| Lagerstroemia indica,also known as purple flower,purple orchid,western water bayberry,hundred day red,etc.,belongs to Myrtales Lythraceae Lagerstroemia,evergreen shrubs or deciduous small trees.Lagerstroemia indica is native to our country,its planting and application has a long history.Our country has rich germplasm resources and breeding materials.However,the research on breeding and cultivation technology of Lagerstroemia indica started late,and there were few varieties with high commercial value and breeding value,and there were relatively few new varieties with special color and leaf color.In this paper,the relationship between light quality and anthocyanin content of leaves and the key genes in anthocyanin synthesis pathway was analyzed by using different wavelengths of light treatment for cuttings of Lagerstroemia indica.At the same time,transcription factors that respond to light regulation and participate in anthocyanin synthesis were cloned for bioinformatics analysis,construction of plant expression vectors,and subcellular localization analysis.Finally,the interaction between the two transcription factors was analyzed.The aim of this study was to further explore the molecular mechanism of light involved in regulating anthocyanin biosynthesis of Lagerstroemia indica,and to provide scientific theoretical basis for molecular breeding of Lagerstroemia indica.The main research results are as follows:(1)Anthocyanin content of a new Lagerstroemia indica variety ’Danhong Ziye’,which was bred by Hunan Academy of Forestry,was measured after 7 days,14 days,21 days and 28 days of white light,red light,blue light and red blue combination light treatment.The results showed that red light and red blue combined light treatment could significantly increase the anthocyanin content in leaves of Lagerstroemia indica.Under the same light quality,the content was the highest at day 14 and the lowest at day 28,among which the content was the highest in the red and blue combined light group at day 14 and the lowest in the white light control group at day 28.The results revealed that different wavelengths of light treatment could promote anthocyanin synthesis in a certain period of time,in which the combination of red and blue light treatment had the most significant effect.Quantitative PCR analysis of genes related to anthocyanin synthesis in leaves of Lagerstroemia indica showed that the combination of red light,blue light and red blue light can improve the expression level of structural genes and LiHY5 in the anthocyanin synthesis pathway,thus promoting anthocyanin synthesis.However,the expression level of LiBBX24 decreased,which may be due to light stress.Correlation analysis further confirmed that LiHY5 helped anthocyanin synthesis,while LiBBX24 inhibited anthocyanin synthesis.(2)To predict cis-acting elements of promoters of anthocyanin related genes.The results illustrated the promoters of anthocyanin structural genes contain a large number of light responsive elements,which can be involved in light regulation.HY5 regulated anthocyanin synthesis by binding to a light-responsive element on the promoter.BBX24 exists in the upstream of HY5.According to qPCR results,it is speculated that BBX24 may affect the expression of HY5 and thus hinder anthocyanin synthesis.(3)Based on the transcriptome data of Lagerstroemia indica,LiHY5 and LiBBX24 genes were cloned successfully.The coding sequence length of LiHY5 gene was 510bp,encoding 169 amino acids.LiHY5 protein contains the bZIP conserved domain and belongs to the bZIP superfamily.The tertiary structure of LiHY5 protein is predicted to be a leucine zipper structure dominated by alpha helices.The coding sequence length of LiBBX24 gene was 729bp and encoded 242 amino acids.LiBBX24 protein contains two B-box domains and belongs to the B-box superfanmily.The prediction of tertiary structure of LiBBX24 protein indicated that it had obvious zinc finger structure.The results of multiple sequence alignment and phylogenetic analysis proved LiHY5 and LiBBX24 were most closely related to Punica granatum.(4)LiHY5 and LiBBX24 were connected with pCAMBIA1300 plant expression vectors,transformed into Agrobacterium Tumefaciens and injected into tobacco leaves for transient expression.The results indicated that the green fluorescence is distributed in the nucleus,which is consistent with the characteristics of transcription factors.(5)In order to study the interaction between LiHY5 and LiBBX24,yeast two-hybrid,bimolecular fluorescence complementation and in vitro protein deposition experiments were used for analysis.The results showed that the yeast cells grew and appeared blue in the tetragonal medium containing X-α-gal、preliminarily demonstrating the interaction between the two.At the same time,bifolecular fluorescence complementing experiment observed yellow fluorescence in tobacco leaf nucleus by confocal microscope,which proved that they can interact in nucleus.Finally,the direct interaction between LiHY5 and LiBBX24 was further demonstrated by GST pull down in vitro.These results demonstrated that LiBBX24 inhibits the normal function of LiHY5 by interacting with LiHY5,thus hindering anthocyanin synthesis.In summary,LiHY5 and LiBBX24 are involved in the regulation of anthocyanins under the influence of light.The results provide new ideas and methods for the synthesis and development of plant secondary metabolites. |
PDF Full Text Request