丹参基因工程体系创新优化及次生代谢调控应用研究
[Abstract]:Saviae Miltiorrhizae Radix is an important medicinal plant model material, and is also a traditional Chinese medicinal material with larger demand, and the effective components of secondary metabolites such as salvianolic acid and tanshinone are anti-oxidation, anti-tumor, anti-virus, antibacterial and anti-inflammatory. Has the effects of reducing blood fat and other medicinal activities and being widely used for preventing and treating diseases such as hyperlipidemia, cardiovascular and cerebrovascular diseases and acute ischemic stroke. With the increase of the incidence of cardiovascular and cerebrovascular diseases and the enhancement of the consciousness of daily health care, the demand of red-rooted salvia is increasing year by year, the resources of the wild red-rooted salvia are exhausted, and the supply and quality of the cultivated red-rooted salvia are difficult to meet, and the feasibility of preparing the secondary metabolites of the red-rooted salvia by chemical synthesis is low. Therefore, based on the genetic engineering strategy, the quality of the red-rooted salvia is improved, and the content of the effective components is increased to cause the researchers' attention. In this paper, we first construct a leaf disc stable transformation and regeneration system based on Bsata herbicide resistance and a hairy root induction culture system based on rolABC, on the basis of which the transcription factor AtEDT1 is overexpressed, in ord to effectively apply that gene engineering strategy to improve the quality of the radix salviae miltiorrhizae, It can effectively meet the supply and supply research ideas, control nodes, key originals and basic materials. The main contents and results are as follows:1. The genetic transformation and regeneration system of the red-rooted salvia with the Basta herbicide as the selection marker is established, firstly, the basta concentration is set by the gradient, the Basta concentration of 0.6 mg/ L is finally determined as the optimal red-rooted salvia selection pressure, the T-DNA framework vector of the 35S promoter is used for driving the Basta resistance gene, the pre-culture and the co-culture are optimized, in that method, the parameters of culture link such as culture, induction regeneration and the like are screened, and the effective transformation and regeneration of the salvia miltiorrhiza on the basi of the selection of the Basta herbicide are realized; the effectiveness of the transformant is confirmed through the T-DNA target PCR amplification and the histochemical staining of the GUS, And provides a base material for the secondary transformation and creation of a double-resistant material. in ord to avoid that interference of the redundant gene of the t-DNA region of the agrobacterium Ri plasmid to the secondary metabolic system of the hairy root of the plant, The secondary metabolic engineering of radix salviae miltiorrhizae was studied by using the hairy root system to the maximum extent. An Arabidopsis transcription factor AtEDT1 (Arabidopsis thaliana Enhanced Droght Toledo 1) over-expression vector was constructed based on the Basta herbicide resistance selection, and an AtEDT1 overexpressing salvia miltiorrhiza conversion material was created by Agrobacterium-mediated genetic transformation. The experiment of cultivation showed that the transformation of the Danshen AEDT1 transformed strain showed obvious growth advantages, especially under the condition of stress stress, the growth rate was significantly better than that of the control material. The results showed that the expression level of the metabolic-related genes (SmTAT, SmHPR, SmPAL, SmC4H, Sm4CL, and SRAS) in the root system of Danshen AEDT1 was significantly higher than that in the root system of AtEDT1. and the synthesis and accumulation of the salvianolic acid compound (rosmarinic acid, purple oxalic acid and salvianolic acid B) in the root system of the transgenic material are correspondingly increased, and the expression level of the SmTAT gene of the transgenic line is up to 6 times of that of the wild-type red-rooted salvia, The content of salvianolic acid B is increased to 61.9 mg/ g, which is about 1.55 times that of wild-type red-rooted salvia. At the same time, the expression of the expression of tanshinone synthetic pathway (SmHMGR, SmMDS, and SmIPPI) in the transgenic lines has various regulation effects, and the synthesis and accumulation of the tanshinone compounds (cryptotanshinone, dihydrotanshinone I and tanshinone IIA) in the transgenic lines also exhibit different accumulation levels. Based on the hairy root induction system of Salvia miltiorrhiza, the effectiveness of miIGS (miIGS) in the study of gene function of Salvia miltiorrhiza is verified. In addition, a single-gene and a multi-gene MGS inhibit expression vector are construct for that synthesis of the bypass gene SmHPPD and the SmCCR, and the effective silence of the target gene is realized based on the hairy root transformation system of the radix salviae miltiorrhizae. Compared with the control materials, SmHPPD and SmCCR inhibit the expression of hairy root morphology and growth without significant difference, but the accumulation level of salvianolic acid (especially the rosmarinic acid) is significantly improved. in that method, the SSAMDC of the key gene of the metabolism of the radix salviae miltiorrhizae is synthesize, the specific expression mode of the tissue is analyze, the overexpressing vector based on the Basta herbicide selection is constructed, the genetic transformation mediated by the agrobacterium is carried out, and the novel salvia miltiorrhiza SmSAMDC overexpressing new material is prepared, An attempt was made to control the production of secondary metabolites of red-rooted salvia by regulating polyamines. The identification of the transgenic molecule and the phenotype indicates that the receptor-red-rooted salvia transformation material has the T-DNA which contains the SSAMDC expression unit, but has no obvious target character expression. Taking into account the ubiquitous transgene silencing phenomenon in the homologous transformation, we carried out the SSAMDC heterologous expression analysis of the tobacco-based transformation receptor material, and found that the tobacco SmSAMDC heterologous expression material is under the condition of drought stress, The relative water content and antioxidant activity of the receptor plant material can be significantly improved by the heterologous expression of SSAMDC, and the basic data is provided for the effective application of the SmSAMDC in the metabolism engineering of the red-rooted salvia. Based on the above research work, this study effectively expanded the genetic transformation platform of Salvia miltiorrhiza, and realized the regulation of the secondary metabolites of the salvia miltiorrhiza based on the overexpression of the transcription factor AtEDT1 and the synthesis of the metabolic bypass gene SmHPPD and the SmHCCR inhibition expression strategy based on the transcription factor AtEDT1. The expression and function of SSAMDC were studied. The research work of this paper provides a feasible transformation platform, a target control site and an application case for the effective application of the salvia miltiorrhiza gene engineering, and provides a new solution for the effective realization of the quality improvement of the red-rooted salvia and the supply of the yield.
【学位授予单位】:电子科技大学
【学位级别】:博士
【学位授予年份】:2017
【分类号】:S567.53
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