Development of an efficient Agrobacterium-mediated transformation method and its application in tryptophan pathway modification in Catharanthus roseus
The biosynthetic pathway of Catharanthus roseus vinca alkaloids has a long research history, including not only identification of metabolic intermediates but also the mechanisms of inter-cellular transport and accumulation of biosynthesized components. Vinca alkaloids pathway begins with strictosidi...
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| Published in | Plant Biotechnology Vol. 40; no. 4; pp. 311 - 320 |
|---|---|
| Main Authors | , , , , , , |
| Format | Journal Article |
| Language | English Japanese |
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Japan
Japanese Society for Plant Biotechnology
25.12.2023
Japan Science and Technology Agency |
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| Abstract | The biosynthetic pathway of Catharanthus roseus vinca alkaloids has a long research history, including not only identification of metabolic intermediates but also the mechanisms of inter-cellular transport and accumulation of biosynthesized components. Vinca alkaloids pathway begins with strictosidine, which is biosynthesized by condensing tryptamine from the tryptophan pathway and secologanin from the isoprenoid pathway. Therefore, increasing the supply of precursor tryptophan may enhance vinca alkaloid content or their metabolic intermediates. Many reports on the genetic modification of C. roseus use cultured cells or hairy roots, but few reports cover the production of transgenic plants. In this study, we first investigated a method for stably producing transgenic plants of C. roseus, then, using this technique, we modified the tryptophan metabolism system to produce transgenic plants with increased tryptophan content. Transformed plants were obtained by infecting cotyledons two weeks after sowing with Agrobacterium strain A13 containing a plant expression vector, then selecting with 1/2 B5 medium supplemented with 50 mg l−1 kanamycin and 20 mg l−1 meropenem. Sixty-eight regenerated plants were obtained from 4,200 cotyledons infected with Agrobacterium, after which genomic PCR analysis using NPTII-specific primers confirmed gene presence in 24 plants with a transformation rate of 0.6%. Furthermore, we performed transformation into C. roseus using an expression vector to join trpE8 and aroG4 genes, which are feedback-resistant mutant genes derived from Escherichia coli. The resulting transformed plants showed exactly the same morphology as the wild-type, albeit with a marked increase in tryptophan and alkaloids content, especially catharanthine in leaves. |
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| AbstractList | The biosynthetic pathway of
Catharanthus roseus
vinca alkaloids has a long research history, including not only identification of metabolic intermediates but also the mechanisms of inter-cellular transport and accumulation of biosynthesized components. Vinca alkaloids pathway begins with strictosidine, which is biosynthesized by condensing tryptamine from the tryptophan pathway and secologanin from the isoprenoid pathway. Therefore, increasing the supply of precursor tryptophan may enhance vinca alkaloid content or their metabolic intermediates. Many reports on the genetic modification of
C. roseus
use cultured cells or hairy roots, but few reports cover the production of transgenic plants. In this study, we first investigated a method for stably producing transgenic plants of
C. roseus
, then, using this technique, we modified the tryptophan metabolism system to produce transgenic plants with increased tryptophan content. Transformed plants were obtained by infecting cotyledons two weeks after sowing with
Agrobacterium
strain A13 containing a plant expression vector, then selecting with 1/2 B5 medium supplemented with 50 mg l
−1
kanamycin and 20 mg l
−1
meropenem. Sixty-eight regenerated plants were obtained from 4,200 cotyledons infected with
Agrobacterium
, after which genomic PCR analysis using
NPTII
-specific primers confirmed gene presence in 24 plants with a transformation rate of 0.6%. Furthermore, we performed transformation into
C. roseus
using an expression vector to join
trpE8
and
aroG4
genes, which are feedback-resistant mutant genes derived from
Escherichia coli
. The resulting transformed plants showed exactly the same morphology as the wild-type, albeit with a marked increase in tryptophan and alkaloids content, especially catharanthine in leaves. The biosynthetic pathway of vinca alkaloids has a long research history, including not only identification of metabolic intermediates but also the mechanisms of inter-cellular transport and accumulation of biosynthesized components. Vinca alkaloids pathway begins with strictosidine, which is biosynthesized by condensing tryptamine from the tryptophan pathway and secologanin from the isoprenoid pathway. Therefore, increasing the supply of precursor tryptophan may enhance vinca alkaloid content or their metabolic intermediates. Many reports on the genetic modification of use cultured cells or hairy roots, but few reports cover the production of transgenic plants. In this study, we first investigated a method for stably producing transgenic plants of , then, using this technique, we modified the tryptophan metabolism system to produce transgenic plants with increased tryptophan content. Transformed plants were obtained by infecting cotyledons two weeks after sowing with strain A13 containing a plant expression vector, then selecting with 1/2 B5 medium supplemented with 50 mg l kanamycin and 20 mg l meropenem. Sixty-eight regenerated plants were obtained from 4,200 cotyledons infected with , after which genomic PCR analysis using -specific primers confirmed gene presence in 24 plants with a transformation rate of 0.6%. Furthermore, we performed transformation into using an expression vector to join and genes, which are feedback-resistant mutant genes derived from . The resulting transformed plants showed exactly the same morphology as the wild-type, albeit with a marked increase in tryptophan and alkaloids content, especially catharanthine in leaves. The biosynthetic pathway of Catharanthus roseus vinca alkaloids has a long research history, including not only identification of metabolic intermediates but also the mechanisms of inter-cellular transport and accumulation of biosynthesized components. Vinca alkaloids pathway begins with strictosidine, which is biosynthesized by condensing tryptamine from the tryptophan pathway and secologanin from the isoprenoid pathway. Therefore, increasing the supply of precursor tryptophan may enhance vinca alkaloid content or their metabolic intermediates. Many reports on the genetic modification of C. roseus use cultured cells or hairy roots, but few reports cover the production of transgenic plants. In this study, we first investigated a method for stably producing transgenic plants of C. roseus, then, using this technique, we modified the tryptophan metabolism system to produce transgenic plants with increased tryptophan content. Transformed plants were obtained by infecting cotyledons two weeks after sowing with Agrobacterium strain A13 containing a plant expression vector, then selecting with 1/2 B5 medium supplemented with 50 mg l−1 kanamycin and 20 mg l−1 meropenem. Sixty-eight regenerated plants were obtained from 4,200 cotyledons infected with Agrobacterium, after which genomic PCR analysis using NPTII-specific primers confirmed gene presence in 24 plants with a transformation rate of 0.6%. Furthermore, we performed transformation into C. roseus using an expression vector to join trpE8 and aroG4 genes, which are feedback-resistant mutant genes derived from Escherichia coli. The resulting transformed plants showed exactly the same morphology as the wild-type, albeit with a marked increase in tryptophan and alkaloids content, especially catharanthine in leaves. The biosynthetic pathway of Catharanthus roseus vinca alkaloids has a long research history, including not only identification of metabolic intermediates but also the mechanisms of inter-cellular transport and accumulation of biosynthesized components. Vinca alkaloids pathway begins with strictosidine, which is biosynthesized by condensing tryptamine from the tryptophan pathway and secologanin from the isoprenoid pathway. Therefore, increasing the supply of precursor tryptophan may enhance vinca alkaloid content or their metabolic intermediates. Many reports on the genetic modification of C. roseus use cultured cells or hairy roots, but few reports cover the production of transgenic plants. In this study, we first investigated a method for stably producing transgenic plants of C. roseus, then, using this technique, we modified the tryptophan metabolism system to produce transgenic plants with increased tryptophan content. Transformed plants were obtained by infecting cotyledons two weeks after sowing with Agrobacterium strain A13 containing a plant expression vector, then selecting with 1/2 B5 medium supplemented with 50 mg l-1 kanamycin and 20 mg l-1 meropenem. Sixty-eight regenerated plants were obtained from 4,200 cotyledons infected with Agrobacterium, after which genomic PCR analysis using NPTII-specific primers confirmed gene presence in 24 plants with a transformation rate of 0.6%. Furthermore, we performed transformation into C. roseus using an expression vector to join trpE8 and aroG4 genes, which are feedback-resistant mutant genes derived from Escherichia coli. The resulting transformed plants showed exactly the same morphology as the wild-type, albeit with a marked increase in tryptophan and alkaloids content, especially catharanthine in leaves. |
| ArticleNumber | 23.0819a |
| Author | Uchiyama, Sato Iyo, Mayu Chin, Dong Poh Miwa, Tetsuya Kisaka, Hiroaki Mii, Masahiro Hirano, Hiroto |
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| Cites_doi | 10.1080/17429145.2013.838312 10.1007/s13205-016-0593-5 10.1186/1471-2229-10-182 10.1074/jbc.M112.359976 10.1021/ja809842b 10.1128/JB.00582-07 10.1104/pp.116.2.853 10.1111/j.1365-313X.2008.03438.x 10.1111/j.1399-3054.1962.tb08052.x 10.1016/0022-2836(80)90029-7 10.1007/s00709-014-0685-1 10.1128/aem.63.2.761-762.1997 10.1038/nplants.2016.208 10.1039/c005378c 10.1007/10_2013_182 10.1016/j.copbio.2012.08.003 10.2174/0929867043455846 10.1073/pnas.1307504110 10.1093/pcp/pcx097 10.1038/ncomms4606 10.1002/bit.20739 10.1186/1471-2229-13-155 10.1007/s00253-010-2794-x 10.1105/tpc.6.10.1441 10.1073/pnas.0812512106 10.1093/jxb/45.5.649 10.1016/0014-4827(68)90403-5 10.1073/pnas.0911451107 10.1073/pnas.1501821112 10.1105/tpc.107.056630 10.1104/pp.107.3.679 10.1186/1472-6750-12-34 10.1002/bit.20081 10.1021/bp050210l |
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| Copyright | 2023 Japanese Society for Plant Biotechnology 2023 Japanese Society for Plant Biotechnology. 2023. This work is published under https://creativecommons.org/licenses/by/4.0/ (the “License”). Notwithstanding the ProQuest Terms and Conditions, you may use this content in accordance with the terms of the License. 2023 Japanese Society for Plant Biotechnology 2023 Japanese Society for Plant Biotechnology |
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| Keywords | Agrobacterium rhizogenes A13 Catharanthus roseus transformation vinca alkaloid tryptophan |
| Language | English Japanese |
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| SubjectTerms | Agrobacterium Agrobacterium rhizogenes A13 Alkaloids Catharanthus roseus Cotyledons E coli Genes Genetic modification Genetic transformation Genomic analysis Hairy root Intermediates Kanamycin Meropenem Metabolism Mevalonate pathway Nucleotide sequence Original Paper Plant bacterial diseases Plants Resistant mutant transformation Transgenic plants Tryptamine Tryptamines Tryptophan vinca alkaloid |
| Title | Development of an efficient Agrobacterium-mediated transformation method and its application in tryptophan pathway modification in Catharanthus roseus |
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| ispartofPNX | Plant Biotechnology, 2023/12/25, Vol.40(4), pp.311-320 |
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