| Abiotic stresses,such as cold and high salinity,are common adverse environmental conditions that can badly limit plant growth,development,and crop production.Recently,many stress-related MYB TF genes in A.thaliana and other species have been successively identified and characterized.However,very little is known of their functions in apple.Apple is a perennial woody plant,which has wide cultivated area.It is very important for the genetic improvement of apple rootstocks and varieties and the cultivation of resistant varieties in the current apple breeding.In the case of apple genetic transformation,the genetic transformation system has been established for the apple variety and apple rootstocks.However,low efficiency and poor reproducibility are not conducive to the acquisition of a large number of apple transformation materials and the follow-up gene function research.High regeneration is the prerequisite for high genetic transformation,and the genotype of apple materials plays a vital important role in regeneration of adventitious buds and genetic transformation of Agrobacterium-mediated.Based on the comparative study on the regeneration ability of 153 apple germplasm resources,in this study,Malus sieversii 31 was chosen for establishment of a high efficiency regeneration and genetic transformation,which provides a theoretical basis for the subsequent apple transgenic research and the functional analysis.In this study,the effective factors of in vitro regeneration and genetic transformation were investigated,and a high efficiency genetic transformation system was established.The results indicated that the regeneration frequency can reach the highest,when the explants were placed for 14 days dark culture with the adaxial contacting medium.1.0 mg·L-1 6-BA and 0.1 mg·L-1 NAA were the most suitable hormone ratio for subculture,and 1/2 MS containing 1.0 mg·L-1 IBA was the optimal medium for rooting.When the explants were immersed with the bacterial(OD600=0.5)for 5 min,they were placed onto the regeneration medium for co-culture,2 d later,they were put onto the regeneration medium with 300 mg·L-1 cefotaxime for 2-4 d.then they were placed onto the regeneration medium with 300 mg·L-1 cefotaxime and 1.5 mg·L-1 hygromycin for selection of resistant buds.Under this condition,the genetic transformation rate can reach up to about 5%.The full-length MdMYB4/44 sequence was obtained from the Apple Genome Database,and leaves from the 'Golden Delicious' shoot culture materials were used for amplification of MdMYB4/44 gene.Cloning and sequencing indicated that the full-length MdMYB4 cDNA was 495 bp long,and further analyses predicted that the gene can encode a protein with 164 amino acid,while the full-length MdMYB44 cDNA was 729 bp long,and this gene can encode a certain protein with 242 amino acid.In relation to other R2R3-MYB TFs in A.thaliana,phylogenetic analysis indicated that MdMYB4,a R2R3 MYB protein that is a homolog of Arabidopsis MYB4,and MdMYB44 was highly homologous with Arabidopsis AtMYB44.In addition,MdMYB4/44 was localized to the nucleus of transformed onion epidermal cells and had a certain transcriptional activation activity by yeast one-hybrid assay.Expression analysis using qRT-PCR showed that MdMYB4/44 was extensively expressed in various apple tissues,among these tissues,the leaf and stem generated the lowest and highest level of MdMYB4 transcripts respectively.Besides,the expression level of MdMYB4 gene can be up-regulated by cold,osmotic,and salt stresses,and over-expression of this gene remarkably enhanced the tolerance of stably transgenic apple calli to high salinity and cold stress.These results suggest that MdMYB4 plays a positive regulatory role in cold and salt stress response.Expression analysis also indicated that MdMYB44 expressed in all tested tissues,among these tissues,the leaf generated the highest level of MdMYB44 transcripts,and quantitative real-time PCR(qPCR)analysis showed that the MdMYB44 expression could be down-regulated by cold,osmotic,and salt stresses.Furthermore,over-expression of the MdMYB44 gene could remarkably alleviate the tolerance of stably transgenic apple calli to high salinity and cold stresses.Therefore,our findings provide evidence that MdMYB44 is involved in tolerance to cold and salt stress in apple calli and this gene act as a negative regulator in plant abiotic stress defense.In this study,the content of malic acid in transgenic and wide type apple calli WT was detected by the method of HPLC.The results showed that there was no significant difference of malic acid content in MdMYB4-transgenic and non-transformed calli.However,the content of malic acid in MdMYB44-transgenic calli was significantly increased,which indicated that over-expression of MdMYB44 gene promoted the accumulation of malic acid.Meanwhile,the expression of genes related to malic acid metabolism was analyzed by qRT-PCR,and the results showed that the expression level of M-PPi gene in MdMYB44-transgenic calli was significantly higher than that of wild-type WT.However,the expression levels of M-ME,MVA-A,CyMDH and PEPC were not significant different.The results indicated that the over-expression of MdMYB44 gene may promote the transportation and accumulation of malic acid in the vacuoles mainly through the up-regulated expression of M-PPi gene. |
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