| Petal spots are a combination of plant evolution,natural selection and artificial selection innovation,and have great significance in the modem flower industry.Senecio cruentus has various bicolor cultivars,which is a good material for studying the regulation mechanism of spot formation.In the previous study of the research group,four genes ScbHLH17,ScbHLH27,ScAG and ScAGL11 differentially expressed in the colored and colorless regions of the carmine bicolor cineraria were identified.In this study,through the expression of these four key genes in the hybrid population,tobacco transgenic,VIGS system establishment and key gene verification,key gene promoter separation analysis and sequence comparisonand following conclusions were finally obtained:(1)Through the hybridization of carmine bicolor and pure carmine variety,it was found that the trait of bicolor was dominant,and the trait separation of F1 generation fellowed Mendel’s law,indicating that the bicolor traits might be controlled by a single gene,while the depth of the flower color and the size of the spot were quantitative traits.The co-segregation of key genes was verified in the hybrid progeny.It was found that ScAG,ScAGL11 and ScbHLH27 expressed in the colorless region,while ScbHLH17 expressed in the colored region,indicating that the expression of ScAG,ScAGL11,ScbHLH27 and ScbHLH17 was correlated with bicolor trait.(2)In order to verify whether the ScAG,ScAGL11,ScbHLH27 and ScbHLH17 genes were key transcription factors that regulated anthocyanin biosynthesis or not,we transformed these three genes into tabacco,and obtained a line of ScAG transgenetic tabacco which’s flower color decreased and a line of ScbHLH17 transgenetic tabacco which’s flower color increased.We confirmed that ScAG had a negative regulation of anthocyanin accumulation,and ScbHLH17 had a positive regulation of anthocyanin accumulation,and speculated that ScAG might be an upstream factor that inhibited the expression of ScbHLH17 gene in the colorless region.However,ScAGL11 and ScbHLH27 did not obtain transgenic tobacco line with significant changes in phenotype.(3)In order to further verify whether the ScAG,ScAGL11,ScbHLH27 and ScbHLH17 gene,were the key transcription factor that regulated structural genes in cineraria or not,we established the VIGS system of cineraria leaves.It was found that when the OD value of the bacterial fluid was 1.5,and the the stage of seedlings were two true leaves of the cineraria,and were cultivated at 8℃ in dark for 3 days,and then cultivated at a humidity of 60%in a climate chamber at 22℃ for short days,the gene silencing efficiency was the highest.The function of four key genes ScAG,ScAGL11,ScbHLH27 and ScbHLH17 were verified by using the VIGS system of cineraria leaves,and it was found that after inhibiting the expression of ScbHLH17 gene,pigment accumulation was inhibited and the expression of structural genes ScANS,ScDFR3,ScF3Hl and ScCHS2 were decreased,but after inhibiting the expression of ScAG gene,pigment accumulation was increased and the expression of structural genes ScANS,ScDFR3,ScF3Hl,ScCHS2 and ScbHLH17 were increased.We finally confirmed that ScbHLHl 7 was a key transcription factor affecting structural gene expression and anthocyanin accumulation.The absence of ScbHLHl 7 gene’s expression in the colorless region directly led to the formation of bicolor trait in cineraria.And ScAG was the key negative transcription factor affecting anthocyanin accumulation,it might be the reason why ScbHLH17 didn’t express in colorless region of bicolor cultivars.(4)In order to find out the reason why key genes expressed differently in bicolor and pure color varieties,the coding regions and promoter sequences of key genes in carmine bicolor and carmine varieties were isolated and compared.We didn’t find out differences in coding regions,but found an extra 20bp TC repeat sequence in ScAG’s promoter in carmine bicolor cultivar.The final conclusion of this study was that the inhibitation of ScbHLH17 gene in the colorless region caused the expression decrease of the structural genes ScANS,ScDFR3,ScF3H1 and ScCHS2,resulting in the formation of spot.The MADS-box transcription factor ScAG gene expressed in the colorless region could negatively regulate the accumulation of anthocyanins. |
