丹参叶遗传转化体系的建立及HMGR基因3’片段的克隆
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摘要
丹参(Salvia miltiorrhiza Bunge)是我国的一种传统中药材。现代药理研究表明,丹参对心血管系统和血液系统的作用十分显著。以丹参为主的多种复方制剂如复方丹参注射液、复方丹参片、复方丹参胶囊和复方丹参滴丸(1997年向美国FDA以治疗药身份申报的品种)等已被广泛用于临床治疗心血管疾病、肾病、肝病及抗感染等。但是,由于丹参生长周期长(2年以上),在传统的栽培模式下,面临着品质严重退化、生产成本相对过高等诸多弊端;离体条件下产生的丹参组培苗,其药用活性成分的积累量还远达不到商业化开发利用的要求。因此,利用现代基因工程技术将丹参药用活性成分生物合成途径中的关键酶基因导入到丹参中,获得转基因的发根、细胞系或再生植株,并进行大规模的培养,是提高丹参药用活性成分的含量和解决丹参药源问题的最佳途径之一。而深入地研究丹参的遗传转化体系,分离克隆其药用活性成分生物合成途径中的关键酶基因,是解决这一问题的前提和关键。
本文以丹参为实验材料,从丹参叶的不同部位对愈伤诱导及分化的影响、不同激素及培养基等对植株生根的影响、丹参毛状根的转化频率的影响因素、毛状根的离体培养条件和植株再生条件、毛状根药用活性成分的积累规律的初探和药用活性成分生物合成途径中的关键酶基因的分离克隆等角度开展了一系列的工作,取得了以下结果:
一、丹参叶再生体系的建立。试验结果表明叶的不同部位对愈伤诱导分化具有差异,其中以叶的形态学下端愈伤诱导率最高,达到了88.9%;其次是叶柄,为80%;而叶的形态学上端最差,仅为61.10%。不同来源的愈伤对丹参的出芽率和平均出芽数都存在极显著的影响,其中叶柄愈伤的出芽率最高,达到了92.6%;平均出芽个数最多,为6.3个;与另外三种培养基(White、1/2MS和MS)相比,B5培养基为促进生根的最适基本培养基;在生根阶段加入一定量的生长激素能有效地提高芽的生根率、平均根数和平均根长,以1mg/L的IBA为最佳。
二、优化了丹参毛状根的诱导条件。实验表明:菌株和外植体类型对丹参毛状根诱导率存在着显著的差异。与A4和R1601相比,C58C1(改造过)为最适菌株;三种外植体(叶片、叶柄和茎段)中,以叶片为外植体更有利于丹参毛状根的产生;最佳共培养时间为3-4天;乙酰丁香酮(AS)浓度为400 uM/L最有利于促进毛状根的产生。
三、研究了丹参毛状根的离体培养条件。实验表明,1/2MS液体培养基最适合丹参毛状根的生长;蔗糖为最适合丹参毛状根生长的碳源,无机铵盐与有机氨(如水解乳蛋白、酵母粉、蛋白胨、牛肉浸膏)相比更适合丹参毛状根的生长; 0.5 mg/L的6-BA有利于促进离体丹参毛状根的生长。
四、进行了稀土元素对丹参毛状根中药用活性成分的诱导合成的初步研究。实验表明,稀土元素处理样中的三种丹参酮的含量皆远大于丹参植株的根和未处理的普通毛状根,其总丹参酮的含量达到了1.800mg/g,分别是植株的根和普通毛状根的1.49倍和4.85倍。
五、研究了丹参毛状根再生条件。实验表明,不同的培养基对丹参毛状根分化出芽存在着显著的影响,MS液体基本培养基与其它三种液体基本培养基相比,更适合丹参毛状根芽的分化。丹参毛状根离体培养在MS液体培养基中15天后,其芽的分化率可以达到37.5%。
六、成功地从丹参中克隆得到了HMGR基因的3’片段,该片段长622bp,通过生物信息学分析,结果表明所得到的序列即为丹参HMGR基因的3’片段。本项研究成功地建立了丹参叶的离体再生系统,为利用根癌农杆菌介导的遗传转化技术进行丹参的品质改良继而获得丹参优良品系提供了转化技术的保障;本文成功地建立了丹参的发根诱导体系,这为丹参的遗传改良提供了另一条候选途径,通过对丹参发根的离体培养条件的优化研究,为今后利用发酵培养技术商业化生产丹参活性成分提供了参考依据;此外,本文还从丹参中克隆了其药用活性成分生物合成途径中的关键酶基因HMGR的3’片段(全长基因正在克隆当中),为利用基因工程手段进行丹参代谢活性成分的分子调控奠定了基础,开展上述研究具有重要的理论和实际意义。
Salvia miltiorrhiza Bunge (Dan-shen) is a famious traditional Chinese medicine in our country. Modern pharmacological and clinical studies have demonstrated that the roots of S. miltiorrhiza possess very effective activities for treatment of cardiovascular and cerebrovascular diseases. Some compound drugs such as injection reagent、troche、capsule、pilula (which has applied for approvement by FDA as the therapeutic medicine 1997) mainly made of Dan-shen, were widely used to cure heart diseases, nephropathy, liver diseases, infection and so on. Whereas, some shortcomings including relative long growth period (more than two years), badly degenerate quality and high cost of production in traditional culture condition, limited the increasing market demanded for Dan-shen. The development of modern biotechn- ology provides one of promising ways to shorten culture period, improve the content of active components and resolve the problem of Dan-shen resources by transferring genes involved in the active components biosynthetic pathway into S. miltiorrhiza and culturing transgenic cell lines,tissues or regenerated plants in a large scale.This, however, significantly relies upon the detailed understanding of the transgenic system and the grope of clone condition of the rate-limiting enzymes on the pathway for active medicine components biosynthesis.
In this paper, in order to establish and optimize genetic transform system of S. miltiorrhiza, several experiments such as the effect of callus inducement and differentiation based on different parts of leaf from S. miltiorrhiza, the fluencing factors on the induction frequency of hairy roots, the culture condition on hairy roots and the accumulation of active medicine components in vitro, the condition on regenerated plants and the reaction condition on cloning of genes encoding the rate-limiting enzymes involved in the active medicine components biosynthetic pathway of S. miltiorrhiza, were performed and the results were reported as follows:
1) The regeneration system from leaf of S. miltiorrhiza was established. The results indicated that calli was induced with various frequency based on different parts of leaf and the highest callus inducement frequency could reach to 88.9% using the morphological underside of leaf as the explant followed by petiole with 80% and then morphological uperside of with 61.10%. Calli from different resoureces displayed various effects to shoot regeneration frequency and numbers among which calli induced from pestiole showed the highest differentiation frequency of 92.6% with the most average number of shoots reaching to 6.3. Compared to other three media (White, 1/2MS and MS), B5 was the most suitable medium to promote root regeneration. Adding some growth hormone to medium could improve the rooting frequence, average number of root and average length of root, and our results showed that the most suitable growth hormone was IBA with concentration of 1mg/L.
2) The inducement condition of hairy roots from S. miltiorrhiza was optimized. The results indicated that remarkable difference was existent using different kinds of bacterium strains and explant types on hairy roots inducement. Compared with A4 and R1601,C58C1 showed much more inducement effect on hairy roots and used as in our subsequent experiment. Lamina was the most suitable explant among the three explants (lamina, petiole and stem) to induce hairy roots and the optimal duration of co-cultivation was about 3-4 days. Acetosyringo (As) of 400uM/L was the most beneficial to promote induct frequencies of hairy roots.
3) The culture condition of hairy roots in vitro was investigated. The results indicated that 1/2MS was the as the most suitable culture medium and sucrose was the optimal carbon source. The growth of hairy roots in the medium containg inorganic ammonium salt as nitrogen source was significantly improved compared to that containg organic ammonia. Adding some growth hormone to medium could significantly accelerate the growth of hairy roots, among which 6-BA with the content of 0.5 mg/L exhibited higher promotion effect compared with other kinds of growth hormone.
4) The effect of rare-earth element on pharmaceutical components in hairy roots of Dan-shen was determinated. The results indicated that the tanshinones content obviously increased and could reach to 1.800mg/g in the roots treated by rare-earth element with the concentration of 10mg/L, which increased 0.49 and 3.85 folds, respectively, compared with ordinary plant roots and hairy roots untreated.
5) The plantlet regeneration condition for hairy roots of S. miltiorrhiza was examined. The results indicated that different media showed notable difference on shoot differentiation from hairy roots of Dan-shen. Compared with the three other kinds of liquid medium, MS was the most suitable medium for shoot differentiation. The regeneration frequency of shoot could reach to 37.5% when hairy roots were cultured in MS liquid medium for 15 days.
6) 3’-end of cDNA encoding 3-hydroxy-3-methylglutaryl-CoA reductase (HMGR) was cloned from S. miltiorrhiza and analysed. The length of fragment isolated was 622bp and bioinformatics analysis results indicated this fragment was a 3’end of HMGR from S. miltiorrhiza.
In summary, a high frequency regeneration system from leaf of S. miltiorrhiza was successfully established, which provided technical support for genetic improvement of S. miltiorrhiza using Agrobacterium tumefaciens mediated transform- ation method. Different factors such as A. rhizogenes strains, explants type, and various concentration of bacterium infected, duration of co-cultivation and acetosyringe(As), were investigated for their influence on hairy root induction from S. miltiorrhiza, which provided another candidate approach for genetic improvement of S. miltiorrhiza. The in vitro culture conditions of hairy roots of S. miltiorrhiza were optimized, which was useful to produce pharmaceutical components of S. miltiorrhiza by ferment culcture technique in large scale. In addition, 3’end of HMGR was cloned from S. miltiorrhiza and the cloning of full-length cDNA was in process, which served the initiation step to molecular regulate pharmaceutical components biosynthetic pathway in the furture.
本文以丹参为实验材料,从丹参叶的不同部位对愈伤诱导及分化的影响、不同激素及培养基等对植株生根的影响、丹参毛状根的转化频率的影响因素、毛状根的离体培养条件和植株再生条件、毛状根药用活性成分的积累规律的初探和药用活性成分生物合成途径中的关键酶基因的分离克隆等角度开展了一系列的工作,取得了以下结果:
一、丹参叶再生体系的建立。试验结果表明叶的不同部位对愈伤诱导分化具有差异,其中以叶的形态学下端愈伤诱导率最高,达到了88.9%;其次是叶柄,为80%;而叶的形态学上端最差,仅为61.10%。不同来源的愈伤对丹参的出芽率和平均出芽数都存在极显著的影响,其中叶柄愈伤的出芽率最高,达到了92.6%;平均出芽个数最多,为6.3个;与另外三种培养基(White、1/2MS和MS)相比,B5培养基为促进生根的最适基本培养基;在生根阶段加入一定量的生长激素能有效地提高芽的生根率、平均根数和平均根长,以1mg/L的IBA为最佳。
二、优化了丹参毛状根的诱导条件。实验表明:菌株和外植体类型对丹参毛状根诱导率存在着显著的差异。与A4和R1601相比,C58C1(改造过)为最适菌株;三种外植体(叶片、叶柄和茎段)中,以叶片为外植体更有利于丹参毛状根的产生;最佳共培养时间为3-4天;乙酰丁香酮(AS)浓度为400 uM/L最有利于促进毛状根的产生。
三、研究了丹参毛状根的离体培养条件。实验表明,1/2MS液体培养基最适合丹参毛状根的生长;蔗糖为最适合丹参毛状根生长的碳源,无机铵盐与有机氨(如水解乳蛋白、酵母粉、蛋白胨、牛肉浸膏)相比更适合丹参毛状根的生长; 0.5 mg/L的6-BA有利于促进离体丹参毛状根的生长。
四、进行了稀土元素对丹参毛状根中药用活性成分的诱导合成的初步研究。实验表明,稀土元素处理样中的三种丹参酮的含量皆远大于丹参植株的根和未处理的普通毛状根,其总丹参酮的含量达到了1.800mg/g,分别是植株的根和普通毛状根的1.49倍和4.85倍。
五、研究了丹参毛状根再生条件。实验表明,不同的培养基对丹参毛状根分化出芽存在着显著的影响,MS液体基本培养基与其它三种液体基本培养基相比,更适合丹参毛状根芽的分化。丹参毛状根离体培养在MS液体培养基中15天后,其芽的分化率可以达到37.5%。
六、成功地从丹参中克隆得到了HMGR基因的3’片段,该片段长622bp,通过生物信息学分析,结果表明所得到的序列即为丹参HMGR基因的3’片段。本项研究成功地建立了丹参叶的离体再生系统,为利用根癌农杆菌介导的遗传转化技术进行丹参的品质改良继而获得丹参优良品系提供了转化技术的保障;本文成功地建立了丹参的发根诱导体系,这为丹参的遗传改良提供了另一条候选途径,通过对丹参发根的离体培养条件的优化研究,为今后利用发酵培养技术商业化生产丹参活性成分提供了参考依据;此外,本文还从丹参中克隆了其药用活性成分生物合成途径中的关键酶基因HMGR的3’片段(全长基因正在克隆当中),为利用基因工程手段进行丹参代谢活性成分的分子调控奠定了基础,开展上述研究具有重要的理论和实际意义。
Salvia miltiorrhiza Bunge (Dan-shen) is a famious traditional Chinese medicine in our country. Modern pharmacological and clinical studies have demonstrated that the roots of S. miltiorrhiza possess very effective activities for treatment of cardiovascular and cerebrovascular diseases. Some compound drugs such as injection reagent、troche、capsule、pilula (which has applied for approvement by FDA as the therapeutic medicine 1997) mainly made of Dan-shen, were widely used to cure heart diseases, nephropathy, liver diseases, infection and so on. Whereas, some shortcomings including relative long growth period (more than two years), badly degenerate quality and high cost of production in traditional culture condition, limited the increasing market demanded for Dan-shen. The development of modern biotechn- ology provides one of promising ways to shorten culture period, improve the content of active components and resolve the problem of Dan-shen resources by transferring genes involved in the active components biosynthetic pathway into S. miltiorrhiza and culturing transgenic cell lines,tissues or regenerated plants in a large scale.This, however, significantly relies upon the detailed understanding of the transgenic system and the grope of clone condition of the rate-limiting enzymes on the pathway for active medicine components biosynthesis.
In this paper, in order to establish and optimize genetic transform system of S. miltiorrhiza, several experiments such as the effect of callus inducement and differentiation based on different parts of leaf from S. miltiorrhiza, the fluencing factors on the induction frequency of hairy roots, the culture condition on hairy roots and the accumulation of active medicine components in vitro, the condition on regenerated plants and the reaction condition on cloning of genes encoding the rate-limiting enzymes involved in the active medicine components biosynthetic pathway of S. miltiorrhiza, were performed and the results were reported as follows:
1) The regeneration system from leaf of S. miltiorrhiza was established. The results indicated that calli was induced with various frequency based on different parts of leaf and the highest callus inducement frequency could reach to 88.9% using the morphological underside of leaf as the explant followed by petiole with 80% and then morphological uperside of with 61.10%. Calli from different resoureces displayed various effects to shoot regeneration frequency and numbers among which calli induced from pestiole showed the highest differentiation frequency of 92.6% with the most average number of shoots reaching to 6.3. Compared to other three media (White, 1/2MS and MS), B5 was the most suitable medium to promote root regeneration. Adding some growth hormone to medium could improve the rooting frequence, average number of root and average length of root, and our results showed that the most suitable growth hormone was IBA with concentration of 1mg/L.
2) The inducement condition of hairy roots from S. miltiorrhiza was optimized. The results indicated that remarkable difference was existent using different kinds of bacterium strains and explant types on hairy roots inducement. Compared with A4 and R1601,C58C1 showed much more inducement effect on hairy roots and used as in our subsequent experiment. Lamina was the most suitable explant among the three explants (lamina, petiole and stem) to induce hairy roots and the optimal duration of co-cultivation was about 3-4 days. Acetosyringo (As) of 400uM/L was the most beneficial to promote induct frequencies of hairy roots.
3) The culture condition of hairy roots in vitro was investigated. The results indicated that 1/2MS was the as the most suitable culture medium and sucrose was the optimal carbon source. The growth of hairy roots in the medium containg inorganic ammonium salt as nitrogen source was significantly improved compared to that containg organic ammonia. Adding some growth hormone to medium could significantly accelerate the growth of hairy roots, among which 6-BA with the content of 0.5 mg/L exhibited higher promotion effect compared with other kinds of growth hormone.
4) The effect of rare-earth element on pharmaceutical components in hairy roots of Dan-shen was determinated. The results indicated that the tanshinones content obviously increased and could reach to 1.800mg/g in the roots treated by rare-earth element with the concentration of 10mg/L, which increased 0.49 and 3.85 folds, respectively, compared with ordinary plant roots and hairy roots untreated.
5) The plantlet regeneration condition for hairy roots of S. miltiorrhiza was examined. The results indicated that different media showed notable difference on shoot differentiation from hairy roots of Dan-shen. Compared with the three other kinds of liquid medium, MS was the most suitable medium for shoot differentiation. The regeneration frequency of shoot could reach to 37.5% when hairy roots were cultured in MS liquid medium for 15 days.
6) 3’-end of cDNA encoding 3-hydroxy-3-methylglutaryl-CoA reductase (HMGR) was cloned from S. miltiorrhiza and analysed. The length of fragment isolated was 622bp and bioinformatics analysis results indicated this fragment was a 3’end of HMGR from S. miltiorrhiza.
In summary, a high frequency regeneration system from leaf of S. miltiorrhiza was successfully established, which provided technical support for genetic improvement of S. miltiorrhiza using Agrobacterium tumefaciens mediated transform- ation method. Different factors such as A. rhizogenes strains, explants type, and various concentration of bacterium infected, duration of co-cultivation and acetosyringe(As), were investigated for their influence on hairy root induction from S. miltiorrhiza, which provided another candidate approach for genetic improvement of S. miltiorrhiza. The in vitro culture conditions of hairy roots of S. miltiorrhiza were optimized, which was useful to produce pharmaceutical components of S. miltiorrhiza by ferment culcture technique in large scale. In addition, 3’end of HMGR was cloned from S. miltiorrhiza and the cloning of full-length cDNA was in process, which served the initiation step to molecular regulate pharmaceutical components biosynthetic pathway in the furture.
引文
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