CN113403323B - Method for improving artemisinin content in sweet wormwood herb - Google Patents

Abstract

The invention discloses a method for improving the content of artemisinin in sweet wormwood herb, which comprises the following steps: and (2) introducing the nucleotide sequence shown in SEQ ID NO.1 into the sweet wormwood to obtain the regenerated transgenic sweet wormwood containing the nucleotide sequence. The nucleotide sequence provided by the invention codes the SPL family transcription factor (AaSPL9), the content of artemisinin in the regenerated transgenic artemisia apiacea obtained by utilizing a transgenic technology is obviously improved, and the nucleotide sequence has important significance for the large-scale production of the artemisinin.

Description

Method for improving artemisinin content in sweet wormwood herb

Technical Field

The invention relates to the technical field of genetic engineering, in particular to a method for improving the content of artemisinin in sweet wormwood herb.

Background

Plant metabolism is divided into primary metabolism and secondary metabolism, wherein primary metabolites (such as saccharides, lipids and nucleic acids) exist in all plants and are necessary for maintaining cell life activities, and plant secondary metabolites refer to a large group of small-molecule organic compounds in plants, which are not necessary for plant growth and development, and the generation and distribution of the small-molecule organic compounds have species, tissue organ and growth and development specificity. The content of secondary metabolites generated in natural plants is extremely low, and the chemical synthesis method is complex in process flow and high in cost. Therefore, researchers have searched for other methods for increasing the content of plant secondary metabolites, and among many methods, increasing the density of secretory glandular hairs, which are a multi-cellular structure specific to plant epidermal cells, is considered as a direct and effective method, and in recent years, researches on glandular hairs have found that many valuable secondary metabolites are specifically synthesized and stored in the structure, such as a large class of natural flavors and fragrances represented by peppermint essential oil and rose essential oil; effective components of Chinese herbal medicine represented by artemisinin; natural plant additives represented by hops.

The secondary metabolites specifically synthesized in glandular hairs have a common feature that the key enzyme genes involved in the synthesis of these secondary metabolites are specifically expressed only in glandular hairs. Therefore, the method for regulating and controlling the glandular hair density by utilizing the genetic engineering method is a plant genetic manipulation means with great application value. In the field of secondary metabolism, few reports about secretory glandular hairs exist, particularly in the research of the traditional Chinese herbal medicine namely artemisia apiacea.

Disclosure of Invention

In view of the above-mentioned drawbacks of the prior art, the technical problem to be solved by the present invention is how to increase the content of artemisinin in artemisia apiacea.

In order to achieve the above object, the first aspect of the present invention provides the use of the nucleotide sequence shown as SEQ ID NO.1 for increasing the content of artemisinin in Artemisia annua.

The second aspect of the invention provides a method for improving the content of artemisinin in sweet wormwood herb, which comprises the following steps: and (2) introducing the nucleotide sequence shown in SEQ ID NO.1 into the sweet wormwood to obtain the regenerated transgenic sweet wormwood containing the nucleotide.

Further, the method comprises the steps of:

step 1, constructing an expression vector according to the nucleotide sequence;

step 2, transferring the expression vector into agrobacterium, and then transferring the agrobacterium into southernwood;

and 3, obtaining the regenerated transgenic southernwood containing the nucleotide through antibiotic screening.

Further, the preparation method of the nucleotide sequence comprises the following steps:

and carrying out reverse transcription on the total RNA of the southernwood to obtain cDNA, and amplifying the cDNA to obtain the nucleotide sequence.

Further, in the nucleotide obtained by the cDNA amplification, a primer has a sequence shown as SEQ ID NO.2 and a sequence shown as SEQ ID NO. 3.

Further, in step 2, the construction of the expression vector according to the nucleotide sequence specifically includes:

BamHI and SpeI enzyme cutting sites are introduced at the 5 'end and the 3' end of the nucleotide sequence, a pHB-GFP vector is subjected to double enzyme cutting to form a linearized vector, and the nucleotide sequence is connected with the pHB-GFP linearized vector through an Infusion seamless cloning technology to obtain an expression vector.

Further, the expression vector is transferred into the agrobacterium by a freeze-thaw method.

Further, the step of transferring the agrobacterium into the sweet wormwood comprises the following specific steps: co-culturing agrobacterium transformed with the expression vector and the explant of the southernwood.

Furthermore, the density of glandular hairs in the regenerative transgenic sweet wormwood is more than or equal to 30 per square millimeter, and the total glandular hairs in the leaves are 96809.

Further, the artemisinin content of the regenerated transgenic sweet wormwood herb is more than or equal to 15mg/g DW.

The nucleotide sequence provided by the invention can code SPL family transcription factor (AaSPL9 for short), and the research shows that AaSPL9 participates in the regulation and control of the density of the glandular hairs of the southernwood, the invention utilizes the transgenic technology to lead the nucleotide sequence into the southernwood to obtain the regenerated transgenic southernwood, by regulating and controlling the density of the glandular hairs of the epidermis of the southernwood, thereby improving the content of artemisinin, specifically, the density of leaf gland hair of non-transgenic sweet wormwood is 20 per square millimeter, the total gland hair quantity of each leaf is 56947, the leaf glandular hair density of the regenerated transgenic sweet wormwood obtained by the application is improved to more than 30 per square millimeter, the total glandular hair number is improved to 96809, the content of the corresponding artemisinin is increased from 11mg/g DW of the non-transgenic artemisia apiacea to more than 15mg/g DW, which shows that the method provided by the application can effectively increase the content of the artemisinin in the artemisia apiacea and has important significance for the large-scale production of the artemisinin.

Drawings

FIG. 1 shows the results of relative expression measurement of AaSPL9 protein in regenerated transgenic Artemisia annua;

FIG. 2A shows the fluorescence microscopic observation of wild type Artemisia annua leaf glandular hair;

FIG. 2B is the fluorescence microscope observation result of regenerated transgenic Artemisia apiacea leaf glandular hairs;

FIG. 3 shows the statistical results of glandular hair density;

FIG. 4 shows the statistics of the total glandular hair number of each leaf;

FIG. 5 shows the analysis results of artemisinin content.

Detailed Description

The technical contents of the preferred embodiments of the present invention will be more clearly and easily understood by referring to the drawings attached to the specification. The present invention may be embodied in many different forms of embodiments and the scope of the invention is not limited to the embodiments set forth herein.

The experimental procedures without specifying the specific conditions in the following examples were carried out according to conventional methods, such as molecular cloning in Sambrook et al: the conditions described in the Laboratory Manual (New York: Cold Spring Harbor Laboratory Press,1989), or according to the manufacturer's recommendations.

Example 1 cloning of the Artemisia apiacea AaSPL9 Gene

1. Extraction of total RNA of sweet wormwood

Taking sweet wormwood leaf tissue, placing the sweet wormwood leaf tissue in liquid nitrogen for grinding, adding the sweet wormwood leaf tissue into a 1.5mL Eppendorf (EP) centrifuge tube containing lysis solution, fully oscillating, and extracting total RNA according to the instruction of a TIANGEN kit.

2. Cloning of Gene encoding AaSPL9 protein

Synthesizing cDNA under the action of PowerScript reverse transcriptase by taking the extracted total RNA as a template; gene-specific primers (SEQ ID NO:2 and SEQ ID NO:3) were designed based on the desired sequence, and the gene encoding AaSPL9 protein was amplified from the total cDNA by PCR and sequenced.

Sequencing results show that the gene for coding AaSPL9 protein is obtained, and the nucleotide sequence of the gene is shown as SEQ ID NO. 1.

Example 2 construction of expression vector

BamHI and SpeI enzyme cutting sites are introduced at the 5 'end and the 3' end of the nucleotide sequence through PCR amplification, the pHB-GFP vector is subjected to double enzyme cutting to form a linearized vector, and the nucleotide sequence is connected with the pHB-GFP linearized vector through an Infusion seamless cloning technology to obtain an expression vector.

Example 3 obtaining of regenerated transgenic Artemisia annua

1. Obtaining of Agrobacterium tumefaciens engineering bacteria containing AaSPL9 overexpression vector

The expression vector obtained in example 2 was transformed into Agrobacterium tumefaciens (e.g., EHA105, available from Cambian, Australia, strain number Gambar 1) by freeze-thaw method, and PCR verification was performed, which indicated that the expression vector was successfully constructed into Agrobacterium tumefaciens strain.

2. Agrobacterium tumefaciens-mediated AaSPL9 gene transformed southernwood

2.1. 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.

2.2. Co-culture of Agrobacterium with explants

And transferring the leaf explant into a co-culture medium (1/2MS + AS 100 mu mol/L), dropwise adding 1/2MS suspension of the activated agrobacterium tumefaciens engineering bacteria containing the AaSPL9 plant over-expression vector, fully contacting the explant with a bacterial solution, and performing dark culture at 28 ℃ for 3 d. Control was leaf explants dropped on 1/2MS liquid medium suspension of Agrobacterium tumefaciens without the gene of interest.

2.3. Selection of resistant regenerated plants

And transferring the southernwood explants which are co-cultured for 3d to a germination screening culture medium (MS +6-BA 0.5mg/L + NAA0.05mg/L + Kan 50mg/L + Cb 500mg/L), performing illumination culture at 25 ℃ for 16h/8h, performing subculture once every two weeks, and performing subculture 2-3 times to obtain Kan-resistant clustered buds. Shearing off the well-grown resistant cluster buds, transferring the cluster buds to a rooting culture medium (1/2MS + Cb 125mg/L) for culturing until the cluster buds grow to root, thereby obtaining a Kan resistant regeneration transgenic southernwood plant.

3. PCR detection of regenerated transgenic sweet wormwood plant

A forward primer meter and a reverse primer are respectively designed according to a 35S promoter region and AaSPL9 at the upstream of an expression cassette where the target gene is positioned, and the target gene is detected. 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 the AaSPL9 plant overexpression vector for transforming artemisia annua, and the constructed agrobacterium tumefaciens strain is used to transform artemisia annua to obtain a regenerated transgenic artemisia annua plant.

Example 4 statistics of epidermal glandular density and Total glandular number of transgenic Artemisia annua

The leaves of non-transgenic Artemisia annua and regenerated transgenic Artemisia annua were observed under excitation light having a wavelength of 450nm to 480nm using a BX51 model microscope from Olympus. Taking the same size of the sweet wormwood leaf leaves, randomly sampling at different 5 positions, and counting the glandular hair density. And measuring the total area of the artemisia apiacea leaves, and calculating to obtain the total glandular hair quantity of each leaf.

In all the figures, CK represents wild type Artemisia annua plants, and OE6\ OE22\ OE32 respectively represent different AaSPL9 overexpression transgenic Artemisia annua strains. Fig. 1 shows the relative expression test results of AaSPL9 protein in the regenerated transgenic artemisia apiacea, fig. 2A shows the fluorescence microscopic observation results of the leaf glandular hairs of the wild-type artemisia apiacea, fig. 2B shows the fluorescence microscopic observation results of the leaf glandular hairs of the regenerated transgenic artemisia apiacea, fig. 3 shows the statistical results of the density of the glandular hairs, and fig. 4 shows the statistical results of the total glandular hairs per leaf, as can be seen from fig. 1-4, when the density of the glandular hairs of the non-transgenic artemisia apiacea is 20 per square millimeter, the density of the leaf glandular hairs of the regenerated transgenic artemisia apiacea is increased to more than 30 per square millimeter, while the total glandular hairs per leaf number is increased from 56947 to 96809, and the excessive expression of AaSPL9 in artemisia apiacea can increase the density of the glandular hairs. X, P <0.01(T test)

Example 5 determination of artemisinin content in transgenic Artemisia annua by HPLC-ELSD

HPLC-ELSD conditions and System applicability and preparation of Standard solutions

HPLC: a water alliance 2695 system was used, the column was a C-18 reverse phase silica column (Symmetry Shield. TM. C18, 5 μm, 250X 4.6mm, Waters) and the mobile phase was methanol: water, methanol: the volume ratio of water is 70:30, the column temperature is 30 ℃, the flow rate is 1.0mL/min, the sample injection amount is 10 muL, the sensitivity (AUFS is 1.0), and the theoretical plate number is not less than 2000 calculated according to the 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 invention, when the mobile phase is methanol (methanol) and water in a ratio of 70% to 30%, 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 contents (X and g) of the standard substance by using a peak area (Y). Through research, the artemisinin in the invention presents a good log-log linear relation in the range of 4-20 g. The log-log linear regression equation for the artemisinin control was: y1.28 e +000X +4.71e +000, R0.979546.

3. Preparation of sample and determination of artemisinin content

2g of fresh leaves of Artemisia annua are taken from the upper part, the middle part and the lower part of the Artemisia annua plant and are baked to constant weight in an oven at the temperature of 45 ℃. Then knocking off leaves and buds from the dried branches, and grinding into powder. Weighing about 0.1g of dry powder into a 2mL Eppendorf tube, adding 2mL of ethanol, treating with 40W of ultrasonic waves for 30min, centrifuging at 5000rpm for 10min, taking supernatant, and filtering with a 0.22 μm filter membrane to obtain the product for measuring the content of artemisinin by HPLC-ELSD.

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.

FIG. 5 shows the analysis result of artemisinin content, as shown in FIG. 5, when the content of non-transformed common Artemisia apiacea (CK) is 11mg/g DW, the content of artemisinin in the transgenic Artemisia apiacea regenerated at the same period is more than 15mg/g DW, and can reach 20mg/g DW at most, and is 1.8 times of the content of non-transformed Artemisia apiacea. Statistical analysis was tested by t-test (. star, P < 0.01).

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.

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Claims (10)

1. The application of the nucleic acid shown as SEQ ID NO.1 in improving the content of artemisinin in artemisia apiacea.

2. A method for improving the content of artemisinin in sweet wormwood herb is characterized by comprising the following steps: and (2) introducing the nucleic acid shown in SEQ ID NO.1 into the artemisia apiacea to obtain the regenerated transgenic artemisia apiacea containing the nucleic acid.

3. The method of claim 2, wherein the method comprises the steps of:

step 1, constructing and obtaining an expression vector according to the nucleic acid;

step 2, transferring the expression vector into agrobacterium, and then transferring the agrobacterium into sweet wormwood;

and 3, obtaining the regenerated transgenic southernwood containing the nucleic acid by screening antibiotics.

4. The method of claim 2, wherein the nucleic acid is prepared by a method comprising:

and carrying out reverse transcription on the total RNA of the sweet wormwood to obtain cDNA, and amplifying the cDNA to obtain the nucleic acid.

5. The method of claim 4, wherein the nucleic acid obtained from the cDNA amplification has primers having the sequences shown in SEQ ID No.2 and SEQ ID No. 3.

6. The method of claim 3, wherein the step 1 of constructing the expression vector based on the nucleic acid specifically comprises:

BamHI and SpeI enzyme cutting sites are introduced into the 5 'end and the 3' end of the nucleic acid, the pHB-GFP vector is subjected to double enzyme cutting to form a linearized vector, and the nucleic acid is connected with the pHB-GFP linearized vector by an Infusion seamless cloning technology to obtain an expression vector.

7. The method of claim 6, wherein said expression vector is transferred into said Agrobacterium by freeze-thaw methods.

8. The method of claim 7, wherein said transforming said agrobacterium into artemisia annua comprises: co-culturing agrobacterium transformed with the expression vector and the explant of the southernwood.

9. The method of any one of claims 2-8, wherein the density of glandular hairs in the regenerated transgenic artemisia apiacea is greater than or equal to 30 per square millimeter.

10. The method of any one of claims 2 to 8, wherein the regenerated transgenic Artemisia annua has an artemisinin content of 15mg/g DW or more.

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