A hybrid Liriodendron chinense LhMIR394A gene and its application

Abstract

The invention discloses a hybrid tulipwood LhMIR394A gene and its application, and belongs to the field of plant molecular biology. The hybrid tulipwood LhMIR394A gene of the invention has a nucleotide sequence as shown in SEQ ID NO.1. The invention uses hybrid tulipwood leaves as materials, obtains the hybrid tulipwood LhMIR394A gene by cloning, constructs its overexpression vector pBI121-LhMIR394A on this basis, transfers it into hybrid tulipwood embryonic callus, obtains transgenic plants, and the results of gene function identification show that the LhMIR394A gene is an important transcription factor that promotes the proliferation of embryonic callus and makes the somatic embryo cotyledons fused in a cup-shaped structure, and has good application prospects in plant growth and development and breeding.

Description

Technical Field

[0001] The invention belongs to the field of plant molecular biology, and more specifically relates to a hybrid Liriodendron chinense LhMIR394A gene and application thereof. Background Art

[0002] Liriodendron is a genus of the Magnoliaceae family. There were dozens of species widely distributed in the Northern Hemisphere. Most of them became extinct after the Quaternary glaciation. Only two natural species remain, namely, Liriodendron chinense and Liriodendron tulipifera. Hybrid Liriodendron chinense × Liriodendron tulipifera was bred by Professor Ye Peizhong, a famous forestry breeder in my country, in 1963. He selected Liriodendron chinense from Lushan Mountain in China as the female parent and hybridized it with Liriodendron tulipifera in North America. It showed obvious hybrid vigor. Hybrid Liriodendron has beautiful tree shape, peculiar leaf shape, large and beautiful flowers, fast growth, and good material. It is an important tree species for garden greening, afforestation and timber. However, hybrid Liriodendron has poor reproductive ability and low fruiting rate, and the seedlings propagated by seeds have large 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] miRNA is widely present in plant genomes. It is a type of non-coding single-stranded RNA molecule with a length of about 21 nucleotides encoded by endogenous genes. It mainly regulates gene expression by binding to target mRNA at the post-transcriptional level, thus playing an important role in plant organ morphology, growth and development, and response to external stress. Among them, miR394 not only affects the stress resistance level of plants, but also plays an important role in plant growth and development. Especially in the regulation of plant shoot apical stem cells, miR394 and its target genes can inhibit the negative feedback regulation of shoot apical stem cell marker genes WUSCHEL and CLV3, thereby maintaining the normal development of plant shoot apical meristems. However, the function of miR394 in somatic embryogenesis has been rarely 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 a hybrid Liriodendron LhMIR394A gene and its application. Another technical problem to be solved by the present invention is to provide an application of the hybrid Liriodendron LhMIR394A gene.

[0005] In order to solve the above technical problems, the technical solution adopted by the present invention is as follows:

[0006] A hybrid Liriodendron chinense LhMIR394A gene, the nucleotide sequence of which is shown in SEQ ID NO.1.

[0007] A vector containing the LhMIR394A gene.

[0008] Furthermore, the vector is a plant expression vector.

[0009] Furthermore, the plant expression vector is pBI121-LhMIR394A.

[0010] The LhMIR394A gene is used in promoting the proliferation of embryonic callus or making the cotyledons of somatic embryos fused into a cup-shaped structure.

[0011] Further, the following steps are included:

[0012] (1) constructing a vector of LhMIR394A gene;

[0013] (2) transforming the constructed LhMIR394A gene vector into hybrid Liriodendron tulipifera embryonic callus;

[0014] (3) Cultivate and screen to obtain proliferating embryonic callus or cotyledon embryos with cotyledons fused in a cup-shaped structure.

[0015] Compared with the prior art, the present invention has the following beneficial effects:

[0016] 1) The present invention uses hybrid tulipwood leaves as materials, obtains the hybrid tulipwood LhMIR394A gene by cloning, and constructs its overexpression vector pBI121-LhMIR394A on this basis, and transfers it into hybrid tulipwood embryonic callus, where the LhMIR394A gene is efficiently expressed in the hybrid tulipwood embryonic callus.

[0017] 2) After 20 days of subculture of transgenic callus tissues of p35S:LhMIR394A, pBI121 empty vector (Empty vector) and untransformed wild type 166302 callus tissues (WT), the embryonic callus tissues of hybrid Liriodendron chinense treated with pBI121 empty vector did not proliferate significantly; while the embryonic callus tissues of transgenic hybrid Liriodendron chinense proliferated significantly, which was significantly higher than the other two control groups.

[0018] 3) After 28 days of somatic embryogenesis induction from transgenic callus containing 35S:LhMIR394A, pBI121 empty vector and WT by liquid suspension system, the cotyledon embryos produced by pBI121 empty vector and WT had two open cotyledons, while the cotyledon embryos of 35S:LhMIR394A had fused cotyledons in a cup-shaped structure. 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 solution of pBI121-LhMIR394A Escherichia coli;

[0021] Figure 3 This is the PCR electrophoresis image of the bacterial solution of a single colony of pBI121-LhMIR394A Agrobacterium;

[0022] Figure 4 This is the PCR verification result of the transgenic callus overexpressing LhMIR394A;

[0023] Figure 5 This is a diagram showing the growth status of callus tissues overexpressing LhMIR394A, pBI121 empty transgenic and wild type;

[0024] Figure 6 This is a diagram showing the somatic embryogenesis of callus tissues overexpressing LhMIR394A, pBI121 empty transgenic and wild type;

[0025] Figure 7 The diagram shows the cotyledon embryo states produced by callus overexpressing LhMIR394A, pBI121 empty transgenic cell and wild type. DETAILED DESCRIPTION

[0026] The present invention is further described below in conjunction with specific examples. In the following examples, the operations not described in detail are all routine biological experimental operations, which can be performed with reference to the molecular biology experiment manual and existing published journals, or according to the kit and product instructions. The materials, reagents, etc. used in the following examples, unless otherwise specified, can all be obtained from commercial sources.

[0027] Example 1: Cloning of hybrid Liriodendron chinense LhMIR394A gene

[0028] 1. Extraction of genomic DNA

[0029] Take finger-sized fresh leaves of hybrid tulipwood plants and grind them thoroughly; after the leaves become powder, add 700μL CTAB lysis solution to a centrifuge tube, stir evenly and place it in a 65℃ water bath or air bath for lysis for 30 minutes, and make sure to shake and mix every 5 minutes; add equal volumes of chloroform and isoamyl alcohol (volume ratio is 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 is 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 2min, remove the supernatant, place in a ventilated place to dry, add 50μL of 65℃ preheated ddH2O, place in a 65℃ warm water bath for 5min to dissolve the DNA in water, and use Nanodrop to detect the extraction concentration and purity after the DNA is dissolved.

[0030] 2. Target gene cloning

[0031] 1) Design primers: Based on the existing genome data of Liriodendron hybridum, BLAST was used for search and comparison. According to the comparison results, Primer 5.0 was used to design the corresponding primers, and Oligo 7.0 was used to analyze the primers. Finally, the primers were determined as follows:

[0032] LhMIR394A-F: 5'-ATCTATCTTTGTCTAGAGGCGGAA-3',

[0033] LhMIR394A-R: 5'-ATCATATCACATGGGTTTCACAA-3'.

[0034] 2) PCR amplification reaction

[0035] Using genomic DNA as a template, PCR was performed using Vazyme's Phanta Max Super-Fidelity DNA Polymerase, and the amplification system was 25 μL 2× Phanta Max Buffer, 1 μL dNTP Mix (10 mM each), 2 μL LhMIR394A-F, 2 μL LhMIR394A-R, 1 μL Phanta Max Super-Fidelity DNA Polymerase, 1 μL hybrid Liriodendron genomic DNA, and 18 μL ddH2O. The 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 stored at 4°C.

[0036] 3) Recovery of target gene

[0037] 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. The steps were consistent with the kit instructions.

[0038] 4) Ligation of target fragment and cloning vector

[0039] TaKaRa's pMD19-T Vector (D102A) was used for the ligation reaction. The reaction system was 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.

[0040] 5) Conversion, screening and sequencing of ligation products

[0041] Transformation: Take out Tiangen DH5α competent cells from -80℃ and thaw on ice; add 10μL of ligation product, flick the bottom of the tube to mix, and place on ice bath for 30min; heat shock at 42℃ for 75s, and then immediately place on ice for 2-3min; add 900μL LB liquid culture medium (without Amp), shake and culture at 37℃, 150rpm for 45min to recover and express the resistance gene; centrifuge at 4000rpm for 2min, discard 800μL supernatant, and resuspend the precipitate in the remaining LB; spread the resuspension from the previous step on a solid LB plate containing Amp, and incubate upside down at 37℃ for 12-16h.

[0042] Positive clone screening: Pick a single clone colony on the LB plate, inoculate it into 800 μL LB liquid culture medium containing Amp antibiotics, and culture it at 37°C, 220 rpm overnight.

[0043] Bacterial liquid PCR identification: The reagent used was 2×T5 Super PCR Mix (Colony) from TSINGKE Biotechnology Co., Ltd. The PCR system was 7.5μL 2×T5 Super PCR Mix, 0.6μL M13F, 0.6μL M13R, 2μL bacterial liquid, 4.3μL ddH2O; the reaction conditions were 98℃3min; 98℃10s, 54℃10s, 72℃2min, 35Cycles; 72℃2min; 4℃ storage.

[0044] 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 and M13R. The sequence is as follows:

[0045] M13-F: 5'-CGCCAGGGTTTTCCCAGTCACGAC-3',

[0046] M13-R: 5'-GAGCGGATAACAATTTCACACAGG-3'.

[0047] Bioinformatics analysis was performed based on the sequencing results to determine that the target gene sequence was as shown in SEQ ID NO.1, and the bacterial solution with the correct sequencing was stored at -80°C.

[0048] Example 2: Construction of hybrid Liriodendron chinense LhMIR394A gene expression vector

[0049] According to 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:

[0050] 35S-F: 5'-TGAAGATAGTGGAAAAGGAAGGTG-3',

[0051] LhMIR394A-F: 5'-ATCTATCTTTGTCTAGAGGCGGAA-3',

[0052] LhMIR394A-R: 5'-ATCATATCACATGGGTTTCACAA-3',

[0053] LhMIR394A+OL-F: 5'-gagaacacgggggactctagaATCTATCTTTGTCTAGAGGCGGAAG-3',

[0054] LhMIR394A+OL-R: 5'-cgatcggggaaattcgagctcATCATATCACATGGGTTTCACAAAG-3'.

[0055] 1. Target gene plus overlap

[0056] The above-mentioned 19T-simple bacterial liquid with the correctly sequenced target gene fragment was shaken, and then the plasmid was extracted using Tiangen's plasmid extraction kit, and the extraction steps were consistent with the instructions.

[0057] The 19T-simple plasmid with the target gene extracted above was used as a template, a pair of primers LhMIR394A+OL-F and LhMIR394A+OL-R were used, and the PCR reaction was performed using Phanta Max Super-Fidelity DNA Polymerase from Vazyme. The amplification system was 25μL 2×Phanta Max Buffer, 1μL dNTP Mix (10mM each), 2μL LhMIR394A+OL-F, 2μL LhMIR394A+OL-R, 1μL Phanta Max Super-Fidelity DNA Polymerase, 1μL 19T-simple plasmid template, and 18μL ddH2O. The PCR reaction conditions were 95℃3min; 95℃15s, 60℃15s, 72℃20s, 35Cycles; 72℃5min; and stored at 4℃.

[0058] The PCR reaction product was electrophoresed on 1% agarose gel, and then the target band was excised and recovered using a DNA gel recovery kit from TSINGKE Biotechnology Co., Ltd. The steps were consistent with the kit instructions.

[0059] 2. Obtaining the linearized vector

[0060] Acquisition of expression vector: Take the DHAα (pBI121 empty vector) bacterial solution stored at -80℃, use an inoculation needle to streak on the LB plate containing kanamycin (50mg / L), and incubate at 37℃ overnight. Then shake the bacterial solution slowly, shake it vigorously, and extract the plasmid in the same way as above.

[0061] Pretreatment of expression vector (enzyme digestion): Two restriction sites, XbaI and SacI, were selected based on the multiple cloning sites on the pBI121 vector, and double restriction digestion was performed on the pBI121 empty vector. The restriction digestion system: 1μL XbaI, 1μL SacI, 10μL plasmid, 5μL10xCutsmart buffer, 33μL ddH2O, incubated at 37℃ for 4 hours; after the restriction digestion, the enzyme was inactivated at 65℃ in a PCR instrument for 30 minutes.

[0062] 3. Connect the target fragment to the linearized vector

[0063] 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×CEIIBuffer, 2 μL of Exnase II, and 9 μL of ddH2O. The mixture was gently mixed by pipetting and then centrifuged briefly to collect the reaction solution to the bottom of the tube and then connected in a PCR instrument at 37°C for 30 minutes.

[0064] 4. Conversion, screening and sequencing of ligation products

[0065] 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 in 800μL 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, LhMIR394A-R (containing a sequence on the vector) and two pairs of primers LhMIR394A-F, LhMIR394A-R 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. The bacterial sample with the correct band size was sent to TSINGKE for sequencing, and the primers used for sequencing were 35S-F and LhMIR394A-R. The bacterial solution of the single colony with the correct sequencing was stored in a -80℃ refrigerator.

[0066] Example 3: Transformation of hybrid Liriodendron chinense embryonic callus with LhMIR394A gene

[0067] In this example, the Agrobacterium infection method was used. The Agrobacterium used was EHA105, which was transformed into hybrid Liriodendron chinense embryonic callus. The genotype of the embryonic callus used was 166302.

[0068] The culture medium formula used is as follows:

[0069] M13 subculture medium: 3 / 4MS+Vc5mg / L+2,4-D 2mg / L+BA0.2mg / L+CH0.5g / L+sucrose 30g / L+agar 2.5g / L.

[0070] 1. Preparation of bacterial solution

[0071] 1) pBI121-LhMIR394A recombinant plasmid transformed into EHA105

[0072] The above sequenced E. coli bacteria with pBI121-LhMIR394A recombinant plasmid were shaken, and then the plasmid was extracted to obtain the pBI121-LhMIR394A recombinant plasmid. The extracted recombinant plasmid was transferred into the Agrobacterium competent cell EHA105, using the EHA105 competent cells of Shanghai Weidi Biological Company, and the transformation method was consistent with the instructions. After a single colony grows, use tweezers to pick up the white pipette tip and dip it into the single colony grown on the plate, put the pipette tip into 800μL LB liquid culture medium containing kanamycin, shake the bacteria at 28℃, shake at 220rpm for 12-16h, and then perform PCR verification of the bacterial solution. The reagent used was 2×T5 Super PCR Mix (Colony) from TSINGKE Biotechnology Co., Ltd., and two pairs of primers, 35S-F, LhMIR394A-R and LhMIR394A-F, LhMIR394A-R, 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 3 shown.

[0073] 2) Preparation of bacterial solution:

[0074] (1) Shake the bacterial solution: Add 2 mL of LB (with 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.

[0075] (2) Propagation of bacterial culture: Add 50 mL of LB (with 50 mg / mL of kanamycin) and 2 mL of bacterial culture to a 250 mL centrifuge tube and shake at 220 rpm and 28°C for 4-6 h. Detect the OD value when it reaches 0.6-1.0.

[0076] (3) Resuspending the bacterial solution: Transfer 40 mL of the 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 culture medium to resuspend the resuspended bacterial solution so that the final OD600 is 0.8. Resuspension formula: measured OD value × 40 mL = 0.8 × volume of M13 liquid culture medium.

[0077] 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.

[0078] 4. Infection and co-cultivation: On a clean bench, take about three dishes of callus and put them in a 100 mL Erlenmeyer flask, add the resuspended bacterial solution, infect for 10-15 min, filter through a 400-mesh sieve, use tweezers to pick up the filter paper to absorb the bacterial solution, transfer the callus to solid M13 medium (+AS, concentration as above), and co-cultivate for 2 days.

[0079] 5. Degerming: Place the co-cultured callus in a 100 mL Erlenmeyer flask and wash it alternately with liquid M13 (+ / -cef) until the callus washing solution 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 allow the degerming time to exceed 10 min.

[0080] 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.

[0081] 7. Callus PCR positive verification: The first subcultured callus is used as a cell line. After the subculture reaches a sufficient number, a callus is taken from each cell line and DNA is extracted by CTAB method. The steps are the same as above. The 2×Rapid TaqMaster Mix reagent of Vazyme is used for transgenic PCR verification. The PCR reaction is carried out using two pairs of primers, 35S-F, LhMIR394A-R and LhMIR394A-F, LhMIR394A-R PCR. The reaction system is 5μL2×Rapid Taq Master Mix, 0.4μLPrimer 1, 0.4μL Primer 2, 3.2μL ddH2O, 1μL DNA template, and the reaction program is 95℃3min; 95℃15s, 54℃15s, 72℃20s, 35Cycles; 72℃5min; 4℃ storage.

[0082] According to the above method, the transgenic callus lines were tested for PCR positive results. Figure 4 . The stripe sizes are as expected.

[0083] 8. Observation of transgenic callus status

[0084] At the same time, the transgenic callus tissues of p35S:LhMIR394A and pBI121 empty vector and the untransformed wild-type 166302 callus tissues (WT) were subcultured, and the weight of the callus at 0d and 20d was weighed, and the weight increase of each callus was calculated. The weight increase of each callus was calculated by ImagJ. The results are shown in Figure 2. Figure 5 As shown. This indicates that LhMIR394A can promote the proliferation of embryonic callus in hybrid Liriodendron chinense.

[0085] Example 4: Somatic Embryo Induction Experiment

[0086] In this example, somatic embryos were induced by liquid suspension system, and the experimental materials were transgenic callus tissues of p35S:LhMIR394A, Emptyvector and WT.

[0087] The culture medium formula used is as follows:

[0088] Liquid suspension medium: 3 / 4MS+Vc5mg / L+2,4-D 2mg / L+BA0.2mg / L+CH0.5g / L+sucrose 30g / L

[0089] Somatic embryo induction medium: 3 / 4MS+Vc5mg / L+ABA2mg / L+Ac2g / L+LH0.2g / L+sucrose40g / L+agar2.5g / L+10mg / LG418 (WT without addition).

[0090] 1. Liquid culture: Choose a 250mL Erlenmeyer flask, use tweezers to pick up about 3.25g of callus on the wall of the flask, slightly flatten it to disperse the cells; use a measuring cylinder to measure 50mL of liquid suspension culture medium, and wash off the callus cells on the wall of the flask; after culturing on a shaker at 23℃ and 95rpm for one week, take out the suspended cells and transfer them to a 50mL measuring cylinder, let it stand until the supernatant is relatively clear and the cells have settled, remove the supernatant, divide the cells into two equal parts and pour them into newly sterilized 250mL Erlenmeyer flasks (about 5mL per bottle); use a clean measuring cylinder to measure 45mL of liquid suspension culture medium and add it to the flask, and continue to culture on a shaker at 23℃ and 95rpm for one week.

[0091] 2. Sieving: Take out the suspension system after subculture, pour the suspended cells in the flask into a 150+400 mesh sieve, and wait for the liquid to flow out; when there is no obvious liquid in the 400 mesh sieve, measure 50 mL of liquid suspension culture medium and flush the single cells on the 400 mesh sieve into a new flask.

[0092] 3. Spread the plate: Use tweezers to spread a piece of filter paper on the cotton, use a pipette to absorb 2 mL of sieved single cells and spread them evenly on the filter paper. After the water on the filter paper is dry, transfer the filter paper to the somatic embryo induction medium, culture in the dark at 23°C for about one month, and then culture under light.

[0093] 4. Observation of somatic embryo induction process: Observe the somatic embryo induction of p35S:LhMIR394A, Empty vector and WT callus cells. Figure 6 A-6C is the somatic embryo induction state at 35 days, and 6D is the statistical result of the somatic embryo number at about 60 days, indicating that LhMIR394A can significantly promote the somatic embryogenesis of hybrid tulipwood. Figure 7 The 28-day somatic embryo development state shows that the cotyledon embryos produced by Empty vector and WT have two open cotyledons, while the cotyledon embryos of p35S:LhMIR394A have fused cotyledons in a cup-shaped structure, indicating that LhMIR394A plays a certain role in the cotyledon development of hybrid tulipwood.

Claims

1. Contains LhMIR394A Application of a gene vector in promoting the proliferation of hybrid tulipwood embryonic callus or making the hybrid tulipwood embryonic cotyledon fused into a cup-shaped structure, the hybrid tulipwood LhMIR394A The nucleotide sequence of the gene is shown in SEQ ID NO.

1.

2. The use according to claim 1, characterized in that: The following steps are involved: (1) Construction LhMIR394A Gene carrier; (2) The constructed LhMIR394A The gene vector was transformed into embryonic callus of hybrid Liriodendron chinense; (3) Cultivate and screen to obtain proliferating embryonic callus tissue or cotyledon embryos with cotyledons fused in a cup-shaped structure.

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