Correlation Study Between Histone Methylation And Chemical Constituents Of Honeysuckle

Lonicerae Japonicae Flos is the dry flower bud or open flower of Caprifoliaceae plant Lonicera japonica Thunb.The major bioactive components of L.japonica are chlorogenic acids,flavonoids and iridoids.It has been widely used in medical and healthcare industry with a wide range of pharmacological activities such as antioxidant,anti-inflammatory,antibacterial,antiviral,heptoprotective effects.The clinical effects of L.japonica and its Chinese patent medicine are closely related to the quality of L.japonica.Therefore,this study puts forward new ideas and methods,combining with the existing quality evaluation system,to radically explain the biosynthetic pathway and signal transduction of chemical composition This paper is of great scientific significance to systematically explain the biological nature of chemical constituents synthesis mechanism of L.japonica.The accumulation of chemical constituents of L.japonica is influenced by both the gene regulation and the external environment.Therefore,this paper combines genetic and environmental factors together.On the one hand,the related functional genes and their regulatory networks are used to systematically explain the biosynthetic pathway of chemical constituents.On the other hand,histone methylation modification which is closely related to the environmental factors is an epigenetic means to explain the synthetic mechanism of chemical constituents.Hence,based on the hypothesis of "histone methylation-transcriptional regulation-gene expression-chemical constituents",combining epigenetics with traditional genetics to analyze the expression level of related genes and the methylation levels of histone H3 at lysine 4(H3K4)and histone H3 at lysine 9(H3K9)were detected,and the correlation between the histone methylation and chemical constituents was further explored,and candidate molecular markers of histone methylation modification related to the chemical constituents of L.japonica were screened out.In this paper,L.japonica,5-azacytidine(5-azaC),histone methyltransferases and demethylases,the contents of chlorogenic acids,flavonoids and iridoids,function gene regulatory networkas are regarded as research materials,pointcut,research object,guide and bridge,respectively.The main findings were as follows(1)Four histone methyltransferases(HMT1,HMT2,HMT3,HMT4)and four histone demethylase(HDT1,HDT2,HDT3,HDT4)were obtained and cloned from the transcriptome of L.japonica,which maintained the stability of histone methylation levels together.(2)The histone methyltransferase and demethylase were analyzed by bioinformatics,the results showed that HMT1 and HMT4 were related to the methylation of H3K9,in addition,HMT4 was also associated with methylation of H3K4.HMT2 and HMT3 were related to the methylation of arginine;HDT1 and HDT2 were related to the demethylation of H3K9,HDT3 was associated with the demethylation of H3K4,and HDT4 had demethylation of H3K4 and H3K9.(3)L.japonica was treated with 20,40,60,80 and 100 ?M 5-azaC,respectively.Fresh leaves were collected after 1,2 and 3 days.The contents of chlorogenic acids,flavonoids and iridoids(chlorogenic acid,neochlorogenic acid,cryptochlorogenic acid,chlorogenic acid A,chlorogenic acid B,chlorogenic acid C,luteolin,luteoloside,loganin,Loganic acid,secoxyloganin and 7-epi-loganin)were analyzed by UPLC-MS/MS,the results showed that the contents of three major chemical constituents of L.japonica leaves treated with 80 ?M 5-azaC for 2 days changed significantly.(4)The contents of three major chemical constituents of L.japonica leaves treated with 80 ?M 5-azaC for 2 days changed significantly.Therefore,leaves treated under this experimental conditions were selected as the materials.Real-time PCR analyzed the expression level of HMT,HDT and related functional genes.The expression levels of HMT1 and HMT4 decreased significantly and HDT2 increased significantly compared with the blank control group.(5)The levels of H3K9 and H3K4 of L.japonica leaves treated under the treatment conditions(4)were analyzed,and the levels of H3K9me 1/2/3 and H3K4me3 significantly decreased compared with the blank control group.(6)H3K9me3 associated with transcriptional inhibition,suggesting that the decrease in H3K9me3 level would cause a decrease in transcriptional inhibition of PAL,CHI,CHS,FNS?,FNS ?2,as a result,the content of the downstream luteolin increased.H3K4me3 associated with transcriptional activation,suggesting that the decline of H3K4me3 level may inhibit the expression of HQT and 8HD1,resulting in the contents of downstream chlorogenic acid,neochlorogenic acid,cryptochlorogenic acid,chlorogenic acid A,chlorogenic acid B,chlorogenic acid C,luteolin,luteoloside,loganin,loganic acid,secoxyloganin and 7-epi-loganin decreased.(7)Based on the obtained results,the correlation between the methylation level and chemical constituents of L.japonica in four different producing areas(Kunming,Beijing,Wudang and Julu)was studied.The results showed that the H3K4me2/3 level of L.japonica in Kunming was lower and the H3K9me3 level of L.japonica in Kunming was higher than that in Beijing,and there were significant differences with H3K4me2/3 and H3K9me3 levels between Kunming and Beijing.The H3K9me3 level of L.japonica in Kunming was higher than that in Julu,and there was significant difference with H3K9me3 level between Kunming and Julu.The relative contents of chlorogenic acids,flavonoids and iridoid terpenoids in Kunming and Beijing,Kunming and Julu were analyzed respectively.The results showed that the contents of chlorogenic acids,flavonoids and iridoids in Kunming were lower than those in Beijing and Julu.Combined with the results of the first four chapters,suggesting that H3K4 methylation may positively regulate the biosynthetic pathway of chlorogenic acids and iridoids,and H3K9 methylation may negatively regulate flavonoids biosynthetic pathway of L.japonica.To sum up,it was found that the H3K9me3 level was negatively correlated with the content of luteolin,and the H3K4me3 level was positively correlated with the contents of chlorogenic acids and iridoids.H3K4me3 and H3K9me3 can be used as candidate molecular markers for the quality evaluation for three major chemical constituents of L.japonica.