Study Anthocyanin Accumulation In Tobacco By Constitutively Expression MYB2,TT8,TTG1 Transcription Factors From Purple Cauliflower

Anthocyanin is a class of flavonoids,and share the biosynthesis pathway of phenanthrene with flavanols.Anthocyanin have a positive effect on plant growth and human health.For plant,Anthocyanin contribute to vegetative organs and reproduction organs as well as fruits bright colors,and contribute to plant adaptation to environmental conditions such as pathogen attacks,intense light irradiation and oxidative stress,and also doubling the shelf life of product.In addition,anthocyanin is safe,non-toxic,resource-rich natural food pigment,and the nutritional and pharmacological effects make it great potential value in food,make-up,medicine industry.In recent years,the molecular mechanism of anthocyanin biosynthesis pathway has become more and more clear.BMW complex consisting of MYB,b HLH and WD40 transcription factors was confirmed wildly involved in the biosynthesis regulation of anthocyanin.However,there were many reports indicated that heterologous expression of the transcription factors show different controlling model compared with their function in original plants,and it is still unclear how these transcription factors regulate the synthesis of anthocyanin in heterologous plants.In this study,three transcription factors,Bo MYB2,BoTT8 and BoTTG1,cloned from a purple cauliflower cultivar “Graffiti”(Brassica oleracea var botrytis),were transformed or co-transformed into tobacco under the control of 35 S promoter,respectively.The effects on morphological characteristics,anthocyanin accumulation pattern and structural genes expression of biosynthesis of anthocyanins were investigated,the functions of three transcription factors in tobacco anthocyanin biosynthesis were revealed.The mainly results are as follows:1.The total RNA in the curd of purple cauliflower Graffiti were extracted and cDNA were synthesized using reverse transcriptase.the specific primers were designed according to the sequence information of GU219986,GU219990 and GU219991 in Genbank.Then 744 bp length of BoMYB2 gene,1027 bp length of BoTTG1 gene and 1574 bp length of BoTT8 gene were amplified from the cDNA,respectively.The analysis results of nucleotide sequence and amino acid sequence and the phylogenetic tree showed that three genes are the expected transcription factors involved in anthocyanin biosynthesis.2.35Spro:MYB2,35Spro:TT8 and 35Spro:MYB2TT8 plant expression vectors with a Bar screen marker gene and 35Spro:TTG1 plant expression vector with a hyg screen marker gene were constructed.These plant expression vectors were transformed or co-transformed into tobacco.Single transcription factors expression plants of 35Spro:MYB2,35Spro:TT8 and 35Spro:TTG1,three double transcription factors expression plants of 35Spro:MYB2TT8,35Spro:MYB2TTG1 and 35Spro:TT8TTG1 and one three transcription factors expression plants of 35Spro:MYB2TT8TTG1 were obtained.No anthocyanin accumulation was observed in callus,root or leaves of 35Spro: TT8,35Spro:TTG1 and 35Spro:TT8TTG1 transgenic lines.But all transgenic tobaccos with BoMYB2 expression accumulate anthocyanin in callus,root and young leaves,and the callus,root and young leaves of 35Spro:MYB2TT8 and 35Spro:MYB2TT8TTG1 transgenic lines are intense purple,show more anthocyanin accumulation than else transgenic lines.The transcript level of structural genes that involved into anthocyanin biosynthesis in leaves were analyzed.The result showed that the transcript levels of the general EBGs,including NtCHS,NtCHI and NtF3 H,were not affected by the transcription factors over-expression,however,the transcript levels of the LBG genes,NtF3 Ha,Nt DFR and NtANS in the leaves of 35Spro:MYB2,35Spro:MYB2TTG1 and 35Spro:MYB2TT8 transgenic lines were higher than in 35Spro:TT8,35Spro:TTG1,35Spro:TT8TTG1 transgenic lines as well as the wild-type.The results showed that the BoMYB2 is responsible and key factor for the anthocyanin biosynthesis in tobacco.3.The floral organs of all transgenic lines expressed BoMYB2 transcription factor led to high levels of anthocyanins accumulation.Despite no anthocyanins accumulation was observed in vegetative organs of 35Spro:TT8 and 35Spro:TTGTTG1 transgenic lines,obvious pigmentation in floral organs was showed.The expression of EBGs in floral organs of all transgenic plants showed no difference,but the expression levels of LBGs(including NtF3 Ha,NtDFR and NtANS)were up-regulated.The results showed that the BoTT8 transcription factor specifically regulates the accumulation of anthocyanins in the floral organ.4.The BoTT8 and BoTTG1 transcription factors have no effect on anthocyanin accumulation on the seed germination and seedling stage.The BoMYB2 gene expressed alone in tobacco promote the accumulation of anthocyanin in cotyledons and young leaves after seed germination.The co-expression of BoMYB2 and BTTG1 inhibited the anthocyanin accumulation in the germination cotyledons but promotes anthocyanin accumulation in young leaves.The co-expression of BoMYB2 and BoTT8 as well as BoMYB2 and BoTT8 and BoTTG1 led to more anthocyanin accumulation in the cotyledons,hypocotyls,root tips after seed germination and seedling.5.The T1 seedling roots length of all transgenic lines and the wild type were investigated,the roots length of 35Spro:TTG1 was longer than wild type,the roots length of the 35Spro:TT8 and the 35Spro:TT8TTG1 were shorter than both the 35Spro:TTG1 and wild type,and the roots length of the 35Spro:MYB2 and 35Spro:MYB2TTG1 were shorter than wild type.The shortest roots length was observed in 35Spro:MYB2TT8 and 35Spro:MYB2TT8TTG1 transgenic lines.The result indicated that heterologous over-expression of BoTTG1 transcription factor could promote the growth of roots,while BoTT8 and BoMYB2 would inhibit the growth of roots.