arabidopsis thaliana.