CN110964724A - Application of cymbidium goeringii miR390c in enhancing cold resistance of plants - Google Patents

Abstract

The invention discloses a Chinese cymbidiummiR390cGenes and application thereof in enhancing cold resistance of plants. The invention is obtained from the cymbidium goeringii cultivar Song plummiR390cThe precursor sequence fragment coding gene is subjected to expression analysis in cymbidium, then the precursor sequence fragment coding gene is constructed to an overexpression vector and is introduced into a target plant to verify the function of the overexpression vector, T3 generation plants are obtained through screening and culturing, the stress at 4 ℃ is carried out on the T3 generation plants, and the overexpression is foundmiR390cThe arabidopsis thaliana plant of the gene has stronger cold resistance than a wild type arabidopsis thaliana plant, so that the gene has wide application in production and breeding of orchids and other horticultural plants.

Description

Application of cymbidium goeringii miR390c in enhancing cold resistance of plants

Technical Field

The invention relates to the technical field of plant genetic engineering, in particular to a non-coding RNA (ribonucleic acid) of cymbidium goeringiimiR390cThe application in enhancing the cold resistance of plants.

Background

The orchid family (Orchidaceae) is one of the largest of the flowering plants, with 25000 varieties worldwide, accounting for approximately 10% of all flowering plants. Goering cymbidium (A. fern)Cymbidium goeringii) Belongs to the floret type of the orchid family, the orchid family is a floret type of the orchid family, the flower type is peculiar, the flower color is elegant, the flower fragrance is delicate,the leaf posture is beautiful, and the ornamental value and the economic value are extremely high. The cymbidium goeringii has high requirements on the growth environment, is very easily influenced by severe environments such as high temperature, low temperature, drought and the like in the growth process, and can cause the reduction of the ornamental quality of gardening and even the death of plants in severe cases. Therefore, the research on the molecular mechanism of the plant for coping with the abiotic stress and the identification of the gene with the stress resistance function have important significance on the breeding, the production and the application of the cymbidium goeringii. miR390 plays an important role in plant stress-resistant response, and can provide important basis for genetic improvement of plants.

MicroRNA390s (miR 390 s) is one of more conservative ancient families in plant microRNAs, can be widely involved in various plant physiological processes by regulating and controlling the expression of target genes, wherein one of hot spots of stress research of plants at present is used for stress response of the plants. Research shows that miR390 can induce cassava, tobacco, arabidopsis thaliana, soybean and the like to respond to drought stress and has tissue specificity. In plants such as corn and sorghum, miR390 can act on inorganic pyrophosphatase to realize salt stress response of plants. In addition, miR390 can regulate and control the processes of plants in coping with hypoxia stress, light stress, hormone stress, heavy metal stress and the like.

miR390 of the potato can influence the expression of downstream target genes (leucine-rich repeat type receptor protein kinase, endo 1,3;1,4- β -D-glucanase, SCRRK 1 and the like) to regulate and control the low-temperature response process of the plant, wherein the miR 390/SCRRK 1 regulation chain not only can enable the potato to respond to low temperature, but also can be regulated and controlled by ABA, GA3, IAA, PEG and other signals, miR390 can up-regulate the process of the plant for responding to cold stress when the bushiza suffers from low temperature, and miR390 of the astragalus can respond to low-temperature signals.

However, the specific mechanism of miR390 for regulating plant response to low-temperature stress is still not clear, so that the miR390 is cloned from cymbidium goeringii by using a genetic engineering technologymiR390cThe precursor gene is transferred into other plants, has important significance for researching the functions of the plants, and has great application prospect.

Disclosure of Invention

The invention provides a Chinese cymbidiummiR390cThe application in enhancing the cold resistance of plants.

A kind of Chinese cymbidiummiR390cUse of plant for enhancing cold resistancemiR390cThe nucleotide sequence of (A) is shown in SEQ ID NO.1 or SEQ ID NO. 2.

Preferably, the plant is cymbidium goeringii.

The specific method comprises the following steps: will contain the cymbidium goeringiimiR390cThe precursor gene is connected to a vector, is transformed into wild arabidopsis thaliana 'Columbia' through agrobacterium-mediated transformation, and is screened and cultured to obtain a transgenic plant.

Since miRNA is short and only about 20bp, and the precursor gene is slightly longer, the connection of the miRNA to a vector is beneficial to the realization of Arabidopsis thaliana transformation, and preferably, the sequence of the precursor gene is shown as SEQ ID No.3 or SEQ ID No. 4.

The invention is prepared by mixing the cymbidium goeringiimiR390cTransforming the gene into wild arabidopsis thaliana 'Columbia', obtaining T3 generation plants through screening and culturing, carrying out stress at 4 ℃ on the plants, and finding over-expressionmiR390cThe arabidopsis thaliana plant of the gene has stronger cold resistance than a wild type arabidopsis thaliana plant, so that the gene has wide application in production and breeding of orchids and other horticultural plants.

Drawings

FIG. 1 ismiR390cExpression in cymbidium. A.miR390cExpression profile in different tissues during vegetative and reproductive growth of cymbidium; B. chinese cymbidiummiR390cExpression at different times under low temperature stress at 4 ℃.

FIG. 2 shows pBI121 and cymbidium goeringiimiR390cGene precursor fragment-encoding gene (MIR390c) A schematic representation of a recombinant vector;

FIG. 3 ismiR390cGel profile of the overexpression vector construction process. A.miR390cA precursor PCR amplification gel map; B. pBI121-MIR390c（miR390cPrecursor) recombinant plasmid bacterial liquid PCR amplification gel map; C. pBI121-MIR390c（miR390cPrecursor) recombinant plasmid restriction map;

FIG. 4 shows the identification results of positive transgenic Arabidopsis plants of the T1 generation. A. Of 9 linesmiR390cA precursor PCR amplification gel map (M: DL2000 Marker; M1: positive control; M2: negative control; M3: blank control); B. of 9 linesmiR390cThe result of the fluorescence quantitative analysis;

FIG. 5 ismiR390cComparison of phenotype before and after stress treatment of over-expressed Arabidopsis with wild type at 4 ℃ (WT: wild type control;35S:MIR390ctransgenic plants).

Detailed Description

The present invention will be further described with reference to the following specific examples.

Example 1 functional prediction of Gene

The material 1 adopted in the embodiment is roots, stems and leaves of the cymbidium goeringii during the vegetative growth period and the reproductive growth period, the material 2 is tender leaves of the cymbidium goeringii during the low-temperature treatment at 4 ℃ for 0h, 2h, 6h, 12h and 24h, and the two materials are quickly frozen in liquid nitrogen after being picked and stored in an ultra-low temperature refrigerator (minus 80 ℃).

1) Extraction of Total Small RNA from various cymbidium tissues

The method is carried out according to the instruction of a TaKaRa plant total RNA extraction kit, and comprises the following specific operations:

rapidly transferring the frozen cymbidium tissue into a mortar precooled by liquid nitrogen, grinding the tissue by a pestle, and continuously adding the liquid nitrogen until the tissue is respectively ground into powder; respectively adding the samples ground into powder into 1.5mL of sterilized tube containing 450 mu l of buffer PE, and repeatedly blowing and beating by using a pipette until no obvious precipitate exists in the lysate; the lysate was centrifuged at 12,000rpm for 5 minutes at 4 ℃; the supernatant was carefully pipetted into a fresh 1.5mL sterile tube. Adding 1/10 volumes of Buffer NB into the supernatant, Vortex mixing, centrifuging at 12,000rpm and 4 ℃ for 5 minutes; carefully sucking the supernatant into a new 1.5mL sterilized tube, adding 450. mu.L Buffer RL, and uniformly mixing the solution by using a pipette; adding absolute ethyl alcohol with the volume 1.5 times of the volume of the mixed solution, uniformly mixing the solution by using a liquid-transferring gun, and immediately transferring the mixed solution into the RNAspin Column;centrifuging at 12,000rpm for 1min, discarding the filtrate, and returning the RNA Spin Column to 2ml Collection Tube; adding 600 μ L of 80% ethanol into RNA Spin Column, centrifuging at 12,000rpm for 30s, and discarding the filtrate; adding 50 mu L of DNase I reaction solution into the center of an RNA Spin Column membrane, and standing for 15 minutes at room temperature; 350 μ L of Buffer RWB was added to the center of the RNA Spin Column membrane, centrifuged at 12,000rpm for 30 seconds, and the filtrate was discarded; adding 600 μ L of 80% ethanol into RNA Spin Column, centrifuging at 12,000rpm for 30s, and discarding the filtrate; the RNA SpinColumn was re-mounted on a 2mL Collection Tube and centrifuged at 12,000rpm for 2 minutes; the RNA SpinColumn was mounted on 1.5mL of RNase Free Collection Tube, and 30. mu.L of RNase Free dH was added to the center of the RNA SpinColumn membrane₂O was left standing at room temperature for 5 minutes, and centrifuged at 12,000rpm for 2 minutes to elute RNA. The obtained RNA is stored in a refrigerator at minus 80 ℃ for later use after concentration and purity detection.

The result of taking 2. mu.L of RNA and detecting by 1% agarose gel electrophoresis shows that 28S and 18S bands are clearer, the brightness of the 28S band is about twice of that of the 18S band, and the RNA quality is better. Detection of RNA purity, OD by means of a micro-accounting protein assay₂₆₀/OD₂₈₀And OD₂₆₀/OD₂₃₀All are between 1.8 and 2.1, have good integrity and can be used for reverse transcription.

2) Reverse transcription and fluorescent quantitative analysis

According to the cymbidium microRNA omics sequencing result, utilizing Primer 5 to design cymbidiummiR390cThe gene stem-loop primer has the reference sequence: 5'-GTCGTATCCAGTGCAGGGTCCGAGGTATTCGCACTG GATACGACAAAACT-3' are provided. Meanwhile, 18S is taken as an internal reference gene, and the primer sequence is as follows: 5'-TCGCAGTGGTTCGTCTTT-3' are provided. Total Small RNAs of the above-mentioned tissues of cymbidium were used as templates, and HiScript III 1 by Vazyme^stThe specification of Strand cDNASynthesis Kit is mixed into a system, cDNA reverse transcription is carried out, and the reverse transcription program comprises the following steps: 15min at 37 ℃ and 5s at 85 ℃.

The reverse transcription product was diluted 10 times, 1. mu.L was used as template, and the fluorescent quantitative primers used were as follows:

miR390c-F：5’-CGCGCGGCTATCTATCCTG-3’

miR390c-R：5’-AGTGCAGGGTCCGAGGTATT-3’

18S-F：5’-GGTCCTATTGTGTTGGCT-3’

18S-R：5’-TCGCAGTGGTTCGTCTTT-3’

the preparation of the reaction solution was carried out using the instructions of the ChamQ ™ Universal SYBR Qpcr Master Mix kit (Vazyme Co.), and the PCR program was run on an Applied Biosystems type real-time fluorescence quantitative analyzer: 5min at 95 ℃; circulating for 40 times at 95 ℃ for 10s and 60 ℃ for 30 s; 95 ℃ for 15s, 60 ℃ for 1min and 95 ℃ for 15 s. Obtaining an amplification curve after the reaction is finished, deriving data through StepOne Software v2.3, analyzing the data by using Excel, and using 2 according to the CT value^-ΔΔCqThe relative expression was calculated by relative quantification, and the data analysis results are shown in FIG. 1.

This example shows that cymbidium goeringii is found based on the analysis of the results of fluorescence quantificationmiR390cThe low temperature treatment at 4 ℃ for 2h has quick response, and then the relative expression amount of the gene is reduced until the relative expression amount is close to an initial value, which indicates thatmiR390cCan regulate the tolerance of the plant to low-temperature stress. And the results of the cymbidium tissue expression analysis show that,miR390cthe expression level was relatively high in roots and pseudobulbs during the vegetative growth period of cymbidium goeringii, so it was presumed thatmiR390cIt is possible to defend plants against low temperatures by regulating certain activities of roots and pseudobulbs.

EXAMPLE 2 cloning and transformation of the Gene

The plant material used in this example was fresh leaves of cymbidium goeringii, songmeiArabidopsis thaliana) The Escherichia coli strain used is Trans5 α for gene cloning and over-expression vector construction, the vector construction is shown in figure 2, the Agrobacterium strain is GV3101 for transforming Arabidopsis thaliana, and the plant expression vector used in the test is pBI121, which are purchased from Protechs Biotech, Inc. and Pulsatis, respectively.

1) Extraction of cymbidium goeringii leaf gDNA

According to the instruction of TaKaRa plant Genomic DNA extraction kit, the specific operation is as follows:

transferring fresh leaf of "Songmei" of spring orchid into a mortar precooled with liquid nitrogen, grinding the tissue with a pestle while continuously adding liquid nitrogen until the ground leaf is powdered; the pulverized sample was quickly added to 1.5mL of sterilized tube containing 500. mu.l Buffer HS I and 10. mu.l of 50 XDTT Buffer mixture, mixed well, to which 10. mu.l RNase A was added, vortexed, mixed well, and incubated at 56 ℃ in a metal bath for 10 min. Adding 62.5 μ l Buffer KAC into the lysed sample, repeatedly blowing and mixing well with a pipette gun, placing on ice for 5min, and centrifuging at 12,000rpm for 5 min; carefully sucking the supernatant, transferring the supernatant into a new 1.5ml of sterilized tube, adding Buffer GB with the same volume as the supernatant, and uniformly mixing the two; transferring the obtained mixed solution into Spin Column of Collection tube, centrifuging at 12,000rpm for 1min, and discarding the filtrate; add 500. mu.l Buffer WA to Spin Column, centrifuge at 12,000rpm for 1min, discard the filtrate; adding 700 ul of Buffer WB along the periphery of the Spin Column wall, centrifuging at 12,000rpm for 1min, and discarding the filtrate; adding 700. mu.l of buffer WB along the periphery of the Spin Column wall, centrifuging at 12,000rpm for 1 minute, and removing the filtrate; centrifuge at 12,000rpm for 2 minutes to ensure no residual liquid remained in the Spin Column. Placing Spin Column in a new 1.5ml sterilized tube, adding 30 μ l of sterile water incubated on a 65 deg.C metal bath to the center of the Spin Column membrane, and standing at room temperature for 5 min; the gDNA was eluted by centrifugation at 12,000rpm for 2 minutes. The obtained gDNA is stored in a refrigerator at-80 ℃ for later use after concentration and purity detection.

2 mu L gDNA is absorbed and detected by 1.5% agarose gel electrophoresis, and the result shows that only one clear macromolecular band is available, and the quality of the whole genome DNA is better. Detection of gDNA purity, OD by means of a micro-accounting protein assay₂₆₀/OD₂₈₀And OD₂₆₀/OD₂₃₀All are between 1.8 and 2.1, have better integrity and can be used for PCR.

2) Design and cloning of target gene primer

Performing Blast homology comparison by using MIR390 related gene sequences of other species according to the existing sequencing data of the cymbidium miRNA group to obtain the sequence containing the cymbidiummiR390cThe precursor sequence of (1). Using Oligo6.0, Prime5.0 in cymbidiummiR390cCorresponding primers are designed at two ends of the hairpin structure of the precursor sequence and addedThe primer sequences of the homologous arms of the selected restriction sites (XbaI and SmaI) and pBI121 in which the two restriction sites are located are as follows:

MIR390c-XbaI-F：5'- GAGAACACGGGGGACTCTAGAACAAGTATGGGAG AACCATTAAAGC-3' (the XbaI cleavage site is underlined),

MIR390c-SmaI-R：5'- ATAAGGGACTGACCACCCGGGAAGAGCAAGAAGA ACCCATAAAACTC-3' (SmaI cleavage site is underlined).

Cloning of the cymbidium MIR390 gene was performed using PrimerStar Max Hi Fidelity enzyme with gDNA as template. The PCR amplification system (50. mu.L) was: 25 μ L PrimerStar Max, 2 μ L Forward Primer, 2 μ L Reverse Primer, 2 μ L lte plate DNA, 19 μ L ddH₂And O. The PCR procedure was: the reaction conditions are pre-denaturation at 94 ℃ for 3min, denaturation at 98 ℃ for 10s, annealing at 60 ℃ for 15s, extension at 72 ℃ for 30s, 32 cycles, total extension at 72 ℃ for 5min and heat preservation at 16 ℃.

After the PCR reaction is completed, all PCR products are taken to be detected by 1.8% agarose gel electrophoresis (the PCR amplification result is shown in figure 3A), target fragments are cut, and the target PCR amplification products are recovered and purified by gel. The DNA gel recovery kit of TransGen company is adopted to purify and recover the target fragment, and the specific operations are as follows: cutting a single target strip from the agarose gel, putting the cut single target strip into a clean centrifugal tube, and weighing the cut single target strip; adding 3 times volume of GSB (300 μ L GSB solution if gel is 0.1g and volume is 100 μ L) into the gel block, standing in 55 deg.C water bath while turning the centrifuge tube up and down continuously and gently until the gel block is completely dissolved; cooling the melted gel solution to room temperature, adding 1 volume of isopropanol (if the gel is 0.1g, 100 μ L of isopropanol), and gently mixing; adding the mixed solution into a centrifugal column, standing at room temperature for 1min, centrifuging at 12000rpm for 1min, discarding the effluent, and then putting the centrifugal column back into the collecting tube; adding 650 μ L of WB solution into the centrifugal column, centrifuging at 12000rpm for 1min, and discarding the effluent; centrifuging at 12000rpm for 2min to remove residual WB as much as possible, placing the adsorption column at room temperature, uncovering, standing for 5min, and air drying completely; placing the centrifugal column into a clean centrifugal tube, suspending and dropwise adding 30 mu L ddH to the middle position of the adsorption film₂O（ddH₂O needs to be put in water at 60-70 ℃ in advanceBath preheat), standing at room temperature for 2min, and centrifuging at 12000rpm for 2min to collect DNA solution. Taking 2 mu L of recovered and purified product, carrying out gel electrophoresis detection by using 1.5% agarose, placing the rest in a refrigerator at the temperature of-20 ℃, and subsequently connecting the product with a pBI121 vector to construct an overexpression vector.

3) And (3) plasmid extraction:

extracting plasmids according to the specification of the small-extraction medium-volume kit of the Tiangen plasmids, and specifically comprising the following steps:

taking 10mL of overnight cultured bacterial liquid, centrifuging at 12000rpm for 1min, and removing supernatant; adding 500 mu L P1 solution (containing RNaseA) into a centrifuge tube with the thallus precipitate, and completely suspending the thallus precipitate by using a vortex apparatus; adding 500 mu L P2 solution into a centrifuge tube, fully cracking thalli when turning the solution gently up and down, adding 700 mu L P3 solution into the centrifuge tube, immediately turning the solution gently up and down, fully mixing the solution, and centrifuging the solution at 12000rpm for 10min when white flocculent precipitates appear; adding 500 μ L of equilibrium liquid BL into adsorption column CP4, centrifuging at 12000rpm for 1min, discarding waste liquid in the collection tube, returning the adsorption column to the collection tube, adding collected supernatant into filtration column CS in batches, centrifuging at 12000rpm for 2min, carefully adding solution collected in the collection tube into adsorption column CP4 in batches, centrifuging at 12000rpm for 1min, discarding waste liquid in the collection tube, and returning adsorption column CP4 to the collection tube; adding 500 μ L deproteinized solution PD into adsorption column CP4, centrifuging at 12000rpm for 1min, discarding waste liquid in the collection tube, and replacing adsorption column CP4 into the collection tube; adding 600 μ l rinsing solution PW (containing anhydrous ethanol) into adsorption column CP4, centrifuging at 12000rpm for 1min, discarding waste liquid in the collection tube, placing adsorption column CP4 back into the collection tube, centrifuging at 12000rpm for 2min, and removing residual rinsing solution in the adsorption column; the adsorption column CP4 was transferred to a new 1.5ml centrifuge tube, and 60. mu.L ddH was added to the middle of the adsorption membrane₂O; standing at room temperature for 2min, centrifuging at 12000rpm for 1min, and collecting the solution in the centrifuge tube as plasmid. Finally, the plasmid concentration was determined and prepared for the next experiment.

4) Double enzyme digestion reaction

The extracted pBI121 plasmid is digested with XbaI and SmaI at 37 ℃ for 30min, and the linear vector is recovered by electrophoresis and stored at-20 ℃ for later use. The double enzyme digestion reaction system is 50 mu L: 20 mu.L of the pBI121 plasmid,5×buffer 5μL，XbaI 1μL，SmaI 1μL，ddH₂O 23μL。

5) recombination reactions

Agarose gel electrophoresis is used for detecting the target gene and the vector pBI121 recovered after enzyme digestion, and reagents are added according to a connection system according to the detected purity and concentration. Wherein, the number of target fragment molecules is: the number of carrier molecules =3: 1-5: 1, and the connection reaction system is as follows: linearized pBI121 vector 7. mu.L, insert 3. mu.L, 5 × CE II buffer 4. mu.L, Exnase II 2. mu.L, ddH₂OUp to 20. mu.L. The reaction was carried out at 37 ℃ for 30min, left at room temperature (without immediate cooling), and transformed to E.coli competent cells after 10 min.

6) Transfer of the ligation product into E.coli

The competent cell Trans5 α strain was taken out from the ultra-low temperature refrigerator, placed on ice to melt, 10. mu.L of recombinant product was taken and added to 100. mu.L of competent cell, the centrifuge tube was placed on ice for 10min, water bath was carried out in a water bath kettle at 42 ℃ for 90s with heat shock without shaking, then immediately placed on ice for 2min, 500. mu.L of liquid medium without antibiotic was added to a super clean bench, thawed by shaking at 37 ℃ and 200 rpm for 25min, centrifuged at 6000 rpm for 1min, 350. mu.L of supernatant was taken out, and the precipitated cells were resuspended, spread on LB plate (Kana concentration 50 mg/L), and cultured overnight at 37 ℃.

7) Identification of recombinants

Single colonies on the plates were picked and inoculated into LB liquid medium containing antibiotic (Kana), and shake-cultured overnight at 200 rpm at 37 ℃. PCR was performed on the bacterial suspension using the full-length primers of the target gene to screen positive clones, and the results of the bacterial assay are shown in FIG. 3B. The positive clones after screening were sent to Nanjing Sipulin for sequencing. And (3) carrying out positive cloning with a correct sequencing result, after amplification culture, extracting plasmids by using a Tiangen plasmid extraction kit, carrying out double enzyme digestion verification, and judging whether the sizes of fragments after enzyme digestion are consistent, wherein the enzyme digestion result is shown in a figure 3C, wherein M: DL2000 Marker; 1:miR390cthe precursor was ligated to pBI121 and digested simultaneously with XbaI and SmaI.

8) Preparation and transformation of Agrobacterium-infected competent cells

This example utilizes Agrobacterium GV3101 toPreparing agrobacterium infection status, and carrying out an infection experiment of arabidopsis; the preparation process of the agrobacterium infection is as follows: selecting an activated agrobacterium single colony, inoculating the agrobacterium single colony in 5mL of liquid LB culture medium, and performing shake culture at 28 ℃ and 250rpm for 20-24 h; 2mL of the bacterial suspension was aspirated, inoculated into a flask containing 50mL of liquid LB medium, and shaken at 28 ℃ and 250rpm to OD₆₀₀The value is about 0.8; placing the expanded bacterial solution on ice for ice bath for 30min, centrifuging at 4 ℃ and 5000 rpm for 5min, and removing the supernatant; 10mL of precooled 0.1 mo1/L CaCl was added₂A solution to fully suspend the precipitated bacteria; centrifuging at 4 deg.C and 5000 rpm for 5min, and discarding supernatant; 1mL of pre-cooled 20 mmo1/L CaCl was added₂The solution fully suspends the thalli to obtain GV3101 competent cells to be prepared, the competent cells are subpackaged into 100 mu L/tube by a centrifuge tube, 20% of sterile glycerol is rapidly added, and the competent cells are placed and stored at minus 80 ℃.

Agrobacterium transformation of recombinants: ice-bath to melt the agrobacterium-infected cells, adding 600ng of the recovered and purified plasmid into 100 mul of agrobacterium-infected cells, mixing the plasmid and the cells gently, and ice-bath for 5 min; quickly freezing with liquid nitrogen for 5min, thermally exciting in metal bath at 37 deg.C for 5min, and rapidly placing on ice for 5 min; adding 800 μ l LB culture medium without any antibiotic, and resuscitating at 28 deg.C and 200 rpm for 2 h; centrifuging at 4000rpm for 3min, and sucking off part of liquid culture medium; mixing the rest bacteria solution with a pipette, and spreading on solid LB medium containing 50 mg/L kanamycin and 100 mg/L gentamicin (GV 3101); and (3) performing inverted culture at 28 ℃ for 30-48 h.

Identification of Agrobacterium recombinants: picking out single colony from the plate culture medium, and inoculating the single colony in a liquid culture medium containing corresponding antibiotics; culturing at 28 deg.C and 200 rpm overnight; use of35SF, respectively matching the following primers to carry out PCR of the bacterial liquid, wherein the sequences of the primers are as follows:

35S-F：5'-GATAGTGGAAAAGGAAGGTG-3'，

35S:MIR390c-R：5'- ATAAGGGACTGACCACCCGGGAAGAGCAAGAAGA ACCCATAAAACTC -3'。

detecting the PCR product by 1.5% agarose gel electrophoresis, identifying whether the PCR product contains the target fragment, adding a proper amount of sterile 50% glycerol into the identified positive clone, and storing at-80 ℃ for later use.

9) Agrobacterium-mediated transformation of Arabidopsis thaliana

The method is characterized in that a target gene is transferred into arabidopsis thaliana by adopting an inflorescence infection method, and the specific operation method comprises the following steps: arabidopsis (col wild type) maintained healthy growth until flowering; activating the agrobacterium GV3101 strain carrying the target gene. Selecting a single colony, inoculating the single colony on 5mL LB culture medium containing kanamycin and gentamicin, and shaking the colony at the temperature of 28 ℃ and the speed of 200 rpm until the bacterial liquid just turns turbid for about 8-10 hours; 1mL of bacterial liquid is sucked and inoculated into a triangular flask (50 mL) for shaking bacteria for 24 hours until the OD value is about 0.8; centrifuging the bacterial liquid at 6000 rpm at room temperature for 5min, removing supernatant, collecting thallus, and suspending with 3% sucrose solution with pH of 5.8; before soaking, adding Silwet L-77 with the concentration of 0.03% (300 mul/L), and shaking out foams; soaking the overground part of arabidopsis in agrobacterium suspension for 1min, and gently shaking the overground part of arabidopsis; laying the soaked arabidopsis thaliana in a tray, covering the tray with a preservative film, sealing the tray with tinfoil paper in the dark, and standing for 24 hours; the tinfoil paper is uncovered, the culture is carried out under a normal condition, and watering is stopped when the seeds are mature.

The 3% sucrose solution resuspension had the following composition: MS culture medium, adding sucrose 30g/L, Silwet-77300 μ L/L. (Note: after preparation, pH was adjusted to 5.8, and after centrifugation and resuspension of the bacterial solution, Silwet L-77 was added, and the conversion relationship between the resuspension solution and the bacterial solution was that the amount of the resuspension solution was OD of the bacterial solution volume =0.8 of the bacterial solution volume).

10) Screening of transgenic plants

The collected seeds of T1 generation transgenic Arabidopsis are sterilized by alcohol and sodium hypochlorite, and the steps are as follows: placing appropriate amount of the obtained transgenic seeds in a 1.5mL centrifuge tube, and soaking for 5min with 0.8% NaClO and ethanol mixed solution (in situ, the volume ratio is 1: 1); sterilizing with 75% alcohol for 5-6 times, each time for 2 min; washing with sterile water for 3-4 times; the suspension was suspended in 0.1% agarose solution.

The sterilized transgenic Arabidopsis seeds are sown on MS solid culture medium containing antibiotics (kanamycin 50 mg/L and cefamycin 100 mg/L), wrapped by tinfoil and placed in a refrigerator at 4 ℃ for vernalization. After 2 days, the medium was removed from the refrigerator and incubated at 22 ℃ under light. After about one week, Arabidopsis thaliana which can grow normally on the medium is transplanted into soil and continues to grow.

11) DNA detection of transgenic plants

Taking a proper amount of T1 generation arabidopsis thaliana and young leaves of transgenic plants, extracting DNA by a CTAB method, and specifically comprising the following operation steps: placing a proper amount of leaves in a sterilized 2mL centrifuge tube, adding 700 mul of CTAB solution, thoroughly grinding by using a ball mill, and standing for 10min at 65 ℃; equal volume of chloroform was added: inverting isoamyl alcohol several times to mix uniformly, and centrifuging at 14000rpm for 10 min; transferring the supernatant into a new sterile centrifuge tube, adding isopropanol with the same volume, reversing and uniformly mixing for several times, standing at room temperature for 2min, centrifuging at 14000rpm for 10min, and pouring off the supernatant; adding 70% anhydrous ethanol, blowing and washing twice by using a liquid transfer gun, centrifuging at 14000rpm for 1min, and removing the supernatant; drying surface liquid, and adding 20 mu L ddH₂And dissolving the O. Taking the DNA of the above-mentioned extracted transgenic and wild type Arabidopsis thaliana, and usingmiR390cPCR detection is carried out by specific primers of the gene.

Chinese cymbidiummiR390cAfter gene transformation of Arabidopsis thaliana, a total of 9 over-expressions were obtainedmiR390cA transgenic Arabidopsis line. The PCR results are shown in FIG. 4A, using the recombinant plasmid as a positive control, the wild type as a negative control, and water as a blank control.

12) Fluorescent quantitative PCR detection of transgenic plants

From the above 9 overexpressing cymbidiummiR390cTotal Small RNA was extracted from young shoots of a transgenic Arabidopsis line, and reverse transcription and fluorescence quantitative primers, methods and procedures were the same as in example 1. U6 was used as an internal reference gene, and the fluorescence quantitative primer was as follows, and the reverse transcription primer was as follows U6-R.

U6-F：5’-GGTGCTAAGAAGAGGAAGAAT-3’

U6-R：5’-CTCCTTCTTTCTGGTAAACGT-3’

After the reaction, the data were analyzed in the same manner as in example 1 using 2 for the CT value^-ΔΔCqThe relative quantitative method was used to calculate the relative expression, and the final data analysis results are shown in FIG. 4B.

This example clones 1 cymbidium goeringiimiR390cPrecursor gene, namedMIR390cNucleotide sequence thereofAs shown in SEQ ID NO.3 or SEQ ID NO.4,MIR390cthe length of the gene nucleotide sequence is 133bp, the gene nucleotide sequence contains 1 stem-loop structure, and a pair of arms are arranged on the gene nucleotide sequencemiR390cA positive/negative complementary sequence, themiR390cThe mature body nucleotide sequence is shown in SEQ ID NO.1 or SEQ ID NO. 2. Then, the Chinese cymbidium already connected with the Chinese cymbidiummiR390cOf precursor genes35S:MIR390cThe over-expression recombinant vector is transferred into a model plant Arabidopsis thaliana, and the result shows that the obtained T1 generation plants are all cymbidium goeringiimiR390cOver-expressing the plants, and of different linesmiR390cThe degree of overexpression varies in young leaves.

Example 3 cymbidiummiR390cPhenotypic identification of enhanced plant cold resistance

1) Obtaining of transgenic homozygous plants: the harvested transgenic T1 generation seeds are sterilized, screened and cultured, and then transplanted into nutrient soil to be cultured at 22 ℃ for 16h in light/8 h in darkness; after detection, retaining the preliminarily confirmed transgenic plants, harvesting seeds of T1 generations after the plants are mature, and numbering to obtain T2 generations; like the T1 generation, seeds of the T2 generation are sterilized and then coated on a screening culture medium containing antibiotics, and the culture medium is placed at 22 ℃ for continuous illumination; performing survival rate statistics on T2 generation seeds with different numbers for about 10 days, selecting plants with survival rate of 75% for transplantation, culturing in nutrient soil at 22 ℃ for 16h in light/8 h in dark, and taking leaves for positive detection; continuously numbering positive T2 generation plants, and collecting seeds to obtain T3 generation seeds; sterilizing the seeds, screening by using a screening culture medium, and placing under the light for continuous illumination culture; around 10 days, different numbered T3 generation plants were observed, all survived and no segregating T3 homozygous plants appeared.

2) Cold stress treatment

Taking 60-day-old wild type transplanted into soil and T3 generation35S:MIR159c3 pots of transgenic arabidopsis thaliana are respectively put into a low-temperature environment at 4 ℃ for culture (water is poured and permeated 1-2 days before culture), and other conditions are as follows: in the first time period, 7000lx of illumination is carried out, the temperature is 4 ℃, the humidity is 75%, and the time duration is 16 h/d; in the second time period, the illumination is 0lx, the temperature is 4 ℃, the humidity is 75 percent, and the time duration is 8 h/d. Three days of culture, photography, observation of phenotype.

The results of the low temperature stress treatment at 4 ℃ are shown in FIG. 5, and the temperature at 4 ℃ isBefore the treatment, the transgenic arabidopsis thaliana and the wild arabidopsis thaliana have no significant difference; after treatment at 4 ℃, compared with the wild type, the cold resistance of the transgenic arabidopsis is enhanced, and the specific expression is as follows: the main inflorescence of the wild type sags, and all the cauline leaves sag and curl; while35S:MIR390cThe whole transgenic plant has small change, the inflorescence still keeps upright, and the stem leaves are curled a few.

This example will turn to cymbidium35S:MIR159cThe T3 generation homozygous plant is obtained by screening and culturing the arabidopsis plant, and after the arabidopsis plant is stressed at low temperature of 4 ℃, the result shows that the arabidopsis plant is over-expressed compared with the wild type35S:MIR159cThe arabidopsis thaliana has stronger cold resistance, which shows thatMIR159cCan help plants improve cold resistance.

Sequence listing

<110> Nanjing university of forestry

Application of <120> cymbidium goeringii miR390c in enhancing cold resistance of plants

<160>14

<170>SIPOSequenceListing 1.0

<210>1

<211>19

<212>RNA

<213> Cymbidium goeringii

<400>1

gcuaucuauc cugaguuuu 19

<210>2

<211>19

<212>DNA

<213> Cymbidium goeringii

<400>2

gctatctatc ctgagtttt 19

<210>3

<211>133

<212>RNA

<213> Cymbidium goeringii

<400>3

acaaguaugg gagaaccauu aaagcucagg agggauagcg ccacggaucg ugagagagcu 60

gauuagcucu uugaggcuuu cucucgcuuc ugugacgcua ucuauccuga guuuuauggg 120

uucuucuugc ucu 133

<210>4

<211>133

<212>DNA

<213> Cymbidium goeringii

<400>4

acaagtatgg gagaaccatt aaagctcagg agggatagcg ccacggatcg tgagagagct 60

gattagctct ttgaggcttt ctctcgcttc tgtgacgcta tctatcctga gttttatggg 120

ttcttcttgc tct 133

<210>5

<211>19

<212>DNA

<213> Artificial sequence (artiartiartifical sequence)

<400>5

cgcgcggcta tctatcctg 19

<210>6

<211>20

<212>DNA

<213> Artificial sequence (artiartiartifical sequence)

<400>6

agtgcagggt ccgaggtatt 20

<210>7

<211>18

<212>DNA

<213> Artificial sequence (artiartiartifical sequence)

<400>7

ggtcctattg tgttggct 18

<210>8

<211>18

<212>DNA

<213> Artificial sequence (artiartiartifical sequence)

<400>8

tcgcagtggt tcgtcttt 18

<210>9

<211>46

<212>DNA

<213> Artificial sequence (artiartiartifical sequence)

<400>9

gagaacacgg gggactctag aacaagtatg ggagaaccat taaagc 46

<210>10

<211>47

<212>DNA

<213> Artificial sequence (artiartiartifical sequence)

<400>10

ataagggact gaccacccgg gaagagcaag aagaacccat aaaactc 47

<210>11

<211>20

<212>DNA

<213> Artificial sequence (artiartiartifical sequence)

<400>11

gatagtggaa aaggaaggtg 20

<210>12

<211>47

<212>DNA

<213> Artificial sequence (artiartiartifical sequence)

<400>12

ataagggact gaccacccgg gaagagcaag aagaacccat aaaactc 47

<210>13

<211>21

<212>DNA

<213> Artificial sequence (artiartiartifical sequence)

<400>13

ggtgctaaga agaggaagaa t 21

<210>14

<211>21

<212>DNA

<213> Artificial sequence (artiartiartifical sequence)

<400>14

ctccttcttt ctggtaaacg t 21

Claims (4)

1. Chinese cymbidiummiR390cThe nucleotide sequence of the application in enhancing the cold resistance of plants is shown in SEQ ID NO.1 or SEQ ID NO. 2.

2. The use according to claim 1, wherein the plant is cymbidium goeringii.

3. The use according to claim 2, comprising: will contain the cymbidium goeringiimiR390cThe precursor gene is connected to a vector, is transformed into wild arabidopsis thaliana 'Columbia' through agrobacterium-mediated transformation, and is screened and cultured to obtain a transgenic plant.

4. Use according to claim 3, characterized in that the cymbidium goeringiimiR390cThe nucleotide sequence of the precursor gene is shown in SEQ ID NO.3 or SEQ ID NO. 4.

Images