| Danshen is the dried plant roots and rhizomes of Salvia miltiorrihiza Bunge, which produced by many places in China. It's a long history to use danshen in China, and it was recorded in many of the herbal information. The main active ingredients of Danshen are divided into fat-soluble terpene quinone ingredients and water-soluble phenolic acid ingredients. The main fat-solube ingredients have anti-inflammatory, anti-myocardial ischemia effects, such as tanshinone, tanshinoneⅡA, cryptotanshinone, and so on; the water-soluble ingredients have anti-ischemic effect, such as Danshensu, protocatechuic aldehyde, caffeic acid, rosmarinic acid, Salvianolic acid B, and so on.The content of the active ingredients of danshen are closely related to genetic background and growth environment. Salvia miltiorrihiza resources in Zhongjiang country, Deyang city of Sichuan province has a good genetic background. A number of studies have shown that environmental factors had significant correlation with both the growth and active ingredients content of danshen, which heat stress is the one of greatest environmental impact factor.In the study, by using of tissue culture and plant regeneration techniques, the Salvia miltiorrihiza groups in Zhongjiang county of Sichuan with the same genetic background was successfully constructed. And simulated with natural conditions of heat stress, the expression analysis of the changes of the transcriptome and the key enzymes gene expression in rosmarinic acid pathway and terpenoid biosynthesis were carried out. It founded the basis proofs for further investigate the relationship between heat stress and the accumulation of active ingredients. Main results are as follows:1. To identify genes involved in heat shock responses in Salvia miltiorrhiza Bunge.The cDNA-AFLP approach was employed to identify genes differentially expressed in leaves at high temperature. Leaves cDNA were digested by EcoR I and Mse I, and two hundred and fifty-six primer combinations were used for selective amplification. One hundred and ninety-six transcription-derived fragment (TDFs) were selected for their differential expression under heat stress. Among of the 196 TDFs,74 TDFs were suppressed and 122 TDFs were up-regulated. All of the TDFs were cloned and sequenced. Compared with the NCBI databases,147 TDFs presented some significant similarity with known plant sequences, whose functions are involved in transcription factor, stress defense, signal transduction, gene regulation and etc.49 TDFs with low identity and no match to known genes may represent novel genes in Salvia miltiorrhiza. All TDFs were analysed and annotated of the Gene Ontology by Blast2go software,86 TDFs were associated with the cellular component,103 TDFs were associated with the molecular function and 98 TDFs were associated with the biological process. Among of all TDFs,8 TDFs were associated with the kinase proteins,7 TDFs were associated with Ca2+-related proteins,14 TDFs were associated with transcription,13 TDFs were associated with sign transfer and 12 TDFs were associated with heat shock protein. This work founded the basis for further study on the relationship between sign transfer, transcription regulation,stress response and the secondary metabolic pathways,and laid the foundation for the interpretation of the relationship between heat stress and the synthesis and accumulation of active constituent.2. The results of Heat Stress impacted on expression of key enzymes of rosmarinic acid biosynthesis of Salvia miltiorrhiza were as follow:rosmarinic acid biosynthesis pathway is the primary way of the water-soluble phenolic acid component synthesis, and heat stress took significant role in biosynthesis of the Salvia miltiorrhiza phenolic acid components. Salvia miltiorrhiza seedlings were stressed with 38℃for along time, The RNA of leaf in 0,2,4,6,12,24,48 hours were extracted. By using qRT-PCR method, the expression profiling of 7 key enzymes included PAL, C4H, 4CL, TAT, HPPD, HPPR and RAS in metabolic pathways of 0-48 hours were constructed with Actin and GAPDH as reference gene.PAL,C4H and RAS were down-regulated; TAT,4CL and HPPD were up-regulated first and down-regulated later;and HPPR were no changed and down-regulated later. The establishment of the expression profiling during heat stress is the basis for the further study on the relationship between the accumulation of the phenolic acid composition and heat stress. 3. The results of Heat Stress impacted on expression of key enzymes of terpenoid biosynthesis of Salvia miltiorrhiza were as follow:terpenoid synthetic pathway is the primary way of the fat-soluble quinone component synthesis, and heat stress took significant role in biosynthesis of the Salvia miltiorrhiza terpenes components. Salvia miltiorrhiza seedlings were stressed with 38 ℃for along time, The RNA of leaf in 0, 2,4,6,12,24,48 hours were extracted. By using qRT-PCR method, the expression profiling of 7 key enzymes included HMGR, DXS, DXR, CMK, IPPI, FPPS and GGPPS in metabolic pathways of 0-48 hours were constructed with Actin and GAPDH as reference gene.During heat stressed, the expression levels of the key enzyme genes of the early terpenoid pathway, HMGR, DXS, DXR, and CMK were up-regulated first and down-regulated later. In DXR pathway, the genes DXS, DXR, and CMK showed temporal expression of the peak with DXS> DXR> CMK.It was speculated that the substrate concentration can induce its expression.The key enzyme genes, IPPI, FPPS and GGPPS in medium-term of terpenoid biosynthesis were affected by heat stress, and their expression were down-regulated first and up-regulated later, but the increase was due to the different gene, the FPPS with the smallest increase; the IPPI normally increase, but not more than the expression of stress before, GGPPS had the largest increase, over the expression of stress before. The establishment of the expression profiling during heat stress is the basis for the further study on the relationship between the accumulation of the terpene quinones composition and heat stress. |
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