Subcellular Localization, The Functional Identification Of Its Promoters And Expression Of Maize Transcription Factor ZmMYB59 In Tobacco

The seed germination can be directly related to the harvest of agricultural production. The study of seed germination characteristics is of great significance to promote agricultural production. In order to study the function of MYB transcription factor gene ZmMYB59 in seed germination, the recombinant plasmid pCAMBIA3301-ZmMYB59-Bar was transformed to tobacco and the expression of ZmMYB59 gene in different sowing depth and during germination was analyzed in our previous study. In this paper,the vectors of ZmMYB59 were constructed and transformed into callus of rice (Oryza sativa L. japonica.cv. Nipponbare), and then the hygromycin resistant transgenic lines were harvested. The expression profiles and function of ZmMYB59, and activity, and the key cis-elements and the germination of ZmMYB59, SOD. POD, CAT, APX activity and MDA. Pro content under abiotic stress were analyzed.The results are as follows:1. The subcellular localization of ZmMYB59 geneThe ZmMYB59 gene was cloned successfully from maize inbred line B73.35S::ZmMYB59-EYFP vector was constructed and transformed into callus of rice for sub cellular localization. The result of sub cellular localization showed that was a nucleus localized transcriptional activator.2. ZmMYB59 promoter gene deletion studiesUsing two fragments of ZmMYB59 promoter, the pCXGUS-MYB-1K,pCXGUS-MYB-2K expression vectors were constructed. GUS staining analysis of these transgenic rice plants (Oryza sativa L. japonica. cv. Nipponbare) will help to analyze the expression profiles and the important motif.Respectively stain Ti seeds, root, stems, and leaves of transfer 1 000 bp and 2 000 bp promoter of ZmMYB59 gene plants at germination period, seedling stage. The T1 seeds, only pCXGUS-MYB-2K endosperm edge could measure the GUS activity. At germination period only shoot tips could measure the GUS activity in pCXGUS-MYB-1K, the GUS expression in pCXGUS-MYB-2K root was week,strong in shoot tips; At seedling stage root, stems, leaves of pCXGUS-MYB-1K and pCXGUS-MYB-2k could measure the GUS activity. But the GUS expression of root, stems, leaves in pCXGUS-MYB-1K were weak.GUS staining results also showed that: pCXGUS-MYB-1K,pCXGUS-MYB-2K can express GUS in the different tissues, such as roots, stems, leaves, indicating promoter of ZmMYB59 gene is not tissue-specific, but also that pCXGUS-MYB-1K is necessary in regulating; Meantime, pCXGUS-MYB-2K combination with fragment pCXGUS-MYB-1K, the stronger GUS staining could be obtained, it seems that pCXGUS-MYB-2K comprises tissue specific cis-acting elements and fragment. In summary,the innteraction and cooperation of multiple cis-acting elements from different regions of promoter were necessary for expression of ZmMYB59.3. Analysis of ZmMYB59 gene function in tobaccoThe germination trend,germination percentage,germination index, the vitality index of ZmMYB59 gene overexpression T2 generation tobacco seeds were analyzed under deep seeding tolerance. SOD, POD,CAT, APX activity and MDA, Pro chlorophyll content of ZmMYB59 gene overexpression T2 generation tobacco seeds were analyzed under abiotic stress. ZmMYB59 gene overexpression T2 generation tobacco seeds were provided by the laboratory.The results are as follows: Compared with wild type tobacco, the root length, seedling height and leaf width of transgenic tobacco were significantly lower. Leaf width and seedling height were significantly different. Compared with wild-type, CAT, POD, SOD and APX enzyme activities activity decreased obviously, and the content of Pro decreased obviously and MDA content was significantly increased by 1.5% NaCl treated. Compared with the wild type, CAT and POD enzyme activities were significantly reduced after 8% PEG treatment for 48h, the activity of SOD was significantly decreased, the activity of APX and the content of Pro was decreased, but the chlorophyll content was significantly decreased. The CAT, POD, APX enzyme activity was significantly lower than that of the wild type, but the SOD activity was significantly decreased, the content of Pro was significantly increased, but the content of MDA was not significantly increased after deep sowing(lcm sand) for 48h. Overexpression of ZmMYB59 gene significantly reduced the activity of ROS scavenging enzymes in tobacco, while the content of MDA increased, indicating that ZmMYB59 maybe is a negative transcription factor in germination under drought and salt stress.These results provide a scientific basis for revealing the molecular mechanism of MYB transcription factor regulating seed germination, and it has important reference value to promote the genetic improvement of seed germination traits.