Effect Of Potassium On Related Genes Expression Of Ferulic Acid Biosynthe-sis In Chinese Angelica

Objective: Angelica sinensis is one of the useful medicines, and ferulic acid from Angelicae sinensis is identified one of the major index of quality. Potassium is activator for 60 kinds of enzymes in plant. It has a positive impact on the growth, yield and quality of crop with the proper used of potassium. There are a lot of study have been reported about Angelicae sinensis, which involved resources evaluation, physiological characteristics, biological medicine and extraction of active ingredients, but there are little study about the gene regulation of active ingredient. This study aimed to clone and express the important genes of ferulic acid from Angelica, and compare different potassium content, to lay the foundation of explore the biosynthesis pathway and nutritional regulation effect of ferulic acid from Angelica, to provide theoretical basis on quality formation and molecular mechanism of Angelica.Method: Designed a pair of degenerate primers referenced the conserved sequence of COMT, 4CL and F5 H genes had been published in Genbank, with the total RNA of Angelica sinensis leaves as the template, used the method of reverse transcription PCR(RT-PCR) to clone the fragment of COMT, 4CL and F5 H genes; then designed a pari of special primers, combined with the method of Rapid Amplification of c DNA Ends(RACR) to cloned other two fragments of COMT and F5 H, spliced the sequence of fragments; at last designed a pair of primers by the sequence spliced, then clone the full-length genes to sequenced, registered in Genbank. Used the method of SYBR Green fluorescence quantitative PCR, with the 18 S gene of Angelicae sinensis as a reference gene, Detecting the expression of COMT, 4CL and F5 H gene in different tissues, different times and different potassium treatment of Angelicae sinensis.Results:(1)Cloning and Analysis the full-length of Caffeic acid-3-O-methyltransferase(COMT) gene in Angelica sinensis(accession number:KP188587): In this study, combining homology cloning and rapid amplification of c DNA ends(RACE) techniques we obtained the c DNA sequence of COMT of Angelica.The results showed The full length of c DNA is 1436 bp, with an open reading frame(ORF) of 1098 bp, and deduced had 365-amino acid polypeptide. Comparing the gene sequence with the 7 other COMT genes from NCBI nucleic acid database, the result shows that it shared high similarity(above 75%).(2) Cloning and Analysis the fragments of 4-Coumarate Coenzyme A Ligase(4CL) gene in Angelica sinensis: In this study, using homology cloning techniques we obtained the c DNA sequence of 4CL of Angelica. The results showed The fragment length of c DNA is 514 bp, and deduced had 170-amino acid polypeptide. Comparing the gene sequence with the 7 other 4CL genes from NCBI nucleic acid database, the result shows that it shared high similarity(above 79%).(3) Cloning and Analysis the full-length of Ferulate-5-Hydroxylas(F5H) gene in Angelica sinensis(accession number:KP257433): In this study, combining homology cloning and rapid amplification of c DNA ends(RACE) techniques we obtained the c DNA sequence of F5 H of Angelica. The results showed The full length of c DNA is 1839 bp, with an open reading frame(ORF) of 1539 bp, and deduced had 512-amino acid polypeptide. Comparing the amino acid sequence with the 7 other F5 H genes from NCBI nucleic acid database, the result shows that it shared high similarity(above 70%).(4) Analysis of COMT’s 、 4CL’s and F5H’s Expression among different Potassium treatment: Fluorescent quantitative PCR analysis showed that the three genes with the extension of timerelative the expression quantity were gradually increased. The m RNA expression of COMT in stems and leaves was higher than that in the root during early stage, but later the relative expression quantity of stems and leaves increased and exceeded the root; 0 mmol/L K+ treatment in stems and roots of COMT has an inhibitory effect. The m RNA expression of 4CL in stems and leaves was higher than that in the root during early stage, but later the relative expression quantity of stems and leaves increased and exceeded the root; 0 mmol/L K+ and 12.0 mmol/L K+ treatment has an inhibitory effect in stems and leaves of 4CL. The expression of the F5 H gene in stems and leaves is higher than the root; In stems and leaves, 0 mmol/L K+ and 12.0 mmol/L K+ treatment has an inhibitory effect early, and late the 12.0 mmol/L K+ potassium treatment reduced the inhibitory effect, but 0 mmol/L K+ treatment still showed the inhibitory effect, But in root, 12.0 mmol/L K+ treatment always showed the inhibitory effect.Conclusion:(1) The full-length of COMT gene、F5H gene and a 4CL gene fragment were cloned from Angelicae sinensis,and registered in Genbank, the accession number of COMT gene was KP188587, the accession number of F5 H gene was KP257433.(2) By studied the expression of genes which related the biosynthesis ferulic acid of Angelicae sinensis at different potassium treatment, it indicated 6.0 mmol/L K+ treatment could accelerates genes expression in stems、leaves and roots of Angelicae sinensis seedlings, may be suitable for the growth of Angelicae sinensis seedlings, but it not necessarily conducived to the accumulation of ferulic acid.