CN102839190A - Method for shortening plant cell suspension culture period - Google Patents
Method for shortening plant cell suspension culture period Download PDFInfo
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- CN102839190A CN102839190A CN201210229723XA CN201210229723A CN102839190A CN 102839190 A CN102839190 A CN 102839190A CN 201210229723X A CN201210229723X A CN 201210229723XA CN 201210229723 A CN201210229723 A CN 201210229723A CN 102839190 A CN102839190 A CN 102839190A
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- 238000004114 suspension culture Methods 0.000 title claims abstract description 18
- 238000000034 method Methods 0.000 title claims abstract description 13
- 238000004904 shortening Methods 0.000 title claims abstract 3
- 239000000725 suspension Substances 0.000 claims abstract description 30
- 206010020649 Hyperkeratosis Diseases 0.000 claims abstract description 18
- 230000012010 growth Effects 0.000 claims abstract description 10
- 241000894006 Bacteria Species 0.000 claims abstract description 8
- 239000012634 fragment Substances 0.000 claims abstract description 8
- 239000013604 expression vector Substances 0.000 claims abstract description 7
- 108700025694 p53 Genes Proteins 0.000 claims abstract description 7
- 230000010261 cell growth Effects 0.000 claims abstract description 6
- 238000005457 optimization Methods 0.000 claims abstract description 6
- 108091030071 RNAI Proteins 0.000 claims abstract description 4
- 238000009825 accumulation Methods 0.000 claims abstract description 4
- 238000000855 fermentation Methods 0.000 claims abstract description 4
- 230000004151 fermentation Effects 0.000 claims abstract description 4
- 230000009368 gene silencing by RNA Effects 0.000 claims abstract description 4
- 241000589155 Agrobacterium tumefaciens Species 0.000 claims abstract 2
- 230000000474 nursing effect Effects 0.000 claims abstract 2
- 239000007788 liquid Substances 0.000 claims description 6
- QVGXLLKOCUKJST-UHFFFAOYSA-N atomic oxygen Chemical compound [O] QVGXLLKOCUKJST-UHFFFAOYSA-N 0.000 claims description 3
- 239000003795 chemical substances by application Substances 0.000 claims description 3
- 238000011081 inoculation Methods 0.000 claims description 3
- 229910052760 oxygen Inorganic materials 0.000 claims description 3
- 239000001301 oxygen Substances 0.000 claims description 3
- 239000003242 anti bacterial agent Substances 0.000 claims description 2
- 229940088710 antibiotic agent Drugs 0.000 claims description 2
- 239000002207 metabolite Substances 0.000 claims description 2
- IJGRMHOSHXDMSA-UHFFFAOYSA-N Atomic nitrogen Chemical compound N#N IJGRMHOSHXDMSA-UHFFFAOYSA-N 0.000 claims 2
- 108091028043 Nucleic acid sequence Proteins 0.000 claims 2
- OKTJSMMVPCPJKN-UHFFFAOYSA-N Carbon Chemical compound [C] OKTJSMMVPCPJKN-UHFFFAOYSA-N 0.000 claims 1
- 108091092195 Intron Proteins 0.000 claims 1
- 229910052799 carbon Inorganic materials 0.000 claims 1
- 238000010276 construction Methods 0.000 claims 1
- 230000006698 induction Effects 0.000 claims 1
- 229910052757 nitrogen Inorganic materials 0.000 claims 1
- 230000001131 transforming effect Effects 0.000 claims 1
- 241000196324 Embryophyta Species 0.000 abstract description 16
- 241000589158 Agrobacterium Species 0.000 abstract description 5
- 238000004519 manufacturing process Methods 0.000 abstract description 5
- 235000001453 Glycyrrhiza echinata Nutrition 0.000 abstract description 3
- 235000006200 Glycyrrhiza glabra Nutrition 0.000 abstract description 3
- 235000017382 Glycyrrhiza lepidota Nutrition 0.000 abstract description 3
- 229940010454 licorice Drugs 0.000 abstract description 3
- 238000011161 development Methods 0.000 abstract description 2
- 240000004670 Glycyrrhiza echinata Species 0.000 abstract 1
- 239000004480 active ingredient Substances 0.000 abstract 1
- 230000000694 effects Effects 0.000 abstract 1
- 230000001404 mediated effect Effects 0.000 abstract 1
- 239000002773 nucleotide Substances 0.000 description 8
- 125000003729 nucleotide group Chemical group 0.000 description 8
- 108090000623 proteins and genes Proteins 0.000 description 4
- 238000012216 screening Methods 0.000 description 4
- 238000007873 sieving Methods 0.000 description 4
- LPLVUJXQOOQHMX-QWBHMCJMSA-N glycyrrhizinic acid Chemical compound O([C@@H]1[C@@H](O)[C@H](O)[C@H](O[C@@H]1O[C@@H]1C([C@H]2[C@]([C@@H]3[C@@]([C@@]4(CC[C@@]5(C)CC[C@@](C)(C[C@H]5C4=CC3=O)C(O)=O)C)(C)CC2)(C)CC1)(C)C)C(O)=O)[C@@H]1O[C@H](C(O)=O)[C@@H](O)[C@H](O)[C@H]1O LPLVUJXQOOQHMX-QWBHMCJMSA-N 0.000 description 3
- 235000013311 vegetables Nutrition 0.000 description 3
- 108090000790 Enzymes Proteins 0.000 description 2
- 102000004190 Enzymes Human genes 0.000 description 2
- 241000202807 Glycyrrhiza Species 0.000 description 2
- 238000012408 PCR amplification Methods 0.000 description 2
- 101000702488 Rattus norvegicus High affinity cationic amino acid transporter 1 Proteins 0.000 description 2
- CKUAXEQHGKSLHN-UHFFFAOYSA-N [C].[N] Chemical compound [C].[N] CKUAXEQHGKSLHN-UHFFFAOYSA-N 0.000 description 2
- 230000003115 biocidal effect Effects 0.000 description 2
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- 108091008146 restriction endonucleases Proteins 0.000 description 2
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- 238000013112 stability test Methods 0.000 description 2
- 238000012546 transfer Methods 0.000 description 2
- VTAJIXDZFCRWBR-UHFFFAOYSA-N Licoricesaponin B2 Natural products C1C(C2C(C3(CCC4(C)CCC(C)(CC4C3=CC2)C(O)=O)C)(C)CC2)(C)C2C(C)(C)CC1OC1OC(C(O)=O)C(O)C(O)C1OC1OC(C(O)=O)C(O)C(O)C1O VTAJIXDZFCRWBR-UHFFFAOYSA-N 0.000 description 1
- 108700008625 Reporter Genes Proteins 0.000 description 1
- 230000015572 biosynthetic process Effects 0.000 description 1
- 239000006285 cell suspension Substances 0.000 description 1
- 238000012258 culturing Methods 0.000 description 1
- 238000013467 fragmentation Methods 0.000 description 1
- 238000006062 fragmentation reaction Methods 0.000 description 1
- LPLVUJXQOOQHMX-UHFFFAOYSA-N glycyrrhetinic acid glycoside Natural products C1CC(C2C(C3(CCC4(C)CCC(C)(CC4C3=CC2=O)C(O)=O)C)(C)CC2)(C)C2C(C)(C)C1OC1OC(C(O)=O)C(O)C(O)C1OC1OC(C(O)=O)C(O)C(O)C1O LPLVUJXQOOQHMX-UHFFFAOYSA-N 0.000 description 1
- 229960004949 glycyrrhizic acid Drugs 0.000 description 1
- UYRUBYNTXSDKQT-UHFFFAOYSA-N glycyrrhizic acid Natural products CC1(C)C(CCC2(C)C1CCC3(C)C2C(=O)C=C4C5CC(C)(CCC5(C)CCC34C)C(=O)O)OC6OC(C(O)C(O)C6OC7OC(O)C(O)C(O)C7C(=O)O)C(=O)O UYRUBYNTXSDKQT-UHFFFAOYSA-N 0.000 description 1
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- 235000019410 glycyrrhizin Nutrition 0.000 description 1
- 230000024053 secondary metabolic process Effects 0.000 description 1
Landscapes
- Breeding Of Plants And Reproduction By Means Of Culturing (AREA)
- Micro-Organisms Or Cultivation Processes Thereof (AREA)
Abstract
一种缩短植物细胞悬浮培养周期的方法，本发明属于生物技术领域。首先，选定p53基因的两个保守区分别为正向片段I、II以及对应的反向序列I’和II’；构建I-GU-II’-II-AG-I’的RNAi片段；筛选出其插在pCAMBIA2300植物表达载体的根瘤农杆菌工程菌。然后，以甘草为例，农杆菌介导法依次获得p53转化的愈伤组织及其稳定表达的悬浮细胞。再次，通过看护培养，利用悬浮细胞，筛选生长速度快、效果好的单细胞系愈伤组织以及由此形成的悬浮细胞系。最后，通过培养工艺优化，获得悬浮细胞生长和产物积累的最佳发酵工艺参数。利用本发明方法获得的培养周期短、密度大、成本低、生产效率高的细胞系，为大规模开发和生产药用植物有效成分提供优质种源。The invention relates to a method for shortening the period of plant cell suspension culture, and the invention belongs to the field of biotechnology. First, the two conserved regions of the selected p53 gene are the forward fragment I, II and the corresponding reverse sequence I' and II'; construct the RNAi fragment of I-GU-II'-II-AG-I'; screen The Agrobacterium tumefaciens engineering bacteria inserted in the pCAMBIA2300 plant expression vector were obtained. Then, taking licorice as an example, p53-transformed calli and its stable-expressing suspension cells were sequentially obtained by the Agrobacterium-mediated method. Thirdly, through nursing culture, the suspension cells are used to screen the single cell line callus with fast growth rate and good effect and the suspension cell line formed thereby. Finally, the optimal fermentation process parameters for suspension cell growth and product accumulation were obtained through the optimization of the culture process. The cell line with short culture period, high density, low cost and high production efficiency obtained by the method of the invention provides high-quality provenance for large-scale development and production of active ingredients of medicinal plants.
Description
Technical field
Patent of the present invention belongs to biological technical field.
Background technology
Cell suspension culture is one of important means of production Secondary Metabolism of Plant thing.But key issues such as the link that the plant suspension cell speed of growth is slow, culture cycle has grown up to cell suspension culture is many, cost height.The present invention utilizes the RNAi principle, through reducing plant p53 expression of gene, shortens the plant suspension cell culture cycle to reach, improves the cell culture density purpose.
Summary of the invention
Make up the vegetable cell expression vector.
The nucleotide sequence of the plant p53 gene through comparison Genbank registration, the nucleotide sequence of selected its two each 30-100 of conserved regions base pairs is respectively forward fragment I and II, and serves as that foundation is provided with its corresponding reverse sequence (I ' and II ') respectively with it; According to two align, antitone sequence designs suitable primer, in the pcr amplification process, makes positive and negative fragment introduce restriction endonuclease sites (EcoR I and Hind III) and corresponding GU-AG intron end sequence respectively.After the process enzyme is cut, connects, transforms, cloned and identifies, obtained four segmental I-AC-II '-II-CT-I ' sequence product; This product is inserted into the pCAMBIA2300 plant expression vector, imports Agrobacterium competence EHA105, filters out the engineering bacteria that can transform plant.
With the Radix Glycyrrhizae is that example obtains the Radix Glycyrrhizae transformant.
Evoked callus from the epicotyl of Radix Glycyrrhizae seedling; Early stage callus and Agrobacterium engineering bacteria are cultivated altogether; In containing antibiotic substratum, select subsequently to cultivate, filter out resistant calli and carry out succeeding transfer culture; With loose resistant calli liquid medium within shaking culture; The culture continued of sieving is carried out suspension culture, obtains stable suspension cell.
The screening of high yield suspension cell line.
Utilize nurse cultivation means, the monoclonal callus of screening fast growth from suspension cell; With shaking culture in this callus liquid medium within; The culture continued of sieving is carried out suspension culture, obtains the suspension system of being made up of with small cell cluster unicellular; Cell growth cycle, the main metabolites content of this suspension system is carried out etc. identifying and stability test that the growth suspension cell of acquisition gets into extensive as seed.
The optimization of cell suspension culture base and culture condition.
Through the optimization of substratum and culture condition, obtain the fermentation parameters such as the righttest carbon nitrogen source, pH, temperature, dissolved oxygen amount and optimum inoculation amount of plant cell growth and product accumulation, for further carrying out the mass cell suspension culture foundation is provided.
Main application
The cell that culture cycle is short, cost is low, production efficiency is high that utilizes the inventive method to obtain will will brought into play keying action aspect large-scale development and the production active components in medicinal plant.
Embodiment
With the Radix Glycyrrhizae is the method that example-interference p53 gene prepares the short cell suspension system of growth cycle
Make up the vegetable cell expression vector
The nucleotide sequence of all the plant p53 genes through Genbank registration, the nucleotide sequence of selected each 50 base pair of its two conserved regions is respectively forward fragment I and II, and serve as that foundation is provided with its corresponding reverse sequence (I ' and II ') respectively with it; According to two align, antitone sequence designs suitable primer, in the pcr amplification process, makes positive and negative fragment introduce restriction endonuclease sites (EcoR I and Hind III) and corresponding GU-AG class intron end nucleotide sequence respectively.The result sees table 1.After the process enzyme is cut, connects, transforms, cloned and identifies, obtained four segmental I-AC-II '-II-CT-I ' sequence product; This product is inserted into the pCAMBIA2300 plant expression vector, imports competence Agrobacterium EHA105, filters out the engineering bacteria pCAM-RNAi-p53 of ability transformed plant cells.
The nucleotide sequence of table 1 p53 gene conservative region dna fragmentation and PCR primer thereof
Annotate: in the frame of table with underscore on Nucleotide be the nucleotide sequence of unexpansive plain gene.
Obtain the licorice cell transformant
Evoked callus from the epicotyl of Radix Glycyrrhizae seedling; Early stage callus and Agrobacterium engineering bacteria EHA105 cultivate altogether; In containing antibiotic substratum, select subsequently to cultivate; The PCR that the resistant calli that forms is carried out GUS histochemical stain, resistant gene identifies; To identifying that the male callus carries out succeeding transfer culture 2-5 time until formation loose type callus; With shaking culture in the loose resistant calli liquid medium within; The culture continued of sieving is carried out suspension culture, obtains stable suspension cell line; The PCR that the suspension cell that forms is carried out the proteic histochemical stain of reporter gene gus, antibiotics resistance gene identifies, to determine whether it is the suspension cell line of conversion.
Screening Potenlini high yield suspension cell line
Utilize the vigorous Radix Glycyrrhizae callus of division on filter paper, to nurse cultivation; The monoclonal callus of screening fast growth from suspension cell; With shaking culture in this callus liquid medium within; Culture carries out suspension culture through the continued of sieving, and obtains the suspension cell line of being made up of with small cell cluster unicellular; Cell growth cycle, the Potenlini equal size of this suspension system such as carried out at mensuration; Then carry out stability test, the final high yield suspension cell line that obtains gets into large scale culturing as seed.The result sees table 2.Can know that by table compare with non-transformed cell, the size of transformant, density, growth cycle and doubling time all are improved, wherein growth cycle shortens nearly 1 time-of-week.
The transformant in table 2 suspension culture and the comparison of contrast
| Average cell diameter/μ m | Cell density/10 5mL -1 | Growth cycle/sky | The doubling time/sky | |
| Non-conversion suspension cell | 47 | 21.64 | 25 | 1.32 |
| Transform suspension cell-1 | 37 | 40.94 | 20 | 0.77 |
| Transform suspension cell-2 | 40 | 30.39 | 17 | 0.91 |
| Transform suspension cell-3 | 33 | 37.99 | 18 | 0.81 |
| Transform suspension cell MV | 36.7* | 36.44** | 18.3** | 0.83** |
Annotate: * * representes to reach utmost point level of signification (p=0.01) with contrast ratio difference; * expression reaches level of signification (p=0.05) with contrast ratio difference.
The optimization of licorice cell suspension culture base and culture condition
Through the optimization of substratum and culture condition, obtain the fermentation parameters such as the righttest carbon nitrogen source, pH, temperature, dissolved oxygen amount and optimum inoculation amount of plant cell growth and product accumulation, for further carrying out the mass cell suspension culture foundation is provided.The result sees table 3.Can be known that by table 3 after substratum and culture condition were optimized, the doubling time significantly shortened, cell density and glycyrrhizic acid content also improve a lot.
The suspension culture base of table 3 transformant and the optimum result of culture condition
Claims (4)
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| CN201210229723XA CN102839190A (en) | 2012-07-05 | 2012-07-05 | Method for shortening plant cell suspension culture period |
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Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103923874A (en) * | 2014-03-25 | 2014-07-16 | 天津大学 | Pseudo-ginseng cell suspension culture method |
| CN111073819A (en) * | 2020-01-16 | 2020-04-28 | 厦门鹭港兆康生物科技有限公司 | Plant cell screening device and method for screening synchronized plant cell lines |
| CN113832176A (en) * | 2020-06-24 | 2021-12-24 | 中国科学院分子植物科学卓越创新中心 | A single-cell genetic transformation method and its application |
-
2012
- 2012-07-05 CN CN201210229723XA patent/CN102839190A/en active Pending
Non-Patent Citations (2)
| Title |
|---|
| 20100630 高洁等 基于植物p53保守序列构建RNAi的DNA片段方法 第19-226页 1 第25卷, 第3期 * |
| 高洁等: "基于植物p53保守序列构建RNAi的DNA片段方法", <天津科技大学> * |
Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103923874A (en) * | 2014-03-25 | 2014-07-16 | 天津大学 | Pseudo-ginseng cell suspension culture method |
| CN111073819A (en) * | 2020-01-16 | 2020-04-28 | 厦门鹭港兆康生物科技有限公司 | Plant cell screening device and method for screening synchronized plant cell lines |
| CN113832176A (en) * | 2020-06-24 | 2021-12-24 | 中国科学院分子植物科学卓越创新中心 | A single-cell genetic transformation method and its application |
| CN113832176B (en) * | 2020-06-24 | 2023-08-01 | 中国科学院分子植物科学卓越创新中心 | Single-cell genetic transformation method and application thereof |
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Application publication date: 20121226 |