Analysis Of DNA Methylation And Related Genes In Different Genetic Backgrounds And At Different Development Stages Of Salvia Miltiorrhiza Using Methylation-sensitive Amplification Polymorphism(MSAP)

Salvia miltiorrhiza Bge.(Labiatae) is a perennial herb, which is widely distributed and be used for a long-time in China. There are differences on the morphological, physiological characteristics, and medicinal ingredients in different sources of S. miltiorrhiza Bge.. DNA methylation is widespread in higher plants, which plays an important role in growth and environmental adaptation. In this study, methylation-sensitive amplified polymorphism (MSAP) technology is used to study the DNA methylation status of S. miltiorrhiza Bge., These were cultivated in the same place with different genetic backgrounds and different growth stages. Sequence Analysis of DNA methylation differences fragment lead to understand the relevant DNA methylation and gene regulation in the growth and development of S. miltiorrhiza Bge.. HPLC was used to determine the active ingredients content in S. miltiorrhiza Bge. with different genetic background, in order to study the relationship between DNA methylation and accumulation of the active ingredient. The results are as follows:1. An optimal reaction system suitable for MSAP analysis was developed to survey the diversity of different genetic backgrounds S. miltiorrhiza Bge.. These S. miltiorrhiza Bge. can be clustered into five categories by UPGMA based on the genetic distance, when the GS value is0.77.2. MSAP was used to analysis the S. miltiorrhiza Bge. with different genetic backgrounds,246methylation sites were obtained and among them the unmethylated bands is the most. On methylation level analysis, S. miltiorrhiza Bge. from Henan has the highest methylation rates, Sichuan ZhongJiang has the lowest rate. Methylation status is different in samples of S. miltiorrhiza Bge. with different genetic backgrounds.13genes were obtained from differences in DNA methylation sequencing fragments which associated with these differentially expressed genes. For instance, GIGANTEA gene, GDSL lipase gene, zinc finger protein, RNA polymerase gene, these may be associated with photoperiodic flowering regulation, stress response and biological metabolism, differentiation and adaptation to adverse environments, Salvia transcription of the genome and primary metabolism.3.MSAP were used to analysis different genetic backgrounds of S. miltiorrhiza Bge.,246methylation sites were obtained and the unmethylated bands is the most;the methylation rate gradually decreased following seedling stage, jointing stage, and flowering stage, but rapidly increased in the maturity stage; From vegetative growth to reproductive growth, the rate of DNA methylation significantly increased and mainly the semi-methylated occurred.The results of the sequence of DNA methylation differences fragments show nine genes associated with these differences. These include the ribulose bisphosphate carboxylase gene, geranyl pyrophosphate synthase gene, phenylalanine metabolism over peroxidase gene, auxin-induced protein gene. These genes may be associated with the growth and development of resistance, the accumulation of fat-soluble ingredients, the accumulation of water-soluble components and root formation, apical dominance, aging and stress adapt to some extent.4. HPLC were used to determination of the active ingredient content in different genetic backgrounds S. miltiorrhiza Bge. which cultivated in the same place. For different genetic backgrounds S. miltiorrhiza Bge., the components of the fat-soluble and water-soluble ingredients are still differences. These differences may be related to GPPS gene, and it also influenced by the regulation of DNA methylation, but whether these differences genes plays a key role in the formation of active ingredient need further study.