| Chrysanthemum(Chrysanthemum × morifolium Ramat.)is a genus of Asteraceae,a perennial herb,and one of the world's famous ornamental plants.Chrysanthemum is divided into summer chrysanthemum,autumn chrysanthemum and winter chrysanthemum,but the main type is autumn chrysanthemum.This fixed flowering period is no longer consistent w ith people's needs to harvest chrysanthemums in four seasons,so research on controlling the chrysanthemum flowering period has attracted much attention.Traditionally,inducing early flowering of chrysanthemum is mainly achieved by changing the ambient temperature and adjusting the photoper iod,and the cost is relatively high.Therefore,more convenient and effective methods to regulate the molecular biology of flowering has more broad prospects and popularization value.At present,there have been some advances in the study of regulating plant flowering through epigenetics.In order to make the method of epigenetic promotion of early flowering of chrysanthemum more suitable for production and application,this study used 5-Azacytidine(5-azaC)and curcumin two methylase inhibitors to set up different treatment methods to deal with 55 Chrysanthemum varieties(including tissue culture plants and cutting plants),then using the regenerated plants of the treatment material as the research object through the methods of growth and development index determination,quantitative PCR,High performance liquid chromatography,MSAP and transcriptome sequencing,the effects of 5-azaC and curcumin on DNA methylation and gene expression in chrysanthemum were studied.The main results are as follows:(1)The treatment of chrysanthemum with 5-azaC and curcumin produced abundant phenotypic variation materials.After treatment with 5-azaC and curcumin for 30 days,the plant height of 7 chrysanthemum cultivar,the root length of 10 chrysanthemum cultivar,the number of leaves of 2 chrysanthemum cultivar and the roots of 10 chrysanthemum cultivar Compared with the control,the number showed a significant decrease;90 days after transplanting,there were signif icant differences in plant height of 6 cultivar compared with the control,and the difference in data from the 30 days of treatment was reduced.However,there were significant differences in the number of leaves of five varieties compared with the control and this difference w as greater than the 30 days data.Using 5-azaC and curcumin to deal with chrysanthemum cutting seedlings,and setting different treatment methods,it was found that the flowering time of some varieties was advanced and delayed,and the flowering time of 10 varieties was about 5-7 earlier than the control.But the total flowering period of all cultivar shows basically no difference.At the same time,it was found that 6 cultivar of treated seedlings showed plant dwarfing mutation,and the dwarfing rate was about 10%.After a period of growth,the development of the foot buds was counted,and it was found that the number of foot buds germinated by the treatment and the control have no difference,indicating that the treatment with these two treatment agents had no obvious effect on the germination of chrysanthemum foot buds.(2)Using MS AP and HPLC methods were conduct the DNA methylation level of the processed materials.By testing the DNA methylation levels of the 10 treated chrysanthemum varieties(the varieties with earlier flowering time after treatment with 5-azaC and curcumin)and the control material,it was found that the treated chrysanthemum materials w ere more methylated than the control materials.The methylation degree is significantly reduced.In order to prove it further,that the advancement of flowering time is related to the reduction of DNA methylation level,the treatment group of ?Zijingling?(the material obtained by treating the seedlings of ?Zijingling? chrysanthemum with 5-azaC and curcumin),the control group and the ?Zijingling? MET1-RNAi transgenic material were used as experimental materials.The flowering time statistics found that the MET1-RNAi transgenic material bloomed first,followed by the 'Purple Elf' treatment group and finally the 'Purple Elf' control material.The DNA methylation level of each material in the flowering period was detected by HPLC.The results showed that the MET1-RNAi transgenic material had the lowest DNA methylation level.The treatment group materials,except for the 5-azaC treatment group,the other three groups compared with the control.Compared with the control group,showed significant decrease in the basification level.The tissue culture seedlings of 6 chrysanthemum varieties were used as the experimental samples,and three treatment methods were set: single 5-azaC treatment,single curcumin treatment and same concentration of 5-azaC and curcumin mixed treatment.HPLC measurement results showed that at 30 days of treatment,except for the mixed treatment group of 'Pushuiliubing',the other treatment groups showed a signif icant decrease in DNA methylation level compared to the control.The difference in DNA methylation level decreased compared to the control,but did not return to the same level as the con trol.At the same time,it was found that the DNA methylation levels of materials of the same variety and different treatment methods decreased in varying degrees.Among them,only the mixed treatment group of 'Nannongliya' had lower methylation levels than their respective 5-azaC and curcumin treatments group.It can be seen that the mixed use of 5-azaC and curcumin to treat chrysanthemums reduced the DNA methylation level of the chrysanthemum,which is not necessarily better than that of a single methylation inhibitor,5-azaC and curcumin.It may not have an additive effect on the reduction of the DNA methylation level of chrysanthemum.(3)Taking the leaves of three chrysanthemum varieties of 'Jinbeidahong','Shenma','Zijingling' and the control material treated with 5-azaC as the experimental samples,the transcriptome sequencing was performed using the Illumina Hiseq sequencing platform.The original data is 191.23 G,with a total of 1,274,873,854 reads.After removing the joints and low-quality data,read and obtain 1,110,456,602 clean reads.The results showed that the 5-azaC-treated 'Jinbeidahong' variety mater ial and the control had 149 differentially expressed genes,mainly involved in the processes of glycerophospholipid metabolism,phenylpropanoid biosynthesis,and interaction of plant pathogenic bacteria.To the cultivar 'Shenma',the treatment group had 956 differentially expressed genes compared with the control,mainly involved in the processes of phenylpropanoid biosynthesis and amino acid biosynthesis.Compared with the control group,there were 1,361 differentially expressed genes in the 'Zijingling' cultivar,which mainly involves plant hormone signal transduction and endoplasmic reticulum protein processing.DNA methyltransferase and flowering-related genes were screened from the above differential genes and it was found that DNA methyltransferase and histone methylase genes showed a downward trend.The flowering-related genes and some important transcription factor genes AP2,MADS,These genes(Cm MYB,Cm WRKY1,Cm WRKY15,Cm WRKY40,Cm WRKY54,Cm EMF2 and Cm EMF4)showed an upward trend.It is speculated that these genes related to flowering may be regulated by DNA methylation.Using real-time fluorescence quantitative PCR method to verify the expression level of four differentially expressed genes,the results were consistent with the transcriptome sequencing results.Based on the above results,this study believes that DNA methylation plays an important role in the process of chrysanthemum flowering,and the external application of 5-azac and curcumin can lead to the change of DNA methylation level and the expression differences of multiple genes in chrysanthemum,thus affecting the flowering time of chrysanthemum.Among them,5-azaC and curcumin were used to treat chrysanthemum cuttings,which is convenient and quick to save money and labor,and more suitable for production applications cessing. |
PDF Full Text Request