A method for improving artemisinin content in Artemisia annua by transfecting iaam gene

Abstract

The invention discloses a method of improving content of artemisinin in artemisia annua by a trans-iaaM gene. The method comprises the following steps: constructing a carrier of the iaaM gene which is specifically expressed in a gland hair cell of artemisia annua, and converting the artemisia annua by means of an agrobacterium tumefaciens mediation method to obtain a transgenetic artemisia annua plant through PCR (Polymerase Chain Reaction) detection; carrying out PCR gene amplification detection on the transgenetic artemisia annua plant and observing and analyzing condition of glandular hair cell of transgenetic artemisia annua and content of artemisinin; screening the transgenetic artemisia annua plant with the content of artemisinin being remarkably improved; and establishing a transgenetic artemisia annua strain with high content of artemisinin through propagation by tissue culture. The content of artemisinin in the transgenetic artemisia annua obtained by the method disclosed by the invention is remarkably improved, reaching 1.5 times of that of uninverted artemisia annua at the highest. The method disclosed by the invention is significant for providing high-yielding and stable new medicine sources to scaled production of artemisinin.

Description

Technical field

[0001] The invention relates to a method for increasing the content of artemisinin belonging to the field of biotechnology, in particular to a method for increasing the content of artemisinin in Artemisia annua by transducing iaaM genes. Background technique

[0002] Artemisinin is a peroxy group-containing sesquiterpene lactone drug extracted from the plant Artemisia annua. It is the most ideal drug for malaria treatment today and can effectively treat Plasmodium infections that are resistant to the traditional drug chloroquine. Artemisinin is mainly obtained by directly extracting from Artemisia annua, or by extracting artemisinic acid, which has a higher content in Artemisia annua, and then semi-synthesized. Except for Artemisia annua and Artemisia annua, no other natural plant resources containing artemisinin have been discovered. Therefore, Artemisia annua is still the only drug source for the production of artemisinin drugs.

[0003] Although Artemisia annua...

Examples

Embodiment 1

[0036] Acquisition of iaaM gene

[0037] Obtain the iaaM gene sequence encoding tryptophan monooxygenase (accession number U83987.1) from the Nucleotide database in GenBank, and send it to Shenzhen Huada Gene Company to synthesize the gene pMD18-iaaM, and transform the gene pMD18-iaaM Store in E.coliDH5α.

[0038] The specific steps are:

[0039] 1) Design specific primers for the iaaM gene to introduce BamHI and PstI restriction sites

[0040] Upstream primer iaaMUP: 5′-GCGGATCCATGTCAGCCTCATCTCTTCT-3′

[0041] Downstream primer iaaMDN: 5'-AACTGCAGTTAATTTCTATTGCGGTAGTTATATC-3'

[0042] 2) Using pMD18-iaaM as template and iaaMUP and iaaMDN as primers, PCR technology was used to amplify the iaaM gene. The PCR reaction system is: pMD-iaaM plasmid (about 100ng) 1uL, 10×Buffer 2.5uL, dNTPs (10mmol / L) 1uL, MgCl 2 (25mmol / L) 1.5uL, iaaMUP (10umol / L) 1uL, iaaMDN (10umol / L) 1uL, TaqDNA polymerase (1U / uL) 1uL, supplement ddH 2 O to 25uL. The reaction procedure is: 95°C pre-denaturation for 4 mi...

Embodiment 2

[0046] Construction of a vector for specific expression of iaaM gene in glandular hair cells of Artemisia annua

[0047] The Arabidopsis Gl2 gene core sequence (accession number: L32873.1) was obtained from the Nucleotide database in GenBank, and the following two specific primers were designed according to its nucleotide sequence:

[0048] The upstream primer is 1Gl2:5′-CCCAAGCTTTTTCCTTCACTATACGTCTTCG-3′

[0049] The downstream primer is 2Gl2:5′-CGCGGATCCCAAATCCTGTCCCTAGCTAG-3′

[0050] The Arabidopsis genomic DNA was amplified to obtain a Gl2 promoter fragment. The iaaM gene was recombined with the Gl2 gene promoter and cloned into the HindIII and PstI sites of the pBI121 plasmid in the T-DNA region of the Ti plasmid. The NPT-II gene was used as the selectable marker gene, and the vector pBI121 was used as the starting vector for transformation and vector construction. The recombinant Ti plasmid of pBI121-Gl2-iaaM was obtained, and the plasmid was extracted for PRC detection and re...

Embodiment 3

[0053] 1. Preparation of engineered Agrobacterium tumefaciens strain

[0054] The constructed recombinant Ti plasmid is transformed into Agrobacterium tumefaciens LBA4404 by the electric shock transformation method. The electric shock transformation method is that 0.1ug of the prepared recombinant Ti plasmid DNA is mixed with 1 mL of competent Agrobacterium tumefaciens and placed in an electric shock cup for electric shock transformation. The instrument performs electrical shock transformation in bacterial transformation mode. The transformed Agrobacterium tumefaciens was spread on YEB medium containing kanamycin 50ug / mL and rifampicin 20ug / mL for selection, and the selected Agrobacterium tumefaciens was cultured in YEB liquid medium at 28°C in a shake flask overnight To the late logarithmic growth period, inoculate the expanded culture system according to 1:10 and continue to shake the flask for 4 hours. The results showed that the constructed vector for specific expression of ...

Technical field

[0001] The invention relates to a method for increasing the content of artemisinin belonging to the field of biotechnology, in particular to a method for increasing the content of artemisinin in Artemisia annua by transducing iaaM genes. Background technique

[0002] Artemisinin is a peroxy group-containing sesquiterpene lactone drug extracted from the plant Artemisia annua. It is the most ideal drug for malaria treatment today and can effectively treat Plasmodium infections that are resistant to the traditional drug chloroquine. Artemisinin is mainly obtained by directly extracting from Artemisia annua, or by extracting artemisinic acid, which has a higher content in Artemisia annua, and then semi-synthesized. Except for Artemisia annua and Artemisia annua, no other natural plant resources containing artemisinin have been discovered. Therefore, Artemisia annua is still the only drug source for the production of artemisinin drugs.

[0003] Although Artemisia annua...

Examples

Embodiment 1

[0036] Acquisition of iaaM gene

[0037] Obtain the iaaM gene sequence encoding tryptophan monooxygenase (accession number U83987.1) from the Nucleotide database in GenBank, and send it to Shenzhen Huada Gene Company to synthesize the gene pMD18-iaaM, and transform the gene pMD18-iaaM Store in E.coliDH5α.

[0038] The specific steps are:

[0039] 1) Design specific primers for the iaaM gene to introduce BamHI and PstI restriction sites

[0040] Upstream primer iaaMUP: 5′-GCGGATCCATGTCAGCCTCATCTCTTCT-3′

[0041] Downstream primer iaaMDN: 5'-AACTGCAGTTAATTTCTATTGCGGTAGTTATATC-3'

[0042] 2) Using pMD18-iaaM as template and iaaMUP and iaaMDN as primers, PCR technology was used to amplify the iaaM gene. The PCR reaction system is: pMD-iaaM plasmid (about 100ng) 1uL, 10×Buffer 2.5uL, dNTPs (10mmol / L) 1uL, MgCl 2 (25mmol / L) 1.5uL, iaaMUP (10umol / L) 1uL, iaaMDN (10umol / L) 1uL, TaqDNA polymerase (1U / uL) 1uL, supplement ddH 2 O to 25uL. The reaction procedure is: 95°C pre-denaturation for 4 mi...

Embodiment 2

[0046] Construction of a vector for specific expression of iaaM gene in glandular hair cells of Artemisia annua

[0047] The Arabidopsis Gl2 gene core sequence (accession number: L32873.1) was obtained from the Nucleotide database in GenBank, and the following two specific primers were designed according to its nucleotide sequence:

[0048] The upstream primer is 1Gl2:5′-CCCAAGCTTTTTCCTTCACTATACGTCTTCG-3′

[0049] The downstream primer is 2Gl2:5′-CGCGGATCCCAAATCCTGTCCCTAGCTAG-3′

[0050] The Arabidopsis genomic DNA was amplified to obtain a Gl2 promoter fragment. The iaaM gene was recombined with the Gl2 gene promoter and cloned into the HindIII and PstI sites of the pBI121 plasmid in the T-DNA region of the Ti plasmid. The NPT-II gene was used as the selectable marker gene, and the vector pBI121 was used as the starting vector for transformation and vector construction. The recombinant Ti plasmid of pBI121-Gl2-iaaM was obtained, and the plasmid was extracted for PRC detection and re...

Embodiment 3

[0053] 1. Preparation of engineered Agrobacterium tumefaciens strain

[0054] The constructed recombinant Ti plasmid is transformed into Agrobacterium tumefaciens LBA4404 by the electric shock transformation method. The electric shock transformation method is that 0.1ug of the prepared recombinant Ti plasmid DNA is mixed with 1 mL of competent Agrobacterium tumefaciens and placed in an electric shock cup for electric shock transformation. The instrument performs electrical shock transformation in bacterial transformation mode. The transformed Agrobacterium tumefaciens was spread on YEB medium containing kanamycin 50ug / mL and rifampicin 20ug / mL for selection, and the selected Agrobacterium tumefaciens was cultured in YEB liquid medium at 28°C in a shake flask overnight To the late logarithmic growth period, inoculate the expanded culture system according to 1:10 and continue to shake the flask for 4 hours. The results showed that the constructed vector for specific expression of ...