Isolation And Functional Characterization Of MYB12 And MYB16 Gene Promoter From ’Gala’ Apple

There is a close relationship between gene expression and the characterization of promoters. To some extent, study in this characterization is helpful to further explore gene function. Previous data in our lab showed that gene silience in commocial harvest ’Gala’ apple can delay apple fruit ripening. In order to lay a foundation for further exploring the functions of MYB12 and MYB16 gene from’Gala’apple, each of genes promoter was cloned by PCR. Promoter fragments were fused with the uidA reporter gene in the binary vector pCAMBI0390 and pBI121. Agrobacterium-mediated transformation of tomato and Arabidopsis was carried out to study the expression characterization of this two promoters. Main results as following:1. A 1290 bp fragment of’Gala’apple MYB12 gene promoter which named ProMdMYB12 was amplified by using primers MYB12-F and MYB12-R. Similarly, ProMdMYB16 984 bp long also cloned by using MYB16-F and MYB16-R. By searching against the PLACE and PlantCARE databases, a lot of cis-elements were found in the sequence. Basal expression elements, tissue-specific elements, phytohormone responsive elements, stress responsive elements, MYB/MYC bounding site and many light responsive elements along with other important cis-elements all be found in both of the promoter sequence. It is helpful for further studying these two promoters expression characterization in plant through analysising cis-elements in promoter sequence.2. Binary vectors were transformed into Agrobacterium tumefaciens strain GV3101 by freeze-thaw. Agrobacterium-mediated transformation of different tissues of tomato. GUS histochemical staining results show that. There is a similarity staining pattern between ProMdMYB12 and ProMdMYB16. Both of them showed high activity in anther and around the seeds, while in the flesh, leaves, petal, calyx, pedicel and so on the activity is no GUS activity. The same expression pattern was also found when quantitative analysis the transient transformation tomato tissues through measuring fluorescence. To research promoter activities around the tomato seeds, paraffin section of seeds treated by ProMdMYB16 was carried out. The results indicated that the staining mainly located around the mucilage and testa.3.12 transgenic plants of ProMdMYB16 were obtained in this study. Different parts at different plant development stages of transgenic Arabidopsis were used for GUS staining. Two typical staining patterns were observed at the very beginning of transgenic Arabidopsis germination. For line 2, GUS staining of 1-day germinated seeds heavily showed in embryo; on the second day located mainly in cotyledon, the conjunction between cotyledon and hypocotyls; on the fifth day it appeared in root but without in root tip and appeared in the conjunction of leaf stalk and base of cotyledon. For line 6, there is no GUS staining at the very beginning; on day 2 GUS activity showed in lower parts of the hypocotyls and the upper parts of root; on day 5 located mainly between root but root tip and lower parts of hypocotyls. Till day 12, the difference of this two lines disappeared. The 12-days transgenic plants GUS staining were found in newly emerged true leave and shoot apical meristem, vascular of hypocotyls, main leaf vein, conjunction of roots and vascular of maturation zone. Moreover, with the help of microscope we focus on the characterization of GUS staining during the lateral root development. Date showed that at the very beginning of lateral root primordial development till the early of lateral root formed the entire lateral tissue showed high GUS activity. With the development, GUS staining mainly located closely to the parent root, after the lateral root formed GUS staining expanded to the vascular of maturation zone. In the vegetative organs ProMdMYB16 activity showed in the main leaf vein of stipules, base of cauline leaf and conjunction of stem and axillary buds. In the productive organs, it was found in pollen, some parts upper or lower of silique during its early development. When the silique grow into mature stage GUS staining disappeared. From these results we believed the cloned ProMdMYB16 has seed embryo, pollen and root tissue specific.4.12-days old seedlings were used for studying ProMdMYB16 responsive to phytohormone and abiotic stresses. Seedlings treated with 200 mM NaCl,20% PEG-6000,100 μM ABA,1 mM SA or 100μM GA respectively showed no obvious difference compared to untreated ones. High temperature enhanced the GUS activity in the conjunction between newly emerged true leave and shoot apical meristem, the most shocking results occurred when 100 μM MeJ was used for treatment as the GUS staining total lost in the whole seedlings. These results showed that ProMdMYB16 activity can be induced by high temperature and reduced by MeJ.