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丹参基因工程体系创新优化及次生代谢调控应用研究

发布时间:2019-04-02 07:22
【摘要】:丹参是重要的药用植物模式材料,也是需求较大的中药材,其丹酚酸和丹参酮类等次生代谢产物有效成分具有抗氧化、抗肿瘤、抗病毒、抗菌消炎、降血脂等多种药用活性并被广泛应用于预防和治疗高血脂、心脑血管和急性缺血性中风等疾病。随着心脑血管疾病发病率升高及日常保健意识的增强,丹参需求量逐年快速增加,野生丹参资源日渐枯竭,而栽培丹参供应及品质难以满足,且通过化学合成制备丹参次生代谢产物可行性较低。因此,基于基因工程策略改善丹参品质、提高有效成分含量日益引起研究者的关注。本文针对丹参基因工程有效实施的关键环节展开工作,首先构建基于Bsata除草剂抗性的叶盘稳定转化再生体系及基于rolABC因子的毛状根诱导培养体系,在此基础上,通过转录因子AtEDT1过表达、丹酚酸合成代谢旁路基因SmHPPD和SmCCR抑制表达及多胺代谢调控基因SmSAMDC过表达的策略,分析丹参酮、丹酚酸等丹参主要次生代谢成分积累规律,为有效应用基因工程策略改善丹参品质、有效满足供给提供研究思路、调控节点、关键原件及基础材料。主要研究内容及结果如下:1.建立了Basta除草剂为选择标记的丹参遗传转化再生体系:首先通过梯度设置Basta浓度,最终确定0.6 mg/L的Basta浓度作为最适丹参选择压;进而构建35S启动子驱动Basta抗性基因的T-DNA骨架载体,并优化预培养、共培养、筛选培养、诱导再生等培养环节参数,实现了基于Basta除草剂选择的丹参农杆菌介导的有效转化再生;经过T-DNA目标PCR扩增及GUS组织化学染色,确认了转化子的有效性,为二次转化创制双抗材料提供了基础材料。2.针对发根质粒诱导毛状根再生的关键基因簇rolABC,构建rolA、rolB、rolC单基因及rolABC多基因丹参毛状根诱导体系,以避免农杆菌Ri质粒T-DNA区域冗余基因对植物毛状根次生代谢系统的干扰,最大限度有效应用毛状根系统进行丹参次生代谢工程研究。3.构建基于Basta除草剂抗性选择的拟南芥转录因子AtEDT1(Arabidopsis thaliana Enhanced Drought Tolerance1)过表达载体,通过农杆菌介导的遗传转化,创制AtEDT1过表达丹参转化材料。栽培实验表明,丹参AtEDT1转化株系表现了明显的生长优势,特别是在逆境胁迫条件下,生长量显著优于对照材料。进一步研究显示,丹参AtEDT1转化材料中,转AtEDT1丹参根系中,丹酚酸合成代谢相关基因(SmTAT、SmHPPR、SmPAL、SmC4H、Sm4CL和SmRAS)的表达水平明显提升。与其对应,转基因材料根系中丹酚酸类化合物(迷迭香酸、紫草酸及丹酚酸B)的合成积累也相应增加,转基因株系SmTAT基因的表达水平高达野生型丹参的6倍,丹酚酸B的含量增加至61.9mg/g约为野生型丹参的1.55倍。同时,过表达AtEDT1也对丹参酮合成途径调控基因(SmHMGR、SmMDS和SmIPPI)表达水平表现为多样调控效果,转基因株系中丹参酮类化合物(隐丹参酮、二氢丹参酮I和丹参酮IIA)的合成积累也呈现不同积累水平。4.基于丹参毛状根诱导体系,验证了MIGS(miRNA-induced gene silencing)技术在丹参基因功能研究中的有效性。进而针对丹参酚酸合成旁路基因SmHPPD和SmCCR构建单基因及多基因MIGS抑制表达载体,基于丹参毛状根转化体系,实现了目标基因的有效沉默。相对于对照材料,SmHPPD和SmCCR抑制表达毛状根形态及生长没有明显差异,但丹酚酸类化合物积累水平(特别是迷迭香酸)显著提高。5.针对丹参多胺合成代谢关键基因SmSAMDC,分析了其组织特异性表达模式,进而构建了基于Basta除草剂选择的过表达载体,经农杆菌介导的遗传转化,创制丹参SmSAMDC过表达新材料,尝试通过调控多胺生成进行丹参次生代谢产物调控。转基因分子、表型鉴定表明,受体丹参转化材料明确整合有包含SmSAMDC表达单元的T-DNA,但没有明显的目标性状体现。考虑到同源转化中普遍存在的转基因沉默现象,我们以烟草为转化受体材料进行SmSAMDC异源表达分析,发现烟草SmSAMDC异源表达材料在干旱胁迫条件下,通过SmSAMDC异源表达可显著提升受体植物材料相对含水量及抗氧化活性,为SmSAMDC在丹参代谢工程中有效应用提供了基础数据。基于以上研究工作,本研究有效拓展了丹参遗传转化平台,实现了基于转录因子AtEDT1过表达及丹酚酸合成代谢旁路基因SmHPPD、SmCCR抑制表达策略的丹参次生代谢产物调控,并对丹参多胺代谢调控基因SmSAMDC的表达特性及功能进行了研究。本文的研究工作为丹参基因工程的有效应用提供了可行转化平台、目标调控位点及实施案例,为有效实现丹参质量提升及产量供应提出了新的解决方案。
[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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