CN106086039A - A kind of Herba Artemisiae Annuae WRKY class transcription factor coded sequence and application - Google Patents

Abstract

The invention discloses a coding sequence of an Artemisia annua WRKY transcription factor, the coding sequence is marked as AaGSW1, its nucleotide sequence is shown in SEQ ID NO:1, and its amino acid sequence is shown in SEQ ID NO:2. The encoding WRKY type transcription factor AaGSW1 of the present invention increases the content of artemisinin by regulating the expression of key enzyme gene for artemisinin synthesis. The content of artemisinin in the leaves of non-transgenic common Artemisia annua was 9 mg/g DW, and the content of artemisinin in the leaves of transgenic Artemisia annua overexpressing AaGSW1 gene expression increased to 20 mg/g DW. The invention is of great significance for providing high-yield and stable new drug sources for large-scale production of artemisinin.

Description

Coding sequence and application of a kind of WRKY transcription factor of Artemisia annua

technical field

The invention relates to the technical field of genetic engineering, in particular to a coding sequence and application of an Artemisia annua WRKY transcription factor.

Background technique

Artemisia annua L. is an annual herb belonging to the family Asteraceae. Artemisinin (artemisinin) is a sesquiterpene lactone compound containing a peroxide bridge structure isolated from its aerial part. It is currently the most effective drug for treating malaria in the world, especially for cerebral malaria and anti-malaria Chloroquine malaria has the characteristics of quick action and low toxicity. Currently, the most effective treatment for malaria recommended by the World Health Organization is artemisinin combination therapy (ACTs). In addition, with the deepening of pharmacological research on artemisinin, scientists have discovered that artemisinin and its derivatives also have anti-inflammatory, anti-schistosome, anti-tumor, and immune-regulating functions. It can be seen that artemisinin is a natural medicine with great potential.

At present, the main source of artemisinin is extracted from the aerial parts of Artemisia annua plants. However, the content of artemisinin in Artemisia annua is very low. The average content of artemisinin in different planting environments and planting varieties is 0.01-1% of the dry weight of leaves of Artemisia annua. , making the large-scale commercial production of this drug limited. Due to the complex structure of artemisinin, the artificial synthesis is difficult, the yield is low, and the cost is high. Although some people have successfully used yeast fermentation to produce artemisinin precursor artemisinin acid, artemisinin still needs to be artificially chemically semi-synthesized to artemisinin, and this research is still in the initial stage of laboratory research and development. There are also attempts to produce artemisinin by tissue culture and cell engineering. However, the content of artemisinin in the callus is less than 0.1% of the dry weight, and the highest in the bud is only 0.16% of the dry weight. The study detected no artemisinin in the roots. Therefore, the feasibility of using tissue culture and cell engineering to produce artemisinin is not high.

Contents of the invention

The object of the present invention is to overcome the deficiencies in the prior art, provide a kind of Artemisia annua AaGSW1 protein coding sequence, this gene encodes WRKY class transcription factor (AaGSW1), and it participates in the density of regulating and controlling the glandular hair of Artemisia annua; Transformation of A. annua with an overexpression vector of AaGSW1 transcription factor can effectively regulate the density of glandular hairs in the epidermis of A. annua, thereby increasing the content of artemisinin. The content of artemisinin has been increased from 9mg/g DW of non-transgenic Artemisia annua to 20mg/g DW (Figure 2). This invention is of great significance for providing high-yield and stable new drug sources for large-scale production of artemisinin.

The present invention is achieved through the following technical solutions:

The present invention provides a coding sequence of an Artemisia annua WRKY transcription factor, the coding sequence is marked as AaGSW1, the nucleotide sequence of the AaGSW1 is shown in SEQ ID NO: 1; its amino acid sequence is shown in SEQ ID NO: 2 Show.

The present invention also provides a polypeptide whose amino acid sequence is shown in SEQ ID NO:2.

The present invention also provides a recombinant expression vector comprising the nucleotide sequence shown in SEQ ID NO:1.

The present invention also provides a recombinant expression transformant comprising the nucleotide sequence shown in SEQ ID NO:1.

The invention also provides the application of the coding sequence AaGSW1 of Artemisia annua WRKY type transcription factors in increasing the content of artemisinin.

Further, the application includes the following steps:

Step 1. Linking the coding sequence AaGSW1 of the Artemisia annua WRKY transcription factor to the plant expression control sequence to construct a plant expression vector containing the coding sequence of the Artemisia annua WRKY transcription factor;

Step 2, transforming the plant expression vector in step 1 into Agrobacterium, and transforming the Agrobacterium into Artemisia annua;

Step 3. Obtain transformed cells containing the coding sequence AaGSW1 of the Artemisia annua WRKY transcription factor through antibiotic selection, and regenerate transgenic plants.

Further, in the above step 2, the freeze-thaw method is used for transfer.

The present invention also provides a method for increasing the content of artemisinin in Artemisia annua, said method comprising the steps of:

Step 1. Analyzing the WRKY transcription factors of Artemisia annua cultivars, and cloning the Artemisia annua WRKY transcription factor AaGSW1 from the Artemisia annua cDNA library;

Step 2, operably linking the AaGSW1 gene to the expression control sequence to form a plant overexpression vector containing the AaGSW1 gene;

Step 3, transforming the plant overexpression vector containing the AaGSW1 gene into Agrobacterium tumefaciens to obtain the Agrobacterium tumefaciens strain with the plant overexpression vector;

Step 4, using the Agrobacterium tumefaciens strain to transform Artemisia annua, screening with hygromycin to obtain resistant seedlings, and then detecting positive plants by PCR are transgenic Artemisia annua seedlings;

Step 5. Analyzing the glandular hair density of the obtained transgenic Artemisia annua, obtaining the transgenic Artemisia annua with significantly increased glandular hair density, and further obtaining the Artemisia annua plants with increased artemisinin content.

Further, the Agrobacterium tumefaciens is EHA105.

Further, in step 4, the transgenic Annua plants detected by PCR refer to the design and synthesis of detection primers for the AaGSW1 gene respectively, DNA amplification, and the positive strains with the target bands observed under ultraviolet light are the transgenic Annua annua plants. For Artemisia plants, the detection of the density of glandular hairs means that under a fluorescent microscope, the secretory glandular hairs of Artemisia annua exhibit bright fluorescence at a specific wavelength, and the number and density are counted after taking pictures.

Wherein, the content of artemisinin in the obtained transgenic Artemisia annua was determined by HPLC-ELSD.

The present invention clones the AaGSW1 gene from Artemisia annua, constructs a plant overexpression vector containing the AaGSW1 gene, uses Agrobacterium tumefaciens as a mediator, and transforms the AaGSW1 gene overexpression vector into Artemisia annua by using the leaf disc method; PCR detects the presence of the exogenous target gene AaGSW1 Integrating the situation, observing and counting the density of glandular trichomes with a fluorescence microscope, the transgenic Artemisia annua with significantly increased glandular trichome density was obtained; the content of artemisinin in Artemisia annua was measured by high-performance liquid chromatography-evaporative light scattering detector (HPLC-ELSD), indicating that The content of artemisinin in the obtained transgenic Artemisia annua whose epidermal gland hair density is significantly increased is also significantly increased.

In the present invention, various vectors known in the art can be used, such as commercially available vectors, including plasmids, cosmids and the like. When producing the Artemisia annua AaGSW1 protein polypeptide of the present invention, the Artemisia annua AaGSW1 protein coding sequence can be operably linked to the expression control sequence, thereby forming an Artemisia annua AaGSW1 protein expression vector.

As used herein, "operably linked" refers to the condition that some portion of a linear DNA sequence is capable of affecting the activity of other portions of the same linear DNA sequence. For example, a signal peptide (secretion leader) DNA is operably linked to a polypeptide DNA if the signal peptide DNA is expressed as a precursor and is involved in the secretion of the polypeptide; if a promoter controls the transcription of the sequence, then it is operably linked to A coding sequence; a ribosome binding site is operably linked to a coding sequence if it is placed in a position to enable its translation. Generally, "operably linked to" means adjacent, and with respect to a secretory leader it means adjacent in reading frame.

The present invention will be further described below in conjunction with the accompanying drawings, in order to fully illustrate the purpose, technical features and technical effects of the present invention.

Description of drawings

Other characteristics, objects and advantages of the present invention will become more apparent by reading the detailed description of non-limiting embodiments made with reference to the following drawings:

Figure 1 shows the results of quantitative PCR detection of AaGSW1 expression in Artemisia annua in a preferred embodiment of the present invention;

Figure 2 shows the results of using HPLC to detect the content of artemisinin in a preferred embodiment of the present invention; *, P<0.01 (T test).

detailed description

The specific embodiments provided by the present invention will be described in detail below in conjunction with the accompanying drawings.

The embodiments of the present invention are described in detail below: the present embodiment is implemented under the premise of the technical solution of the present invention, and detailed implementation and specific operation process are provided, but the protection scope of the present invention is not limited to the following implementation example. The experimental method that does not indicate specific conditions in the following examples is usually according to conventional conditions, such as molecular cloning such as Sambrook: the conditions described in the laboratory manual (New York: Cold Spring Harbor Laboratory Press, 1989), or according to the manufacturer's instructions suggested conditions.

Embodiment 1, cloning of Artemisia annua AaGSW1 gene

1. Extraction of Total RNA from Artemisia annua Genome

Take the leaf tissue of Artemisia annua, grind it in liquid nitrogen, add it to a 1.5mL Eppendorf (EP) centrifuge tube filled with lysate, shake it fully, and extract total RNA according to the instructions of the TIANGEN kit. The quality of total RNA was identified by formaldehyde denaturing gel electrophoresis, and then the RNA content was determined on a spectrophotometer.

2. Cloning of Artemisia annua AaGSW1 gene

Using the extracted total RNA as a template, cDNA was synthesized under the action of PowerScript reverse transcriptase; gene-specific primers (SEQ ID NO:3 and SEQ ID NO:4) were designed according to the sequence of the AaGSW1 gene, and the total cDNA was obtained by PCR. The AaGSW1 gene was amplified and sequenced.

Through the above steps, the full-length coding sequence (SEQ ID NO: 1) of the transcription factor in Artemisia annua was obtained and its protein coding sequence (SEQ ID NO: 2) was deduced, wherein, the start codon is ATG, and the stop codon Sub is TAG.

Example 2, Construction of plant binary interference expression vector containing AaGSW1 gene

In order to study the effect of AaGSW1 gene on the development of secretory glandular hairs in Artemisia annua, an overexpression vector PHB-AaGSW1 was constructed to overexpress AaGSW1. In order to facilitate the construction of the expression vector, the restriction site of BamH1 was introduced into the forward primer, and the restriction site of Sac1 was introduced into the reverse primer. The primers are shown in Table 1;

Table 1 PCR primers constructed by PHB-AaGSW1 vector

In this example, the Artemisia annua AaGSW1 gene is operably linked to the expression control sequence, and the vector can be used to regulate the content of artemisinin in Artemisia annua through a developmental regulation strategy.

Example 3. Agrobacterium tumefaciens-mediated AaGSW1 interference vector genetically transforms Artemisia annua to obtain transgenic Artemisia annua plants

1. Acquisition of Agrobacterium tumefaciens Engineering Bacteria Containing AaGSW1 Overexpression Vector

The plant binary overexpression vector containing AaGSW1 in Example 2 was transformed into Agrobacterium tumefaciens (such as EHA105, which is a publicly available biological material in the market, which can be purchased from CAMBIA Company in Australia, and the strain number is Gambar 1 ), and validated by PCR. The results showed that the plant binary interference expression vector containing AaGSW1 had been successfully constructed into the Agrobacterium tumefaciens strain.

2. Agrobacterium tumefaciens mediated AaGSW1 gene transformation of Artemisia annua

2.1. Preculture of explants

Artemisia annua seeds were soaked in 75% ethanol for 1 min, then soaked in 20% NaClO for 20 min, rinsed with sterile water 3-4 times, blotted the surface moisture with sterile absorbent paper, and inoculated in hormone-free MS (Murashige and Skoog, 1962) Cultivate in solid medium at 25°C under 16h/8h (light/dark) light to obtain sterile seedlings of Artemisia annua. After the seedlings grow to about 5cm, cut the explants of the leaves of the sterile seedlings for transformation.

2.2. Co-cultivation of Agrobacterium and explants

The leaf explants were transferred to the co-cultivation medium (1/2MS+AS 100 μmol/L), and the Agrobacterium tumefaciens engineering bacteria containing the activated AaGSW1 plant binary overexpression vector were added dropwise. 1/2MS suspension, so that the explants fully contacted with the bacterial solution, and cultured in the dark at 28°C for 3 days. Take the leaf explants dripped in the 1/2MS liquid medium suspension of Agrobacterium tumefaciens without the target gene as a control.

2.3. Screening of resistant regenerated plants

The Artemisia annua explants of described co-cultivation 3d were transferred to the germination selection medium (MS+6-BA 0.5mg/L+NAA0.05mg/L+Hyg 50mg/L+Cb 500mg/L) at 25 ℃, 16h/8h light culture, subculture once every two weeks, after 2-3 subcultures, Hyg-resistant clustered buds can be obtained. The well-grown resistant clustered shoots were cut off and transferred to rooting medium (1/2MS+Cb 125 mg/L) for cultivation until rooting, thereby obtaining Hyg-resistant regenerated Artemisia annua plants.

3. PCR detection of transgenic Artemisia annua plants

According to the 35S promoter region and AaGSW1 upstream of the expression cassette where the target gene is located, the forward primer design and reverse primer were designed to detect the target gene. The results showed that specific DNA fragments could be amplified by using the designed PCR specific primers. However, when non-transformed Artemisia annua genomic DNA was used as a template, no fragment was amplified.

In this example, the plant expression vector was transformed into Agrobacterium tumefaciens to obtain the Agrobacterium tumefaciens strain containing the AaGSW1 plant binary overexpression vector used for transformation of Artemisia annua, and the constructed Agrobacterium tumefaciens strain was used to transform Artemisia annua , to obtain transgenic Artemisia annua plants detected by PCR. The acquisition of transgenic Artemisia annua plants provides direct materials for the screening of Artemisia annua strains with higher artemisinin content.

Example 4. Determination of artemisinin content in transgenic Artemisia annua using HPLC-ELSD

1. HPLC-ELSD conditions and system suitability and preparation of standard solutions

HPLC: adopt water alliance 2695 system, chromatographic column is C-18 reverse phase silica gel column (SymmetryShieldTM C18, 5 μm, 250 * 4.6mm, Waters), mobile phase is methanol: water, the volume ratio of methanol: water is 70:30, The column temperature is 30°C, the flow rate is 1.0mL/min, the injection volume is 10μL, the sensitivity (AUFS=1.0), and the number of theoretical plates is not less than 2000 based on the artemisinin peak.

ELSD: Water alliance 2420 system is adopted, the drift tube temperature of the evaporative light scattering detector is 40°C, the amplification factor (gain) is 7, and the carrier gas pressure is 5bar;

Accurately weigh 2.0 mg of artemisinin standard (Sigma Company) and dissolve it completely in 1 mL of methanol to obtain a 2 mg/mL artemisinin standard solution, which is stored at -20°C for future use.

In the present invention, the mobile phase is methanol (methanol):water, and when the ratio is 70%:30%, the retention time of artemisinin is 5.1min, and the peak shape is good. The number of theoretical plates is not less than 2000 based on the calculation of artemisinin.

2. Preparation of standard curve

Inject 2 μl, 4 μl, 6 μl, 8 μl, and 10 μl of the reference solution under corresponding chromatographic conditions to record the spectrum and chromatographic parameters, and perform regression analysis on the standard content (X, μg) with the peak area (Y) respectively. Through research, artemisinin in the present invention exhibits a good log-log linear relationship in the range of 4-20 μg. The log-log linear regression equation of the artemisinin reference substance is: Y=1.28e+000X+4.71e+000, R=0.979546.

3. Sample Preparation and Determination of Artemisinin Content

A total of 2 g of fresh leaves of Artemisia annua were taken from the upper, middle and lower parts of the plants, and dried in an oven at 45°C until constant weight. The leaves are then tapped from the dried shoots and ground into a powder. Weigh about 0.1g of dry powder into a 2mL Eppendorf tube, add 2mL of ethanol, treat with 40W ultrasonic wave for 30min, centrifuge at 5000rpm for 10min, take the supernatant and filter it with a 0.22μm filter membrane, then use HPLC-ELSD to determine the content of artemisinin.

The content of artemisinin was determined by HPLC-ELSD, the sample injection volume was 20 μl, and the content of artemisinin (mg) in the sample was calculated according to the peak area into the linear regression equation, and then divided by the dry weight of Artemisia annua leaves (g) , so as to calculate the content of artemisinin in Artemisia annua plants.

The transgenic plants transformed with the AaGSW1 overexpression vector in the present invention can significantly increase the content of artemisinin in Artemisia annua. When the content of non-transformed common Artemisia annua was 9 mg/g DW, the average content of artemisinin in Artemisia annua transformed with AaGSW1 overexpression vector reached 20 mg/g DW at the same time, which was 2.2 times the content of non-transformed Artemisia annua, as shown in Figure 2 As shown, artemisinin was detected by HPLC in transgenic AaGSW1 overexpressed Artemisia annua, and the results are the average value of three repetitions, and the error bars represent the standard deviation. Statistical analysis was performed by t-test (*, P<0.01).

In this example, the HPLC-ELSD method was used to measure the content of artemisinin in transgenic Artemisia annua, and the metabolic engineering strategy of transforming AaGSW1 overexpression vector was used to find that the expression of AaGSW1 gene had a clear correlation with the content of artemisinin. The overexpression study to increase the content of artemisinin in Artemisia annua provides strong experimental evidence.

The Artemisia annua WRKY transcription factor coding sequence AaGSW1 of the present invention can increase the content of artemisinin in plants, connect the coding sequence to the plant expression regulation vector, and construct the plant expression vector containing the coding sequence; transfer the expression vector to into Agrobacterium, and Agrobacterium was transformed into Artemisia annua; transformed cells containing the coding sequence were obtained by antibiotic screening, and transgenic plants were regenerated; the content of artemisinin in the transgenic Artemisia annua obtained by the present invention was significantly regulated, and in non-transformed When the content of common Artemisia annua was 9mg/g DW, the average content of artemisinin in Artemisia annua transformed with AaGSW1 overexpression vector was 20mg/gDW at the same time, which was 2.2 times that of non-transformed Artemisia annua. The invention provides a transcription factor coding sequence for regulating the content of artemisinin in Artemisia annua, which lays a solid foundation for large-scale production of artemisinin by using the coding sequence.

The preferred specific embodiments of the present invention have been described in detail above. It should be understood that those skilled in the art can make many modifications and changes according to the concept of the present invention without creative efforts. Therefore, all technical solutions that can be obtained by those skilled in the art based on the concept of the present invention through logical analysis, reasoning or limited experiments on the basis of the prior art shall be within the scope of protection defined by the claims.

Claims (10)

1. a Herba Artemisiae Annuae WRKY class transcription factor coded sequence, it is characterised in that described coded sequence is designated as AaGSW1, described The nucleotide sequence of AaGSW1 is as shown in SEQ ID NO:1.

2. a Herba Artemisiae Annuae WRKY class transcription factor coded sequence, it is characterised in that the aminoacid sequence of described AaGSW1 coding is such as Shown in SEQ ID NO:2.

3. a peptide species, it is characterised in that the aminoacid sequence of described polypeptide is as shown in SEQ ID NO:2.

4. a recombinant expression carrier, it is characterised in that described recombinant expression carrier comprises the nucleoside as shown in SEQ ID NO:1 Acid sequence.

5. a recombinant expressed transformant, it is characterised in that described recombinant expressed transformant comprises as shown in SEQ ID NO:1 Nucleotide sequence.

Herba Artemisiae Annuae WRKY class transcription factor coded sequence AaGSW1 the most according to claim 1 and 2 is improving artemislnin content In application.

Application the most according to claim 6, it is characterised in that described application comprises the following steps:

Step 1, Herba Artemisiae Annuae WRKY class transcription factor coded sequence AaGSW1 is connected on plant expression regulation sequence, builds containing described The plant expression vector of Herba Artemisiae Annuae WRKY class transcription factor coded sequence；

Step 2, the described plant expression vector in step 1 is proceeded to Agrobacterium, described Agrobacterium is proceeded to Herba Artemisiae Annuae；

Step 3, pass through antibiotic-screening, it is thus achieved that the conversion containing described Herba Artemisiae Annuae WRKY class transcription factor coded sequence AaGSW1 is thin Born of the same parents, regeneration of transgenic plant.

Application the most according to claim 7, it is characterised in that in described step 2, uses freeze-thaw method to proceed to.

9. the method improving content of artemisinin in sweet wormwood, it is characterised in that described method comprises the steps:

Step 1, to Herba Artemisiae Annuae cultigen H-ZIP IV class Transcription factor analysis, from Herba Artemisiae Annuae cDNA library clone obtain Herba Artemisiae Annuae WRKY Class transcription factor AaGSW1；

Step 2, AaGSW1 gene is operatively connectable to expression regulation sequence, forms the plant containing described AaGSW1 gene Overexpression vector；

Step 3, by containing described AaGSW1 gene plant overexpression vector convert Agrobacterium tumefaciems, it is thus achieved that have described plant surpass The Agrobacterium tumefaciens strain of expression vector；

Step 4, utilize described Agrobacterium tumefaciens strain convert Herba Artemisiae Annuae, obtain resistance Seedling through hygromycin selection, then be detected as through PCR Positive plant is transgene abrotanum Seedling；

Step 5, the transgene abrotanum obtained is carried out trichome density analysis, it is thus achieved that transgenic that trichome density significantly improves is blue or green Artemisia, and then obtain the Herba Artemisiae Annuae plant that artemislnin content improves.

Method the most according to claim 9, it is characterised in that described Agrobacterium tumefaciems is EHA105.