CN105695507B - Method for improving artemisinin content in sweet wormwood herb by transferring ICS1 gene - Google Patents
Method for improving artemisinin content in sweet wormwood herb by transferring ICS1 gene Download PDFInfo
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- CN105695507B CN105695507B CN201610272336.2A CN201610272336A CN105695507B CN 105695507 B CN105695507 B CN 105695507B CN 201610272336 A CN201610272336 A CN 201610272336A CN 105695507 B CN105695507 B CN 105695507B
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- C12N9/90—Isomerases (5.)
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- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
- A01H—NEW PLANTS OR NON-TRANSGENIC PROCESSES FOR OBTAINING THEM; PLANT REPRODUCTION BY TISSUE CULTURE TECHNIQUES
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- C12N15/09—Recombinant DNA-technology
- C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
- C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
- C12N15/82—Vectors or expression systems specially adapted for eukaryotic hosts for plant cells, e.g. plant artificial chromosomes (PACs)
- C12N15/8201—Methods for introducing genetic material into plant cells, e.g. DNA, RNA, stable or transient incorporation, tissue culture methods adapted for transformation
- C12N15/8202—Methods for introducing genetic material into plant cells, e.g. DNA, RNA, stable or transient incorporation, tissue culture methods adapted for transformation by biological means, e.g. cell mediated or natural vector
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- C12Y—ENZYMES
- C12Y504/00—Intramolecular transferases (5.4)
- C12Y504/04—Intramolecular transferases (5.4) transferring hydroxy groups (5.4.4)
- C12Y504/04002—Isochorismate synthase (5.4.4.2)
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Abstract
The invention relates to a method for improving the content of artemisinin in sweet wormwood herb by transferring an ICS1 gene, belonging to the technical field of biology. The invention clones isochorismate synthase ICS1 gene from Artemisia annua, constructs plant expression vector containing ICS1 gene, transfers ICS1 gene into Artemisia annua and regenerates plant by mediating with agrobacterium tumefaciens, detects integration condition of exogenous target gene ICS1 by PCR, measures content of artemisinin in transgenic Artemisia annua by high performance liquid chromatography and evaporative light scattering detector (HPLC-ELSD), and screens to obtain transgenic Artemisia annua plant with increased content of artemisinin. The artemisinin content in the transgenic artemisia apiacea obtained by the method is remarkably improved and reaches 1.90 times of that of a non-transformed control plant at most, so that the method for improving the artemisinin content in the artemisia apiacea is provided, and a foundation is laid for large-scale production of artemisinin by utilizing the transgenic artemisia apiacea.
Description
Technical Field
The invention relates to a method for improving artemisinin content in the field of biotechnology, in particular to a method for improving artemisinin content in sweet wormwood herb by transferring ICS1 gene.
Background
Artemisia annua L is an annual herb of Artemisia of Compositae. The sesquiterpene lactone oxide containing peroxide bridge extracted from the aerial parts of the plant, namely artemisinin, is the antimalarial drug which is the most widely applied and has the best curative effect at present, and is particularly more effective for treating cerebral malaria and chloroquine malaria. Artemisinin Combination Therapies (ACTs) are currently the most effective treatment for malaria recommended by the world health organization. With the progress of pharmacological research on artemisinin, scientists found that artemisinin and its derivatives also have anti-inflammatory, anti-tumor, anti-cancer and immunomodulatory effects.
However, the content of artemisinin in the plant artemisia apiacea is very low, the large-scale commercial production is limited, and the global market demand cannot be completely met. The artemisinin has complex structure, high artificial synthesis difficulty, low yield, high cost and no feasibility. The production of the artemisinin by the yeast engineering has the disadvantages of high early investment cost and limited yield and cannot meet the requirements. The prior art shows that plant genetic engineering provides a feasible method for improving the content of artemisinin in sweet wormwood herb.
Artemisia annua has secretory glandular hairs (glandular trichomes) and non-secretory glandular hairs (nonglandular trichomes). Secretory glandular hairs are abundantly present on the front and back of the leaves of Artemisia annua, stems, flowers, where a large amount of secondary metabolites accumulate, and artemisinin is also thought to be stored.
Salicylic Acid (SA) is an important plant hormone, plays a very important role in the growth and development of plants, and is closely related to the disease-resistant defense of plants. Isochorismate synthase (ICS) is one of the key enzymes in the salicylic acid synthesis pathway in plants, and is responsible for converting chorismate into Isochorismate, which is then further synthesized into the final product salicylic acid. The ICS1 gene is associated with stress resistance regulated by salicylic acid. The ICS1 mutant study shows that the endogenous salicylic acid content of the plant with deletion of the ICS1 gene is also extremely low. Therefore, cloning the ICS1 gene and transforming the artemisia apiacea has important significance for researching the metabolic coordination of a salicylic acid synthesis pathway and an artemisinin pathway and breeding by genetic engineering.
The invention aims to obtain the sweet wormwood plant with high artemisinin yield by adopting a genetic engineering means and provides a new way for large-scale production of artemisinin.
Disclosure of Invention
The invention aims to overcome the defects in the prior art and provides a method for improving the content of artemisinin in sweet wormwood herb by transferring an ICS1 gene. The gene cloning, vector construction, genetic transformation, molecular detection, artemisinin extraction and content determination are used in the invention, so that a method for stably improving the content of artemisinin in artemisia apiacea is established, and a foundation is laid for large-scale production of artemisinin by using artemisia apiacea.
The invention provides a method for improving the content of artemisinin in sweet wormwood herb by transferring an ICS1 gene, which comprises the following steps:
(1) obtaining an ICS1 gene by adopting a gene cloning method;
(2) connecting the ICS1 gene to an expression regulatory sequence to construct a plant expression vector containing an ICS1 gene;
(3) transforming the plant expression vector containing the ICS1 gene into agrobacterium tumefaciens to obtain an agrobacterium tumefaciens strain containing the plant expression vector containing the ICS1 gene;
(4) and transforming the sweet wormwood by using the agrobacterium tumefaciens strain containing the ICS1 gene plant expression vector to obtain a transgenic sweet wormwood plant which is detected by PCR and integrates the exogenous target gene ICS 1.
Further, the DNA sequence of the ICS1 gene is shown in SEQ ID NO. 1.
Further, the amino acid sequence encoded by the ICS1 gene is shown in SEQ ID NO. 2.
Further, in the step (1), the gene cloning method comprises the steps of: extracting total genome RNA of the southernwood, carrying out reverse transcription on the obtained total genome RNA of the southernwood by a reverse transcriptase XL to obtain a first strand cDNA, and carrying out PCR amplification by taking the first strand cDNA as a template and taking an upstream primer and a downstream primer as a primer pair, wherein the DNA sequence of the upstream primer is shown as SEQ ID NO.3, and the DNA sequence of the downstream primer is shown as SEQ ID NO. 4.
Preferably, in the step (1), the gene cloning method comprises the steps of: extracting the total RNA of the genome of the southernwood, carrying out reverse transcription on the obtained total RNA of the genome of the southernwood by a reverse transcriptase XL to obtain a first strand cDNA, designing and amplifying an upstream primer and a downstream primer of a complete coding frame according to a DNA sequence shown by SEQ ID No.1, wherein the upstream primer is the DNA sequence shown by SEQ ID No.3, the downstream primer is the DNA sequence shown by SEQ ID No.4, the cDNA of the first strand is used as a template, the upstream primer and the downstream primer are used as a primer pair, and sequencing is carried out after PCR amplification.
Further, in the step (2), the construction of the plant expression vector containing the ICS1 gene comprises the following steps: firstly constructing an intermediate vector PJET-ICS1, then carrying out enzyme digestion on the intermediate vector PJET-ICS1 and an expression vector pHB-n-flag, recovering an ICS1 gene segment and a pHB-n-flag vector large segment, carrying out connection transformation, selecting a single clone, extracting a plasmid, and carrying out PCR detection and enzyme digestion verification to obtain the plant expression vector containing the ICS1 gene. pHB-n-flag is obtained by inserting flag into pHB201 expression vector after 35s promoter, and storing.
Further, in the step (2), the construction of the plant expression vector containing the ICS1 gene comprises the following steps: firstly, connecting an ICS1 gene fragment obtained by gene cloning in the step (1) to a vector PJET through ligase to obtain an intermediate vector PJET-ICS 1; designing an upstream primer and a downstream primer for introducing a restriction enzyme site Sac I, wherein the DNA sequence of the upstream primer for introducing the restriction enzyme site Sac I is shown as SEQ ID NO.5, the DNA sequence of the downstream primer for introducing the restriction enzyme site Sac I is shown as SEQ ID NO.6, and carrying out PCR amplification by taking the intermediate vector PJET-ICS1 as a template and the upstream primer and the downstream primer for introducing the restriction enzyme site Sac I as a primer pair to obtain a product 1; carrying out enzyme digestion on the product 1 and an expression vector PHB-n-flag by using Sac I to obtain an ICS1 gene segment with an enzyme digestion site Sac I and a PHB-n-flag vector large segment; and recovering the ICS1 gene segment with the restriction enzyme site Sac I and the PHB-n-flag vector large segment, connecting and transforming, selecting a single clone, and extracting a plasmid for PCR detection and restriction enzyme digestion verification to obtain the plant expression vector containing the ICS1 gene.
Further, in the step (4), the converting includes the steps of: pre-culturing explants; co-culturing agrobacterium and explants; screening resistant regeneration plants; wherein,
the pre-culture comprises the following steps: soaking herba Artemisiae Annuae seed in 75% ethanol for 1min, soaking in 20% NaClO for 20min, washing with sterile water for 3-4 times, removing surface water with sterile absorbent paper, inoculating in hormone-free MS solid culture medium, culturing at 25 deg.C under illumination to obtain herba Artemisiae Annuae sterile seedling, and cutting sterile seedling leaf explant for transformation when the seedling grows to about 5 cm;
the co-culture comprises the following steps: transferring the leaf explant into a co-culture medium, dropwise adding 1/2MS suspension of the agrobacterium tumefaciens engineering bacteria containing the activated plant binary expression vector containing the ICS1 gene to ensure that the explant is fully contacted with a bacterial liquid, carrying out dark culture at 28 ℃ for 3 days, and taking the leaf explant dropwise added to 1/2MS liquid culture medium suspension of agrobacterium tumefaciens without a target gene as a control;
the screening comprises the following steps: transferring the sweet wormwood herb explants cultured for 3 days in the co-culture process to a germination screening medium for illumination culture at 25 ℃, carrying out subculture once every two weeks, obtaining hygromycin (Hyg) resistant cluster buds after carrying out subculture for 2-3 times, shearing the well-grown Hyg resistant cluster buds, transferring the sheared cluster buds to a rooting medium for culture until rooting, and obtaining the Hyg resistant regeneration sweet wormwood herb plants.
Further, in the step (4), the PCR detection includes the steps of: designing a forward primer and a reverse primer according to the expression cassettes p35s-ICS1-nos sequence p35s and ICS1 of the target gene respectively; carrying out DNA amplification; observing under ultraviolet rays, and if the target strip is positive, the plant line is the transgenic southernwood plant; the DNA sequence of the forward primer is shown as SEQ ID NO.5, and the DNA sequence of the reverse primer is shown as SEQ ID NO. 4.
Further, the method also comprises the step (5) of carrying out high performance liquid chromatography and evaporative light scattering detector determination (HPLC-ELSD) determination on the artemisinin content in the transgenic artemisia apiacea, and screening to obtain transgenic artemisia apiacea plants with increased artemisinin content.
Preferably, in the step (5), the HPLC-ELSD assay comprises the following conditions: the chromatographic column is a C-18 reverse phase silica gel column, the mobile phase adopts methanol and water with the volume ratio of 70:30, the column temperature is 30 ℃, the flow rate is 1.0mL/min, the sample injection amount is 20 mu L, the temperature of a drift tube of an evaporative light scattering detector is 40 ℃, the amplification factor is 7, and the carrier gas pressure is 5 bar.
The method for improving the content of the artemisinin in the artemisia apiacea by transferring the ICS1 gene adopts a genetic engineering method to introduce the isochorismate synthetase ICS1 gene into artemisia apiacea plants to obtain transgenic artemisia apiacea strains with the high artemisinin content remarkably improved, the content of the artemisinin in the artemisia apiacea transferred with the ICS1 gene can reach 15.2mg/g of dry weight to the maximum and is 1.90 times of that of non-transformed common artemisia apiacea (8mg/g), and the method has important significance for providing a high-yield and stable new medicine source for large-scale production of the artemisinin.
Drawings
FIG. 1 is a graph showing the results of measurement of artemisinin content in Artemisia annua carrying ICS1 transgene and non-transformed normal Artemisia annua.
Detailed Description
The technical contents of the present invention will be further described with reference to the accompanying drawings and examples. The following examples will assist those skilled in the art in further understanding the invention, but are not intended to limit the invention in any way. Experimental procedures without specific conditions noted in the following examples, generally followed by conventional conditions, such as molecular cloning in Sambrook et al: a Laboratory manual is described in New York, Cold Spring Harbor Laboratory Press, 1989 edition, or as recommended by the manufacturer.
The agrobacterium tumefaciens EHA105 related to the invention is already in Huang Yali, Jiang Jing Liang, Tian Yun Long, Guo Ping and Zhu Chang Xiong; study of agrobacterium tumefaciens-mediated genetic transformation of trichoderma harzianum, journal of biological engineering of china, 2008, 28 (3): 38-43, respectively. Agrobacterium tumefaciens EHA105 is commercially available from publicly available sources, such as the CAMBIA corporation, Australia, under the strain designation Gambar 1.
Example 1
Step one, cloning of an ICS1 gene of Artemisia apiacea
(1) Extraction of total RNA of sweet wormwood genome
The total RNA of Artemisia apiacea genome was extracted using an RNA kit from TIANGEN. Collecting 50-100mg of tender leaves of herba Artemisiae AnnuaeThe tablets were rapidly ground to a powder in liquid nitrogen, 550. mu.L of lysis solution RL (with mercaptoethanol added) was added, vortexed and shaken vigorously to mix. Centrifuging at 9800rpm for 5min, sucking 450 μ L, transferring to a filtration column CS, placing CS in a collection tube, centrifuging at 12000rpm for 5min, carefully sucking 400 μ L of supernatant in the collection tube into a RNase-free centrifuge tube, slowly adding anhydrous ethanol with the volume 0.5 times of that of the supernatant, mixing uniformly, transferring to an adsorption column CR3, centrifuging at 12000rpm for 1min, discarding waste liquid, and placing CR3 back into the collection tube. 350 μ L of deproteinizing solution RW1 was added to CR3, centrifuged at 12000rpm for 1min, the waste liquid was discarded, and CR3 was returned to the collection tube. 80 μ L of DNase1 working solution (10 μ L of Nase1 stock solution +70 μ L of LRDD solution, gently mixed and mixed) was added to the center of CR3, and the mixture was allowed to stand at room temperature for 15 minutes. 350 μ L of deproteinizing solution RW1 was added to CR3, centrifuged at 12000rpm for 1min, the waste liquid was discarded, and CR3 was returned to the collection tube. Add 500. mu.L of rinsing solution RW (ethanol added) to CR3, let stand at room temperature for 2 minutes, centrifuge at 12000rpm for 1min, remove the waste from the collection tube, replace CR3 back into the collection tube, and repeat once. Centrifuge at 12000rpm for 5 min. Standing CR3 at room temperature for 5min, adding CR3 into new RNase-free, and adding 70 μ LRNase-free dd H dropwise into the middle part of the adsorption membrane2And O, standing at room temperature for 2min, and centrifuging at 12000rpm for 2min to obtain an RNA solution. Putting 10 mu L of the gel into an RNase-free tubule for gel running and concentration measurement; the remaining 60. mu.L was stored in RNase-free centrifuge tubes at-80 ℃.
(2) Cloning of Artemisia apiacea ICS1 Gene
The obtained total RNA of the genome of Artemisia annua is subjected to reverse transcription by reverse transcriptase XL (AMV) to obtain first strand cDNA, and an upstream primer (a DNA sequence shown in SEQ ID NO. 3) and a downstream primer (a DNA sequence shown in SEQ ID NO. 4) of a complete coding frame are designed and synthesized according to a coding sequence (a DNA sequence shown in SEQ ID NO. 1) of the Artemisia annua ICS1 gene. And (3) taking the first strand cDNA as a template, and carrying out sequencing after PCR amplification. DNA sequencing was performed by Shanghai Sangni Biotech Ltd. The sequencing result showed that the cloned sequence (DNA sequence shown in SEQ ID NO. 1) had nonsense point mutation with the coding sequence of Artemisia annua ICS1 gene reported in GenBank, but they were completely identical in amino acid sequence.
The example adopts a gene cloning method to obtain the Artemisia carvifolia chalcone isomerase ICS1 gene with correct sequence from the Artemisia carvifolia, and provides an important gene for improving the content of artemisinin in the Artemisia carvifolia by transferring the ICS1 gene.
Step two, construction of plant binary vector containing ICS1 gene
(1) Construction of intermediate vector PJET-ICS1
An intermediate vector PJET-ICS1 is constructed by using a PJET vector (Takara, Inc., Dalian) as a basic element. Specifically, the ICS1 gene fragment (the DNA sequence of which is shown in SEQ ID NO. 1) obtained in the first step is connected to a PJET vector through ligase, and the correctness of the gene is confirmed by sequencing of Shanghai Sangni Biotech Co.
(2) Construction of plant expression vector containing ICS1 Gene
The ICS1 gene is linked to the corresponding restriction enzyme site position by using pHB-n-flag as an expression vector. Specifically, designing and synthesizing an upstream primer and a downstream primer which are introduced with a restriction enzyme site Sac I, wherein the DNA sequence of the upstream primer is shown as SEQ ID NO.5, the DNA sequence of the downstream primer is shown as SEQ ID NO.6, and taking the upstream primer and the downstream primer as a primer pair, and carrying out PCR amplification by taking the intermediate vector PJET-ICS1 as a template to obtain a product 1; carrying out enzyme digestion on the product 1 and an expression vector PHB-n-flag by using Sac I to obtain an ICS1 gene segment with an enzyme digestion site Sac I and a PHB-n-flag vector large segment; and recovering the ICS1 gene segment with the restriction enzyme site Sac I and the PHB-n-flag vector large segment, connecting and transforming, selecting a single clone, and extracting a plasmid for PCR detection and restriction enzyme digestion verification to obtain the plant expression vector containing the ICS1 gene.
In the embodiment, a salicylic acid synthesis pathway key enzyme gene ICS1 is operably connected with an expression regulatory sequence to form a plant expression vector containing an ICS1 gene, and the expression vector can be used for improving the content of artemisinin in artemisia apiacea by a metabolic engineering strategy.
Step three, obtaining the Agrobacterium tumefaciens of the binary plant expression vector containing the ICS1 gene
The plant binary expression vector containing the ICS1 gene is transferred into agrobacterium tumefaciens (such as EHA105, a biological material publicly sold in the market, which can be purchased from CAMBIA corporation of Australia, and the strain number is Gambar 1) and PCR verification is carried out.
Step four, agrobacterium tumefaciens mediated ICS1 gene transformation southernwood
(1) Pre-culture of explants
Soaking herba Artemisiae Annuae seed in 75% ethanol for 1min, soaking in 20% NaClO for 20min, washing with sterile water for 3-4 times, blotting surface water with sterile absorbent paper, inoculating in hormone-free MS (Murashige and Skoog, 1962) solid culture medium, and culturing at 25 deg.C under light/8 h (light/dark) to obtain herba Artemisiae Annuae aseptic seedling. After the seedling grows to about 5cm, shearing a sterile seedling leaf explant for transformation.
(2) Co-culture of Agrobacterium with explants
Transferring the leaf explant to a co-culture medium (1/2MS + AS100 mu mol/L) added with Acetosyringone (AS), dropwise adding 1/2MS suspension of the activated Agrobacterium tumefaciens engineering bacteria containing the plant binary expression vector of the ICS1 gene, fully contacting the explant with a bacterial solution, and performing dark culture at 28 ℃ for 3 days. Control was leaf explants dropped on 1/2MS liquid medium suspension of Agrobacterium tumefaciens without the gene of interest.
(3) Selection of resistant regenerated plants
Transferring the sweet wormwood herb explants cultured for 3 days in a co-culture manner to a germination screening medium (MS +6-BA0.5mg/L + NAA 0.05mg/L + Kan50mg/L + cb 500mg/L) added with 6-benzylaminopurine (6-BA), naphthylacetic acid (NAA), kanamycin (Kan) and carbenicillin (cb) for light culture at 25 ℃ for 16h/8h (light/dark), subculturing once every two weeks, obtaining Hyg resistant multiple shoots after subculturing for 2-3 times, cutting the well-grown resistant multiple shoots, transferring the cut resistant multiple shoots to a rooting medium (1/2MS + cb 125mg/L) for rooting, and obtaining Hyg resistant regenerated sweet wormwood herb plants.
Step five, PCR detection of transgenic southernwood plant
A forward primer (a DNA sequence shown as SEQ ID NO. 5) and a reverse primer (a DNA sequence shown as SEQ ID NO. 4) are respectively designed according to an expression cassette p35s-ICS1-nos sequence p35s and ICS1 where the target gene is located to detect the target gene. The result shows that the designed PCR specific primer can amplify specific DNA segment, and when non-transformed southernwood genome DNA is used as a template, no segment is amplified.
In this embodiment, the plant expression vector is transformed into agrobacterium tumefaciens to obtain an agrobacterium tumefaciens strain containing an ICS1 gene plant expression vector for transforming artemisia apiacea, and the constructed agrobacterium tumefaciens strain is used to transform artemisia apiacea to obtain a transgenic artemisia apiacea plant detected by PCR. The acquisition of transgenic southernwood plants provides direct materials for screening southernwood strains with higher artemisinin content.
Step six, determining the artemisinin content in the transgenic sweet wormwood herb by using HPLC-ELSD
(1) HPLC-ELSD conditions and system applicability and preparation of standard solution
HPLC: a water alliance 2695 system is adopted, a chromatographic column is a C-18 reverse phase silica gel column (symmeryshelled TM C18, 5 mu m, 250 multiplied by 4.6mm, Waters), a mobile phase is methanol and water, the volume ratio of the methanol to the water is 70:30, the column temperature is 30 ℃, the flow rate is 1.0mL/min, the sample injection amount is 10 mu L, the sensitivity (AUFS is 1.0), and the theoretical plate number is not lower than 2000 calculated according to an artemisinin peak.
ELSD: adopting a water alliance 2420 system, wherein the temperature of a drift tube of the evaporative light scattering detector is 40 ℃, the amplification factor (gain) is 7, and the carrier gas pressure is 5 bar;
accurately weighing 2.0mg of artemisinin standard (Sigma company), dissolving completely with 1mL of methanol to obtain 2mg/mL of artemisinin standard solution, and storing at-20 deg.C for use.
In the embodiment, the mobile phase is methanol and water, the volume ratio is 70 percent to 30 percent, the retention time of the artemisinin is 5.1min, and the peak pattern is good. The theoretical plate number is not less than 2000 calculated by artemisinin.
(2) Preparation of Standard Curve
And respectively injecting 2 mu L, 4 mu L, 6 mu L, 8 mu L and 10 mu L of the reference substance solution under corresponding chromatographic conditions to record a chromatogram and chromatographic parameters, and respectively performing regression analysis on the content (X, mu g) of the standard substance by using a peak area (Y). Through studies, artemisinin in this example exhibited a good log-log linear relationship in the 4-20 μ g range. The log-log linear regression equation of artemisinin reference substance is that Y is 5.404e +0000X +1.858e +0000, R2＝0.999184。
(3) Preparation of sample and determination of artemisinin content
Drying fresh folium Artemisiae Annuae in oven at 55 deg.C, grinding, adding 0.1g sample into 2mL EP tube, adding 1mL methanol, ultrasonic treating for 30min, centrifuging at 12000rpm for 10min, and collecting supernatant in new EP tube; adding 1mL of methanol into the precipitate, continuing ultrasonic treatment for 30min, centrifuging at 12000rpm for 10min, sucking the supernatant, mixing the two supernatants uniformly, and suction-filtering 1mL of the supernatant in a new EP tube.
And (3) measuring the content of artemisinin by adopting HPLC-ELSD, wherein the sample injection volume is 20 mu L, substituting the peak area into a linear regression equation to calculate the content (mg) of artemisinin in the sample, and dividing by the dry weight (g) of the artemisia apiacea leaves of the sample so as to calculate the content of artemisinin in the artemisia apiacea plants.
In the embodiment, the ICS1 gene remarkably improves the content of artemisinin in the sweet wormwood herb. As shown in FIG. 1, the content of artemisinin in the ICS1 transgenic Artemisia annua can reach 15.2mg/g of dry weight at most and is 1.90 times of that in non-transformed common Artemisia annua (8mg/g dry weight). CK in the figure represents non-transformed normal Artemisia annua; pHB-AaICS1-6, 15, 30 and 31 represent different transgenic lines.
In the embodiment, the content of artemisinin in transgenic artemisia apiacea is measured by an HPLC-ELSD method, and the artemisia apiacea plant with high artemisinin yield is obtained by a metabolic engineering strategy of transforming ICS1 gene, so that an ideal method is provided for large-scale production of artemisinin.
The foregoing detailed description of the preferred embodiments of the invention has been presented. It should be understood that numerous modifications and variations could be devised by those skilled in the art in light of the present teachings without departing from the inventive concepts. Therefore, the technical solutions available to those skilled in the art through logic analysis, reasoning and limited experiments based on the prior art according to the concept of the present invention should be within the scope of protection defined by the claims.
Claims (9)
1. A method for improving the content of artemisinin in sweet wormwood herb by transferring ICS1 gene is characterized by comprising the following steps:
(1) obtaining an ICS1 gene by adopting a gene cloning method; the DNA sequence of the ICS1 gene is shown as SEQ ID NO. 1;
(2) connecting the ICS1 gene to an expression regulatory sequence to construct a plant expression vector containing an ICS1 gene;
(3) transforming the plant expression vector containing the ICS1 gene into agrobacterium tumefaciens to obtain an agrobacterium tumefaciens strain containing the plant expression vector containing the ICS1 gene;
(4) and transforming the sweet wormwood by using the agrobacterium tumefaciens strain containing the ICS1 gene plant expression vector to obtain a transgenic sweet wormwood plant which is detected by PCR and integrates the exogenous target gene ICS 1.
2. The method for improving the content of artemisinin in artemisia apiacea by transferring ICS1 gene as claimed in claim 1, wherein the amino acid sequence coded by ICS1 gene is shown as SEQ ID No. 2.
3. The method for improving the content of artemisinin in artemisia apiacea by transferring ICS1 gene as claimed in claim 1, wherein in the step (1), the gene cloning method comprises the following steps: extracting total genome RNA of the southernwood, carrying out reverse transcription on the obtained total genome RNA of the southernwood by a reverse transcriptase XL to obtain a first strand cDNA, and carrying out PCR amplification by taking the first strand cDNA as a template and taking an upstream primer and a downstream primer as a primer pair, wherein the DNA sequence of the upstream primer is shown as SEQ ID NO.3, and the DNA sequence of the downstream primer is shown as SEQ ID NO. 4.
4. The method for improving the content of artemisinin in artemisia apiacea by transferring ICS1 gene as claimed in claim 1, wherein in the step (1), the gene cloning method comprises the following steps: extracting the total RNA of the genome of the southernwood, carrying out reverse transcription on the obtained total RNA of the genome of the southernwood by a reverse transcriptase XL to obtain a first strand cDNA, designing and synthesizing an upstream primer and a downstream primer of a complete coding frame according to a DNA sequence shown by SEQ ID NO.1, wherein the upstream primer is the DNA sequence shown by SEQ ID NO.3, the downstream primer is the DNA sequence shown by SEQ ID NO.4, the cDNA of the first strand is used as a template, the upstream primer and the downstream primer are used as a primer pair, and sequencing is carried out after PCR amplification.
5. The method for improving the content of artemisinin in artemisia apiacea by transferring ICS1 gene as claimed in claim 1, wherein in the step (2), the construction of the plant expression vector containing ICS1 gene comprises the following steps: firstly constructing an intermediate vector PJET-ICS1, then carrying out enzyme digestion on the intermediate vector PJET-ICS1 and an expression vector pHB-n-flag, recovering an ICS1 gene segment and a pHB-n-flag vector large segment, carrying out connection transformation, selecting a single clone, extracting a plasmid, and carrying out PCR detection and enzyme digestion verification to obtain the plant expression vector containing the ICS1 gene.
6. The method for improving the content of artemisinin in artemisia apiacea by transferring ICS1 gene as claimed in claim 1, wherein in the step (2), the construction of the plant expression vector containing ICS1 gene comprises the following steps: firstly, connecting an ICS1 gene fragment obtained by gene cloning in the step (1) to a vector PJET through ligase to obtain an intermediate vector PJET-ICS 1; designing an upstream primer and a downstream primer for introducing a restriction enzyme site Sac I, wherein the DNA sequence of the upstream primer for introducing the restriction enzyme site Sac I is shown as SEQ ID NO.5, the DNA sequence for introducing the restriction enzyme site Sac I is shown as SEQ ID NO.6, and carrying out PCR amplification by taking the intermediate vector PJET-ICS1 as a template and the upstream primer and the downstream primer for introducing the restriction enzyme site Sac I as a primer pair to obtain a product 1; carrying out enzyme digestion on the product 1 and an expression vector PHB-n-flag by using Sac I to obtain an ICS1 gene segment with an enzyme digestion site Sac I and a PHB-n-flag vector large segment; and recovering the ICS1 gene segment with the restriction enzyme site Sac I and the PHB-n-flag vector large segment, connecting and transforming, selecting a single clone, and extracting a plasmid for PCR detection and restriction enzyme digestion verification to obtain the plant expression vector containing the ICS1 gene.
7. The method for transforming ICS1 gene to increase the content of artemisinin in Artemisia annua as claimed in claim 1, wherein in the step (4), the transformation comprises the following steps: pre-culturing explants; co-culturing agrobacterium and explants; screening resistant regeneration plants; wherein,
the pre-culture comprises the following steps: soaking herba Artemisiae Annuae seed in 75% ethanol for 1min, soaking in 20% NaClO for 20min, washing with sterile water for 3-4 times, removing surface water with sterile absorbent paper, inoculating in hormone-free MS solid culture medium, culturing at 25 deg.C under illumination to obtain herba Artemisiae Annuae sterile seedling, and cutting sterile seedling leaf explant for transformation when the seedling grows to about 5 cm;
the co-culture comprises the following steps: transferring the leaf explant into a co-culture medium, dropwise adding 1/2MS suspension of the agrobacterium tumefaciens engineering bacteria containing the activated plant binary expression vector containing the ICS1 gene to ensure that the explant is fully contacted with a bacterial liquid, carrying out dark culture at 28 ℃ for 3 days, and taking the leaf explant dropwise added to 1/2MS liquid culture medium suspension of agrobacterium tumefaciens without a target gene as a control;
the screening comprises the following steps: transferring the sweet wormwood herb explants cultured for 3 days in the co-culture process to a germination screening medium, performing illumination culture at 25 ℃, performing subculture once every two weeks, performing subculture for 2-3 times to obtain Hyg-resistant cluster buds, shearing the Hyg-resistant cluster buds growing well, transferring the Hyg-resistant cluster buds to a rooting medium, and culturing to root, thereby obtaining Hyg-resistant regeneration sweet wormwood herb plants.
8. The method for improving the content of artemisinin in artemisia apiacea by transferring ICS1 gene as claimed in claim 1, wherein in the step (4), the PCR detection comprises the following steps: designing a forward primer and a reverse primer according to the expression cassettes p35s-ICS1-nos sequence p35s and ICS1 of the target gene respectively; carrying out DNA amplification; observing under ultraviolet rays, and if the target strip is positive, the plant line is the transgenic southernwood plant;
the DNA sequence of the forward primer is shown as SEQ ID NO.5, and the DNA sequence of the reverse primer is shown as SEQ ID NO. 4.
9. The method for improving the content of artemisinin in artemisia apiacea by transferring the ICS1 gene as claimed in claim 1, further comprising the step of (5) carrying out HPLC-ELSD measurement on the content of artemisinin in the transgenic artemisia apiacea, and screening to obtain transgenic artemisia apiacea plants with improved content of artemisinin.
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