Isolation And Functional Analysis Of Malus Baccata(L.) Borkh MTB Transcription Factors MbMYB4and MbMYB108

In the natural environment,plants often face unfavorable factors such as drought,cold,and freezing,which have an impact on growth and yield.When plants are under adversity stresses,they will adjust internal mechanisms to deal with adverse external conditions at the physiological,biochemical and molecular levels.Therefore,the internal regulation mechanism and growth characteristics determine the distribution of plants.M.baccata(L.)Borkh.is mainly distributed in northeast of my country and Inner Mongolia,can tolerate low temperature and drought stresses.MYB transcription factor is a very critical part in the process of coping with environmental stresses.However,there are few researches on the stress resistance of MYB TF in M.baccata.which severely hinders the selection of stress-resistant varieties in M.baccata.In this study,we cloned two MYB transcription factor genes from M.baccata and studied their functions.The results are as follows:(1)We cloned two R2R3-MYB transcription factor genes: MbMYB4 and MbMYB108.The c DNA fragment of MbMYB4 contains 762 bases and can be translated into a protein encoded by253 amino acids;and the c DNA fragment of MbMYB108 contains 903 bases and can be translated into a protein encoded by 299 amino acids.Sequence alignment results and predictions of these two protein structures indicate that they both contained the conserved MYB domain.MbMYB4 has the highest homology with Md MYB4,and MbMYB108 has the highest homology with Md MYB108.(2)The subcellular localization results show that both MbMYB4 and MbMYB108 are localized in the nucleus,which is consistent with their functions as transcription factors.(3)q-PCR results showed that the expression levels of MbMYB4 and MbMYB108 genes in different parts of M.baccata were different,the parts with higher expression of MbMYB4 are in new leaves and roots;the parts with higher expression of MbMYB108 are in new leaves and old leaves.Environmental stresses such as cold,salt stress,dehydration,heat and ABA can induce the expression of MbMYB4 and MbMYB108,but these two genes are more sensitive to low temperature and drought stresses.(4)MbMYB4 and MbMYB108 were introduced into wild-type Arabidopsis and make these two genes overexpress in transgenic Arabidopsis,expose all strains including wild-type Arabidopsis(WT),unloaded Arabidopsis(UL)and transgenic plants to cold and water-deficient environments.The results showed that the morphology and survival rate of transgenic Arabidopsis were better than WT and UL.(5)Measure the physiological and biochemical indexes of all lines.Under low temperature and drought stresses,the proline and chlorophyll level and the levels of POD and CAT enzyme activities in transgenic plants are higher,while MDA content and relative conductivity have a low rise,indicating that the plasma membrane of transgenic Arabidopsis was less damaged.(6)MbMYB4 and MbMYB108 can activate At CBF1 and At CBF3 transcription factors,and positively regulate the expression of At COR15 a and At RD29 a,two key genes that response to low temperature stress.Therefore,MbMYB4 and MbMYB108 can enhance the cold tolerance of transgenic Arabidopsis through the CBF-dependent pathway.(7)MbMYB4 and MbMYB108 can induce the up-regulated expression of At NCED3(9-cis-epoxy carotenoid dioxygenase 3)and the ABA signal transduction gene At Sn RK2.4(sucrose non-glycolytic protein kinase 2.4).It shows that MbMYB4 and MbMYB108 can participate in the response of plants to drought stress through two pathways: regulating ABA synthesis and ABA signal transduction.In addition,the expression of At CAT1 and At P5 CS genes increased significantly under drought stress.This indicates that MbMYB4 and MbMYB108 can regulate the key genes of drought stress to improve the scavenging ability of reactive oxygen species(ROS),thereby enhancing the drought resistance of plants.The results of the study laid a molecular foundation for revealing the mechanism of MYB transcription factor in the process of low temperature and drought resistance in apples.At the same time,it provides new candidate genes for the cultivation of cold and drought resistant apple rootstocks.The research results will enrich the control mechanism of plant’s cold resistance and drought resistance,which is of great significance for improving apple resistance and improving breeding efficiency.