CN116334109A - Application of overexpression of AaMAPK6 gene in sweet wormwood herb in improving artemisinin content and method - Google Patents
Application of overexpression of AaMAPK6 gene in sweet wormwood herb in improving artemisinin content and method Download PDFInfo
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Abstract
The invention discloses an application of overexpressing AaMAPK6 gene in sweet wormwood herb in improving the content of artemisinin and a method thereof, belonging to the technical field of plant genetic engineering, wherein the method comprises the following operation steps: constructing an AaMAPK6 gene on a plant binary expression vector, then transforming sweet wormwood under the mediation of agrobacterium tumefaciens, detecting by fluorescent quantitative RCR, measuring the content of sweet wormwood, and screening to obtain a transgenic plant with the increased content of sweet wormwood, wherein the nucleotide sequence of the AaMAPK6 gene is shown as SEQ ID NO.3, and the content of sweet wormwood in the plant over-expressing the AaMAPK6 gene is obviously increased by 1.26-1.56 times compared with the wild type plant.
Description
Technical Field
The invention relates to the technical field of plant genetic engineering, in particular to application of overexpression of an AaMAPK6 gene in sweet wormwood in improving the content of artemisinin and a method for improving the artemisinin in sweet wormwood.
Background
Artemisinin is a sesquiterpene lactone compound containing a peroxidation bridge structure and plays an important role in treating chloroquine-resistant malaria. The world health organization recommends artemisinin combination therapy as the first method of treatment of malaria. The artemisinin content in the sweet wormwood plants is too low to meet the treatment requirements of patients, especially patients in developing countries. Therefore, how to increase the production of artemisinin becomes a great problem in artemisinin production and a research hot spot at home and abroad. The Mitogen-activated protein kinase (MAPK) cascade is a highly conserved signaling pathway in eukaryotes and is a phosphorylated signaling pathway composed of MAPKKK, MAPKK, MAPK tertiary protein kinases. Is involved in regulating the growth and development of plants, immune response, adversity stress and other processes [ Zhao et al 2017]. Several transcription factors have been reported in Artemisia annua to be strongly induced to express by JA and to be involved in regulating artemisinin biosynthesis, such as ORA, bHLH112, NAC1, etc. Whether the presence of MAPK kinase in artemisia annua responds to induction of phytohormones and interacts with transcription factors that regulate artemisinin biosynthesis, thereby affecting artemisinin biosynthesis? Thus, the identification of MAPK kinase genes in the medicinal plant Artemisia annua is a method that attempts to increase artemisinin production.
Disclosure of Invention
In view of the above, it is an object of the present invention to provide an application of overexpressing AaMAPK6 gene in artemisia apiacea in increasing artemisinin content; the second object of the present invention is to provide a method for increasing the content of artemisinin in artemisia annua.
In order to achieve the above purpose, the present invention provides the following technical solutions:
1. the application of the overexpression AaMAPK6 gene in the sweet wormwood herb in improving the content of artemisinin is provided, and the nucleotide sequence of the AaMAPK6 gene is shown as SEQ ID NO. 3.
2. A method for increasing the content of artemisinin in artemisia apiacea, comprising the steps of: through over-expression of the AaMAPK6 gene in sweet wormwood, the nucleotide sequence of the AaMAPK6 gene is shown as SEQ ID NO. 3.
Preferably, the method for over-expressing the AaMAPK6 gene in sweet wormwood specifically comprises the following steps: constructing an AaMAPK6 gene on a plant binary expression vector, then transforming sweet wormwood under the mediation of agrobacterium tumefaciens, screening a transgenic plant, and screening the transgenic plant with the increased artemisinin content.
Preferably, the plant binary expression vector takes a pHB vector as a skeleton vector.
Preferably, the plant binary expression vector is constructed by constructing an AaMAPK6 gene on a pHB vector through BamHI and XbaI sites.
Preferably, the agrobacterium is EHA105.
Preferably, the method for screening the transgenic plants adopts fluorescence quantitative RCR detection.
Preferably, the upstream primer of the fluorescence quantitative RCR is shown as SEQ ID NO.10, and the downstream primer is shown as SEQ ID NO. 11.
In a preferred embodiment of the present invention, the reaction conditions of the fluorescent quantitative RCR are: pre-denaturation at 94℃for 3min; denaturation at 94℃for 10s, annealing at 58℃for 20s, extension at 72℃for 20s,40 amplification cycles, fluorescence was collected after 72℃extension for each cycle.
The invention has the beneficial effects that: the invention clones the coding sequence of the sweet wormwood AaMAPK6 gene, the nucleotide sequence of the coding region of the AaMAPK6 gene is shown as SEQ ID NO.3, and the amino acid sequence obtained after translation is shown as SEQ ID NO. 4; aaMAPK6 gene is constructed on a plant binary expression vector pHB through BamHI and XbaI loci, then sweet wormwood is transformed under the mediation of agrobacterium EHA105, and after fluorescence quantitative RCR detection and sweet wormwood content measurement, transgenic plants with improved sweet wormwood content are obtained through screening, and in the plants over-expressing AaMAPK6 gene, the sweet wormwood content is obviously improved by 1.26-1.56 times compared with the wild type, so that the method has important significance for cultivating new varieties with high sweet wormwood content and large-scale production.
Drawings
In order to make the objects, technical solutions and advantageous effects of the present invention more clear, the present invention provides the following drawings for description:
FIG. 1 is the AaMAPK6 gene sequence;
FIG. 2 is a pHB vector map;
FIG. 3 is a graph showing the gene expression level of transgenic plants;
FIG. 4 is a graph showing artemisinin content in AaMAPK6 transgene.
Detailed Description
The present invention will be further described with reference to the accompanying drawings and specific examples, which are not intended to limit the invention, so that those skilled in the art may better understand the invention and practice it.
Example 1 AaMAPK6 Gene acquisition
Extracting the RNA of the sweet wormwood, reversing the RNA into cDNA, and designing an upstream primer and a downstream primer of AaMAPK6 according to the genome of the sweet wormwood:
the upstream primer is as follows: 5'-atggatccatcaaatcaacaacc-3' (SEQ ID NO. 1);
the downstream primer is: 5'-cagattctggtactcgggat-3' (SEQ ID NO. 2);
then, carrying out PCR amplification by taking the sweet wormwood cDNA as a template and using an AaMAPK6 upstream primer and a AaMAPK6 downstream primer, wherein the amplification condition is that the pre-denaturation is carried out for 5 minutes at 95 ℃; denaturation at 95℃for 30 seconds, annealing at 56℃for 30 seconds, extension at 72℃for 105 seconds, for a total of 32 cycles; extending at 72deg.C for 2 min, preserving at 4deg.C, performing PCR amplification, recovering amplified product, and sequencing to obtain AaMAPK6 gene sequence (figure 1), wherein the nucleotide sequence of coding region is shown as SEQ ID NO.3, and the amino acid sequence obtained after translation is shown as SEQ ID NO. 4.
Example 2 construction of AaMAPK6 Gene plant expression vector
1. Construction of the overexpression vector pHB-AaMAPK6
According to the cloned AaMAPK6 gene sequence, analyzing the restriction enzyme digestion site of the coding region and the multiple cloning site on a pHB vector (figure 2), selecting BamHI and SpeI as restriction enzyme digestion sites constructed by the vector, introducing a BamHI restriction enzyme digestion site upstream of the full length of AaMED25, and introducing a SpeI restriction enzyme digestion site downstream of AaMED25, wherein the primers are as follows:
AaMAPK6-F-BamH1:5’-tgcagcccgggggatccATGGATCCATCAAATCAACA-3’(SEQ ID NO.5)
AaMAPK-R-SpeI：5’-cgataccgtcactagtCAGATTCTGGTACTCGGGAT-3’(SEQ ID NO.6)
the primers were used to amplify AaMAPK6, and the amplified AaMAPK6 sequence and pHB were then digested simultaneously with the endonucleases BamHI and XbaI and subjected to homologous recombination ligation.
2. Construction of interference expression vector pBin19-AaMAPK6
A300 bp specific fragment (SEQ ID NO. 7) on an AaMAPK6 genome is selected as an interference fragment, a pair of primers is designed, and HindIII and XbaI enzyme cutting sites are respectively introduced into the 5' ends of the upstream primer and the downstream primer:
imapk6-F：5’-gcggtaccaagcttTATCTAACATCACTGCACGATATCAG-3’(SEQ ID NO.8)；
imapk6-R：5’-gcctcgagtctagaTATTTGGTTGCCATGACCTAACC-3’(SEQ ID NO.9)；
forward cloning the fragment onto the interference vector pHANNIBAL; continuing to introduce KpnI and XhoI restriction sites at the 5' end of the upstream and downstream primers respectively, reversely cloning the fragments onto an interference vector pHANNIBAL introduced with forward fragments, digesting the vector with SacI and SpeI, recovering an interference expression frame containing forward and reverse AaMAPK6 fragments, and connecting the fragments to a large skeleton of a plant expression vector pBin19 digested with SacI and XbaI through T4 ligase to finally form an RNAi vector pBin19-AaMAPK6 of AaMAPK6.
Example 3 obtaining transgenic Artemisia annua
1. Preparing sweet wormwood seedlings:
(1) Taking a proper amount of sweet wormwood seeds in an Ep tube with the volume fraction of 75% of 1mL, adding 1mL of alcohol, soaking for 30min, then sucking the alcohol, and washing with sterile water for 3-5 times;
(2) Adding 1mL sodium hypochlorite solution with volume fraction of 10%, mixing, sterilizing in dark for 10min, and washing with sterile water for 3-5 times;
(3) Sucking 1mL of sterile water, sucking the seeds into a pipetting gun together, uniformly coating on an MS culture medium, sucking off excessive water, and drying. Vernalizing at 4deg.C for 2d, and culturing in an illumination culture room. (culture conditions are that the temperature is 25 ℃, the photoperiod is 16h light/8 h dark, and the illumination intensity is 80-220 mu mo.m) -2 ·s -1 Culturing for about one week under the humidity of 60%), and cutting the first pair of true leaves at the base of the petiole for genetic transformation of sweet wormwood when the seedlings grow out of the first pair of true leaves;
2. genetic transformation of Artemisia annua:
(1) Respectively converting the constructed over-expression vector (pHB-AaMAPK 6) and the interference expression vector pBin19-AaMAPK6 into agrobacterium EHA105;
(2) Screening on a resistance plate (Rif+Kan), and selecting monoclonal bacterial plaques for PCR detection;
(3) And (3) performing expansion culture: 50mL of YEP (Rif+Kan) solution is added into a 250mL triangular flask, 100 mu L of positive bacteria solution is added, the temperature is 28 ℃, the rpm is 200, and the culture is carried out overnight;
(4) Culturing the engineering bacteria to OD 600 When the ratio is about 0.6, the ratio is 4000rpm, and the centrifugation is performed for 5 minutes, thereby collecting the bacterial cells. The bacterial pellet was resuspended to OD with 1/2MS liquid medium 600 About 0.5, at 28℃and 200rpm for 30min;
(5) The cultured bacterial liquid is infected with the artemisia annua leaf explant for 20min, and the artemisia annua leaf explant is spread on an MS plate (MS+NAA 0.1mg/L+6-BA 1.0 mg/L). After 2d, dark culture at 25℃it was transferred to a differentiation plate (MS+NAA+6-BA+hygror). Changing the screening culture medium every 10 days, and growing a large number of cluster buds from the differentiation culture substrate after one month;
(6) Cutting off the cluster buds, and sequentially transferring to a strong seedling culture medium and a rooting culture medium until a complete plant is formed;
(7) Transplanting the rooted sweet wormwood plants into soil for growth;
3. gene expression level detection of positive plants
Detecting the gene expression quantity of the positive transgenic plant by using a fluorescence quantitative method, wherein Actin is used as an internal reference gene, and a primer for fluorescence quantitative detection is used as a primer:
qAaMAPK6-F：5’-GAACATCATGCCCCAACC-3’(SEQ ID NO.10)；
qAaMAPK6-R：5’-GGATTGTTTATATACAACAAACAGGAAG-3’(SEQ ID NO.11)；
qActin-F：5’-CCAGGCTGTTCAGTCTCTGTAT-3’(SEQ ID NO.12)；
qActin-R：5’-CGCTCGGTAAGGATCTTCATCA-3’(SEQ ID NO.13)：
reaction conditions: pre-denaturation at 94℃for 3min, denaturation at 94℃for 10s, annealing at 58℃for 20s, extension at 72℃for 20s,40 amplification cycles, fluorescence was collected after 72℃extension for each cycle.
The results are shown in FIG. 3, wherein the graph A shows the relative expression amount of the AaMAPK6 gene in the AaMAPK6 transgenic sweet wormwood (imapk 6-1, imapk6-2, imapk6-3, imapk 6-4) and wild-type sweet wormwood (CK), and the expression amount of the AaMAPK6 gene in the AaMAPK6 transgenic sweet wormwood strain is reduced to different degrees; panel B shows the relative expression levels of the AaMAPK6 gene in the over-expressed AaMAPK6 transgenic sweet wormwood (OX-AaMAPK 6-5, OX-AaMAPK6-7, OX-AaMAPK6-8, OX-AaMAPK 6-11) and wild-type sweet wormwood (CK), and shows that the expression levels of the AaMAPK6 gene in the over-expressed AaMAPK6 transgenic sweet wormwood strain are increased to different degrees.
Example 4 AaMAPK6 overexpression and interference expression determination of artemisinin content in Artemisia annua plants
1. Extraction of artemisinin
(1) Collecting aerial parts of herba Artemisiae Annuae, baking in oven at 40deg.C until completely drying, and grinding into powder;
(2) Preheating petroleum ether in a water bath kettle with constant temperature of 60 ℃;
(3) 0.2g of the powder is weighed into a 50mL Ep tube, 25mL of petroleum ether is added, and 80Hz ultrasonic treatment is carried out for 40min;
(3) Filtering with filter paper, and collecting filtrate in a 50mL small beaker; washing residues on the filter paper with 25mL petroleum ether, and collecting filtrate again in the same beaker;
(4) The liquid was transferred to a 100mL rotary evaporation flask and rotary evaporated under reduced pressure in a 50 ℃ water bath until petroleum ether was completely evaporated.
(5) Adding 1mL chromatographic grade methanol into a rotary evaporation bottle, and performing ultrasonic treatment for 1min; transfer the resuspended suspension to a 1.5mL Ep tube, centrifuge at 12000rpm for 10min; filtering the supernatant with 0.22 μm filter membrane, and measuring arteannuin content of the filtrate by high performance liquid chromatography combined with evaporative light scattering detection (HPLC-ELSD);
2. HPLC (high Performance liquid chromatography) determination of artemisinin content
The HPLC instrument was an SPD20A system controller, evaporative light scattering detector (Evaporative Light-Scattering Detector, ELSD). Using a Waters C18 column, mobile phase: acetonitrile and water, volume ratio of 60%
40%, flow rate: 1mL/min; the ELSD detection system is water alliance 2420, evaporative light scattering detectionThe temperature of the drift tube is 40 ℃, and the pressure of carrier gas is 5bar; standard samples were sampled at 20 μl, each at 20 μl, and 3 injections were repeated. The peak time of the artemisinin standard product is 9.703min, the retention time of the sample is 9.673min, the content of artemisinin in the sample is calculated according to the concentration and peak area of the standard product, and the content of artemisinin in the dry weight of the sample is calculated by dividing the content by the dry weight of the artemisinin powder.
The result of the artemisinin content detection of the transgenic plants is shown in fig. 4, wherein the graph A shows the comparison of the artemisinin content in the transgenic artemisia annua of the interference AaMAPK6 and the wild type artemisia annua, and the graph B shows the comparison of the artemisinin content in the transgenic artemisia annua of the overexpression AaMAPK6 (imapk 6-1, imapk6-2, imapk6-3 and imapk 6-4) and the wild type artemisia annua (CK), which shows that the artemisinin content in the plants of the interference AaMAPK6 gene is not obviously increased, and the artemisinin content in the plants of the overexpression AaMAPK6 gene is obviously increased by 1.26-1.56 times compared with the wild type.
The above-described embodiments are merely preferred embodiments for fully explaining the present invention, and the scope of the present invention is not limited thereto. Equivalent substitutions and modifications will occur to those skilled in the art based on the present invention, and are intended to be within the scope of the present invention. The protection scope of the invention is subject to the claims.
Claims (9)
1. The application of the overexpression of AaMAPK6 gene in sweet wormwood in improving the content of artemisinin is characterized in that: the nucleotide sequence of the AaMAPK6 gene is shown as SEQ ID NO. 3.
2. A method for increasing the content of artemisinin in artemisia annua, comprising the steps of: through over-expression of the AaMAPK6 gene in sweet wormwood, the nucleotide sequence of the AaMAPK6 gene is shown as SEQ ID NO. 3.
3. The method for increasing the content of artemisinin in artemisia annua according to claim 2, which is characterized in that: the method for over-expressing the AaMAPK6 gene in sweet wormwood comprises the following steps: constructing an AaMAPK6 gene on a plant binary expression vector, then transforming sweet wormwood under the mediation of agrobacterium tumefaciens, screening a transgenic plant, and screening the transgenic plant with the increased artemisinin content.
4. A method for increasing the content of artemisinin in artemisia annua according to claim 3 which is characterized in that: the plant binary expression vector takes pHB vector as skeleton vector.
5. A method for increasing the content of artemisinin in artemisia annua according to claim 3 which is characterized in that: the plant binary expression vector is formed by constructing an AaMAPK6 gene on a pHB vector through BamHI and XbaI sites.
6. A method for increasing the content of artemisinin in artemisia annua according to claim 3 which is characterized in that: the agrobacterium is EHA105.
7. A method for increasing the content of artemisinin in artemisia annua according to claim 3 which is characterized in that: the method for screening the transgenic plants adopts fluorescent quantitative RCR detection.
8. The method for increasing the content of artemisinin in artemisia annua according to claim 7, which is characterized in that: the upstream primer of the fluorescence quantitative RCR is shown as SEQ ID NO.10, and the downstream primer is shown as SEQ ID NO. 11.
9. The method for increasing the content of artemisinin in artemisia annua according to claim 7, which is characterized in that: the reaction conditions of the fluorescence quantitative RCR are as follows: pre-denaturation at 94℃for 3min; denaturation at 94℃for 10s, annealing at 58℃for 20s, extension at 72℃for 20s,40 amplification cycles, fluorescence was collected after 72℃extension for each cycle.
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