Qiang Live Full-length Transcriptome Analysis And Gene Cloning And Function Analysis Of Key Enzymes In Phenylalanine Metabolism

Notopterygium incisum is a traditional Chinese medicinal herb with anti-inflammatory,sedative and cardiac arrhythmia treatment effects.At present,the main way to obtain Notopterygium incisum is wild-harvesting.However,wild Notopterygium incisum resources can hardly meet the current market demand,and over-harvesting has led to the depletion of wild Notopterygium incisum resources.On the other hand,artificially grown Notopterygium incisum faces problems such as long dormancy time of seeds and low germination rate.The active ingredients of Notopterygium incisum are mainly coumarin-like substances and volatile oils.These compounds are basically produced by a series of enzymatic reactions through the phenylalanine metabolic pathway of the plant.In this study,the full-length transcriptome of Notopterygium incisum was sequenced and analyzed using third-generation sequencing technology,and the key enzyme genes PAL,C4H and4CL of Notopterygium incisum phenylalanine metabolism were cloned.bioinformatics and functional analysis of the enzyme proteins were performed.In order to improve the yield of active substances in artificially grown Notopterygium incisum and to provide a research basis for its molecular breeding.1.Full-length transcriptome sequencing was performed on the roots of5-year-old Notopterygium incisum using Pac Bio triple sequencing platform,and268,196 readable sequences were obtained.Among the readable sequences,217,322were full-length non-chimeric sequences.After clustering and correction,a total of24,730 HQ isoform sequences were obtained.After redundancy removal,11,533Unigene were obtained for subsequent analysis.The obtained Unigene was used for functional annotation analysis.There were 10046 Unigene annotations to the NR database,and the results showed that carrot had the most similar sequences to Notopterygium incisum.There were 5607 Unigene annotated to the GO database.The results showed the most Unigene annotated to the cellular process branch of biological processes.There are 5465 genes annotated to the KEGG database.As a result,the functions of most genes are unknown,and the remaining known genes are involved in regulating various processes of plant growth and development.It indicates that Notopterygium incisum genes have high species specificity and their functions may be somewhat differentiated during the evolutionary process.2.The sequenced sequences were Blast compared in the NCBI website.The clones obtained for PAL,C4H,4CL-1,and 4CL-3 were relatively conserved evolutionarily.The sequence similarity between Notopterygium incisum C4H gene and C4H gene of various Umbelliferae was high;the sequence similarity between Notopterygium incisum 4CL-1 and 4CL-3 genes showed that there was a high sequence similarity with carrot,and the 4CL-1 gene might have a partial functional divergence.The sequencing results of Notopterygium incisum 4CL-2 gene showed that it did not match any plant 4CL gene,indicating that the sequenced spliced sequence of this gene might be wrong.3.When the cloned genes were subjected to prokaryotic induction of expression,it was found that the expression of Notopterygium incisum PAL protein was induced by the addition of IPTG at three temperature gradients of 18℃,28℃and 37℃.As the culture temperature increased,the protein expression also accumulated significantly.The expression of Notopterygium incisum C4H protein was induced by the addition of IPTG at two culture temperatures of 28℃and 37℃,and the amount of protein induced at 37℃induction conditions was significantly higher than that at 28℃induction conditions,but no significant protein accumulation was observed at 18℃induction conditions.No significant accumulation of Notopterygium incisum 4CL protein was observed under the three different temperature incubation conditions even with the addition of IPTG.4.Clustering analysis of the three enzyme proteins showed that PAL proteins are relatively conserved in evolution,but the PAL proteins of Notopterygium incisum may be functionally specific to those of other medicinal plants.The C4H proteins of Notopterygium incisum also became a small subbranch alone and differed evolutionarily from those of plants in the same genus of medicinal herbs,such as Notopterygium incisum and Angelica.Some plants of the Erythrinaceae family have some functional similarity with the C4H proteins of the Umbelliferae family.The Notopterygium incisum 4CL protein in Umbelliferae has some evolutionary similarity with the 4CL protein of Lonicerae in Umbelliferae.5.The proteins encoded by Notopterygium incisum PAL,C4H,4CL-1 and 4CL-3genes were analyzed.The results showed that the molecular formula of Qianglong PAL protein is C₃₄₁₇H₅₄₈₁N₉₄₁O₁₀₄₈S₃₂,consisting of 712 amino acids;the theoretical molecular weight is:77539.70 Da;it is a relatively stable acidic hydrophilic protein.The secondary structure is rich in a large number ofα-helical structures.Notopterygium incisum PAL protein contains obvious cleavage enzyme structural domain,which proves its function as an aminolytic enzyme.PAL protein is localized in the cytoplasm and has no typical transmembrane structural domain.PAL protein N-terminal has no signal peptide.The molecular formula of C4H protein is C₂₆₄₀H₄₁₇₁N₇₁₁O₇₂₃S₁₇;it consists of505 amino acids.The theoretical molecular weight is:57984.51 Da;it is an unstable basic hydrophilic protein.The secondary structure of Notopterygium incisum C4H protein is rich in a large number ofα-helical structures,and its predicted protein structure is relatively loose.The predicted protein structural domain analysis of C4H protein belongs to the P450 protein family,and eukaryotic P450 proteins are associated with microsomal membranes.C4H protein is localized in the endoplasmic reticulum and contains a typical transmembrane structural domain,which belongs to transmembrane proteins.A signal peptide sequence exists at the N-terminal end.The molecular formula of 4CL-1 protein is C₂₇₀₉H₄₃₃₉N ₆₉₃O ₈₀₂S₁₈;it consists of552 amino acids.The theoretical molecular weight is 60026.49 Da;the theoretical isoelectric point is 7.63,which is a relatively stable basic hydrophobic protein.The molecular formula of 4CL-3 protein is C₂₆₄₉H₄₁₉₆N₆₈₈O₇₈₅S₂₀;it consists of536 amino acids with theoretical molecular weight of 58883.80 Da;it is a relatively stable alkaline hydrophilic protein.The secondary structure of Notopterygium incisum4CL protein contains a large number of irregular convolutions,and the number ofα-helices is reduced compared with PAL and C4H.The 4CL proteins all contain an adenylate-forming domain,and the family of proteins that possess this domain includes acyl and aryl coenzyme a ligases,which catalyze ATP-dependent reactions.4CL proteins are localized in the peroxisomes.4CL contains a typical transmembrane domain and is a transmembrane protein.4CL proteins have no signal peptide at the N-terminal end.