Phenylalanine Ammonia-lyase Gene Cloning During Stress Responses Against Salicylic Acid And Methyl Jasmonate In Bletilla Striata

Objective:In this study,the traditional Chinese herbal medicine Bletilla striata was used as source material.We explored the changes in the synthesis and accumulation of important signaling molecules,SA and MeJA,and their inhibition on B.striata major secondary metabolites?e.g.phenols,dactylorhin A,4-hydroxybenzyl alcohol?,and the effects of signal molecules on antioxidant index and Proline content.We also studied phenylalanine ammonia-lyase?PAL,EC.4.3.1.5?,a key enzyme in the synthesis of metabolites such as phenols and bibenzyls,and the tissue expression specificity of each gene family member under different stress treatments.We screened and cloned the full-length cDNA sequence of the candidate major PAL member from the root.The PAL gene sequence was used for bioinformatic analyses and stress response expression change detection.Methods:?1?B.striata was treated with SA and SA inhibitor paclobutrazol(PP₃₃₃);and MeJA and MeJA inhibitor ibuprofen?IBU?(control group:SA group,SA+PP₃₃₃group,MeJA group,MeJA+IBU group).Changes of antioxidant enzyme activities?SOD,CAT?and MDA and Proline contents in each treatment group under different treatment concentrations and time points were observed.The optimal concentration of SA and MeJA stress treatment was screened out.?2?The changes of secondary metabolites in SA and MeJA treatments were detected by HPLC.?3?The kit was used to detect the changes of total phenolic content and phenylalanine ammonia lyase activity under SA and MeJA treatments.?4?Detection of PALs expression specificity in roots,stems and leaves by Real-time PCR.The relative number of PALs gene changes under different treatment conditions was observed.Screening for members with significant expression in the roots of medicinal parts under different stress treatments.?5?The full-length cDNA of BsPAL gene was obtained by RT-PCR and RACE cloning from B.striata root PAL.Bioinformatics software such as ProtParam,SOPMA and SWISS-MODEL were used to compute and predict the physical and chemical properties and domains of the encoded protein.DNAMAN and MEGA were utilized for amino acid sequence alignment and phylogenetic analysis.Results:?1?When treated with different concentrations of SA and MeJA for 24 h,the activity of CAT and SOD in SA?100?mol/L?and MeJA?100?mol/L?treatment groups was significantly higher than that of the control,with higher Proline content and lower MDA level.It suggests that this concentration can be used as a post-stress treatment.?2?B.striata was treated with SA,SA+PP₃₃₃,MeJA,MeJA+IBU.The results show that the content of white and total phenols initially increased reaching a peak at 24 h,and then decreased,under SA treatment.The same trend was observed under SA+PP₃₃₃ treatment,but peaked at 48 h,with significantly higher total phenolic content at 48 h and 72 h than that of SA treatment.The PAL enzyme activity peaked at 6 h under SA treatment and then gradually decreased.After the addition of inhibitor PP₃₃₃,the enzyme activity peaked at 24 h,then decreased,but it was significantly different from what was observed with CK treatment.Under MeJA treatment,B.striata and total phenolic content and PAL activity were significantly higher than the control and co-treatment groups.The total phenolic content and PAL activity change under MeJA+IBU treatment was significantly reduced than that observed with MEJA treatment,but the change trend was similar.?3?Under the treatment of SA and MeJA,the content of B.striata and medium p-hydroxybenzyl alcohol was 48 h>120 h,and it was also higher than that of the corresponding CK treatment group.The change in trend of the content was similar as that of the total phenol,whereby it initially increased and then decreased;and the content of dactylorhin A was different.Even though the difference was not significantly large,the content under SA treatment is the highest at 48 h.?4?The expression of different PAL family members in B.striata different tissues shows that the expression of BsPAL1-5 in B.striata root was significantly higher than that in leaves and stems,while the expression of BsPAL4 and BsPAL5 genes in roots was significantly higher than other family members.When treated with MeJA and SA,the expression of BsPAL1-5 was diverse,and the level of change among members was also different.However,the relative expression of BsPAL1-5 with different treatments shows that MeJA group>SA group>CK group,while for BsPAL4 the expression level is higher at each sampling time point under both treatments.We propose that PAL gene family can be used as a candidate for the study of metabolic pathways of B.striata.?5?The full-length cDNA of BsPAL4 gene was 2438 bp,encoding a polypeptide consisting of708 amino acids.The relative molecular mass of the predicted protein was 76.5 kDa with isoelectric point of 6.39.The full-length cDNA of BsPAL5 gene was 2708 bp,encoding a 797 amino acid peptide.The composition of the polypeptide predicted a relative molecular mass of 86.2 kDa with an isoelectric point of 6.24.Both BsPAL4and BsPAL5 have the typical domain and active site of plant PAL enzyme,without any transmembrane domain,and is closely related to the PAL gene of Dendrobium candidum and Phalaenopsis.Conclusion:?1?Both exogenous SA and MeJA are able to promote the accumulation of total phenol and 4-hydroxybenzyl alcohol as the important secondary metabolites of B.striata,yet it has little effect on the accumulation of dactylorhin A.?2?Under SA and MeJA stress treatment,showing B.striata and different response patterns to SA and MeJA.?3?Members of the BsPALs gene family are tissue-specific,with higher expression levels of BsPAL4 in roots under the different treatments,possibly in response to stress response elements in the root family.?4?The BsPAL4and BsPAL5 genes have the typical structural features of the PAL gene family.They are highly conserved in evolution.