Gene Cloning And Functional Study Of Flower Color Related Transcription Factor HymMYB2 In Hydrangea Macrophylla

Research on plant color has always been a hotspot of people's research,and it plays an important role in the cultivation of new plant varieties.In higher plants,the R2R3-MYB transcription factor is an important transcription factor for anthocyanin biosynthetic pathways in plants,and is the most widely studied transcription factor.They regulate the synthesis of anthocyanins by regulating the expression of a single gene or multiple genes in the anthocyanin biosynthetic pathway,thereby making the flower colors appear different colors.Hydrangea macrophylla has the characteristics of large flowers and beautiful colors.It has been loved by people and has become one of the world's most promising flower plants.At present,there are few molecular mechanisms for the formation of the flower color of Hydrangea macrophylla.In this study,gene cloning and functional research on flower color-related transcription factors of Hydrangea macrophylla have important implications for elucidating the molecular mechanism of flower color formation and flower color improvement of Hydrangea macrophylla.The main results of this study are as follows:(1)The primers were designed based on the Unigene of HymMYB2 gene in the transcriptome database of Hydrangea macrophylla's sterile flower,The full-length sequence of the HymMYB2 gene was successfully obtained using the cDNA and DNA of the blue hydrangea variety 'Endless Summer' infertile flowers at the full bloom period.The gene does not contain introns,has an open reading frame of 939bp and can encode 312 amino acids.Bioinformatics analysis showed that HymMYB2 transcription factor contains R2 and R3 domains,which is a typical R2R3 type MYB transcription factor.The amino acid sequence of HymMYB2 protein has the highest homology with the MYB2 sequence of Kivifruit.(2)In order to explore the expression mechanism of HmMYB2 gene in Hydrangea macrophylla,the tissues and organs of Hydrangea macrophylla were analyzed by real-time fluorescence quantitative PCR.The results showed that the HmMYB2 gene was expressed in all tissues and organs of Hydrangea macrophylla,and the relative expression levels in each tissue and organ were fertile flowers>infertile flowers>petioles>roots>buds>leaf petioles>stems.In the different stages of infertile flower formation,the expression of HymMYB2 gene in the flowering stage was significantly higher than that in other stages.The fertile flowers,infertile flowers and flower stalks of the Hydrangea macrophylla variety 'endless summer'are all blue,which indicates that the HmMYB2 gene may have an important effect on the formation of blue anthocyanins.In different flower color Hydrangea macrophylla varieties,the HymMYB2 gene showed a trend of first increase and then decrease with the development of Hydrangea macrophylla's infertile flowers,and it was the highest during the flowering period.The expression levels of HymMYB2 gene in the sterile flowers of three Hydrangea macrophylla varieties in the flowering stage were 'endless summer'(blue)?'Emilia'(white)?'pink'(red).The color difference of Hydrangea macrophylla varieties may be the reason that the relative expression of HymMYB2 gene is different in the infertile flowers of Hydrangea macrophylla varieties of different color in the flowering period.(3)The results of measuring the total anthocyanin content of the Hydrangea macrophylla showed that in the Hydrangea macrophylla variety 'Endless Summer',the total content of anthocyanins first increased and then decreased,which was highly consistent with the flower color.In different colors of Hydrangea macrophylla,the order of the total content of anthocyanins is 'Pink Daisies'?'Endless Summer'?'Emilia'.The results of Pearson correlation analysis showed that the expression level of HymMYB2 gene was extremely significantly correlated with the total anthocyanin content of infertile flowers of the blue Hydrangea macrophylla variety 'Endless Summer',and the correlation coefficient was as high as 0.971.However,it is irrelevant among different color Hydrangea macrophylla varieties.Correlation analysis with the key genes in the anthocyanin biosynthesis pathway of Hydrangea macrophylla showed that the relative expression levels of HymMYB2 were extremely significant with the relative expression levels of C3'5'H,DFR,and ANS,and significantly correlated with the relative expression levels of F3H and BZ1 in infertile flowers of blue Hydrangea macrophylla variety 'Endless Summer',it was suggested that the HymMYB2 gene may have regulatory effects on C3'5'H,DFR,ANS,F3H and BZ1.It can be inferred that HymMYB2 has a regulatory effect on the formation of blue anthocyanins in Hydrangea macrophylla,and its regulation may be achieved by regulating the expression of C3'5'H,DFR,ANS,F3H,and BZ1.(4)The prokaryotic expression vector pET28a(+)-HymMYB2 was successfully constructed and the protein was successfully expressed in E.coli BL21.The molecular weight of the expressed protein was basically the same as the predicted molecular weight of the protein.(5)The plant overexpression vector pEGAD-HymMYB2 was constructed and successfully transformed into Agrobacterium GV3101.The inflorescence of Arabidopsis thaliana was infected by inflorescence infusion method,and transgenic plants were successfully obtained.T1 generation transgenic plant seedlings were compared with wild-type Arabidopsis.It was found that the HymMYB2 transgenic Arabidopsis seedlings were more robust than wild-type Arabidopsis seedlings,and the number of lateral roots and leaves was significantly increased.The number of lateral roots is about eight times that of wild-type Arabidopsis plants,and the number of leaves is about twice that of wild-type Arabidopsis plants.In addition,there was a color change in the petiole of the transgenic plant seedlings,and anthocyanin substances were accumulated in the petiole.This shows that HymMYB2 gene can not only promote the increase of the number of lateral roots and leaves,but also promote the accumulation of anthocyanins.This indicates that the heterologous expression of HymMYB2 gene in Arabidopsis can not only promote the increase of the number of lateral roots and leaves,but also promote the accumulation of anthocyanin at the petiole.