| Polyphenols is a class of important secondary metabolites in Pinus koraiensis with physiological function. It can improve the antioxidative activity and Helps to prevent cancer and cardiovascular disease,which have attracted increasing attention in the fields of nutrition,health and medicine.Therefore,it is very important to study pine polyphenols synthesis metabolic pathway and manipulation measure.In the study,using adventitious buds inducted from zygote embryo of Pinus koraiensis as experimental material,8 elicitors(PHE,CA,MeJA,SA,CTS,YE,La,Eu)as study subjects and contents of polyphenols and proanthocyanidin as indexes, the optimum elicitor and the optimun combination of induction were established, the effect of elictors on defense system against oxidative stress of Pinus koraiensis was analysed. We have carried out the analysis of the transcriptome of Pinus koraiensis using the Illumina HiSeq method, analysed polyphenols biosynthesis metabolic pathways and related functional genes. The effect of elicitors on functional gene related polyphenols biosynthesis expression was analysed by RT-PCR. Mechanism of elicitor in the polyphenols boisynthesis was explored. The main findings were as follows.Different elicitors had different physiological function on the growth of pine adventitious buds within the tested concentrations of this experiment. PHE, CTS, YE, La and Eu could significantly promote the growth in proper concentration range,CA, MeJA and SA could obviously inhibit its growth, High concentration of PHE and CTS could inhibit growth. In proper concentration range,7 elicitors (PHE, MeJA, SA, CTS, YE, La, Eu) could significantly improve the accumulation of polyphenols and procyanidins, could promote the polyphenols synthesis.The optimum induced concentration respectively were:2mmol/L PHE,10μmol/L MeJA,40μmol/L SA,100 mg/L CTS,100mg/L YE,100 μmol/L La and 400 μmol/L Eu。Plackett-Burman design test showed:the significant factors to influence the accumulation of polyphenols were following order:CTS>La>MeJA>PHE>Eu>SA>YE.3 kinds of key factors which influence the synthesis of polyphenols were established:CTS,La and MeJA. The induciton conditions with 3 kinds of key factors were optimized by Box-Benhnken Design test. The optimal induction condition was follows:CTS concentration 79.58 mg/L,La concentration 42.21 μmol/L and MeJA 16.63 umol/L.The predicted value of the accumulation of polyphenols was 14.28mg/g FW and its experimental value was 13.42 mg/g FW. Compared with the control group, the content of polyphenols was increased by 122.19%. CTS,La and MeJA showed synergetic effect on promotion of polyphenols biosynthesis.CTS,MeJA,La,and CTS+ La+MeJA could significantly activate antioxidant defense system and phenylpropanoid pathway,and promote the accumulation of antioxidant compounds polyphenols and proanthocyanidins in Pinus koraiensis,but inducted response time and intensity were different.CTS+La+MeJA combined treatment was more effective than elictors(CTS,La,MeJA). By using the synergistic effect,CTS+ La+MeJA could obviously be alleviated the inhibition of adventitious buds growth,be more improved antioxidant enzymes of defense system against oxidative stress(SOD,POD,CAT,PAL,C4H) activities and increase the accumulation of polyphenols compounds. CTS,MeJA,La and CTS+ La+MeJA as forign elicitor could send inducing signal inside the cell through signal teansduction pathway,quickly stimulate defense system against oxidative stress of Pinus koraiensis,activate phenypropanoid metabolic pathway,promote synthesis and accumulation of antioxidant polyphenols.Transcriptome sequencing analysis of pine adventitious buds by Illumina HiSeq technology was performed.13369359 contig and 172608 unigenes were obtained through sequence assembly. A preprocessed database of unigene sequences was created, blasting with NR,GO,KOG and KEGG databases.58406(33.84%) unigeges were annotated,17996 unigenes were involved in 48 GO-terms,26026 unigenes were classified into 25 function categories of KOG database,9846 unigenes distributed in 334 pathways in KEGG database,997 unigenes involved in the biosynthesis of secondary metabolism.Through function annotation we have found that,49 unigenes involved pine polyphenols biosynthesis,related to 5 secondary metabolism biosynthetic pathway(Phenylpropanoid biosynthesi,Flavonoid biosynthesis,Flavone and flavonol biosynthesis,Anthocyanin biosynthesis,Stilbenoid, diarylheptanoid and gingerol biosynthesis) and 4 functional genes (PAL,C4H,4CL,CHS,F3H,F3’H,FLS,DFR,ANR,LAR,UFGT,3-OMT,CCoAOMT,HCT).Comparison of the two samples(CTS+La+MeJA and CK) showed 5713differntially expressed unigene,including 3449 up-regulated unigene and 2264 down-regulated unigene. These differential expressed unigenes were associated with Ribosome,Metabolic pathway and Biosynthesis of secondary metabolited.23 selected differential expressed unigenes may encode 8 key functional genes (PAL,4CL,CHS,F3H,DFR,ANR,LAR,UFGT) in polyphenols biosynthetic pathway. Through RT-PCR analysis,it was found that elicitors could improve the expression of 8 functional genes,especially the CHS expression, then the ANR and LAR expression. The result intially confirmed,CHS,ANR and LAR are the key genes regulating pine polyphenols biosynthesis. CTS+La+MeJA could effectively up regulate CHS,ANR and LAR expression in cells,which are 185,25 and 21 times of control group.CTS+La+MeJA could significantly mprove the content of 4 polyphenols monomers (Catechin,Epicatechin,Procyanidin B2 and Taxifolin) during the increase of polyphenols and proanthocyanidins. The high expression of CHS,ANR and LAR by CTS+La+MeJA was correlated positively with the increase of polyphenols component.When elicitors and induced combination interacted with Pinus koraiensis cells, could quickly activate defense system against oxidative stress,specificity and selectivity induce key genes(CHS,LAR,ANR) of pine polyphenols biosynthesis erpression,finally promote biosynthesis and accumulation of secondary metabolites polyphenols to achieve regulation of polyphenols biosynthesis. |
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