Cloning Of Maize ZmMYB59 Gene And Its Priliminary Functional Study

Maize is one of the most important crops in China. Seeds can make full use of water under deep soil layer to germination after deep-sowing. However, the existing varieties have poor germination ability from the deep soil layer. It is of great significance to find the key genes controlling seed germinaiton tolerant to deep-sowing and breed new varieties. In our previous study, ZmMYB59 possiblely played an important role in the process of seed germinstion. The ZmMYB59 gene was cloned successfully with specific primers and its nucleotide and promoter sequences were analyzed. On this basis, the gene expression in different sowing-depths, different treatments, different germination stages and different tissues was further analyzed using Real-Time PCR. Finally, the reeombinant expression vector pCAMBIA3301-Bar-ZmMYB59 was introduced into tobacco. The main conclusions were listed as follows:1. The ZmMYB59 gene was cloned successfully from maize inbred line B73. The ORF contained 687 nucleotides and encoded 288 amino acids. ZmMYB59 protein had two typical MYB domains and belonged to R2R3-MYB transcription factor family by the analysis of phylogenetic relationship of amino acid sequence and phylogenetic tree. The MYB domain sequence was widespread and conservative in both single cotyledon and dicotyledonous. The Perl program was employed to take out 2000 bp promoter sequence and analyze the cis-elements.The results showed that many cis-elements were found to bind MYB sites responsive to hormones(especially GA).2. The results of Real-Time PCR analysis showed that: ZmMYB59 gene expression in maize inbred line B73 with strong deep-sowing germinaiton ability was decreased since the sowing depth was increased, and its expression in maize inbred line X178 with poor deep-sowing germinaiton ability was decreased firstly and then increased before embryo germination. After embryo germination, there were no obvious changes of ZmMYB59 gene expression in plumules and roots of B73 with the enhancement of sowing depth while its expression in mesocotyls was gradually decreased. In addition, ZmMYB59 gene expression in GA3-treated mesocotyls was lower than that in the corresponding plumule and root, which showed that the parallel change to deep-sowing. The ZmMYB59 expression in UCZ 、IAAand ABA-treated mesocotyls was betweenthat in the corresponding plumule and root. Our previous study showed that mesocotyl elongation was the main contributors to deep-sowing seed germinaiton. Therefore, it is speculated that ZmMYB59 may negatively regulate seed germinaiton tolerant to deep-sowing and its functions are likely to be related with GA3 signal pathway.3. ZmMYB59 DNA sequence was connected to pCAMBIA3301 vector. The recombinant plasmid pCAMBIA3301-Bar-ZmMYB59 was transformed to tabacoo. After selecting in culture medium containing antibiotics, 29 ZmMYB59 transgenic plants were successfully gained by PCR characterization. The results of phenotypic identification showed that ZmMYB59 transgenic plants had lower height and smaller leaves than wide-type tabacoo, suggesting that the growth of ZmMYB59 transgenic plants was inhibited to some extent.And then analysis of the germination of ZmMYB59 transgenic tobacco seeds under abiotic stresse. The results of ANOVA analysis showed that : the overexpression of ZmMYB59 gene significantly decreased the seeds germination under standard and deep-sowing conditions.