The Dynamic Accumulation Of(-)-patchoulol And The PTS Gene Cloning And Expression During The Development Of Pogostemon Cablin

(-)-Patchoulol, one of the active constituents of Pogostemon cahlin, has widely been used in pharmaceuticals, food and cosmetics because of its antibacterial and antiviral activities, which gives rise to an increasing demand. In China,(-)-patchoulol was used as a chemical marker required by law for the quality control of P. cablin herba and patchouli oil. It was reported that the content of (-)-patchoulol in patchouli oil was very little, which limited the large-scale production and development and utilization of (-)-patchoulol. Therefore, it is necessary to study the biosynthesis and acculation rules of (-)-patchoulol in order to implement effective control means. There was very little information about the dynamic accumulation of (-)-patchoulol and the biological mechanisms during the maturity of P. cablin. In this study, the dynamic accumulation mode and tissue distribution pattern of (-)-patchoulol during the development of P. cablin were tested. Moreover, patchoulol synthase (PTS) was cloned and the PTS gene expression patterns during patchouli development was detected. The main results were as follows:(1) Patchouli plant growth and maturity lasted for eight months, and could be divided into four principal phases according to the changes of plant size, color and biomass of the plant:1st) slow growth period, this phase occurred during the first three months;2nd) fast growth period, this phase occurred from the fourth month to the sixth month;3rd) ripe period, this phase occurred during the seventh month;4th) fully ripe period, this phase occurred during the last month. The stems of patchouli had similar essential oil content with the roots, but significantly less than that of the leaves; and essential oil showed the similar accumulation trends in leaves, stems and roots, essential oil contents in all the three parts showed growing trends from120to210days after cultivation (DAC) and reached their maximum levels at210DAC, and then no longer change at the last phase.(2)(-)-Patchoulol contents was analyzed by GC-MS during the maturity of P. cablin. The results showed that (-)-patchoulol was in higher quantity in leaves (mean value is42.73%) than those in stems (mean value is36.55%) and roots (mean value is24.31%).(-)-patchoulol contents showed there was a similar accumulation trend in leaves and stems, but significantly different from roots.(-)-Patchoulol contents in leaves and stems showed a slight decrease trend at the beginning, followed by a growing phase and reached their maximum levels at210and180DAC respectively, finally ended by a dramatically declining phase. Nevertheless, the accumulation of (-)- patchoulol in roots presented oscillations alternated with maximums and minimums of (-)-patchoulol levels, and reached the maximum level at210DAC.(3) Five kinds of methods that including Trizol method, modified Trizol method, CTAB method, modified CTAB method and guanidine isothiocyanate-SDS method for RNA extraction from roots, stems, leaves, and calluses of P. cablin were tested in this study. The results showed that modified Trizol method, modified CTAB method, Trizol method and modified CTAB method were the most preferable methods for the RNA extraction from roots, stems, leaves and calluses of P. cablin respectively.(4) Patchoulol synthase (PTS) was cloned from Chinese patchouli in this study, with a length of1659bp, the enzyme consists of552amino acids, GenBank accession number is KP694233. Alignment revealed53and14varying nucleotides from the sequence coding patchoulol synthase (India patchouli) AY508730and KF98351respectively. These variations resulted in26and8differing amino acids (aa) in the expressed protein respectively. Sequence alignment revealed the changes of4.7%and1.4%of the aa in comparison to the previously published sequences AHL24448and Q49SP3respectively.(5) Partial sequences of GAPDH,18S rRNA and β-actin were cloned in this study with the lengths of496bp,440bp and599bp respectively, GenBank accession numbers were KP676601, KP694234and KP676600respectively. The appropriateness of three housekeeping genes as internal controls for quantitative real-time PCR (RT-qPCR) analysis of gene expression of P. cablin was evaluated. The result showed that18S rRNA was the most stable gene, followed by β-actin. The optimum gene that might be used as reference for different ontogenetic stages was18S rRNA.(6) A study of PTS gene expression patterns by RT-qPCR showed that there was the same change trends between the expression profiles and the (-)-patchoulol accumulations at most periods, but no correlation at some periods during patchouli development, especially in leaves and stems. In addition, the PTS gene was expressed at the highest level in the leaf among the three patchouli parts at225DAC, but there is no significant differences in stems and roots.