丹參基因工程體系創(chuàng)新優(yōu)化及次生代謝調(diào)控應用研究
發(fā)布時間：2019-04-02 07:22
【摘要】：丹參是重要的藥用植物模式材料,也是需求較大的中藥材,其丹酚酸和丹參酮類等次生代謝產(chǎn)物有效成分具有抗氧化、抗腫瘤、抗病毒、抗菌消炎、降血脂等多種藥用活性并被廣泛應用于預防和治療高血脂、心腦血管和急性缺血性中風等疾病。隨著心腦血管疾病發(fā)病率升高及日常保健意識的增強,丹參需求量逐年快速增加,野生丹參資源日漸枯竭,而栽培丹參供應及品質(zhì)難以滿足,且通過化學合成制備丹參次生代謝產(chǎn)物可行性較低。因此,基于基因工程策略改善丹參品質(zhì)、提高有效成分含量日益引起研究者的關注。本文針對丹參基因工程有效實施的關鍵環(huán)節(jié)展開工作,首先構建基于Bsata除草劑抗性的葉盤穩(wěn)定轉(zhuǎn)化再生體系及基于rolABC因子的毛狀根誘導培養(yǎng)體系,在此基礎上,通過轉(zhuǎn)錄因子AtEDT1過表達、丹酚酸合成代謝旁路基因SmHPPD和SmCCR抑制表達及多胺代謝調(diào)控基因SmSAMDC過表達的策略,分析丹參酮、丹酚酸等丹參主要次生代謝成分積累規(guī)律,為有效應用基因工程策略改善丹參品質(zhì)、有效滿足供給提供研究思路、調(diào)控節(jié)點、關鍵原件及基礎材料。主要研究內(nèi)容及結(jié)果如下:1.建立了Basta除草劑為選擇標記的丹參遺傳轉(zhuǎn)化再生體系:首先通過梯度設置Basta濃度,最終確定0.6 mg/L的Basta濃度作為最適丹參選擇壓;進而構建35S啟動子驅(qū)動Basta抗性基因的T-DNA骨架載體,并優(yōu)化預培養(yǎng)、共培養(yǎng)、篩選培養(yǎng)、誘導再生等培養(yǎng)環(huán)節(jié)參數(shù),實現(xiàn)了基于Basta除草劑選擇的丹參農(nóng)桿菌介導的有效轉(zhuǎn)化再生;經(jīng)過T-DNA目標PCR擴增及GUS組織化學染色,確認了轉(zhuǎn)化子的有效性,為二次轉(zhuǎn)化創(chuàng)制雙抗材料提供了基礎材料。2.針對發(fā)根質(zhì)粒誘導毛狀根再生的關鍵基因簇rolABC,構建rolA、rolB、rolC單基因及rolABC多基因丹參毛狀根誘導體系,以避免農(nóng)桿菌Ri質(zhì)粒T-DNA區(qū)域冗余基因?qū)χ参锩珷罡紊x系統(tǒng)的干擾,最大限度有效應用毛狀根系統(tǒng)進行丹參次生代謝工程研究。3.構建基于Basta除草劑抗性選擇的擬南芥轉(zhuǎn)錄因子AtEDT1(Arabidopsis thaliana Enhanced Drought Tolerance1)過表達載體,通過農(nóng)桿菌介導的遺傳轉(zhuǎn)化,創(chuàng)制AtEDT1過表達丹參轉(zhuǎn)化材料。栽培實驗表明,丹參AtEDT1轉(zhuǎn)化株系表現(xiàn)了明顯的生長優(yōu)勢,特別是在逆境脅迫條件下,生長量顯著優(yōu)于對照材料。進一步研究顯示,丹參AtEDT1轉(zhuǎn)化材料中,轉(zhuǎn)AtEDT1丹參根系中,丹酚酸合成代謝相關基因(SmTAT、SmHPPR、SmPAL、SmC4H、Sm4CL和SmRAS)的表達水平明顯提升。與其對應,轉(zhuǎn)基因材料根系中丹酚酸類化合物(迷迭香酸、紫草酸及丹酚酸B)的合成積累也相應增加,轉(zhuǎn)基因株系SmTAT基因的表達水平高達野生型丹參的6倍,丹酚酸B的含量增加至61.9mg/g約為野生型丹參的1.55倍。同時,過表達AtEDT1也對丹參酮合成途徑調(diào)控基因(SmHMGR、SmMDS和SmIPPI)表達水平表現(xiàn)為多樣調(diào)控效果,轉(zhuǎn)基因株系中丹參酮類化合物(隱丹參酮、二氫丹參酮I和丹參酮IIA)的合成積累也呈現(xiàn)不同積累水平。4.基于丹參毛狀根誘導體系,驗證了MIGS(miRNA-induced gene silencing)技術在丹參基因功能研究中的有效性。進而針對丹參酚酸合成旁路基因SmHPPD和SmCCR構建單基因及多基因MIGS抑制表達載體,基于丹參毛狀根轉(zhuǎn)化體系,實現(xiàn)了目標基因的有效沉默。相對于對照材料,SmHPPD和SmCCR抑制表達毛狀根形態(tài)及生長沒有明顯差異,但丹酚酸類化合物積累水平(特別是迷迭香酸)顯著提高。5.針對丹參多胺合成代謝關鍵基因SmSAMDC,分析了其組織特異性表達模式,進而構建了基于Basta除草劑選擇的過表達載體,經(jīng)農(nóng)桿菌介導的遺傳轉(zhuǎn)化,創(chuàng)制丹參SmSAMDC過表達新材料,嘗試通過調(diào)控多胺生成進行丹參次生代謝產(chǎn)物調(diào)控。轉(zhuǎn)基因分子、表型鑒定表明,受體丹參轉(zhuǎn)化材料明確整合有包含SmSAMDC表達單元的T-DNA,但沒有明顯的目標性狀體現(xiàn)�？紤]到同源轉(zhuǎn)化中普遍存在的轉(zhuǎn)基因沉默現(xiàn)象,我們以煙草為轉(zhuǎn)化受體材料進行SmSAMDC異源表達分析,發(fā)現(xiàn)煙草SmSAMDC異源表達材料在干旱脅迫條件下,通過SmSAMDC異源表達可顯著提升受體植物材料相對含水量及抗氧化活性,為SmSAMDC在丹參代謝工程中有效應用提供了基礎數(shù)據(jù)�；谝陨涎芯抗ぷ�,本研究有效拓展了丹參遺傳轉(zhuǎn)化平臺,實現(xiàn)了基于轉(zhuǎn)錄因子AtEDT1過表達及丹酚酸合成代謝旁路基因SmHPPD、SmCCR抑制表達策略的丹參次生代謝產(chǎn)物調(diào)控,并對丹參多胺代謝調(diào)控基因SmSAMDC的表達特性及功能進行了研究。本文的研究工作為丹參基因工程的有效應用提供了可行轉(zhuǎn)化平臺、目標調(diào)控位點及實施案例,為有效實現(xiàn)丹參質(zhì)量提升及產(chǎn)量供應提出了新的解決方案。
[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.
【學位授予單位】：電子科技大學
【學位級別】：博士
【學位授予年份】：2017
【分類號】：S567.53
[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.
【學位授予單位】：電子科技大學
【學位級別】：博士
【學位授予年份】：2017
【分類號】：S567.53
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