CN113584034A

CN113584034A - miRNA related to artemisinin biosynthesis, miRNA precursor and application thereof

Info

Publication number: CN113584034A
Application number: CN202110988015.3A
Authority: CN
Inventors: 郭志英
Original assignee: Fujian Polytechnic Normal University
Current assignee: Fujian Polytechnic Normal University
Priority date: 2021-08-26
Filing date: 2021-08-26
Publication date: 2021-11-02
Anticipated expiration: 2041-08-26
Also published as: CN113584034B

Abstract

The invention relates to miRNA related to artemisinin biosynthesis, miRNA precursors and application thereof. The miRNA related to artemisinin biosynthesis is an Artemisia annua miRNA-miR160 gene, the sequence is shown as SEQ ID NO:1 in the sequence table, and the sequence is as follows: TGCCTGGCTCCCTGTATGCCA are provided. A precursor of miRNA related to artemisinin biosynthesis, wherein the precursor sequence of miRNA related to artemisinin biosynthesis is shown in SEQ ID NO.2 in the sequence table; according to the invention, the content of artemisinin in the artemisia annua can be increased by utilizing miRNA related to artemisinin biosynthesis, and the transgenic artemisia annua with high artemisinin content is cultivated, so that high-yield and stable plant materials are provided for large-scale production of artemisinin.

Description

miRNA related to artemisinin biosynthesis, miRNA precursor and application thereof

Technical Field

The invention relates to miRNA related to artemisinin biosynthesis, miRNA precursors and application thereof.

Background

Artemisinin is the main active ingredient of the well-known medicinal plant Artemisia annua (Artemisia annua L.) and Artemisinin-based combination therapies (ACTs) are the current world-wide remedies for malaria. Artemisinin is mainly derived from artemisia annua at present, but the content of artemisinin in artemisia annua plants is very low, so that the cost and the yield of the production process are limited. Genetic modification of artemisia annua through a secondary metabolic engineering strategy is a recognized most potential technical means capable of improving the content of artemisinin. The artemisinin synthesis pathway is clear at present, but how to perform gene regulation on the synthesis pathway according to needs remains a hotspot and difficulty of research in the academia. microRNA (miRNA) is known to be an important molecule involved in regulation of secondary metabolism genes, but the type and molecular mechanism of miRNA playing a role in regulating artemisinin biosynthesis are yet to be researched.

Disclosure of Invention

The invention provides miRNA related to artemisinin biosynthesis, miRNA precursors and application thereof. According to the invention, the content of artemisinin in the artemisia annua can be increased by utilizing miRNA related to artemisinin biosynthesis, and the transgenic artemisia annua with high artemisinin content is cultivated, so that high-yield and stable plant materials are provided for large-scale production of artemisinin.

In order to achieve the purpose, the technical scheme of the invention is as follows:

a miRNA related to artemisinin biosynthesis is an artemisia annua miRNA-miR160 gene, the sequence of the miRNA is shown as SEQ ID NO 1 in a sequence table, and the sequence is as follows: TGCCTGGCTCCCTGTATGCCA are provided.

A precursor of miRNA related to artemisinin biosynthesis, wherein the precursor sequence of miRNA related to artemisinin biosynthesis is shown in SEQ ID NO.2 in the sequence table;

the sequence is as follows:

ataTGCCTGGCTCCCTGTATGCCAtttgtagagctgatcatcgacatatcgatggcctccgtggatgGCGTATGAGGAGCCAAGCATAttcc。

application of miRNA related to artemisinin biosynthesis in culturing high-content artemisinin transgenic plants.

Further, the transgenic plant with high content of artemisinin is an artemisia annua plant.

An application of miRNA related to artemisinin biosynthesis in cultivation of transgenic plants with high content of artemisinin is disclosed, wherein the miRNA related to artemisinin biosynthesis is transgenic into artemisia annua, and the expression of the miRNA is inhibited in the artemisia annua, so that transgenic artemisia annua with high artemisinin content is cultivated.

A method for culturing transgenic plants with high content of artemisinin comprises the steps of transgenically introducing the miRNA related to artemisinin biosynthesis into artemisia annua L, and inhibiting the expression of the miRNA in the artemisia annua L, so as to culture the transgenic artemisia annua L with high content of artemisinin.

In particular to a method for cultivating transgenic plants with high content of artemisinin,

1) amplifying a precursor gene sequence by using a PCR method by taking wild artemisia annua DNA as a template; the sequence of the precursor gene is shown as SEQ ID NO. 2;

2) performing a connection reaction on a precursor gene DNA and a Blunt Zero vector, converting a connection product into an escherichia coli Trans1-T1 competent cell, screening and picking out a positive clone through Kan, performing PCR reaction verification and sequencing identification on the picked positive clone, and selecting a correctly identified bacterial liquid to extract a plasmid Blunt Zero-miR 160;

3) carrying out PCR amplification and gel recovery on the plasmid Blunt Zero-miR160 extracted in the step 2) by using a forward primer STTM160-PHB-F containing a BamHI enzyme cutting site and a reverse primer STTM160-PHB-R containing a SpeI enzyme cutting site to obtain a DNA fragment with the enzyme cutting site,

then connecting the obtained DNA fragment with the enzyme cutting site with a PHB-flag linearized vector by adopting a seamless cloning method;

transforming the product obtained by connection into escherichia coli, selecting a monoclonal colony for amplification culture, performing bacteria detection by using an STTM160 forward primer STTM160-PHB-F and a specific primer rbcsr on the vector, then selecting positive bacteria for cloning and sequencing verification, wherein the bacterial strain with correct sequencing is the miR160 suppression expression vector STTM 160-PHB;

the forward primer STTM160-PHB-F containing a BamHI enzyme cutting site is tctctctctaagcttGGATCctggcatacaggctagagcca, the reverse primer STTM160-PHB-R containing a SpeI enzyme cutting site is cttatcgataccgtcACTAGTtgcctggctctagcctgtatgccaattcttcttctt, and the PHB-flag linearization carrier is obtained by carrying out double enzyme cutting on the PHB-flag by BamHI and SpeI produced by New England Biolabs company and then carrying out glue recovery;

the specific primer rbcsr on the carrier is attaacttcggtcattagaggc;

4) transforming the miR160 inhibition expression vector STTM160-PHB obtained in the step 3) into artemisia annua callus through an agrobacterium-mediated method; then, carrying out selective culture on the obtained callus until granular resistant callus grows out; transferring the vigorous resistant callus to a differentiation culture medium containing hygromycin for culturing into regenerated plantlets, then transferring the regenerated plantlets to a rooting culture medium for strengthening seedlings, then carrying out seedling hardening and transplanting, selecting normally growing plants, carrying out hygromycin PCR screening to obtain positive T0 generation plants, harvesting seeds from mature T0 generation plants, carrying out T1 generation seed reproduction, harvesting seeds from mature T1 generation plants, carrying out T2 generation seed reproduction, and finally identifying the seeds harvested from T2 generation plants by hygromycin PCR, wherein the positive is a homozygous transgenic plant with high content of artemisinin.

Compared with the prior art, the invention has the following beneficial effects: the miRNA related to artemisinin biosynthesis can improve the content of artemisinin in artemisia annua and culture transgenic artemisia annua with high artemisinin content, so that high-yield and stable plant materials are provided for large-scale production of artemisinin.

Drawings

FIG. 1 is a schematic view of the structure of STTM 160.

FIG. 2 is a stem-loop structure of a precursor sequence of miRNA (aan-miR160) related to artemisinin biosynthesis according to the invention.

FIG. 3 shows the results of real-time fluorescence quantitative PCR analysis of the expression of miRNA (miR160) related to artemisinin biosynthesis in transgenic Artemisia annua with different overexpression and suppression expressions.

FIG. 4 shows the result of detecting the content of artemisinin in transgenic Artemisia annua plants by liquid chromatography-mass spectrometry (LC-MS/MS).

Detailed Description

The present invention will be described in detail below with reference to examples and drawings, but the present invention is not limited to the examples.

One) detailed description of the invention

the sequence is as follows:

the miRNA related to artemisinin biosynthesis is applied to culturing high-content artemisinin transgenic plants.

the specific primer rbcsr on the carrier is attaacttcggtcattagaggc;

Example 1

the specific primer rbcsr on the carrier is attaacttcggtcattagaggc;

In particular, the amount of the solvent to be used,

miR160 expression inhibition vector references Tang G, Yan J, Gu Y, et al.construction of short distance target mix (STTM) to block the functions of plant and animal microRNAs [ J ]. Methods,2012,58(2) are designed, the STTM framework sequence STTM is a nucleotide fragment with the length of 48bp, and the sequences at the two ends are the corresponding target miR160 binding sites which are 196bp in total: TGGCATACAGGctaGAGCCAGGCAGTTGTTGTTGTTATGGTCTAATTTAAATATGGTCTAAAGAAGAAGAATTGGCATACAGGctaGAGCCAGGCA, the structural schematic is shown in the following figure and designated as STTM 160. This short "gene" was obtained synthetically by Jinweizhi Biometrics. The STTM can effectively inhibit the combination of the miRNA and the target gene, resulting in the accumulation of the target gene, and thus plays an important role in studying the function of the key miRNA. And (3) constructing the STTM160-PHB vector according to the construction method of the miR160 overexpression vector. The insert STTM160 homologous amplification forward primer is STTM 160-PHB-F: tctctctctaagcttGGATCctggcatacaggctagagcca (containing BamHI enzyme cutting site), and the reverse primer is STTM 160-PHB-R: cttatcgataccgtcACTAGTtgcctggctctagcctgtatgccaattcttcttctt (containing SpeI enzyme cutting sites), and inserting the STTM160 fragment into the PHB-flag vector in a seamless cloning manner to complete the construction of the miR160 inhibition expression vector. (as shown in the schematic diagram of the STTM160 structure in FIG. 1). The strain with the correct sequencing is the successfully constructed expression vector STTM160-PHB for miR160 inhibition.

Screening of artemisia annua miR160

Method for treating artemisia annua with methyl jasmonate (MeJA): spraying MeJA solution with concentration of 100 μ M on multiple Artemisia annua seedlings which grow for 1 month and have consistent size and growth vigor, culturing under normal illumination, taking off the overground part of the Artemisia annua after 1h, 2h, 4h, 8h and 12h respectively, quickly freezing with liquid nitrogen immediately, and storing in a refrigerator at-80 ℃;

a method for treating artemisia annua with Salicylic Acid (SA) comprises the following steps: spraying SA solution with concentration of 1.0mM on multiple Artemisia annua seedlings which grow for 1 month and have consistent size and growth vigor, culturing under normal illumination, taking off the overground part of the Artemisia annua after 1h, 3h, 5h, 7h and 9h respectively, quickly freezing with liquid nitrogen immediately, and storing in a refrigerator at-80 deg.C;

a method for treating artemisia annua with abscisic acid (ABA) comprises the following steps: spraying ABA solution with the concentration of 10 MuM on a plurality of artemisia annua seedlings which grow for 1 month and have the same size and growth vigor, culturing under normal illumination, taking off the overground part of the artemisia annua after 1h, 2h, 4h, 8h and 12h respectively, quickly freezing by using liquid nitrogen immediately, and storing in a refrigerator at-80 ℃;

samples were taken as blank controls before the three hormone treatments, and three biological replicates were taken at each time point.

The RNA of the samples was extracted and purified by the method of TRIzol (Invitrogen, Carlsbad, Calif., USA). Then the concentration and purity of RNA are controlled by using NanoDrop. After the RNA of the artemisia annua is extracted, the quality is qualified by quality detection, and then the artemisia annua is directly used for constructing a small RNA (sRNA) library.

Using wild artemisia annua DNA as a template, amplifying to a precursor gene sequence by using a PCR method, designing a primer, amplifying, connecting a target segment with a carrier to obtain a transgenic plant, and detecting.

And screening miRNA with obvious expression difference compared with a blank control after hormone treatment according to the sequencing result of sRNA, and screening miR 160. The sequence is as follows: TGCCTGGCTCCCTGTATGCCA are provided.

sRNA sequencing library preparation A TruSeq Small RNA Sample Prep Kits was used. The reference document of the miRNA library construction process is German Marcelo A, et al.Global identification of microRNA-target RNA pairs and minor analysis of RNA ends [ J ]. NATURE BIOTECHNOLOGY,2008. sRNA (16-28nt) is separated from RNA by polyacrylamide gel, the construction and sequencing of sRNA library of MeJA, SA and ABA on the overground part of Artemisia annua in the research are committed to be completed by Union Chun GmbH, and a sequencing instrument is Illumina Hiseq 2500.

The method for extracting the total RNA of the artemisia annua specifically comprises the following steps:

total RNA (containing miR160) of artemisia annua is extracted by adopting an EnxWeiji corporation miRNA Isolation kit.

The method comprises the following specific steps:

the water saturated phenol is put at room temperature in advance to avoid flocculent precipitation, and then a phenol chloroform reagent (water saturated phenol: chloroform: isoamyl alcohol 125: 24: 1) is prepared and extracted according to the following steps:

a. calculating or estimating the mass of the sample;

b. grinding artemisia annua leaves in a precooled mortar by using liquid oxygen, and adding powder of a sample into a lysine/Binding Buffer by using a precooled spoon to mix uniformly;

c. adding 1/10 volume of miRNA Homogenate Additive, mixing by vortexing or reversing for several times, and placing the mixture on ice for 10 min;

d. adding phenol-chloroform with the same volume as the lysine/Binding Buffer, and uniformly mixing by swirling for 30-60 sec;

e.10000 Xg centrifuging at room temperature for 5min to separate organic phase and water phase;

f. transferring the water phase to a new centrifuge tube, and adding 1.25 times of volume of absolute ethyl alcohol into the water phase transferred to the new test tube;

g. adding the lysine/Binding Buffer mixed solution into a centrifugal column, centrifuging at 10000 Xg for 15sec, and discarding the filtrate;

h. adding 700mL of miRNA Wash Solution 1, and centrifuging for 5-10 sec;

i. 500mL of Wash Solution 2/3 was added and the filtrate was discarded (run twice);

j. discarding the filtrate, centrifuging for 1min to remove the excessive liquid;

k. placing the centrifugal column into a new centrifugal tube, adding 100mL of ribozyme-free double distilled water preheated at 95 ℃ in advance into the middle of the centrifugal tube, and centrifuging at the maximum rotating speed for 20-30sec to collect the total RNA of the artemisia annua.

The method for extracting the artemisia annua DNA specifically comprises the following steps:

extracting DNA of artemisia annua leaves by adopting a radix puerariae Plant Genomic DNAkit kit:

the method comprises the following specific steps:

adding 0.1 percent beta-mercaptoethanol into preheated GP 1; adding a specified volume of absolute ethyl alcohol into the buffer GD and the rinsing liquid PW, and extracting according to the following steps:

a. adding 700 mu L of buffer solution GP1 preheated at 65 ℃ into a 1.5mL centrifuge tube, grinding blades by liquid nitrogen, quickly transferring the blades into the centrifuge tube, uniformly mixing by vortex, and placing the mixture in a 65 ℃ water bath for 20 min;

b. adding chloroform solution with the same volume as GP1, mixing uniformly by vortex, and centrifuging at 12000rpm for 5 min;

c. transferring the aqueous phase solution to a new centrifuge tube, adding 700 mu L of buffer solution GP2, adding into an adsorption column after vortex mixing, centrifuging, and discarding waste liquid;

d. washing the adsorption column with 500 μ L buffer GD and 600 μ L rinsing solution PW (twice), centrifuging at 12000rpm for 30sec each time, and pouring off waste liquid;

e. placing adsorption column CB3 back into the collecting tube, centrifuging, discarding waste liquid, and standing at room temperature for 5 min;

f. transferring the adsorption column CB3 into a new centrifuge tube, adding 50 μ L of 65 deg.C preheated double distilled water dropwise into the adsorption membrane, standing at room temperature for 5min, centrifuging, and collecting filtrate, and storing at-20 deg.C.

The miR160 precursor sequence amplification specifically comprises the following steps:

the method comprises the following specific steps:

extracting a sequence with the length of about 500bp containing a miR160 precursor (about 150 bp) from artemisia annua genome data to obtain a miR160 gene, and designing a forward primer (miR160-F) by taking the sequence as a template: ggagggtgaaggaatcaaca, reverse primer (miR 160-R): ccgctacccttcaattaacc, performing PCR amplification by taking artemisia annua DNA as a template to obtain a miR160 precursor sequence;

(1) PCR amplification

Reaction system: 1 μ L of Artemisia annua DNA, 2.5 μ L of miR160-F, 2.5 μ L of miR160-R, 10 μ L of 5 × Phusion HF Buffer, 0.5 μ L of Phusion DNApolymerase, and finally adding ddH₂O to 50 μ L;

PCR reaction process): 30sec at 98 deg.C, 35 amplification cycles (first denaturation at 98 deg.C for 10 sec; then annealing at 60 deg.C for 30sec), 50sec at 72 deg.C extension, and 8min at 72 deg.C extension,

(2) electrophoresis

Adding loading buffer into the PCR product obtained by PCR amplification, placing in 1% agarose gel at 150V for electrophoresis for 20min, detecting under an ultraviolet lamp, and cutting gel according to the size of the strip.

(3) Glue recovery

The gene segments after electrophoresis and gel cutting were recovered according to the instructions of the gel recovery kit of the whole-body gold company.

(4) Connection of

According to the complete formula gold pEasy^TMThe Blunt Zero kit ligation protocol ligated the gel-recovered product to the vector in the following reaction system: mu.L of the gel recovered in step (3), 1. mu.L of pEASY-Blunt Zero Cloning, vector, and ddH₂The volume of the mixture is between O and 5 mu L,

(5) transformation of Escherichia coli

1) And (4) taking out the competent cells from a refrigerator at the temperature of-80 ℃, adding the products connected in the step (4), and gently mixing.

2) Ice-cooling for 30min, heat-shocking for 90sec in 42 deg.C water bath, and ice-cooling for 2 min.

3) Add 500. mu.L of antibiotic-free LB medium to the clean bench, activate at 37 ℃ and 200rpm/min for 1 h.

4) Applying the bacterial liquid on Kan⁺75mg/L LB solid plate, 37 degrees C overnight inverted culture.

(6) PCR bacterial detection system and program

Randomly picking a plurality of monoclonal bacterial plaques from the plate, respectively adding 500 mu L of LB liquid culture medium containing 75mg/L Kan antibiotics, culturing for 4h at 37 ℃ and 200rpm/min, taking bacterial liquid as a template, and using a specific primer M13F on Blunt Zero: gtaaaacgacggccagt, respectively; M13R: caggaaacagctatgac PCR detection.

The PCR bacterial detection reaction system is as follows: 1 μ L of bacterial liquid, 1 μ LM13F, 1 μ LM13R, 10 μ L of 2 XpreMix rTaq, and ddH₂The volume of the mixture is between O and 20 mu L,

the reaction conditions were as follows: pre-denaturation at 94 ℃ for 4min, 30 amplification cycles (denaturation at 94 ℃ for 30sec, annealing at 55 ℃ for 30sec), extension at 72 ℃ for 50sec, extension at 72 ℃ for 10min,

(7) sequencing

Detecting PCR products by using agarose gel electrophoresis, picking out strains with the size consistent with that of target fragments, taking out 200 mu L of bacterial liquid, sending the bacterial liquid to Suzhou Jinweizhi technology service company Limited for sequencing to be miR160 precursor, and storing the residual bacterial liquid at 4 ℃. And (4) storing the bacterial liquid obtained by comparing the sequencing result with the related sequence in the artemisia annua genome for subsequent experiments.

The method for synthesizing the first strand cDNA of miR160 by adopting a stem-loop method specifically comprises the following steps

And (3) synthesizing the first strand cDNA of miR160 by adopting a Shanghai-produced miRNA first strand cDNA synthesis (stem-loop method) kit.

The method specifically comprises the following steps:

1. the following reaction mixture was added to an ice-bath centrifuge tube for reverse transcription: 2 XmiRNA L-RT Solution mix (10. mu.L), miRNAL-RT Enzyme mix (1.5. mu.L), Total RNA (3-4. mu.g), 10. mu.M Stem-loop primer (1. mu.L) and RNase-free water to 20. mu.L;

2. mixing, centrifuging for 30min at 16 deg.C, heating for 30min at 37 deg.C, heating at 85 deg.C for 5min to inactivate enzyme, and storing at-20 deg.C.

Synthesizing first strand cDNA, performing fluorescent quantitative PCR experiment, and analyzing miR160 expression level

Comparative example 1

3) carrying out PCR amplification and gel recovery on the plasmid Blunt Zero-miR160 extracted in the step 2) by using a forward primer miR160-PHB-F containing a BamHI enzyme cutting site and a reverse primer miR160-PHB-R containing a SpeI enzyme cutting site to obtain a DNA fragment with the enzyme cutting site,

transforming the product obtained by connection into escherichia coli, selecting a monoclonal colony for amplification culture, detecting the colony by using a forward primer miR160-PHB-F and a specific primer rbcsr on the carrier, selecting a positive clone, sequencing and verifying, wherein the strain with correct sequencing is an miR160 overexpression carrier miR 160-PHB;

the forward primer miR160-PHB-F containing BamHI enzyme cutting sites is tctctctctaagcttGGATCCcacccactaccatcacactc; the reverse primer miR160-PHB-R containing the SpeI enzyme cutting site is cttatcgataccgtcACTAGTttacaccgctacccttcaat;

the PHB-flag linearized vector is obtained by carrying out double enzyme digestion on the PHB-flag by BamHI and SpeI produced by New England Biolabs company and then recovering glue;

the specific primer rbcsr on the carrier is attaacttcggtcattagaggc.

4) The miR160 overexpression vector miR160-PHB obtained in the step 3) is transformed into artemisia annua callus through an agrobacterium-mediated method; then, carrying out selective culture on the obtained callus until granular resistant callus grows out; selecting vigorous resistant callus, transferring the vigorous resistant callus to a differentiation culture medium containing hygromycin for culture, transferring the vigorous resistant callus to a rooting culture medium after a regenerated plantlet grows to form a strong seedling, picking out a transgenic seedling for hardening and transplanting, selecting a normally grown plant, and carrying out hygromycin PCR screening to obtain a positive T0 generation plant; seeds of T1 generation are harvested and identified until T2 generation, and homozygous transgenic plants are obtained.

Specifically, miR160 overexpression vector construction

(1) Obtaining the insert

According to a PHB-flag carrier sequence and a DNA sequence of a miR160 precursor, introducing a linearized carrier terminal homologous sequence before a digestion site at the 5 ' end of a miR160 precursor DNA sequence fragment in the forward direction and the reverse direction of an amplification primer, so that the 5 ' and 3 ' extreme ends of the amplified miR160 precursor sequence fragment are respectively provided with homologous recombination sequences corresponding to the two ends of the linearized cloning carrier. Designing a forward primer for inserting a DNA sequence fragment of the miR160 precursor into a PHB-flag carrier according to the primer design principle, wherein the forward primer is miR 160-PHB-F: tctctctctaagcttGGATCCcacccactaccatcacactc (containing BamHI enzyme cutting sites), and the reverse primer is miR 160-PHB-R: cttatcgataccgtcACTAGTttacaccgctacccttcaat (containing SpeI enzyme cutting sites), PCR amplification was performed using a bacterial solution containing the DNA sequence of miR160 precursor correctly sequenced as a template.

The PCR reaction system is as follows: 1 mul bacterial liquid, 2.5 mul miR160-PHB-F, 2.5 mul miR160-PHB-R, 10 mul 5 XPhusion HF Buffer, 0.5 mul Phusion DNA polymerase, adding water to 50 mul,

the PCR reaction conditions are as follows:

amplification pre-denaturation at 98 ℃ for 30sec, 35 amplification cycles (98 ℃ 10 sec; 60 ℃ 30sec), extension at 72 ℃ for 50sec, extension at 72 ℃ for 8min,

and (3) performing electrophoresis and glue recovery after PCR amplification.

(2) Preparation of linearized vector PHB-flag

Carrying out double enzyme digestion on a PHB-flag vector by using BamHI and SpeI restriction enzymes, and loading the sample according to the following system components: 2 μ g PHB-flag plasmid, 1.5 μ L of LBamHI-HF, 1.5 μ L of SpeI-HF, 5 μ L of 10 XCutsmart, plus ddH₂And (3) performing enzyme digestion for 30min at 37 ℃ when the volume of O is 50 mu L, and then performing gel recovery on an enzyme digestion product to obtain a linear carrier PHB-flag.

(3) Ligation of vector and target fragment

Connecting the PCR gel recovery product in the step (1) with the gel recovery enzyme digestion product in the step (2);

reference is made in particular to the near shore company

plus One step PCR Cloning Kit (NR005-01A) instructions:

the reaction system is as follows: step (1) PCR glue recovery product, linear carrier PHB-flag, 1 uL NovoRec plus recombinase, 4 uL 5 x buffer, then ddH is added₂O to 20 mu L, wherein the molar ratio of the PCR glue recovery product in the step (1) to the linear carrier PHB-flag is 2: 1;

before connection, the components are mixed according to a molar ratio of 2: 1, adding a PCR glue recovery product and a linear carrier PHB-flag, slightly mixing, carrying out short centrifugation, and then incubating for 10min at 50 ℃.

And (3) transforming the ligation product into escherichia coli, selecting a monoclonal colony for amplification culture, and then performing amplification culture on the colony by using an insert miR160 forward primer miR160-PHB-F and a carrier specific primer rbcsr: attaacttcggtcattagaggc bacteria detection, selecting positive clone, sending to company for further verification. The strain with correct sequencing is the miR160-PHB successfully constructed as the miR160 overexpression vector.

Obtaining transgenic artemisia annua plants

Adopting agrobacterium-mediated artemisia annua genetic transformation, and infecting artemisia annua leaf discs with EHA105 agrobacterium containing PCR (polymerase chain reaction) bacteria detection positive recombinant expression vectors miR160-PHB, STTM160-PHB and a PHB-flag empty vector respectively, wherein the experimental method comprises the following steps: zhang L, sting F, Li F, et al.development of transgenic Artemisia annua (Chinese word) plants with an enhanced content of artemisinins, an effective anti-malarial drug, by hairpin-RNA-mediated gene immunization [ J ]. Biotechnology and Applied Biochemistry,2009. transgenic Artemisia annua seedlings were removed from culture flasks, planted in pots, and covered with preservative film around the pots to prevent water loss from the seedlings. The growth is carried out under the illumination condition of 16h/8h at 28 ℃. Selecting strains with good growth state and vigorous vitality for numbering,

the PHB-flag is induced to be a no-load transgenic plant, and the numbers of the plant are Vector-1, Vector-2, Vector-3 and … …;

the miRNA overexpression transgenic plant induced by EHA105-miR160-PHB is numbered as follows: 35S is miR160-1, 35S is miR160-2, 35S is miR160-3 and … …;

miR160 induced by STTM160-PHB inhibits expression of transgenic plants, and the number is as follows: STTM160-1, STTM160-2, STTM160-3, … …;

meanwhile, wild type artemisia annua is taken as negative control, and the numbers of the wild type artemisia annua are WT-1, WT-2, WT-3 and … ….

Identification of transgenic plants

For Hyg screening positive transgenic plants, further confirmation of the gene integration into the Artemisia annua genome by PCR is required in order to eliminate false positive. Taking out 100mg of each serial number of transgenic artemisia annua seedlings, and extracting transgenic artemisia annua DNA by referring to the previous step. The PHB-flag vector has a hygromycin resistance gene (hpt), and PCR amplification identification is carried out by using the hpt gene, a vector construction gene forward primer and a vector specific reverse primer rbscr, hpt-F (CGATTTGTGTACGCCCGACAGTC), hpt-R (CGATGTAGGAGGCGTGGATATG).

qRT-PCR analysis of miRNA expression level

The real-time fluorescent quantitative PCR reaction uses TransStart Top Green qPCR super Mix of the whole gold company to analyze the expression level of miRNA in artemisia annua, takes the artemisia annua Actin gene as an internal reference, takes miR160 first strand cDNA as a template, adopts a two-step method to amplify a PCR program, amplifies and pre-denatures 10sec at 95 ℃, performs 40 amplification cycles (95 ℃ 5 sec; 60 ℃ 30sec), and separates the reaction (95 ℃ 15 sec; 60 ℃ 30 sec; 95 ℃ 30 sec).

Determination of artemisinin content in transgenic artemisia annua by LC-MS/MS

Sample pretreatment: respectively collecting WT, Vector, 35S, miR160 and STTM160 transgenic Artemisia annua fresh leaf material, and oven drying in an oven at 50 deg.C overnight; grinding the material with a grinder, adding a proper amount of methanol according to a method of extracting 500g of methanol per 0.05g of the material; carrying out ultrasonic extraction for 30min in 40W ultrasonic after uniform oscillation; centrifuging the sample at 10000 rpm for 20 min; extracting twice, centrifuging, mixing the supernatants, rotary evaporating to dry, dissolving in methanol, diluting by 500 times, collecting supernatant, and performing content determination by liquid chromatography-tandem mass spectrometry (LC-MS/MS).

An Agilent 1200 liquid chromatograph-G6410A triple quadrupole mass spectrometer was used, comprising a G1311A infusion pump, a G1329A autosampler, and a G1316C column incubator. The liquid chromatographic column is a ZORBAX SB-C18 silica gel column with 2.1 AX SB-C18 spectral content of 3.5 AX; the mobile phase was acetonitrile-0.1% aqueous formic acid (70: 30 ratio); the flow rate is 0.3 ml/min; the detection time is 4.2 min; the sample injection volume is 5 samples. The mass spectrum was measured using an electrospray ionization (ESI) ion source in positive ion mode at a nebulizer gas pressure of 40psi, a temperature of 350si, a gas flow rate of 10L/min and a capillary voltage of 4000V. MassHunter software controls the system and data processing workstation (Agilent, USA).

The reagents and starting materials used in the present invention are commercially available or can be prepared according to literature procedures. Experimental procedures without specific conditions noted in the following examples, generally following conventional conditions such as Sambrook et al molecular cloning: the conditions described in the Laboratory Manual (New York: Cold Spring Harbor Laboratory Press, 1989), either according to conventional conditions or according to the manufacturer's recommendations;

the overexpression vector (PHB-Flag) plasmid used is constructed with reference to Reining C, Xianghui C, Tingting Z, et al. Integrated Transcript and Metabolite Profiles, derived That EbHI Plays an antigen Role in cloning and expression in Erigeron Britain Hairy Roots [ J ]. Frontiers in Plant Science,2018,9:789-, and the suppression expression vector (STTM) plasmid used is constructed with reference to Guiliang T, Jun Y, Yiou G, et al. construction of short target plasmid (STTM) to 2012 of vectors and antigens RNAs [ J ]. method 58(2): 125. Unless otherwise indicated, percentages and parts are by weight.

As shown in FIG. 2, the stem-loop structure of the precursor sequence of miRNA (aan-miR160) related to artemisinin biosynthesis of the invention is shown. The precursor sequence is folded into a stable stem-loop structure, belongs to the typical secondary structure of the miRNA precursor, and accords with the structural characteristics of the miRNA precursor.

FIG. 3 shows the results of real-time fluorescence quantitative PCR analysis of the expression of miRNA (aan-miR160) related to artemisinin biosynthesis in transgenic Artemisia annua with different overexpression and suppression expression. Wherein, the wild type artemisia annua (WT), the no-load (Vector), miR160 overexpression (35S: miR160) and miR160 inhibition expression (STTM160) plants have miR160 expression level. Actin acts as an internal control.

The result shows that the expression level of miR160 in the overexpression transgenic line is 1.57-4.58 times of that of wild artemisia annua; the expression level is reduced by 27-85% when the transgenic artemisia annua is inhibited and expressed, which indicates that the expression quantity of the aan-miR160 in the transgenic artemisia annua is successfully over-expressed or inhibited and the transgenic artemisia annua with lower miR160 expression level and higher miR160 expression level are respectively screened for artemisinin content determination.

FIG. 4 shows the result of detecting the content of artemisinin in transgenic Artemisia annua plants by liquid chromatography-mass spectrometry (LC-MS/MS). Wherein, the content of artemisinin in wild artemisia annua (WT), no-load (Vector), miR160 overexpression (35S: miR160) and miR160 suppression expression (STTM160) plants.

The artemisinin content of miR160 after overexpression is remarkably reduced, compared with the artemisinin content of wild artemisia annua, the artemisinin content of an overexpression strain is reduced to 30.1-58.9% of the original artemisinin content, wherein the minimum content of miR160-7 artemisinin is only 2.81mg/g in 35S; on the contrary, the content of artemisinin in the transgenic artemisia annua for inhibiting and expressing the miR160 is obviously increased, and compared with the wild type, the content of artemisinin in the transgenic artemisia annua strain for inhibiting and expressing the miR160 is increased by 65.5-81.0%, wherein the highest content is 17.20mg/g of STTM160-7 strain.

While the preferred embodiments of the present invention have been described in detail, it will be understood by those skilled in the art that the invention is not limited thereto, and that various changes and modifications may be made without departing from the spirit of the invention, and the scope of the appended claims is to be accorded the full scope of the invention.

<110> institute of Fujian technology

<120> miRNA related to artemisinin biosynthesis

<160>15

<170>PatentIn version 3.4

<210>1

<211>21

<212>RNA

<213> Artemisia annua L (Artemisia annua L.)

<400>1

TGCCTGGCTCCCTGTATGCCA

<210>2

<211>92

<212>PRT

<213> Artemisia annua L (Artemisia annua L.)

<400>1

ataTGCCTGGCTCCCTGTATGCCAtttgtagagctgatcatcgacatatcgatggcctccgtggatgGCGTATGAGGAGCCAAGCATAttcc

Sequence listing

<110> institute of Fujian technology

<120> miRNA related to artemisinin biosynthesis, miRNA precursor and application thereof

<160> 2

<170> SIPOSequenceListing 1.0

<210> 3

<211> 21

<212> DNA

<213> Artemisia annua (Artemisia annua)

<400> 3

tgcctggctc cctgtatgcc a 21

<210> 2

<211> 92

<212> DNA

<213> Artemisia annua (Artemisia annua)

<400> 2

atatgcctgg ctccctgtat gccatttgta gagctgatca tcgacatatc gatggcctcc 60

gtggatggcg tatgaggagc caagcatatt cc 92

Claims

1. An miRNA associated with artemisinin biosynthesis, wherein: the miRNA related to artemisinin biosynthesis is an Artemisia annua miR160 gene, the sequence is shown as SEQ ID NO:1 in a sequence table, and the sequence is as follows: TGCCTGGCTCCCTGTATGCCA are provided.

2. The precursor of a miRNA associated with artemisinin biosynthesis according to claim 1, wherein: the precursor sequence of the miRNA related to artemisinin biosynthesis is shown as SEQ ID NO.2 in the sequence table;

the sequence is as follows:

3. use of the miRNA related to artemisinin biosynthesis of claim 1 in cultivation of transgenic plants with high content of artemisinin.

4. The use of the miRNA related to artemisinin biosynthesis in cultivation of transgenic plants with high artemisinin content in the plant cultivation method as claimed in claim 3, wherein the miRNA comprises the following steps: the transgenic plant with high content of artemisinin is an artemisia annua plant.

5. The use of miRNA associated with artemisinin biosynthesis in breeding of transgenic plants with high artemisinin content according to claim 4, wherein the miRNA comprises the following components: and (3) transgenically introducing the miRNA related to artemisinin biosynthesis into the artemisia annua, and inhibiting the expression of the miRNA in the artemisia annua so as to culture the transgenic artemisia annua with high artemisinin content.

6. A method for cultivating a transgenic plant with high content of artemisinin is characterized in that: transgenic artemisia annua containing high content of artemisinin is cultivated by transferring the miRNA related to artemisinin biosynthesis in the artemisia annua of claim 1 into the artemisia annua and inhibiting the expression of the miRNA in the artemisia annua.

7. The method for cultivating transgenic plant with high content of artemisinin as claimed in claim 6, wherein:

transforming the product obtained by connection into escherichia coli, selecting a monoclonal colony for amplification culture, performing bacteria detection by using an STTM160 forward primer STTM160-PHB-F and a specific primer rbcsr on the carrier, then selecting positive bacteria for cloning and sequencing verification, wherein the bacterial strain with correct sequencing is an miR160 suppression expression carrier STTM 160-PHB;

the specific primer rbcsr on the carrier is attaacttcggtcattagaggc;