The Ectopic Expression Of EsMyb90 Promotes Anthocyanin Synthesis In Transgenic Plants

Anthocyanins are water-soluble flavonoid pigments commonly found in higher plants.They offer red,blue and purple to fruits,vegetables,flowers and seeds,and have antioxidant activity,thus which play an important role in the prevention of cardiovascular diseases,cancer and diabetes.The genes regulating anthocyanin biosynthesis are included in MYB,basic helix-loop-helix(b HLH)and WD40 proteins,which interact to form MYB-b HLH-WD40(MBW)complex,and MYB is the most critical determinant to regulate plant anthocyanin synthesis.R2R3-MYB is the largest subfamily.The studies shown that many R2R3-MYB proteins are involved in plant metabolism such as regulation of anthocyanins.In this study,a R2R3-MYB family member EsMyb90 gene was cloned from Eutrema Salsugineum,a salt-tolerant model plant.Further,35S:EsMyb90 overexpression vector was constructed and introduced into tobacco and Arabidopsis,respectively.And the phenotype of transgenic plants was observed.The anthocyanin content and the expression of anthocyanin synthesis structural gene in different tissues of transgenic plants,which revealed that EsMyb90 ectopic expression promoted the synthesis of anthocyanins in transgenic tobacco and Arabidopsis stems,leaves and flowers.In addition,the activity of SOD,CAT and APX were detected in transgenic tobacco,and the results showed that EsMyb90 gene significantly increased the antioxidant activity of transgenic plants.1.Sequence analysis of EsMyb90EsMYB90 have R2 and R3 DNA binding domains,belonging to R2R3-MYB family.Phylogenetic analysis revealed that EsMYB90 is in the same evolutionary branch with Arabidopsis At MYB75,At MYB90 and At MYB114,thus they has the most relationship in evolutionary.Additionally,EsMYB90 has some homology with Antirrhinum majus Am ROSEA1(44%),Malus x domestica Md MYB10(42%),Vitis vinifera Vv MYBA1(40%)and Muscari armeniacum Ma AN2(39%).The above R2R3-MYBs have all reported to involve in the regulation of anthocyanins synthesis in plants.In our study,EsMYB90 has no more than 80%homology with other MYBs,EsMYB90 may have different anthocyanin regulation mechanisms from other R2R3-MYBs.2.Construction of 35S:EsMyb90 overexpression vector and acquisition of transgenic plantsThe pCAMBIA3301H-35S:EsMyb90 overexpression vector was constructed and introduced into tobacco and Arabidopsis by Agrobacterium-mediated transformation.The ectopic EsMyb90 transgenic tobacco and Arabidopsis were obtained by the bastar screening and PCR molecular identification.The initially screened transgenic plants were subjected to obtain homozygous transgenic plants.3.EsMyb90 is localized to the nucleusThe pCAT-35S:EYFP-EsMyb90 fusion expression vector was constructed,and was introduced into the onion epidermal cells by plasmid bombardment.The result showed that EsMyb90 was localized in the nucleus according to the observation of fluorescence microscopy.4.Phenotypic observation of EsMyb90 transgenic tobacco and Arabidopsis plantsEsMyb90 transgenic tobacco and Arabidopsis plants have strong anthocyanin accumulation.In EsMyb90 transgenic tobacco,the anthocyanin accumulation is mainly in stems,leaves,flowers and fruit pods.In EsMyb90 transgenic Arabidopsis,the anthocyanins accumulation is not only in stems,leaves,flowers and fruit pods,but also in the roots and seeds.Thus,the expression patters of EsMyb90 gene are different in tobacco and Arabidopsis.5.The anthocyanin contents were increased significantly in the EsMyb90 transgenic plantsThe detection of anthocyanin content showed that the anthocyanin pigmentation of EsMyb90 transgenic tobacco and Arabidopsis significantly increased.Compared with wild type,the contents of anthocyanins were increased 48.79,95.17,45.67,4.94,17.75 and 2.58 times in the stems,young leaves,mature leaves,flowers,fruit pods and seeds of EsMyb90-4 lines,respectively.Meanwhile,compared with wild type,the contents of anthocyanins were increased46.88,64.32,24.21,41.48,81.88 and 36.55 times in roots,stems,leaves,flowers,fruit pods and seeds of Arabidopsis EsMyb90-3 lines,respectively.In summary,the anthocyanin contents were increased significantly in EsMyb90 transgenic tobacco and Arabidopsis.6.The expression of anthocyanin biosynthesis genes were significantly up-regulated in EsMyb90 transgenic plantsThe anthocyanin biosynthtic genes mainly include Nt PAL,Nt CHS,Nt CHI,Nt F3 H,Nt F3'H,Nt DFR,Nt LDOX and Nt UFGT in tobacco.q RT-PCR analysis revealed that these anthocyanin biosynthtic genes except for Nt F3 H and Nt F3'H,were up-regulated in stem,leaf and flower tissues of EsMyb90 transgenic tobacco compared with wild type.Particularlly,the late anthocyanin biosynthesis genes Nt DFR,Nt LDOX and Nt UFGT were significantly up-regulated.Compare with wild type,the expression levels of Nt LDOX in stems,leaves and flowers of EsMyb90-4 lines were up-regulated by 3795.39,14489.79 and 4.93 times,respectively.7.EsMyb90 transgenic tobacco showed enhanced antioxidant activityUnder normal growth conditions and 150 m M Na Cl treatment,the antioxidant enzyme activities of SOD,CAT and APX in EsMyb90 transgenic tobacco were significantly higher than that of wild type.The results suggested that the antioxidant activity of EsMyb90 transgenic tobacco was enhanced compared with wild type.In conclusion,the ectopic expression of EsMyb90 in tobacco and Arabidopsis thaliana significantly enhanced the pigmentation of transgenic plants,increased the anthocyanin content of transgenic plants and the expression of anthocyanin synthesis structural gene,and enhanced antioxidant capacity of transgenic plants.This indicates that EsMyb90 plays an important role in the regulation of anthocyanin synthesis,but the regulation mechanism of EsMyb90 needs further research.