Cloning And Expression Analysis Of Dfchss, A Chalcone Synthase Gene Families From Dryopteris Fragrans(L.)Schott

Dryopteris fragrans (L) Schott is Dryopteridaceae Dryopteris perennial herbaceous plants. In our country,the center distribution is heilongjiang province,and the most widespread regional distribution is wudalianchi. The habitat of Dryopteris fragrans is very special, it grows in the eruption of a crack in the rock, and it also has certain adaptability to hot and cold environment, was praised as "the bane of skin", it is the rare natural folk medicinal plant resource. Researchers’ study of Dryopteris fragrans are mainly concentrated in cultivation, physiology, chemical composition analysis, drug analysis, etc., but the exploration on its molecular mechanism of flavonoids biosynthesis in benzene propane metabolic pathways is less, and the studies also not deep enough. CHS as the first key enzymes of flavonoids biosynthesis pathway.it lies in the early stage of the flavonoids biosynthesis, and is very important for the regulation of the secondary metabolism of flavonoids. The diversity of CHS gene and the complexity of gene family make the expression of genes regulated by different development stages, tissue specificity and the outside bad environment.This thesis mainly use Dryopteris fragrans cultured tissue as material, through the transcriptome sequencing result, RT-PCR and RACE technology cloning to get synthetase chalcone gene families in Dryopteris fragrans.Based on bioinformatics analysis, the genetic structure, protein sequence, and the analysis of the physical and chemical properties were studied, the construction of the evolutionary tree determines the evolution position of Dryopteris fragrans. Through relative fluorescence quantitative techniques and ultraviolet spectrophotometer method, respectively detect in different parts, under high temperature and low temperature treatment and the conditions of UV, CHS gene specificity expression and total contents of flavonoids in Dryopteris fragrans, analysising the relationship between gene expression and the accumulation of flavonoids matter, explore the role these structural gene took in flavonoids biosynthesis pathway of Dryopteris fragrans, increasing the understanding in molecular level, to provide sufficient theoretical basis and experimental materials to truly achieve flavonoids biosynthesis gene engineering in Dryopteris fragrans applications of active substances to improve production quality, finally lay the foundation to achieve Dryopteris fragrans flavonoids content in breeding and secondary metabolites factory production.In this study the main results are as follows:1. This essay through use RT-PCR and RACE methods, first isolated from Dryopteris fragrans cultured tissue breedings to get synthetase chalcone family members of the three genes, after sequence analysis,these three members of the family are synthetase chalcone supergene family proteins, and respectively named as DfCHS1, D/CHS2, DJCHS3.2. The total length of DfCHSl gene sequence is1710bp, the open reading frame is1209bp, totally encoding403amino acids (GeneBank serial number is KF530802). DfCHS1protein molecular formula, the molecular weight and isoelectric point are C1965H3094N526O574S25,44.07kD and6.33respectively, belongs to instability hydrophilic protein; secondary structure is given priority to alpha helix and beta folding, is the betaprotein; Tertiary structure prediction is the same as secondary structure result; through signal peptide and protein curly structure prediction and transmembrane structure analysis showed that, DfCHSl protein which contains no signal peptide, belongs to non-secretory protein, not curly spiral structure, does not exist trans-membrane structure domain; Glycosylation sites and phosphorylation sites forecast show that DfCHSl protein exists3potential N-glycosylation sites,46potential O-glycosylation sites,17protein kinase phosphorylation sites,10serine sites,4threonine sites and3tyrosine sites. Sequence analysis illustrates that it has higherhomology with Ceratopteristhalictroides Brongn., Equisetumarvense, Piceaasperata,78%,74%and72%respectively.The total length D/CHS2gene sequence is1695bp, the open reading frame is1113bp, encoding371amino acids (GeneBank serial number is KF530813). DfCHS2protein molecular formula, molecular weight and isoelectric point are C1806H2856N5300522S16,40.86kD and9.34respectively, belongs to instability hydrophilic protein; Secondary structure is given priority to alpha helix and beta folding, for beta protein; Tertiary structure prediction is the same as secondary structure result; through signal peptide and protein curly structure prediction and transmembrane structure analysis showed that, DfCHSl protein which contains no signal peptide,belongs to non-secretory protein, not curly spiral structure, does not exist transmembrane structure domain; Glycosylation sites and phosphorylation sites forecast show that DfCHS2protein exists3potential N-glycosylation sites,38potential O-glycosylation sites,15protein kinase phosphorylation sites,12serine sites,1threonine sites and2tyrosine sites. Sequence analysis illustrates that it has higher homology with Ceratopteristhalictroide sBrongn.,Equisetumarvense,ginkgo,72%,69%and69%respectively.The total length of DfCHS’3gene sequence is1341bp, the open reading frame is960bp, encoding320amino acids (GeneBank serial number is KF530615). DfCHSl protein molecular formula, molecular weight and isoelectric point are C1490H2408N428O449S20,34.14kD and8.29respectively, belongs to instability hydrophilic protein; Secondary structure is given priority to alpha helix and beta folding, for beta protein; Tertiary structure prediction is the same as secondary structure result through signal peptide and protein curly structure prediction and transmembrane structure analysis showed that, DfCHSl protein which contains no signal peptide,belongs to non-secretory protein, not curly spiral structure, does not exist transmembrane structure domain; Glycosylation sites and phosphorylation sites forecast show that DfCHS2protein has no potential N-glycosylation sites,37potential O-glycosylation sites,12protein kinase phosphorylation sites,9serine sites,2 threonine sites and1tyrosine sites. Sequence analysis illustrates that it has higher homology with Ceratopteristhalictroides Brongn.,Equisetumarvense ginkgo,12%,68%and69%respectively.3. DfCHSl, D/CHS2, D/CHS3gene protein sequence with other representative plant, a total of32species CHS genes and their family members build the system tree, CHS exists obvious species specificity, the CHS of ferns have the same origin with the others, and have the closer evolution relationship with gymnosperms.4.Detecting Dryopteris fragrans DfCHSl, D/CHS2, D/CHS3gene in sporophyte, gametophyte, spores, leaves and petioles through method of relative fluorescence quantitative PCR,they are all have expression, but its expression level have obvious difference, DfCHSl had the highest amount of expression in the petiole, about4-0.7times than in other tissue; DfCHS2had the highest amount of expression in the gametophyte, and almost undetectable in petiole; DfCHS3expressed in the highest amount of expression in the sporophyte, quantity is3times than in gametophyte, is9times than in leaf. Overall, DfCHSl quantity has the widest expression in different organizations, followed by DfCHS2, DfCHS3(alpha<0.05) and the lowest expression quantity.5. Detection by using the method of relative fluorescence quantitative PCR, Dryopteris fragrans DfCHSl, DfCHS2, DfCHS3gene expression quantity have the same change trend under the condition of low temperature of4℃treatment, all showed that increase first, at the time of12h reached the highest expression quantity, achieving20times than in control group.In48h-60h met minimum,72h rose again.The effect that low temperature induction treatment has on the amount of expression of the CHS gene family members is raletive complex and a significant difference.Under the condition of high temperature of35℃treatment, suggesting that genes exist certain similarity between family members. The change of express quantity in0h and24h was relatively stable, no significant variation, after36h, gene expression showed a sharp rise, is about35times than in the control group.High temperature induction treatment has significant impact on the expression of CHS gene family, this may be related to the synthesis of active ingredients.Under the condition of UV is not the same, only DfCHSl expression quantity appeared a rise under UV irradiation volume, reaching the highest in18h,express quantity is3times than in the control group; DfCHS2and DfCHS3decreased with the extension of time.This phenomenon is opposite with previous studies, the decline of expression may be associated with the physiological characteristics of plant itself, ane also can be related with other genes correlation.6. By using ultraviolet spectrophotometry to detect the flavonoids content in corresponding condition of Dryopteris fragrans, the results showed that in different parts of Dryopteris fragrans, in CHS gene family members, DfCHSl gene has the closest relationship with flavonoids content and presents a positive correlation, DfCHS2and DfCHS3showed no obvious correlation. Under the condition of low temperature of4℃, the change of flavonoids content presents W type trend with the extension of time, and showed no correlation with the amount of CHS gene expression. Under the condition of high temperature35℃, flavonoids content and gene expression illustrates no correlation. Under the condition of ultraviolet (UV), with the extension of processing time, the total flavonoids content and gene expression quantity both appeared relative complex change trend, and there is no regular changes.