Cloning And Analysis Of Genes Involved In Tyrosine-Branched Pathway Of Rosmarinic Acid Biosynthesis From Pogostemon Cablin

Pogostemon cablin is in the family of Lamiaceae,the genus in Pogostemon,growing as an aromatic perennial herb or shrub.Pogostemon cablin has the effects of eliminating turbid pathogens with aromatics,appetizing,antiemetic and heat relieving.Before the Ming Dynasty,it was introduced to China from Southeast Asia.At present,the propagation of Pogostemon cablin in China is mainly based on cuttings,with long growth cycle,reproductive degradation and rare flowering.Therefore,it is urgent to explore more efficient propagation methods of Pogostemon cablin.Rosmarinic acid is a kind of secondary metabolite with high yield in the organism,which is widely distributed in plants such as Lamiaceae and Boraginaceae.It is the main component of many kinds of medicinal materials and Chinese patent medicines,and has antioxidant,anti-inflammatory,anti-tumor and other pharmacological activities.At present,little information about the enzymes involved in tyrosine-branched pathway is available in Pogostemon cablin,so the cloning of key tyrosine branch enzyme genes in the biosynthetic pathway of Pogostemon cablin rosmarinic acid was performed.This paper discusses the optimal time for sterilization of Pogostemon cablin explants,the optimal medium for callus induction,the optimal medium for adventitious bud induction and the optimal medium for root induction.In this paper,TAT and HPPR from the tyrosine branch of the Pogostemon cablin rosmarinic acid biosynthetic pathway were cloned and bioinformatically analysed,mainly by homologous cloning,with the following results:1.The disinfection time of the explants had some influence on the survival rate of Pogostemon cablin explants.The best disinfection method was 75%ethanol for 30s and0.1%mercuric chloride for 8min.Optimal medium for callus induction:MS+1 mg/L2,4-D+1.5 mg/L NAA+1 mg/L 6-BA.Optimal medium for adventitious bud induction:MS+0.5 mg/L 6-BA.Optimal medium for root induction:MS+1 mg/L IBA.2.Comparison of Pogostemon cablin RNA extraction methods:compared with the CTAB method,the RNA extracted by the Trizol method has good integrity,no obvious degradation,and no protein and DNA contamination and high purity.3.The conserved region of Pc TAT was amplified by designing degenerate primers based on the known TAT gene sequence of the species,and a conserved region of 645 bp in length was obtained;RACE primers were designed based on the sequence of the conserved region,a 3’RACE fragment of 926 bp and a 5’RACE fragment of 571 bp were obtained after PCR amplification.A full-length c DNA sequence of 1583 bp was acquired after assembling the conservative fragment,the 5’RACE fragment and the 3’RACE fragment,including 65 bp of the 5’UTR,276 bp of the 3’UTR and a complete open reading frame,which is located at 66-1307 bp of the gene.Analysis of the Pc TAT coding region using bioinformatics software showed that the open reading frame of Pc TAT is 1242 bp and encodes 413 amino acids;based on the conserved domain,TAT belongs to the PLP-dependent Asp aminotransferase superfamily（AAT-like proteins）;predicted molecular weight 45.33 k Da;predicted isoelectric point7.84;a molecular formula C₂₀₃₈H₃₂₃₃N₅₃₅O₅₈₃S₂₄;an unstable protein;aliphatic index95.62;a hydrophobic protein;no signal peptide;no transmembrane helix structure;subcellular localization in the cytoplasm.The secondary structure prediction shows that the protein is dominated by Alpha helix（Hh）43.58%and Random coil（Cc）34.62%,Extended strand（Ee）at 14.77%and Beta turn（Tt）at the lowest level of 7.02%;further prediction of its high level structure yielded a three dimensional model of Pogostemon cablin TAT protein;TAT amino acid multiple sequence alignment showed the highest similarity between Pogostemon cablin and Perilla frutescens at 81.3%,and the sequence identity with Coleus scutellarioides、Petunia x hybrid、Salvia miltiorrhiza、Scutellaria baicalensis、Agastache rugosa、Solanum pennellii、Prunella vulgaris、Salvia splendens and Actinidia chinensis var.chinensis was high at 71.9-80.3%.4.The conserved region of Pc HPPR was amplified by designing degenerate primers based on the known HPPR gene sequence of the species,and a conserved region of Pc HPPR was 708 bp;RACE primers were designed based on the sequence of the conserved region,a 3’RACE fragment of 837 bp and a 5’RACE fragment of 595 bp were obtained after PCR amplification respectively.A full length c DNA of 1158 bp was acquired after assembling the conservative fragment,the 5’RACE fragment and the3’RACE fragment,including 75 bp of the 5’UTR,141 bp of the 3’UTR and a complete open reading frame,which is located at 76-1017 bp of the gene.Analysis of the Pc HPPR coding region using bioinformatics software showed that the open reading frame of Pc HPPR is 942 bp and encodes 313 amino acids;the protein is classified as a 2-hydroxyacid dehydrogenase,being specifically matched to HPPR,belonging to the NADB Rossmann Superfamily;predicted molecular weight 34.10 k Da;predicted isoelectric point 5.37;molecular formula C₁₅₁₈H₂₄₄₀N₄₁₄O₄₅₂S₁₂;a stable protein;aliphatic index 104.63;a hydrophobic protein;no signal peptide;no transmembrane helix structure;subcellular localization in the nucleus;the secondary structure prediction shows that the protein is dominated by Alpha helix（Hh）at 43.45%and Random coil（Cc）at 33.23%,Extended strand（Ee）at 16.29%and Beta turn（Tt）at the lowest level of7.03%.The 3D model of the HPPR protein of Pogostemon cablin was further predicted,and the HPPR amino acid multiple sequence alignment showed the highest similarity between Pogostemon cablin and Scutellaria baicalensis at 88.5%,and the sequence identity with Salvia miltiorrhiza,Salvia officinalis,Agastache rugosa,Coleus scutellarioides,Prunella vulgaris,Melissa officinalis,Mentha longifolia and Perilla frutescens at 84%-87.9%,and the lower identity with Arabidopsis thaliana and Nicotiana tabacum at 74.8%and 82.7%.Analysis of the Pc HPPR gene by using q RT-PCR showed that the greatest transcript abundance was found in root tissues,followed by leaves and the lowest in stems.