Application of LhMIR394B gene in enhancing somatic embryogenesis efficiency in hybrid Liriodendron chinense
By constructing and expressing an overexpression vector of the hybrid tulipwood LhMIR394B gene, the problem of low somatic embryogenesis efficiency in hybrid tulipwood was solved, and the number of somatic embryos generated was significantly increased.
Patent Information
- Application Number
- CN202211742742.2
- Authority / Receiving Office
- CN · China
- Patent Type
- Patents(China)
- Current Assignee / Owner
- Filing Date
- 2022-12-30
- Publication Date
- 2025-09-30
- Estimated Expiration
- 2042-12-30
AI Technical Summary
During somatic embryogenesis, hybrid tulip trees have poor reproductive capacity, low fruit set rate and large genetic variation. The existing molecular regulatory mechanism of somatic embryogenesis is not in-depth enough, and there is little research on the function of miR394 in somatic embryogenesis.
By cloning the LhMIR394B gene of hybrid Liriodendron chinense, an overexpression vector pBI121-LhMIR394B was constructed, and the gene was transformed into embryonic callus by Agrobacterium infection to achieve efficient expression of the LhMIR394B gene.
The efficiency of somatic embryogenesis was significantly improved, and the number of somatic embryos generated was significantly increased compared with the untransformed wild type.
Smart Images
Figure CN116103295B_ABST
Abstract
Description
Technical Field
[0001] The invention belongs to the field of plant molecular biology, and more particularly relates to the application of hybrid Liriodendron tulipifera LhMIR394B gene in enhancing the efficiency of somatic embryogenesis. Background Art
[0002] Liriodendron, a genus of the Magnoliaceae family, once had dozens of species widely distributed throughout the Northern Hemisphere. However, most became extinct after the Quaternary glaciation, leaving only two extant species: Liriodendron chinense and Liriodendron tulipifera. The hybrid Liriodendron (L. chinense × L. tulipifera) was developed in 1963 by renowned Chinese forest breeder Professor Ye Peizhong, using Liriodendron tulipifera from Lushan, China as the female parent and crossing it with Liriodendron tulipifera from North America. The hybrid exhibits significant hybrid vigor. The hybrid boasts an attractive tree shape, unique leaves, large, beautiful flowers, rapid growth, and excellent wood quality, making it an important species for garden landscaping, afforestation, and timber production. However, the hybrid has poor reproductive capacity and low fruit set, and its seedlings propagated by seed exhibit significant genetic variation. The somatic embryogenesis pathway has the advantages of rapid reproduction and genetic stability. Hybrid tulip tree already has a mature somatic embryogenesis system, but the molecular regulatory mechanism during its somatic embryogenesis process needs to be further studied.
[0003] MiRNAs are ubiquitous in plant genomes. They are a class of non-coding, single-stranded RNA molecules approximately 21 nucleotides long, encoded by endogenous genes. They regulate gene expression primarily at the post-transcriptional level by binding to target mRNAs, thereby playing a vital role in plant organ morphogenesis, growth and development, and responses to external stresses. Among them, miR394 not only influences plant stress tolerance but also plays a crucial role in plant growth and development. In particular, miR394 and its target genes can inhibit the negative feedback regulation of the apical stem cell marker genes WUSCHEL and CLV3, thereby maintaining normal development of the plant apical meristem. However, the function of miR394 in somatic embryogenesis has been little studied. Summary of the Invention
[0004] In view of the above problems existing in the prior art, the technical problem to be solved by the present invention is to provide an application of the hybrid tulipwood LhMIR394B gene in enhancing the efficiency of somatic embryogenesis.
[0005] In order to solve the above technical problems, the technical solutions adopted by the present invention are as follows:
[0006] The invention discloses an application of the hybrid Liriodendron chinense LhMIR394B gene in enhancing the efficiency of somatic embryogenesis. The nucleotide sequence of the hybrid Liriodendron chinense LhMIR394B gene is shown in SEQ ID NO.1.
[0007] Furthermore, the application includes the following steps:
[0008] (1) Construction of a vector for the LhMIR394B gene;
[0009] (2) transforming the constructed LhMIR394B gene vector into plants or plant tissues;
[0010] (3) Cultivate and screen plants or plant tissues with enhanced somatic embryogenesis efficiency.
[0011] Furthermore, the plant is a hybrid tulip tree.
[0012] Furthermore, the plant tissue is hybrid tulip tree embryonic callus.
[0013] Furthermore, the transformation is carried out by Agrobacterium infection.
[0014] Furthermore, the vector is a plant expression vector.
[0015] Furthermore, the plant expression vector is pBI121-LhMIR394B.
[0016] Compared with the prior art, the present invention has the following beneficial effects:
[0017] (1) The present invention uses hybrid tulip leaves as material and clones the hybrid tulip leaf LhMIR394B gene. Simultaneously, an overexpression vector pBI121-LhMIR394B is constructed based on this gene and transferred into hybrid tulip leaf embryonic callus. The LhMIR394B gene is efficiently expressed in the hybrid tulip leaf embryonic callus.
[0018] (2) p35S: LhMIR394B and untransformed wild-type 166302 callus (WT) were induced to produce somatic embryos by liquid suspension system. Two months later, the number of somatic embryos produced by transgenic callus overexpressing LhMIR394B was significantly increased compared with that of wild-type. BRIEF DESCRIPTION OF THE DRAWINGS
[0019] Figure 1 This is the PCR result diagram of the cloned target gene fragment;
[0020] Figure 2 This is the PCR electrophoresis diagram of a single colony of Escherichia coli containing pBI121-LhMIR394B;
[0021] Figure 3This is the PCR electrophoresis image of a single colony solution of Agrobacterium pBI121-LhMIR394B;
[0022] Figure 4 This is a diagram showing the PCR verification results of transgenic calli overexpressing LhMIR394B;
[0023] Figure 5 This is a diagram showing the somatic embryo induction of callus tissues overexpressing LhMIR394B transgene and wild type;
[0024] Figure 6 This is a statistical analysis of the number of somatic embryos produced in callus tissues overexpressing the LhMIR394B transgene and the wild type. DETAILED DESCRIPTION
[0025] The present invention is further described below with reference to specific examples. In the following examples, any operations not described in detail are routine biological experimental procedures and can be performed with reference to molecular biology laboratory manuals and existing published journals, or according to the kits and product instructions. Unless otherwise specified, the materials and reagents used in the following examples are all commercially available.
[0026] Example 1: Cloning of the hybrid Liriodendron chinense LhMIR394B gene
[0027] 1. Extraction of genomic DNA
[0028] Take finger-sized fresh leaves of hybrid tulip trees and grind them thoroughly; after the leaves become powder, add 700μL CTAB lysis buffer to the centrifuge tube, stir evenly and place it in a 65℃ water bath or air bath for 30 minutes, making sure to shake and mix every 5 minutes; add equal volumes of chloroform and isoamyl alcohol (volume ratio of 24:1) to the centrifuge tube, vortex and mix, and centrifuge at 12000rpm for 5 minutes; take the supernatant and add equal volumes of chloroform and isoamyl alcohol (volume ratio of 24:1), and repeat the previous step; take the supernatant to another clean 1.5mL centrifuge tube, add 1.5 times the volume of ice ethanol, and place it at 4℃ for 30 minutes to allow the DNA to precipitate at the bottom of the centrifuge tube; after precipitation, centrifuge at 12000rpm for 5 minutes, remove the supernatant, and add 700μL Wash with 70% or 75% alcohol, shake gently upside down, centrifuge at 12000rpm for 2 minutes, remove the supernatant, place in a ventilated place to dry, add 50μL of 65℃ preheated ddH2O, place in a 65℃ warm water bath for 5 minutes to dissolve the DNA in water. After the DNA is dissolved, use Nanodrop to detect the extraction concentration and purity.
[0029] 2. Cloning of target genes
[0030] 1) Primer Design: Based on the existing hybrid Liriodendron chinense genome data, BLAST search and comparison were performed. According to the comparison results, corresponding primers were designed using Primer 5.0. The primers were analyzed using Oligo 7.0. The final primers were determined as follows:
[0031] LhMIR394B-F: 5'-CCTATAAATACACCTCCCTACCTC-3',
[0032] LhMIR394B-R: 5'-AGCGAAATTTACAGAACTTGCTT-3'.
[0033] 2) PCR amplification reaction
[0034] PCR was performed using genomic DNA as a template using Vazyme's Phanta Max Super-Fidelity DNA Polymerase. The amplification system consisted of 25 μL 2× Phanta Max Buffer, 1 μL dNTP Mix (10 mM each), 2 μL LhMIR394B-F, 2 μL LhMIR394B-R, 1 μL Phanta Max Super-Fidelity DNA Polymerase, 1 μL hybrid Liriodendron genomic DNA, and 18 μL ddH₂O. PCR reaction conditions were: 95°C for 3 min; 95°C for 15 s, 54°C for 15 s, 72°C for 20 s, 35 cycles; 72°C for 5 min; and storage at 4°C.
[0035] 3) Recovery of target genes
[0036] The above PCR reaction products were electrophoresed on 1% agarose gel. Figure 1 As shown, the target band was excised and recovered using a DNA gel recovery kit from TSINGKE Biotechnology Co., Ltd., following the same steps as the kit instructions.
[0037] 4) Ligation of target fragment and cloning vector
[0038] TaKaRa's pMD19-T Vector (D102A) was used for ligation reaction. The reaction system consisted of 5 μL solution, 1 μL pMD19-T Vector, 2 μL PCR purified product, and 2 μL H2O. The mixture was mixed by pipetting, centrifuged at low speed, and ligated in a water bath at 16°C overnight.
[0039] 5) Conversion, screening and sequencing of ligation products
[0040] Transformation: Remove Tiangen DH5α competent cells from -80°C and thaw on ice. Add 10 μL of the ligation product, gently flick the bottom of the tube to mix, and place on ice for 30 minutes. Heat shock at 42°C for 75 seconds, then immediately place on ice for 2-3 minutes. Add 900 μL of liquid LB medium (without Amp) and incubate at 37°C with shaking at 150 rpm for 45 minutes to allow the resistance gene to express. Centrifuge at 4000 rpm for 2 minutes, discard 800 μL of the supernatant, and resuspend the pellet in the remaining LB. Spread the resuspension from the previous step onto a solid LB plate containing Amp and incubate inverted at 37°C for 12-16 hours.
[0041] Positive clone screening: Pick a single colony from the LB plate and inoculate it into 800 μL LB liquid medium containing Amp antibiotics, and culture it at 37°C and 220 rpm overnight.
[0042] Bacterial liquid PCR identification: TSINGKE Biotechnology Co., Ltd. 2×T5 Super PCR Mix (Colony) was used. The PCR system consisted of 7.5 μL 2×T5 Super PCR Mix, 0.6 μL M13F, 0.6 μL M13R, 2 μL bacterial liquid, and 4.3 μL ddH2O. The reaction conditions were 98°C for 3 min, 98°C for 10 s, 54°C for 10 s, 72°C for 2 min, 35 cycles, 72°C for 2 min, and storage at 4°C.
[0043] After the reaction, 5 μL of PCR product was taken for agarose gel electrophoresis. Figure 2 The bacterial solution with the correct band after verification was sent to Qingke Biotechnology Co., Ltd. for sequencing. The sequencing used universal primers M13-F and M13-R. The sequence is as follows:
[0044] M13-F: 5'-CGCCAGGGTTTTCCCAGTCACGAC-3',
[0045] M13-R: 5'-GAGCGGATAACAATTTCACACAGG-3'.
[0046] Bioinformatics analysis was performed based on the sequencing results to determine that the target gene sequence was as shown in SEQ ID NO. 1. The bacterial solution with the correct sequencing was stored at -80°C.
[0047] Example 2: Construction of Hybrid Liriodendron LhMIR394B Gene Expression Vector
[0048] Based on the sequencing results of 19T-simple, appropriate primers were designed using Oligo7.0 and NEB μLider Assembly Tool (http: / / nebuilder.neb.com / ). The primer sequences are as follows:
[0049] 35S-F: 5'-TGAAGATAGTGGAAAAGGAAGGTG-3',
[0050] LhMIR394B-F: 5'-CCTATAAATACACCTCCCTACCTC-3',
[0051] LhMIR394B-R: 5'-AGCGAAATTTACAGAACTTGCTT-3',
[0052] LhMIR394B+OL-F: 5'-gagaacacgggggactctagaCCTATAAATACACCTCCCTACCTCTTT-3',
[0053] LhMIR394B+OL-R: 5'-cgatcggggaaattcgagctcAGCGAAATTTACAGAACTTGCTTG-3'.
[0054] 1. Target gene plus overlap
[0055] The 19T-simple bacterial suspension containing the target gene fragment that was sequenced correctly was shaken vigorously, and then the plasmid was extracted using the Tiangen plasmid mini-extraction kit, and the extraction steps were consistent with the instructions.
[0056] The 19T-simple plasmid carrying the target gene extracted above was used as a template, and a pair of primers, LhMIR394B+OL-F and LhMIR394B+OL-R, were used with Vazyme's Phanta Max Super-Fidelity DNA Polymerase for PCR. The amplification system consisted of 25 μL 2× Phanta Max Buffer, 1 μL dNTP Mix (10 mM each), 2 μL LhMIR394B+OL-F, 2 μL LhMIR394B+OL-R, 1 μL Phanta Max Super-Fidelity DNA Polymerase, 1 μL 19T-simple plasmid template, and 18 μL ddH2O. PCR reaction conditions were: 95°C for 3 min; 95°C for 15 s, 69°C for 15 s, 72°C for 20 s, 35 cycles; 72°C for 5 min; and storage at 4°C.
[0057] The PCR reaction products were electrophoresed on a 1% agarose gel, and the target band was then excised and recovered using a DNA gel recovery kit from TSINGKE Biotechnology Co., Ltd., following the same steps as in the kit instructions.
[0058] 2. Obtaining linearized vectors
[0059] To obtain the expression vector: Take a DHAa (pBI121 empty vector) bacterial suspension stored at -80°C and streak it onto an LB plate containing kanamycin (50 mg / L) using an inoculating needle. Incubate the plate in an inverted position at 37°C overnight. Shake the suspension gently and vigorously, and extract the plasmid using the same method as above.
[0060] Expression vector pretreatment (enzyme digestion): Two enzyme digestion sites, XbaI and SacI, were selected based on the multiple cloning site on the pBI121 vector. The pBI121 empty vector was double-digested with the following enzyme digestion system: 1 μL XbaI, 1 μL SacI, 10 μL plasmid, 5 μL 10× Cutsmart buffer, 33 μL ddH2O, and incubated at 37°C for 4 hours. After the enzyme digestion, the enzyme was inactivated at 65°C in a PCR instrument for 30 minutes.
[0061] 3. Connect the target fragment to the linearized vector
[0062] Vazyme's ClonExpress II One Step Cloning Kit was used for the ligation reaction. The ligation system was 1 μL of the target gene insert, 4 μL of the linearized pBI121 vector, 4 μL of 5×CEII Buffer, 2 μL of Exnase II, and 9 μL of ddH2O. The mixture was gently pipetted and mixed, and then the reaction solution was collected to the bottom of the tube by brief centrifugation and ligated in a PCR instrument at 37°C for 30 minutes.
[0063] 4. Conversion, screening and sequencing of ligation products
[0064] The recombinant plasmid was transformed into DH5α competent cells of TIANGEN, and the transformation method was consistent with the instructions. After the colonies grow, use tweezers to pick up the white pipette tip and pick up the single colony grown on the plate, and place the pipette tip into 800μL of LB liquid culture medium containing kanamycin, shake the bacteria at 37°C and 250rpm for 12-16h. Then perform bacterial liquid PCR verification. The 2×T5 Super PCR Mix (Colony) reagent used was TSINGKE Biotechnology Co., Ltd., and 35S-F, LhMIR394B-R (containing a sequence on the vector) and LhMIR394B-F, LhMIR394B-R two pairs of primers were used for bacterial liquid PCR. The PCR system and reaction procedure were the same as above. After the PCR reaction, agarose gel electrophoresis was performed, and the bands were as follows. Figure 2 As shown. Bacterial samples with the correct band size were sent to TSINGKE for sequencing using primers 35S-F and LhMIR394B-R. Single colony samples with the correct sequencing results were stored at -80°C.
[0065] Example 3: Transformation of Hybrid Liriodendron Embryogenic Callus with LhMIR394B Gene
[0066] In this example, the Agrobacterium infection method was used. The Agrobacterium used was EHA105, which was transformed into embryonic callus of hybrid Liriodendron chinense. The genotype of the embryonic callus used was 166302.
[0067] The culture medium formula used is as follows:
[0068] M13 subculture medium: 3 / 4MS + Vc 5 mg / L + 2,4-D 2 mg / L + BA 0.2 mg / L + CH 0.5 g / L + sucrose 30 g / L + agar 2.5 g / L.
[0069] 1. Bacterial liquid preparation
[0070] 1) Transformation of EHA105 with pBI121-LhMIR394B recombinant plasmid
[0071] The above sequenced correctly E. coli bacteria carrying the pBI121-LhMIR394B recombinant plasmid were shaken, and then the plasmid was extracted to obtain the pBI121-LhMIR394B recombinant plasmid. The extracted recombinant plasmid was transformed into Agrobacterium competent cells EHA105, using EHA105 competent cells from Shanghai Weidi Biotechnology Co., Ltd. The transformation method was consistent with the instructions. After a single colony grew, use tweezers to pick up a single colony grown on the plate with a white pipette tip. Place the pipette tip in 800 μL of LB liquid medium containing kanamycin, shake the bacteria at 28°C and 220 rpm for 12-16 hours, and then perform PCR verification of the bacterial solution. 2×T5 Super PCR Mix (Colony) from TSINGKE Biotechnology Co., Ltd. was used for bacterial PCR using two pairs of primers: 35S-F, LhMIR394B-R and LhMIR394B-F, LhMIR394B-R. The PCR system and reaction procedures were the same as above. After the PCR reaction, agarose gel electrophoresis was performed. The bands were as follows. Figure 3 shown.
[0072] 2) Preparation of bacterial solution:
[0073] (1) Shake the bacterial solution: Add 2 mL of LB (containing 50 mg / mL of kanamycin) and 20 μL of bacterial solution to a 10 mL centrifuge tube and shake at 220 rpm and 28°C for 12-16 h.
[0074] (2) Propagation of bacterial culture: Add 50 mL of LB (added with 50 mg / mL of kanamycin) and 2 mL of bacterial culture to a 250 mL centrifuge tube. Shake at 220 rpm and 28°C for 4-6 h. Detect the OD value when it reaches 0.6-1.0.
[0075] (3) Resuspending the Bacterial Solution: Transfer 40 mL of the above bacterial solution to a 50 mL centrifuge tube and centrifuge at 5000 rpm for 10 min. Discard the supernatant and add an appropriate amount of M13 liquid medium to resuspend the suspension to a final OD600 of 0.8. Resuspension formula: measured OD value × 40 mL = 0.8 × volume of M13 liquid medium.
[0076] 3. Pre-culture of infected materials: Take callus about 18 days after subculture and pre-culture it on M13 medium containing AS (acetosyringone, 100umol / L) for 4-6 days.
[0077] 4. Infection and co-cultivation: In a clean bench, take about three dishes of callus and place them in a 100 mL Erlenmeyer flask. Add the resuspended bacterial solution and infect for 10-15 min. Pass the solution through a 400-mesh sieve and filter it. Use tweezers to pick up the filter paper and absorb the bacterial solution. Transfer the callus to solid M13 medium (+AS, concentration as above) and co-cultivate for 2 days.
[0078] 5. Degerming: Place the co-cultured callus in a 100 mL Erlenmeyer flask and wash it alternately with liquid M13 (+ / -cet) until the callus washing liquid is clear. Collect the callus with a 400-mesh sieve and culture it on solid M13 (+cef, concentration of 400 mg / mL) for recovery culture. Be careful not to degerming for more than 10 minutes.
[0079] 6. Screening: After one week of sterilization and recovery culture, the infected calli were placed on solid M13 (+cef) supplemented with G418 (75 mg / L) for screening and culture.
[0080] 7. Callus PCR Verification: The first subcultured callus was designated as a cell line. After sufficient subcultures were achieved, a callus was collected from each cell line and DNA was extracted using the CTAB method, following the same procedures as above. Transgene PCR verification was performed using Vazyme's 2× Rapid Taq Master Mix. PCR reactions were performed using two primer pairs, 35S-F, LhMIR394B-R, and LhMIR394B-F, LhMIR394B-R. The reaction system consisted of 5 μL 2× Rapid Taq Master Mix, 0.4 μL Primer 1, 0.4 μL Primer 2, 3.2 μL ddH2O, and 1 μL DNA template. The reaction procedure was: 95°C for 3 min, 95°C for 15 s, 54°C for 15 s, 72°C for 20 s, 35 cycles, 72°C for 5 min, and storage at 4°C.
[0081] According to the above method, the transgenic callus lines were tested for PCR positive results. Figure 4 The stripe sizes are as expected.
[0082] Example 4: Somatic Embryo Induction Experiment
[0083] In this example, somatic embryos were induced by liquid suspension system. The experimental materials were p35S:LhMIR394B and untransformed wild-type 166302 callus (WT).
[0084] The culture medium formula used is as follows:
[0085] Z14 medium: 3 / 4MS + Vc 5mg / L + ABA 2mg / L + Ac 2g / L + CH 0.2g / L + sucrose 40g / L + agar 2.5g / L;
[0086] Seedling selection culture medium: 3 / 4MS+Vc 5mmg / L+sucrose 30g / L+agar 8g / L.
[0087] 1. Liquid culture: Select a 250 mL Erlenmeyer flask and use tweezers to pick up about 3 dishes of callus on the wall of the flask. Slightly flatten the flask to disperse the cells to an area of about 5 square centimeters. Use a graduated cylinder to measure 45 mL of M13 liquid medium and flush the callus cells on the wall of the flask to the bottom of the flask. Incubate the flask on a shaker at 23°C and 95 rpm for one week. Check the cell status regularly to avoid browning. Remove the suspended cells that have been cultured on the shaker for one week and transfer them to a 50 mL graduated cylinder. Let it stand until the supernatant is relatively clear. After the cells have settled, remove the supernatant. At this time, the cell volume is about 10 mL. Divide it into two subcultures, each containing 5 mL of cells, and pour them into two clean Erlenmeyer flasks respectively. Use a clean graduated cylinder to measure 45 mL of M13 liquid medium and add it. Incubate the flask on a shaker at 23°C and 95 rpm for another week.
[0088] 2. Sieving: Take out the suspension system after subculture, pour the suspended cells in the triangular flask into a 150+400 mesh sieve, and wait for the liquid to flow out (two bottles of the same suspended cells are sieved together); gently tap the sieve to make the liquid in the sieve flow out quickly, measure 50 mL of liquid M13, and flush the single cells on the 400 mesh sieve into a new triangular flask.
[0089] 3. Spread the plate: Use tweezers to spread a piece of filter paper on the cotton. Depending on the single-cell concentration, use a pipette to absorb 1.5-2 mL of sieved single cells and spread them evenly on the filter paper. After the water on the filter paper is dry, use another pair of tweezers to place the filter paper on Z14 (G41810 mg / L) solid culture medium (be careful not to have bubbles between the filter paper and the culture medium). During this period, make sure to shake the flask from time to time. After the end, place the spread plate in the dark at 23°C for about one month, and then transfer it to light for culture for about one month. At this time, the seedlings with two cotyledons can be transferred to the seedling selection culture medium.
[0090] 4. Observation of somatic embryo induction process: Observe the somatic embryo induction of p35S:LhMIR394B and untransformed wild-type callus cells, such as Figure 5 As shown in Figure 2, compared with the untransformed wild-type callus, we found that the somatic embryogenesis of the cell line overexpressing LhMIR394B was relatively slow, while the number of somatic embryos was significantly increased. Two months later, the somatic embryogenesis efficiency of the transgenic callus overexpressing LhMIR394B and the wild type was statistically analyzed, and it was found that the number of somatic embryos in the transgenic callus overexpressing LhMIR394B was significantly increased compared with the wild type. The results are shown in Figure 2. Figure 6 shown.
Claims
1. Hybrid tulip tree LhMIR394B The use of genes in enhancing the efficiency of somatic embryogenesis comprises the following steps: (1) Construction LhMIR394B Gene carriers; (2) The constructed LhMIR394B The gene vector is transformed into plant tissue; (3) Cultivate and screen plant tissues with enhanced somatic embryogenesis efficiency; The plant tissue is embryonic callus of hybrid Liriodendron chinense; The hybrid Liriodendron LhMIR394B The nucleotide sequence of the gene is shown in SEQ ID NO.
1.
2. The use according to claim 1, characterized in that The transformation is carried out by Agrobacterium infection.
3. The use according to claim 1, characterized in that The vector is a plant expression vector.
4. The use according to claim 3, characterized in that The plant expression vector is pBI121- LhMIR394B .