Cloning And Functional Analysis Of MdWRKY33 Gene In Apple

Apple is a perennial woody plant and it is one of the most common fruit trees cultivated.Various environmental stress factors affect the apple industry,resulting in a decrease in apple production.Therefore,one of the main goals of current apple breeding is to cultivate varieties containing comprehensive resistance.Through transcriptome sequencing analysis,it was found that the expression level of Md WRKY33 gene,which may be related to stress resistance,was significantly increased in homologous tetraploid materials with better comprehensive stress resistance compared with common diploid apple.Therefore,the full length of the MdWRKY33 gene was cloned and cloned in 'Hanfu' apple for the first time.Overexpression vectors and silencing vectors were constructed.Agrobacterium-mediated transformation of apple 'GL-3',transgenic plants were obtained,and the function of MdWRKY33 gene was verified by stress treatment,which provided a theoretical reference for apple disease resistance breeding research.The main findings of this experiment are as follows:1.For the first time,the CDS sequence of MdWRKY33 gene was cloned from the 'Hanfu' apple.The full-length CDS sequence was 1716 bp.Contains two WRKY domains and belongs to the WRKY transcription factor family of class I.2.Phylogenetic analysis revealed the highest homology with white pear in Rosaceae.Subcellular localization of the Md WRKY33 gene showed that the MdWRKY33 gene was located in the nucleus.3.The overexpression vectors and silencing vectors of the apple Md WRKY33 gene were constructed.Apple ‘GL-3' was transformed by Agrobacterium-mediated method and 5 overexpressing lines and 3 silencing lines were obtained.4.Stress treatment of overexpressed and silenced plants with NaCl and SA respectively showed that Md WRKY33 overexpressed plants showed strong resistance to silencing plants and controls,indicating that overexpression of MdWRKY33 showed resistance to salt stress.Overexpression of Md WRKY33 was enhanced under SA simulated stress conditions.5.The over-expressed plants and the silenced plants were inoculated with Alternaria graminis.It was found that the plants over-expressing Md WRKY33 were less susceptible than the control plants,while the silenced plants were more severe than the control plants,indicating that Md WRKY33 had significant disease-resistant functions.