Effect Of Biological Clock Regulation On Secondary Metabolites Produced By Longan Cells Culture

Longan（Dimocarpus longan Lour）is a characteristic fruit tree in China of Tropical and Subtropical.Its fruit is rich in various secondary metabolites such as flavonoids and carotenoids which has high edible and medicinal value.According to the theory of plant cell pluripotency,callus can also be used to produce of secondary metabolites.Embryogemic callus of Dimocarpus logan has many advantages such as fast growth rate,large proliferation coefficient and strong regeneration ability.The Circadian Clock is involved in regulating almost all growth and metabolism in plants,light signals as one of the factors can also affect plant metabolism.In this study,the embryogemic callus of Logan and Longan embryogenic suspension cells were used to study the effect of circadian clock callus and MeJA on the accunulatio of flavonoids and carotenoids and the expression of their synthesis-related genes.The effects of different photoperiod treatments on the protective enzyme activities of each cell were studied.The expressions of the PRRs family genes of the important clock genes of the circadian clock oscillator under the photoperiod,MeJA and somatic embryogenesis were studied.The bioinformatics of PRRs family genes was studied.In order to lay the foundation for the study of the application of biological clock regulation in the production of secondary metabolites such as flavonoids and carotenoids in longan embryogenic calli.The main results of this test are as follows:1 Effects of photoperiod of circadian clock on the accumulation of flavonoids and carotenoids in embryogenic calli of longanLoose embryogenic callus of logan（Red Nucleon cultivar LC2 cell line）was used to study the effects of different photoperiods on the accumulation of flavonoids and carotenoids in embryogenic callus of longan.There are five kinds of photoperiods:full light 24 h/0 h L/D,long light 18 h/6h L/D,half-light 12 h/12 h Effects of L/D,short-light 6 h/18 h L/D and total darkness 0 h/24 h L/D.The results showed that:The content and yield of flavonoids in longan embryogenic callus under the short-light 6 h/18 h L/D treatment were the highest which were 4.733mg/g（DW dry weight）and 0.2993 mg/bottle（DW dry weight）and the lowest were 24h light which were only 2.800 mg/g（DW dry weight）and0.1766 mg/bottle（DW dry weight）.The effects of different photoperiod treatments on the content and yield of flavonoids in longan embryogenic callus were consistent,and the order was:6h>12h>18h>0h>24h.The content and yield of carotenoid in longan embryogenic callus under the full light 24 h/0 h L/D treatment were the highest which were 51.25μg/g（DW dry weight）and 3.2697μg/bottle（DW dry weight）and the lowest were semi-light 12 h/12 h L/D which were only 41.25μg/g（DW dry weight）and 2.7772μg/bottle（DW dry weight）.The order of flavonoid content and yield in longan embryogenic callus under different photoperiod treatments was:24h>0h>6h>18h>12h.2 Effect of photoperiod of circadian clock on cell protective enzyme activity in embryogenic callus of longanThe longan loose embryogenic callus（Red Nucleon cultivar LC2cell line）which were experimental material was used to study the protective enzymes of SOD,POD,PRO,H₂O₂ and PAL in longan embryogenic callus,which were deal with different photoperiod for 20days.The results showed that the activity of SOD in longan embryogenic callus which were deal with different photoperiod was as follows:12h>18h>24h>0h>6h;the activity of POD enzyme was0h>12h>18h>6h>24h;the PRO content was 24h>0h>18h>6h>12h;the H₂O₂ content was 12h>18h>0h>24h>6h and the PAL enzyme activity was as follows:6h>18h>0h>24h>12h.3 Bioinformatics Analysis of PRRs Family Genes and Their Expression Analysis in Longan Somatic EmbryogenesisThe longan genome database（NCBI accession number:BioProject PRJNA305337）was used to identify and functionally analyze the longan PRRs gene family that five family members were obtained,namely PRR1,PRR3,PRR5,PRR7 and PRR9.Phylogenetic tree analysis indicated that the PRRs gene family in longan be divided into three subgroups.The first group was PRR1,second group were PRR3 and PRR7,the third group were PRR5 and PRR9.Conservative domain analysis revealed that the longan PRRs family genes had REC conserved domains at the N-terminus and had CCT modules at C-terminals.The genes of this family all had three identical protein motifs.Protein analysis indicated that the family genes were unstable hydrophilic proteins and acidic proteins.One thing worth paying attention to this family had multiple transmembrane structures in the membrane and in the membrane.Gene structure analysis showed that members of the PRRs family all had introns.Subcellular localization analysis showed that PRR1 localizes to the nucleus and mitochondria but the other four genes were localized to mitochondria.The results of the promoter cis-acting element analysis indicated that the promoter region of PRRs not only contains a large number of TATA-box and CAAT-box core components,but also contains a large number of photoresponsive components,some of which have elements that regulate hormone levels and low temperature,drought,and anaxia.Oxygen induction and day and night regulation and other components.Real-time PCR expression of longan PRRs gene family members in different somatic embryogenesis can be obtained:there were two expression patterns of PRRs family members,one of which was an inverted"V"-shaped expression pattern of PRR1,3,7,9 and another was a linear descending expression pattern of PRR5.The expression level of PRR1/3/7 was the highest expression level in the IcpEC stage.The longan somatic embryogenesis process was a complex process of gene differential expression,material energy change and morphological process.The transcriptional levels of the PRRs family genes were also complex which had a certain time-phase effect.In the four stages of somatic embryogenesis,PRR1/3/7/9 rapidly increased in the early stage and then decreased rapidly,while PRR5 showed a downward state.In the process,there was no obvious change in the exterior of the plant,but the expression of the internal gene changes drastically.Under the treatment of different somatic embryogenesis,the expression patterns of members of the PRRs family can be divided into two types,one was parallel line type which basically no change,and the another was"M"type which first rise,then fall,then rise and then fall.This indicates that the longan PRRs family genes had different expression levels in different somatic embryogenesis stages.4 Effect of MeJA on Proliferation of Longan Embryogenic Suspension Cells and Accumulation of Flavonoids and CarotenoidsThe results showed that MeJA inhibited the proliferation of longan suspension cells.When the concentration of MeJA was 200μmol/L,the flavonoid content was up to 3.11 mg/g（DW dry weight）.The highest yield was 0.82 mg/bottle（DW dry weight）when the concentration of MeJA was 0μmol/L.When the concentration of MeJA was 400μmol/L,the carotenoid content was the highest which was 44.12μg/g（DW dry weight）and It was significantly higher than the control group.The highest carotenoid yield was 7.11μg/bottle（DW dry weight）at a concentration of 300μmol/L.5 Expression Analysis of PRRs Family Genes in Longan Biological Clock under Photoperiod and MeJA TreatmentThe results showed that the five members of the PRRs family have different expression patterns in different photoperiod treatments which can be divided into three different expression patterns.PRR1 was the inverted"V"expression pattern.PRR3 was a"V"expression pattern,which was a specific component of the circadian clock.PRR5,7,and 9were all"M"expression patterns,which were part of the morning cycle of the biological clock.PRR5 and 9 not only highly related members,but also their expression patterns were consistent.PRR3 and 7 were two highly related members but their expression patterns were not the same.The expression patterns of PRR7 and PRR5,9 were the same,while PRR3 was a single expression pattern.In addition to the expression pattern,the relative expression of PRRs family genes also had a certain regularity which the relative expression levels of PRR1,5,and 9 were the highest at 6h.The expression patterns of the five members of the PRRs family were different under different concentrations of MeJA,but the gene expression was highest at a MeJA concentration of 400μmol/L.6 Expression Analysis of Genes Related to Flavonoids and Carotenoids in Longan by Photoperiod and MeJA TreatmentThe results showed that the flavonoid synthesis genes CHI and CHS were“M”expression patterns under photoperiod treatment,then DFR and F3H were inverted"V"expression patterns.The four gene expression patterns used for carotenoid synthesis vary.The flavonoid biosynthesis genes CHI,CHS and DFR under different concentrations of MeJA were all in the"W"expression pattern,and F3H was firstly increased and then decreased in the rising expression pattern.The four genes of GGPS,PSY,PDS and ZDS used in carotenoid synthesis were not only all"W"expression patterns,but also the expression levels were similar.