CN110904106A - Application of cymbidium goeringii miR159b in enhancing plant cold sensitivity - Google Patents

Abstract

The invention discloses a Chinese cymbidiummiR159bGenes and their use in enhancing cold sensitivity in plants. The invention is obtained from the cymbidium goeringii cultivar Song plummiR159bThe gene encoding the precursor sequence fragment of (a)MIR159bExpression analysis is carried out in cymbidium goeringii, then the cymbidium goeringii is constructed until an overexpression vector is introduced into a target plant to verify the function of the overexpression vector, T3 generation plants are obtained through screening and culture, and the root growth speed of the seedling stage of the T3 generation plants is obviously accelerated compared with that of the wild plants; wild type to be transplanted into soil for 60 days and35S：MIR159bafter the transgenic plant is stressed at the low temperature of 4 ℃, the phenotype of the transgenic plant, such as droop of main inflorescence, no re-erection, droop and curling of cauline leaves, yellowing of basal leaves and the like, is found, and the gene is shown in orchidAnd other horticultural plants.

Description

Application of cymbidium goeringii miR159b in enhancing plant cold sensitivity

Technical Field

The invention relates to the technical field of plant genetic engineering, in particular to a non-coding RNA (ribonucleic acid) of cymbidium goeringiimiR159bThe application in enhancing the cold sensitivity 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 species of floret type Geshenlan in orchid family, and has peculiar flower type, light flower color, delicate flower fragrance, beautiful leaf appearance and high ornamental value and economic value. 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 plants for coping with abiotic stress and the identification of genes with adversity response function have important significance on the breeding, production and application of cymbidium goeringii. miR159 plays an important role in plant stress-resistant response and can provide an important basis for genetic improvement of plants.

MicroRNA159s (miR 159 s) is one of more conservative ancient families in plant microRNAs, and can participate in various life activity processes such as plant growth and development, plant secondary metabolism, plant response biology and abiotic stress. At present, research on miR159 focuses on growth and development, and adverse stress response and the like are relatively less concerned. miR159 can regulate plant response to heat stress, e.g., overexpression of miR159 can increase heat sensitivity of wheat. miR159 in cotton plays an important role in the process of crops for resisting pathogenic bacteria. The miR159 of the sweet potato can regulate the sweet potato to respond to salt stress. miR159 is also a key factor for crops such as corn, rice and tomato to respond to drought stress, but the specific regulation and control way in rice is related to variety, and the regulation and control modes of miR159 in different varieties are different. At present, the action of miR159 on the aspect of plant response to cold stress is not reported, so that the miR159 is cloned from cymbidium goeringii by using a genetic engineering technologymiR159bThe 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 cymbidiummiR159bThe application in enhancing the cold sensitivity of plants.

A kind of Chinese cymbidiummiR159bUse in enhancing cold sensitivity of plants, said plantsmiR159bThe 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 goeringiimiR159bThe 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 goeringiimiR159bTransforming the plant into wild arabidopsis thaliana 'Columbia', obtaining T3 generation plants through screening and culturing, and finding that the root growth speed of the plant in the seedling stage is obviously accelerated compared with that of the wild arabidopsis thaliana; wild type to be transplanted into soil for 60 days and35S： MIR159bafter the transgenic plant is stressed at the low temperature of 4 ℃, the phenotype of the transgenic plant, such as droop of main inflorescence, no re-erection, droop and curling of cauline leaves, yellowing of basal leaves and the like, is found, and the gene has wide application in the production of orchids and other horticultural plants.

Drawings

FIG. 1 ismiR159bExpression in cymbidium. A.miR159bExpression profile in different tissues during vegetative and reproductive growth of cymbidium; B. chinese cymbidiummiR159bExpression at different times under low temperature stress at 4 ℃.

FIG. 2 shows pBI121 and cymbidium goeringiimiR159bGene precursor fragment-encoding gene (MIR159b) A schematic representation of a recombinant vector;

FIG. 3 ismiR159bGel profile of the overexpression vector construction process. A.miR159bA precursor PCR amplification gel map; B. pBI121-MIR159b (miR159bPrecursor) recombinant plasmid bacterial liquid PCR amplification gel map; C. pBI121-MIR159b (miR159bPrecursor) recombinant plasmid restriction map;

FIG. 4 shows transgenic Arabidopsis thaliana positive for the T1 generationAnd (5) identifying the plants. A. Of 9 linesmiR159bA precursor PCR amplification gel map (M: DL2000 Marker; M1: positive control; M2: negative control; M3: blank control); B. of 9 linesmiR159bThe result of the fluorescence quantitative analysis;

FIG. 5 ismiR159bComparative root phenotype map of overexpression Arabidopsis (WT: wild-type control; 35S:MIR159btransgenic plants);

FIG. 6 ismiR159bComparison of phenotype before and after stress treatment of overexpression Arabidopsis with wild type at 4 ℃ (WT: wild type control; 35S:MIR159btransgenic 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 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 ultralow-temperature refrigerator at (-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 anhydrous ethanol with the volume of 1.5 times of the mixed solution to the mixtureAfter the solution is uniformly mixed by a liquid transfer gun, all the mixed solution is immediately transferred 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 better 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 cymbidiummiR159bThe gene stem-loop primer has the reference sequence: 5'-GTCGTATCCAGTGCAGGGTCCGAGGTATTCGCACTG GATACGACTAGAGC-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:

miR159b-F：5’- CGCGTCTGGATGTCAGGGA -3’

miR159b-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 quantificationmiR159bThe response was fast after a treatment at 4 ℃ for 6h, and then the relative expression decreased until the initial value was approached, indicating thatmiR159bCan regulate plant response to low temperature stress. And the results of the cymbidium tissue expression analysis show that,miR159bin both vegetative growth period and reproductive growth period of cymbidium goeringii, the expression level in roots is higher than that in leaves and pseudobulbs, so it is presumed thatmiR159bIt is possible to make plants respond to low temperatures by regulating certain activities of the roots.

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

Based on the existing sequencing data of the cymbidium miRNA group, the method utilizes other speciesMIR159Performing Blast homology comparison on related gene sequences to obtain the product containing cymbidium goeringiimiR159bThe precursor sequence of (1). Using Oligo6.0, Prime5.0 in cymbidiummiR159bCorresponding primers are designed at two ends of the hairpin structure of the precursor sequence, homologous arms of the two enzyme cutting sites on the selected enzyme cutting sites (XbaI and SmaI) and pBI121 are added, and the primer sequences are as follows:

MIR159b-XbaI-F：5'- GAGAACACGGGGGACTCTAGATAGAGCTCCCTGACATCCAGA AG-3' (the XbaI cleavage site is underlined),

MIR159b-SmaI-R：5'- ATAAGGGACTGACCACCCGGGTGGAGCTCCCTTC AATCCAA-3' (SmaI cleavage site is underlined).

Using gDNA as template, and using PrimerStar Max high fidelity enzyme to make cymbidiumMIR159bCloning of the gene. The PCR amplification system (50. mu.L) was: mu.l PrimerStar Max, 2. mu.l Forward Primer, 2. mu.l Reverse Primer, 2. mu.l Template DNA, 19. mu.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 column into a clean centrifuge tubeSuspending and dropping 30 mu L ddH to the middle position of the adsorption film₂O（ddH₂And preheating O in a water bath at 60-70 ℃ in advance), standing at room temperature for 2min, and centrifuging at 12000rpm for 2min to collect a 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 was digested with XbaI and SmaI at 37 ℃ for 30min, recovering the linear carrier by electrophoresis, and storing at-20 ℃ for later use. The double enzyme digestion reaction system is 50 mu L: pBI121 plasmid 20. mu.L, 5 XBuffer 5. mu.L, XbaI 1. mu.L, SmaI 1. mu.L, 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 90 s 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:miR159bthe precursor was ligated to pBI121 and digested simultaneously with XbaI and SmaI.

8) Preparation and transformation of Agrobacterium-infected competent cells

In the embodiment, agrobacterium GV3101 is used for preparing agrobacterium competence for 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:MIR159b-R：5'- ATAAGGGACTGACCACCCGGGTGGAGCTCCCTTCA ATCCAA -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 usingmiR159bPCR detection is carried out by specific primers of the gene.

Chinese cymbidiummiR159bAfter gene transformation of Arabidopsis thaliana, a total of 9 over-expressions were obtainedmiR159bA 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 cymbidiummiR159bTotal Small RNA was extracted from young stem leaves (same height) of the transgenic Arabidopsis line, and the reverse transcription and fluorescence quantitative primers, method and procedure 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 goeringiimiR159bPrecursor gene, namedMIR159bThe nucleotide sequence is shown as SEQ ID NO.3 or SEQ ID NO.4,MIR159bthe length of the gene nucleotide sequence is 214bp, the gene nucleotide sequence contains 1 stem-loop structure, and a pair of arms are arranged on the gene nucleotide sequencemiR159bA positive/negative complementary sequence, themiR159bThe mature body nucleotide sequence is shown as SEQ ID No.1 or SEQ ID number 2. Then, the Chinese cymbidium already connected with the Chinese cymbidiummiR159bOf precursor genes35S:MIR159bThe 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 goeringiimiR159bOver-expressing the plants, and of different linesmiR159bThe degree of overexpression varies in young leaves.

Example 3 cymbidiummiR159bPhenotypic identification of enhanced plant cold sensitivity

1) Obtaining of transgenic homozygous plants and observation of phenotype: 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.

The transgenic plants with obvious phenotype are selected for observation, and the result is shown in figure 5, compared with the wild type, the transgenic arabidopsis plants grow faster in the seedling stage, the root system length is about 0.5cm when the seeds are poured into the screening culture medium for 12 days, and the root system of the wild type is about 0.1 cm. In addition, the overground part of the transgenic plant at the seedling stage has a significantly higher growth speed than that of the wild type, the true leaves of the transgenic plant reach 4 at day 12, and only 2 true leaves of the wild type grow.

2) Cold stress treatment

Taking 60-day-old wild type transplanted into soil and T3 generation35S:MIR159b3 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 result of the 4 ℃ low-temperature stress treatment is shown in FIG. 5, and the transgenic Arabidopsis and the wild Arabidopsis have no significant difference before the treatment at 4 ℃; after treatment at 4 ℃, compared with the wild type, the cold sensitivity of the transgenic arabidopsis is obviously enhanced, and the cold sensitivity is specifically shown as follows: the main inflorescence of the transgenic type sags, does not stand again, the cauline leaves sag and curl, and the basal leaves turn yellow; while the wild type inflorescence only slightly sags at the top, the stem leaves are not curled obviously, and the basal leaves are not yellow.

This example will turn to cymbidium35S:MIR159bScreening and culturing Arabidopsis plants to obtain T3 generation homozygous plants, and finding that the root growth speed of the Arabidopsis plants in the seedling stage is remarkably increased compared with that of wild plants; wild type to be transplanted into soil for 60 days and35S：MIR159bafter the transgenic plant is stressed at the low temperature of 4 ℃, the result shows that the transgenic plant is over-expressed compared with the wild type35S:MIR159bThe Arabidopsis thaliana has stronger cold sensitivity, which shows thatMIR159bCan help plant improve cold sensitivity.

Application of <120> cymbidium miR159b in enhancing plant cold sensitivity

<160>14

<170>SIPOSequenceListing 1.0

<210>1

<211>20

<212>RNA

<213> Cymbidium goeringii

<400>1

uagagcuccc ugacauccag 20

<210>2

<211>20

<212>DNA

<213> Cymbidium goeringii

<400>2

tagagctccc tgacatccag 20

<210>3

<211>214

<212>RNA

<213> Artificial sequence (artiartiartifical sequence)

<400>3

uagagcuccc ugacauccaa aguaaggccu uugagggcuu guuauugcug cuggguuagg 60

aauuccacaa uucuauuuca uuaaaauccc ucaaauuggc auaacacaau gguguaaaaa 120

uaauauauug gaaauauggg gcagacuugc ggguuucgug accaaggagc uauuggcaag 180

ucuuaagagc uuguuuggau ugaagggagc ucca 214

<210>4

<211>214

<212>DNA

<213> Artificial sequence (artiartiartifical sequence)

<400>4

tagagctccc tgacatccaa agtaaggcct ttgagggctt gttattgctg ctgggttagg 60

aattccacaa ttctatttca ttaaaatccc tcaaattggc ataacacaat ggtgtaaaaa 120

taatatattg gaaatatggg gcagacttgc gggtttcgtg accaaggagc tattggcaag 180

tcttaagagc ttgtttggat tgaagggagc tcca 214

<210>5

<211>19

<212>DNA

<213> Artificial sequence (artiartiartifical sequence)

<400>5

cgcgtctgga tgtcaggga 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>44

<212>DNA

<213> Artificial sequence (artiartiartifical sequence)

<400>9

gagaacacgg gggactctag atagagctcc ctgacatcca gaag 44

<210>10

<211>41

<212>DNA

<213> Artificial sequence (artiartiartifical sequence)

<400>10

ataagggact gaccacccgg gtggagctcc cttcaatcca a 41

<210>11

<211>20

<212>DNA

<213> Artificial sequence (artiartiartifical sequence)

<400>11

gatagtggaa aaggaaggtg 20

<210>12

<211>41

<212>DNA

<213> Artificial sequence (artiartiartifical sequence)

<400>12

ataagggact gaccacccgg gtggagctcc cttcaatcca a 41

<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 cymbidiummiR159bThe nucleotide sequence of the application in enhancing plant cold sensitivity is shown as SEQ ID NO.1 or SEQ ID NO. 2.

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

3. The method of claim 2The method is characterized by comprising the following steps: will contain the cymbidium goeringiimiR159bThe 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 goeringiimiR159bThe nucleotide sequence of the precursor sequence is respectively shown as SEQ ID NO.3 or SEQ ID NO. 4.

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