Method for establishing hybrid liriodendron somatic embryogenesis system based on LhAHL10 / 15 gene

Abstract

The invention discloses a method for establishing a hybrid liriodendron somatic embryogenesis system based on an LhAHL10 / 15 gene, and relates to the technical field of plant genetic engineering. According to the method for establishing the hybrid liriodendron somatic embryogenesis system based on the LhAHL10 / 15 gene disclosed by the invention, the purpose of promoting the generation of the hybrid liriodendron somatic embryogenesis is achieved by promoting the LhAHL15 gene or inhibiting the expression of the LhAHL10 gene; the nucleotide sequences of the LhAHL15 gene and the LhAHL10 gene are respectively as shown in SEQ ID NO. 3 and SEQ ID NO. 1. The result shows that overexpression of the LhAHL15 in the hybrid liriodendron is beneficial for improving the indirect somatic embryogenesis efficiency of the hybrid liriodendron. The overexpression of the LhAHL10 in the hybrid liriodendron can also improve the somatic embryo generation efficiency, but can influence the modeling development process of somatic embryos, so that the development of the somatic embryos is stagnated in the globular embryo stage.

Description

Technical Field

[0001] The invention belongs to the technical field of plant genetic engineering, and more specifically, relates to a method for establishing a hybrid tulipwood somatic embryogenesis system based on LhAHL10 / 15 gene. Background Art

[0002] Hybrid tulip tree (Liriodendron sino-americanum PCYieh ex Shang etZ.R.Wang), also known as hybrid tulip tree or hybrid magnolia wood, is a plant of the genus Liriodendron in the Magnoliaceae family. It is obtained through artificial hybridization, with Liriodendron sino-americanum as the female parent and Liriodendron sino-americanum as the male parent. The leaves of hybrid tulip tree are shaped like magnolias, so it is also called hybrid magnolia wood. Its flowers are large, yellow, and fragrant. They grow solitarily at the top of the branches and resemble tulips, so it is also called "woody tulips". It grows fast and has good wood quality. It is a precious commercial tree species. The leaf shape is like a magnolia and the flowers are like marigolds. It is an excellent garden greening tree species. It is an ancient relic plant that was once widely distributed in the temperate regions of the northern hemisphere. The North American tulip tree is native to southeastern North America and is also distributed in Qingdao, Lushan, Nanjing, Guangzhou and other places in my country. The inconsistent flowering period of male and female tulip trees results in low natural reproduction efficiency. Currently, asexual reproduction is generally used for the reproduction of tulip trees, including cuttings, grafting, tissue culture, etc. However, artificial reproduction methods such as cuttings and grafting are difficult and have low survival rates.

[0003] Somatic embryogenesis is a manifestation of plant cell totipotency, a way for somatic cells or other cells to develop into embryos without fertilization. Somatic embryogenesis is regulated by many aspects, such as transcription factors, hormones, epigenetic modifications, etc. Ectopic expression of specific transcription factors or the loss of certain chromatin modifying proteins can cause somatic cells to acquire totipotency and transform into embryonic cells. Previous studies have reported that many single gene ectopic expression can directly induce somatic embryogenesis, such as BBM (Baby Boom), WUS (WUSCHEL), LEC 1 (LEAFY COTYLEDON 1), AHL15 (AT-HOOK MOTIF NUCLEAR LOCALED 15), etc.

[0004] During embryonic development in Arabidopsis, AHL genes have high expression levels from the early embryonic development, indicating that AHL15 is involved in embryonic development in the early embryonic development. Until the globular embryo stage, AHL15 expression is distributed throughout the embryo, and the expression level of AHL15 in heart-shaped embryos and cotyledon-shaped embryos is high, which also plays an important role in the formation of embryo morphology. These studies have shown that the AHL gene family plays a vital role in the embryonic development process and somatic embryo development. In 2,4-D-induced somatic embryogenesis, AHL genes may act downstream of 2,4-D and upstream of YUC-mediated auxin biosynthesis. In addition, BBM and AHL act synergistically on somatic embryogenesis and can activate the expression of downstream YUC genes. In addition to providing technical means for biotechnology and molecular breeding, somatic embryogenesis also provides an important research basis for exploring the plant embryonic development process and molecular regulation mechanism.

[0005] At present, most of the research on somatic embryos of forest trees is focused on optimizing and improving the conditions for somatic embryogenesis, and there are not many studies on the molecular mechanism of somatic embryogenesis. Strengthening the research on the regulatory mechanism of somatic embryogenesis will help to further provide reference genes for plants with low somatic embryogenesis ability and improve the ability and efficiency of somatic embryogenesis. As a plant with great potential economic value, hybrid tulipwood has a low natural fruiting rate, poor reproductive ability, and a shortage of hybrids. Research on improving its somatic embryogenesis and plant regeneration efficiency is of great significance for the preservation, genetic improvement and rapid reproduction of its germplasm resources. Summary of the invention

[0006] 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 method for establishing a hybrid tulipwood somatic embryogenesis system based on the LhAHL10 / 15 gene, which is used to regulate the formation and development of hybrid tulipwood somatic embryos.

[0007] In order to solve the above technical problems, the technical solution adopted by the present invention is as follows:

[0008] A method for establishing a hybrid tulipwood somatic embryogenesis system based on the LhAHL15 gene, which achieves the purpose of regulating the hybrid tulipwood somatic embryogenesis by regulating the expression of the LhAHL15 gene; the nucleotide sequence of the LhAHL15 gene is shown in SEQ ID NO.3.

[0009] The regulating LhAHL15 gene expression is to promote LhAHL15 gene expression.

[0010] The method for establishing a somatic embryogenesis system of hybrid tulipwood based on the LhAHL15 gene comprises:

[0011] 1) constructing an overexpression vector of the hybrid Liriodendron chinense LhAHL15 gene;

[0012] 2) transforming the constructed overexpression vector of hybrid tulipwood LhAHL15 gene into hybrid tulipwood;

[0013] 3) Cultivate, screen and obtain transgenic hybrid tulipwood plants with significantly improved somatic embryogenesis efficiency.

[0014] A method for establishing a hybrid tulipwood somatic embryogenesis system based on the LhAHL10 gene, by regulating the expression of the LhAHL10 gene, thereby achieving the purpose of regulating the somatic embryogenesis of the hybrid tulipwood; the nucleotide sequence of the LhAHL10 gene is shown in SEQ ID NO.1

[0015] The regulating LhAHL10 gene expression is inhibiting LhAHL10 gene expression.

[0016] The method for establishing a somatic embryogenesis system of hybrid tulipwood based on the LhAHL10 gene comprises:

[0017] 1) Construction of editing vector for hybrid Liriodendron chinense LhAHL10 gene;

[0018] 2) Transforming the constructed editing vector of hybrid tulipwood LhAHL10 gene into hybrid tulipwood;

[0019] 3) Cultivate, screen and obtain transgenic hybrid tulipwood plants with significantly improved somatic embryogenesis efficiency.

[0020] Application of hybrid tulipwood LhAHL15 gene in regulating the expression of genes related to somatic embryogenesis in hybrid tulipwood.

[0021] The somatic embryogenesis-related genes are BBM, LEC1, PIN1, and PLT2.

[0022] Application of hybrid tulipwood LhAHL10 gene in regulating the expression of genes related to somatic embryogenesis in hybrid tulipwood.

[0023] The somatic embryogenesis-related genes are LEC1, PIN1 and PLT2.

[0024] Compared with the prior art, the present invention has the following beneficial effects:

[0025] 1) The present invention achieves the purpose of promoting somatic embryogenesis of hybrid tulipwood by promoting the expression of LhAHL15 gene, the nucleotide sequence of LhAHL15 gene is shown in SEQ ID NO.3; constructing an overexpression vector of hybrid tulipwood LhAHL15 gene; transforming the constructed overexpression vector of hybrid tulipwood LhAHL15 gene into hybrid tulipwood; cultivating, screening and obtaining transgenic hybrid tulipwood plants with significantly improved somatic embryogenesis efficiency.

[0026] 2) The present invention achieves the purpose of promoting somatic embryogenesis of hybrid tulipwood by inhibiting the expression of LhAHL10 gene, the nucleotide sequence of LhAHL10 gene is shown in SEQ ID NO.1; constructing an editing vector of hybrid tulipwood LhAHL10 gene; transforming the constructed editing vector of hybrid tulipwood LhAHL10 gene into hybrid tulipwood; cultivating, screening and obtaining transgenic hybrid tulipwood plants with significantly improved somatic embryogenesis efficiency.

[0027] 3) The results of the present invention show that on a culture medium without exogenous hormones, overexpression of LhAHL15 promotes somatic embryogenesis, while the number of somatic embryos in the gene-edited strain (ahl15-ko) is reduced, and most of them stagnate at the globular embryo stage, and a small number of somatic embryos can continue to develop. Overexpression can form complete cotyledon embryos, while gene editing will cause somatic embryo development abnormalities and fail to form normal plants. Compared with the control group, the number of somatic embryos of LhAHL10-OE increased significantly, but the development of somatic embryos stagnated at the globular embryo stage, and the volume of the globular embryos was significantly larger than that of the somatic embryos in the control group. The number of somatic embryos induced by ahl10-ko increased significantly compared with the control and was similar to the number of somatic embryos induced by overexpression.

[0028] 4) The results of the present invention show that the base of the hypocotyl of the somatic embryo induced by the callus of the ahl15-ko mutant is swollen and the radicle is missing. In the plantlet period, the root tip development is stagnant and callus accumulation occurs. The hypocotyl of the cotyledon embryo overexpressing LhAHL15 is thicker and the cotyledon is fleshy compared to the wild type and the empty load. In the seedling stage, the overexpression of LhAHL15 has a thick stem and fleshy leaves. The volume of the somatic embryo induced by the positive callus of the ahl10-ko mutant is significantly smaller than that of the control group, but the embryonic development is complete without obvious defects. The cotyledon embryo overexpressing LhAHL10 has disordered development, no obvious embryonic structure, and cannot develop into a plantlet in the later stage. The plant transformation rate of LhAHL10-OE is close to zero, but the plant transformation rate of ahl10-ko is not significantly different from that of the control group.

[0029] 5) The results of the present invention show that overexpression of LhAHL15 significantly increases the expression level of BBM, while overexpression of LhAHL10 has no significant effect on the expression level of BBM. Overexpression of LhAHL15 can significantly increase the expression level of LEC1, while overexpression of LhAHL10 has no significant effect on the expression level of LEC1, and the expression level of LEC1 is significantly increased after knocking out LhAHL10. Overexpression of LhAHL15 significantly increases the expression level of PIN1, gene editing of LhAHL15 significantly reduces the expression level of PIN1, and the expression level of PIN1 is significantly increased after knocking out LhAHL10. Overexpression of LhAHL15 significantly increases the expression level of the PLT2 gene. Knockout of LhAHL15 significantly reduces the gene expression level of PLT2. However, knocking out LhAHL10 significantly increases the gene expression level of PLT2. BRIEF DESCRIPTION OF THE DRAWINGS

[0030] Figure 1 This is a positive identification image of LhAHL10 / 15 transgenic callus;

[0031] Figure 2 This is the result of qRT-PCR of LhAHL10 / 15 transgenic callus;

[0032] Figure 3 Figure 1 shows the positive identification of ahl10 / 15-ko transgenic callus and the results of first-generation sequencing (a is the positive callus of ahl10-ko transgenic and the results of second-generation sequencing and the peak diagram; b is the sequence result of ahl10-ko gene editing protein, and the terminator is indicated in the red box; c is the positive callus of ahl15-ko transgenic and the results of second-generation sequencing and the peak diagram; d is the sequence result of ahl15-ko gene editing protein, and the terminator is indicated in the red box);

[0033] Figure 4 Diagram of somatic embryogenesis induction (a is somatic embryo induced by wild-type callus; b is transgenic Pbi121 positive callus; c is transgenic CRISPR / Cas9 positive callus; d is somatic embryo induced by 35S:LhAHL15 transgenic callus; e is somatic embryo induced by ahl15-ko transgenic callus; f is the statistical result of somatic embryo induction data);

[0034] Figure 5 Figure 1 shows the induction of somatic embryogenesis (a is the somatic embryo induced by wild-type callus; b is the transgenic Pbi121 positive callus; c is the transgenic CRISPR / Cas9 positive callus; d is the somatic embryo induced by 35S:LhAHL10 transgenic callus; e is the somatic embryo induced by ahl10-ko transgenic callus; f is the statistical results of somatic embryo induction data;

[0035] Figure 6 The phenotypes of ahl15-ko knockout mutant and LhAHL15-OE at the cotyledon embryo stage and plantlet stage (a is the phenotype of ahl15-ko at the cotyledon embryo stage; b is the phenotype of ahl15-ko at the plantlet stage; c is the cotyledon embryo overexpressing LhAHL15; d is the plantlet overexpressing LhAHL15; e is the plant transformation rate of different transgenic lines; f is the statistical diagram of plant transformation rate);

[0036] Figure 7 The pictures of cotyledon embryos of ahl10-ko knockout mutant and LhAHL10-OE and the statistical diagram of plant transformation rate; a is the picture of cotyledon embryos of LhAHL10-OE and ahl10-ko; b is the statistical diagram of cotyledon embryo size; c is the statistical diagram of the effect of LhAHL10 gene on plant transformation rate;

[0037] Figure 8 The diagram shows the effect of LhAHL10 / 15 on embryonic callus induction (a is CRISPR / Cas9 empty callus, ahl15-ko, ahl0-ko single embryo induced callus; b is pBI121 empty callus, LhAHL15-OE, LhAHL10-OE single embryo induced callus; cd are schematic diagrams of non-embryonic callus; e is a schematic diagram of embryonic callus; f is a statistical diagram of the induction efficiency of LhAHL10 on embryonic callus; g is a statistical diagram of the induction efficiency of LhAHL15 on embryonic callus);

[0038] Fig. 9 This is a graph showing the effect of LhAHL10 / 15 on the expression of genes related to somatic embryogenesis. DETAILED DESCRIPTION

[0039] In order to make the purpose, technical scheme and advantages of the present invention clearer, the present invention is further described below in conjunction with specific embodiments. Unless otherwise specified in the following embodiments, the technical means used are conventional means well known to those skilled in the art.

[0040] The material used in the present application is embryonic callus induced from immature embryos, which is cultured in a constant temperature incubator at 22° C. in the dark.

[0041] The pBI121, pYLsgRNA-AtU3b, pYLsgRNA-AtU3d, pYLsgRNA-AtU6-1, and pYLCRISPR_Cas9P35S-N used in this application were stored by the Molecular Laboratory of Nanjing Forestry University, and pClone007 Blunt was purchased from Beijing Qingke Biological Company; the Escherichia coli strain was Trelief TM5αChemically Competent Cell was purchased from Beijing Qingke Biotechnology Co., Ltd., and Agrobacterium strain EHA105 was purchased from Shanghai Weidi Biotechnology Co., Ltd.

[0042] The RNA extraction kit (Vazyme) used in this application; RNA reverse transcription kit (Vazyme); restriction endonucleases EcoRⅠ, KpnⅠ, HindⅢ, XbaⅠ (NEB); ClonExpress ll One Step Cloning Kit (Vazyme); Phanta Max Super-Fidelity DNA Polymerase (Vazyme); Gel recovery kit (Qingke); Plasmid mini-extraction kit (TIANGEN); AceQ qPCR SYBR Green Master Mix (Vazyme). Antibiotics: Geneticin (G418); Cefpiramide (Cef); Kanamycin (Kan); Ampicillin (Amp).

[0043] The culture medium used in this application is as follows:

[0044] M13 callus subculture medium: 3 / 4MS+2,4-D 1mg / L+6-BA 0.2mg / L+Vc 5mg / L+CH 0.5g / L+sucrose 30g / L+agar 2.5g / L; the pH value of the culture medium is 5.72-5.74.

[0045] Z36 suspension culture medium: 3 / 4MS+Vc 5mg / L+KT 0.5mg / L+BA 0.2mg / L+NAA0.2mg / L+CH0.5g / L+sucrose 50g / L+agar 2.4g / L; the pH value of the culture medium is 5.72-5.74.

[0046] Z14 somatic embryo induction medium: 3 / 4MS+Vc 5mg / L+LH 0.2g / L+sucrose 40g / L+agar 2.4g / L; the pH value of the culture medium is 5.72-5.74.

[0047] Embryogenic callus induction medium: 3 / 4MS+2,4-D 2mg / L+6-BA 0.2mg / L+Vc 5mg / L+CH 1g / L+sucrose 40g / L+agar 3.6g / L; the pH value of the culture medium is 5.72-5.74.

[0048] LB liquid medium: NaCl (10 g / L) + tryptone 10 g / L + yeast extract 5 g / L.

[0049] Example 1

[0050] 1. Total RNA extraction and cDNA acquisition

[0051] RNA was extracted from embryonic callus tissue of hybrid Liriodendron chinense using RNA extraction kit (Vazyme). The extracted total RNA was used as a template and reverse transcription was performed using RNA reverse transcription kit (Vazyme) to obtain cDNA. The product was stored at -20℃ to reduce the number of repeated freeze-thaw cycles.

[0052] 2. LhAHL10 / 15 gene cloning

[0053] Using the CDS sequence of the LhAHL10 / 15 gene as a reference sequence, the LhAHL10 / 15 gene cloning primer sequence was designed using the Oligo7 website. The sequence information is as follows:

[0054] pLhAHL10-clone-F: 5'-ACTACCCACTCACTCCCTTG-3',

[0055] pLhAHL10-clone-R: 5'-TTACATGTGGGCCTTGACCT-3';

[0056] pLhAHL15-clone-F: 5'-TCCTCCTCACAAAACACCAGA-3',

[0057] pLhAHL15-clone-R: 5'-TATGCACAGCCACAACATCG-3'.

[0058] Using hybrid Liriodendron cDNA as template, prepare premix reaction system, and place the mixture in PCR instrument for reaction. All operations must be performed on ice, and each component must be fully mixed and centrifuged to the bottom of the tube.

[0059] PCR reaction system: ddH 2 O 17μL, 2×Phanta Max Buffer 25μL, dNTP Mix 1μL, Primer-F 2μL, Primer-R 2μL, Phanta Max Super-Fidelity DNA Polimerase 1μL, cDNA 2μL.

[0060] The PCR reaction program was as follows: pre-denaturation at 95°C for 3 min; denaturation at 95°C for 15 sec, annealing at 60°C for 15 sec, and extension at 72°C for 3 min, for 35 cycles; and complete extension at 72°C for 5 min.

[0061] The PCR amplification product band was clear and bright, and the target fragment was recovered using the DNA gel recovery kit (TSP602-200) of TSINGKE Company. The gel-cutting recovered fragment was connected to the pClone007 intermediate vector.

[0062] The ligation reaction system is: 3 μL of target fragment, 1 μL of pClone007 Blunt Vector, 1 μL of 10×Topo Mix, 1 μL of ddH 2 Add all the solution directly to the bottom of the PCR tube and mix it by gently pipetting.

[0063] The connection reaction procedure is: 22-30℃ room temperature reaction. Do not place on ice after the reaction is completed to avoid reducing the connection efficiency.

[0064] The intermediate vector with the target fragment was transformed into DH5α E. coli, and the bacterial solution was verified by PCR. The bacterial solution with correct PCR verification was sent to the company for sequencing. The DH5α E. coli with correct sequencing was extracted with the Tiangen Plasmid Mini-Extraction Kit (DP106). The prepared intermediate vector plasmid containing the target fragment was placed in a -20℃ refrigerator.

[0065] The CDS sequence of the LhAHL10 gene finally cloned is shown in SEQ ID NO.1, and the amino acid sequence of its expressed protein is shown in SEQ ID NO.2; the CDS sequence of the LhAHL15 gene is shown in SEQ ID NO.3, and the amino acid sequence of its expressed protein is shown in SEQ ID NO.4.

[0066] Example 2

[0067] 1. Construction of overexpression vector

[0068] The homology arm is added to the target sequence, and the homology arm primer sequence for constructing the vector is as follows:

[0069] OL-LhAHL10-F:

[0070] 5'-tggagagaacacgggggactATGTCGGGTAGAGAGTCCTTTGG-3',

[0071] OL-LhAHL10-R:

[0072] 5'-cgatcggggaaattcgagctTTATTTCCATGGCATGTTCGCCAAG-3';

[0073] OL-LhAHL15-F:

[0074] 5'-tggagagaacacgggggactATGGGAGGAGTCGATCTGTCG-3',

[0075] OL-LhAHL15-R:

[0076] 5'-cgatcggggaaattcgagctTTAGTAAGATGGTGGTGGCCTAGT-3'.

[0077] The pBI121 plasmid vector was digested with restriction enzymes at the XbaⅠ and SacⅠ sites, and the vector digestion products were recovered using a DNA recovery kit.

[0078] The enzyme digestion reaction system was: 10×cutsmart 5μL, XbaⅠ1μL, SacⅠ1μL, plasmid 7μL, ddH 2 O36μL

[0079] The enzyme digestion reaction program was: 37°C for 30 min, 80°C for 20 min, and 4°C infinity.

[0080] The linearized vector and the target gene fragment recovered after gel excision were homologously recombined using ClonExpress II One Step Cloning Kit.

[0081] The homologous recombination ligation reaction system is: 5×CE II Buffer 4μL, II 2μL, linearized vector 4μL, target fragment 2μL, ddH 2 2. 4% dHO: 8 μL.

[0082] The homologous recombination ligation reaction procedure was: 37°C for 60 min, 4°C infinity.

[0083] The homologous recombination ligation reaction products of the two genes were transformed into DH5α E. coli, and the bacterial solution was verified by PCR. The bacterial solution verified by PCR was sent to the company for sequencing. The DH5α E. coli with correct sequencing was extracted with the Tiangen Plasmid Mini-Prep Kit (DP106) and then transformed into Agrobacterium. The specific steps are as follows.

[0084] Take the competent Agrobacterium stored in the ultra-low temperature refrigerator and put it in the room temperature to melt. After melting, put it in the ice box to keep it low temperature; add about 1 μg of plasmid DNA to 100 μL competent cells, gently stir the bottom of the centrifuge tube by hand to fully mix the plasmid and bacterial solution, let it stand on ice for 5 minutes, then put it in liquid nitrogen for quick freezing for 5 minutes, immediately put it in a 37℃ water bath for 5 minutes, and then let it stand in ice for 5 minutes; add about 700 μL of LB liquid culture medium without antibiotics to the tube, and put it in a shaking shaker at 28℃ (150rpm) for 3 hours; centrifuge at 5000rpm for about 1 minute to collect bacteria, then take about 100 μL of supernatant and gently blow to resuspend the bacteria, spread the bacteria on a plate made of LB solid culture medium containing the corresponding antibiotics with a sterile coating rod, and invert it in a 28℃ incubator for 3 days. You can pick a single clone and proceed to the next experiment.

[0085] 2. Transformation and screening of positive callus of hybrid Liriodendron chinense

[0086] 1) Pre-culture of hybrid tulipwood embryonic callus

[0087] Pre-culture on 3 / 4 medium with an AS concentration of 1 mg / mL for 2-3 weeks is used to allow the embryonic callus to grow most vigorously, which is conducive to Agrobacterium infection.

[0088] 2) Preparation of Agrobacterium culture solution

[0089] Pick a single colony of Agrobacterium grown on a culture medium containing kan. Inoculate it into 700 μL of liquid LB culture medium and culture it at 28°C, 220 rpm on a shaker for 4-6 hours. After verifying the positivity of the culture by PCR, take 20 μL of the culture and add it to 2 mL of LB culture medium and culture it at 28°C, 220 rpm on a shaker for 12 hours. Then expand the culture at a ratio of 1:25 for 4 hours, detect the OD value of the culture with a UV spectrophotometer, and control the OD value of the culture between 0.6-0.8. Centrifuge at room temperature at 5000 rpm for 10 minutes, remove the supernatant, and collect the bacteria. Resuspend the bacteria in a certain volume of 3 / 4MS liquid culture medium (AS concentration is 1 mg / mL) and prepare it for use.

[0090] 3) Infection and co-cultivation

[0091] Place the hybrid tulipwood callus tissue pre-cultured for 18 days in a 250mL conical flask, gently crush it with sterilized tweezers, and then slowly flush the callus tissue to the bottom of the conical flask using the bacteria cultured in the previous step. Place the conical flask on a low-speed shaker at 90rpm for 10 minutes to allow the bacterial solution to fully contact the callus tissue. Subsequently, filter the callus tissue with a 400-mesh cell sieve, and use sterilized dry filter paper and cotton to absorb the bacterial solution on the callus surface. Place the callus tissue on a 3 / 4 medium with an AS concentration of 1 mg / mL and co-culture for 2 days.

[0092] 4) Degerming and screening positive callus

[0093] The hybrid tulipwood callus tissue after co-cultivation was washed at least 3 times with MS liquid medium supplemented with 1000 mg / L of cephalosporin (about 5 minutes each time), and then sterilized with ddH 2 O rinse 3 times, then use sterilized cotton and filter paper to absorb the water, and place the callus tissue on the recovery medium supplemented with Cef (400 mg / L). After 7 days, the medium is replaced with the basic medium supplemented with Cef (400 mg / L) and G418 (90 mg / L). The medium is replaced every 21 days until yellow-white callus grows on the callus, and then the callus is expanded.

[0094] 5) Identification of transgenic positive callus

[0095] Three overexpression transgenic lines were randomly selected to extract DNA, and the transgenic callus was positively identified by PCR reaction. pBI121 empty transgenic and WT were used as control groups ( Figure 1 ). Subsequently, RNA was extracted from the positive callus lines, converted to cDNA, and then qRT-PCR was performed to detect its expression level. Figure 2 As shown, the expression levels of LhAHL10 or LhAHL15 genes in the transgenic lines were significantly higher than those in the wild type and empty transgenic callus (control group).

[0096] 3. Induction of transgenic positive somatic embryo seedlings

[0097] 1) Induction of somatic embryogenesis

[0098] Take 1g of positive callus tissue in a 250mL conical flask, add 50mL of M13 liquid culture medium, and culture at 90rpm shaking for 14 days, during which the culture medium is replaced once; filter the callus that has been suspended for 14 days with a cell sieve with a 150-mesh upper layer and a 400-mesh lower layer; retain the single cells on the 400-mesh cell sieve, use 50mL of Z36 culture medium to backwash into the conical flask, and culture at 90rpm shaking for 2 days; use a 1mL pipette to draw 1mL of the suspended culture material into a centrifuge tube. Invert and mix thoroughly, then draw 10μL on a slide, draw a straight line, and count the number of cells under a microscope. Repeat 3 times for each sample and finally take the average value. Use Z36 culture medium to dilute the suspended material to ensure that there are 8000-10000 cells on each filter paper.

[0099] 2) Direct induction of somatic embryos from callus

[0100] Take 0.1g of callus tissue in a 10mL centrifuge tube, add 3mL of M13 liquid medium, and mix thoroughly. Pipette 70μL of the mixed solution onto filter paper padded with cotton. After the cotton absorbs the moisture on the filter paper, use tweezers to transfer the filter paper to Z14 medium and culture for 28 days. Select 3 strains for each gene, and replicate 5 dishes for each strain.

[0101] Example 3

[0102] 1. Construction of sgRNA expression cassette

[0103] 1) 1 round of PCR

[0104] Dilute AtU3d, AtU3b, and AtU6-1 plasmids to 2-5 ng / μL, and use primers UF / 3d-R, grT1 / GR-R, UF / 3b-R, grT2 / GR-R, UF / 6-1-R, and grT3 / GR-R to amplify 3d, 3b, and 6-1 plasmids by PCR. The primer sequences are as follows:

[0105] UF:

[0106] 5'-CTCCGTTTTACCTGTGGAATCG-3',

[0107] 3d-R:

[0108] 5'-ATCGCAACCATCGATGCATATgaccaatggtgctttg-3',

[0109] grT1:

[0110] 5'-TATGCATCGATGGTTGCGATgttttagagctagaaat-3';

[0111] GR-R:

[0112] 5'-CGGAGGAAAATTCCATCCAC-3',

[0113] 3b-R:

[0114] 5'-GTGGGCGGCCCCTTGCTTTTgaccaatgttgctcc-3',

[0115] grT2:

[0116] 5'-AAAGCAAGGGGCCGCCCACgttttagagctagaaat-3';

[0117] 6-1-R:

[0118] 5'-TGTTGGACGACCGGTGTGGCaatcactacttcgtct-3',

[0119] grT3:

[0120] 5'-CCACACCGGTCGTCCAACAgttttagagctagaaat-3'.

[0121] PCR reaction system: ddH 2 O 8.5μL, 2×Phanta Max Buffer 12.5μL, dNTP Mix 0.5μL, Primer-F 1μL, Primer-R 1μL, Phanta Max Super-Fidelity DNA Polimerase 0.5μL, DNA 1μL.

[0122] The PCR reaction program was as follows: pre-denaturation at 95°C for 3 min; denaturation at 95°C for 15 sec, annealing at 56°C for 15 sec, and extension at 72°C for 30 sec, for 35 cycles; and complete extension at 72°C for 5 min.

[0123] 2) 2 rounds of PCR

[0124] The primers for the three target sites are all UF / GR-R, and the template is the PCR product diluted 10 times from the previous step. The primer sequences are as follows:

[0125] UF:

[0126] 5'-CTCCGTTTTACCTGTGGAATCG-3',

[0127] GR-R:

[0128] 5′-CGGAGGAAAATTCCATCCAC-3′.

[0129] PCR reaction system: ddH 2 O 17μL, 2×Phanta Max Buffer 25μL, dNTP Mix 1μL, Primer-F 2μL, Primer-R 2μL, Phanta Max Super-Fidelity DNA Polimerase 1μL, and 1μL of each PCR product from the previous round.

[0130] The PCR reaction program was as follows: pre-denaturation at 95°C for 3 min; denaturation at 95°C for 15 sec, annealing at 56°C for 15 sec, and extension at 72°C for 36 sec, for 35 cycles; and complete extension at 72°C for 5 min.

[0131] 2% agarose gel was used for electrophoresis detection, and the fragments were cut and recovered to obtain AtU3d, AtU3b, and AtU6-1 with the target gene added, which were named AtU-t1-3d, AtU-t2-3b, and AtU-t3-6-1, respectively.

[0132] 3) 3 rounds of PCR

[0133] The products AtU-t1-3d, AtU-t2-3b, and AtU-t3-6-1 recovered from the previous round of gel excision were diluted to 30 ng / μL. The primers are as follows:

[0134] U-GAL:

[0135] 5'-ACCGGTAAGGCGCGCCGTAGTGCTCGACTAGTATGGAATCGGCAGC AAAGG-3',

[0136] pgs-GA2:

[0137] 5'-CAGGGAGCGGATAACAATTTCACACAGGCACATCCACTCCAAGCTCTTG-3',

[0138] U-GA2:

[0139] 5'-GTGCCTGTGTGAAATTGTTATCCGCTCCCTGGAATCGGCAGCAAAG G-3',

[0140] pgs-GA3:

[0141] 5'-CCACGCATACGATTTAGGTGACACTATAGCGCATCCACTCCAAGCTCTTG-3',

[0142] U-GA3:

[0143] 5'-CGCTATAGTGTCACCTAAATCGTATGCGTGGTGGAATCGGCAGCAAA GG-3'

[0144] pgs-GAR:

[0145] 5'-TAGCTCGAGAGGCGCGCCAATGATACCGACGCGTATCCATCCACTCC AAGCTCTTG-3'.

[0146] PCR reaction system: ddH 2 O 17μL, 2×Phanta Max Buffer 25μL, dNTP Mix 1μL, Primer-F 2μL, Primer-R 2μL, Phanta Max Super-Fidelity DNA Polimerase 1μL, and 2μL of the product recovered from the previous round of gel excision.

[0147] The PCR reaction program was as follows: pre-denaturation at 95°C for 3 min; denaturation at 95°C for 15 sec, annealing at 56°C for 15 sec, and extension at 72°C for 42 sec, for 35 cycles; and complete extension at 72°C for 5 min.

[0148] 2% agarose gel was used for electrophoresis detection, and the fragments were cut and recovered, and sent for bidirectional detection. At this time, AtU-t1-3d, AtU-t2-3b, and AtU-t3-6-1 were named oT1, oT2, and oT3, respectively.

[0149] 2. Connect sgRNA expression cassette with CRISPR / Cas9 to construct gene editing vector

[0150] The plasmid was digested with enzyme (Cas9 plasmid concentration = 373.9 ng / μL).

[0151] The enzyme digestion reaction system is: 10×cutsmart 5μL, Bsa1-HFV 2 1μL, plasmid 7μL, ddH 2 O 37 μL.

[0152] The enzyme digestion reaction program was: 37°C for 30 min, 80°C for 20 min, and 4°C infinity.

[0153] The linearized vector and the target gene fragment recovered after gel excision were homologously recombined using ClonExpress II One Step Cloning Kit.

[0154] The homologous recombination ligation reaction system is: oT1 1μL, oT2 1μL, oT3 1μL, linearized vector 100ng3μL, Gibson Assembly Master Mix 7μL, ddH 2 O 7 μL.

[0155] The homologous recombination ligation reaction procedure was: 50°C for 30 min, 4°C infinity.

[0156] The homologous recombination ligation reaction products (gene editing vectors) were transformed into DH5α Escherichia coli, and the bacterial solution was verified by PCR. The bacterial solution verified by PCR was sent to the company for sequencing. The DH5α Escherichia coli that was sequenced and correct was extracted with the Tiangen Plasmid Mini-Extraction Kit (DP106) and then transformed into Agrobacterium. The gene editing vector was transformed into hybrid Liriodendron callus tissue, and positive callus was identified.

[0157] 3. Identification of transgenic positive callus

[0158] Yellow, delicate callus tissue grew on the dark brown callus. Three callus tissues after transgenic recovery were randomly selected to extract DNA. Then, the transgenic positive identification of different strains was performed by PCR reaction. The fragment cloning primers were designed on the genome, and the primer sequences are as follows:

[0159] Target-F:

[0160] 5'-ATGTCGGGTAGAGAGTCCTTT-3',

[0161] Target-R:

[0162] 5'-CCAAAGCAACCATCTGCTTC-3',

[0163] Using DNA as a template, the sequences of the three targets were amplified. The PCR products were then connected to the intermediate vector 007B, and five single clones were selected from each strain for first-generation sequencing.

[0164] The results are as follows Figure 3As shown, ahl10-ko has a single base insertion and deletion site, and ahl15-ko has a small fragment deletion and a single base insertion site. The results of protein sequence analysis showed that the sequencing peaks and protein sequences showed that the protein sequences of ahl10-ko and ahl15-ko showed premature termination. In summary, the gene editing events of LhAHL10 and LhAHL15 in hybrid Liriodendron chinense were successful.

[0165] Example 4

[0166] 1. Effect of LhAHL10 / 15 on the efficiency of somatic embryo induction

[0167] 1) Using LhAHL15-OE, ahl15-ko and control group callus tissues as experimental materials, the effect of LhAHL15 on somatic embryogenesis was further explored by inducing somatic embryogenesis.

[0168] The results are as follows Figure 4 As shown in the figure, on a medium without exogenous hormones, overexpression of LhAHL15 promoted somatic embryogenesis, while the number of somatic embryos in the gene-edited line (ahl15-ko) decreased, and most of them stagnated at the globular embryo stage, with a small number of somatic embryos continuing to develop. Overexpression can form complete cotyledonary embryos, while gene editing will cause somatic embryo development to be malformed and unable to form normal plants.

[0169] 2) Using LhAHL10-OE, ahl10-ko and control group callus tissues as experimental materials, the effect of LhAHL10 on somatic embryogenesis was further explored by inducing somatic embryogenesis.

[0170] The results are as follows Figure 5 As shown in the figure, compared with the control, the number of somatic embryos of LhAHL10-OE increased significantly, but the development of somatic embryos was arrested at the globular embryo stage, and the volume of globular embryos was significantly larger than that of somatic embryos in the control group. The number of somatic embryos induced by ahl10-ko increased significantly compared with the control and was similar to that of overexpressed somatic embryos.

[0171] 2. Effect of LhAHL10 / 15 on somatic embryo morphology

[0172] 1) The result is as follows Figure 6 As shown, the hypocotyl base of the somatic embryos induced by callus of the ahl15-ko mutant was swollen and the radicle was missing. At the plantlet stage, the root tip development was arrested and callus accumulation occurred. Compared with the wild type and the empty vector, the cotyledon embryos overexpressing LhAHL15 had thicker hypocotyls and fleshy cotyledons. At the seedling stage, overexpressing LhAHL15 had thick stems and fleshy leaves.

[0173] 2) The results are as follows Figure 7As shown in the figure, the volume of somatic embryos induced by positive callus of ahl10-ko mutant was significantly smaller than that of the control group, but the embryonic development was complete without obvious defects. The cotyledon embryos overexpressing LhAHL10 had disordered development, no obvious embryonic structure, and could not develop into plantlets at the later stage. The plant transformation rate of LhAHL10-OE approached zero, but the plant transformation rate of ahl10-ko was not significantly different from that of the control group.

[0174] 3. Effect of LhAHL10 / 15 on embryonic callus induction

[0175] The results are as follows Figure 8 As shown, AHL genes promote the induction of embryonic callus from cotyledon embryos. The induction efficiency of embryonic callus by LhAHL10-OE is as high as 90%, while ahl10 inhibits the induction of embryonic callus, with the induction efficiency of embryonic callus ranging from 10% to 20%. LhAHL15-OE promotes the induction of callus from cotyledon embryos, while ahl15 inhibits the induction of embryonic callus.

[0176] 4. Effects of LhAHL10 / 15 on genes related to somatic embryogenesis

[0177] Ectopic overexpression of BBM can promote the formation of callus and ectopic buds, changes in leaf morphology, and hormone-free regeneration of explants; PLT2 controls cell division and amyloplast formation to regulate root cap development and gravitropism; in the embryo, LEC1 regulates seed development by interacting with other transcription factors, and LEC1 is required for embryo maturation; PIN1 is important for auxin transport during somatic embryo development.

[0178] Overexpressed and gene-edited transgenic positive callus tissue was used as experimental material. RNA was extracted and converted into cDNA, and the expression of BBM, LEC1, PIN1, and PLT2 genes was detected using fluorescent quantitative PCR technology.

[0179] The results are as follows Fig. 9As shown in the figure, overexpression of LhAHL15 significantly increased the expression level of BBM, while overexpression of LhAHL10 had no significant effect on the expression level of BBM. Overexpression of LhAHL15 can significantly increase the expression level of LEC1, while overexpression of LhAHL10 had no significant effect on the expression level of BBM. The expression level of LEC1 was significantly increased after knocking out LhAHL10. Overexpression of LhAHL15 significantly increased the expression level of PIN1, gene editing of LhAHL15 significantly decreased the expression level of PIN1, while knocking out LhAHL10 significantly increased the expression level of PIN1. Overexpression of LhAHL15 significantly increased the expression of PLT2, while knocking out LhAHL15 significantly decreased the gene expression of PLT2. However, knocking out LhAHL10 significantly increased the gene expression of PLT2.

[0180] The above description is only illustrative rather than restrictive of the present invention. Those skilled in the art will understand that many modifications, changes or equivalents may be made without departing from the spirit and scope defined by the appended claims, but all will fall within the scope of protection of the present invention.

Claims

1. A method for establishing a somatic embryogenesis system of hybrid Liriodendron chinense based on LhAHL15 gene, characterized in that: By regulating the expression of the LhAHL15 gene, the purpose of regulating the somatic embryogenesis of hybrid tulipwood is achieved; the nucleotide sequence of the LhAHL15 gene is shown in SEQ ID NO.

3.

2. The method for establishing a somatic embryogenesis system of hybrid Liriodendron chinense according to claim 1, characterized in that: The regulating LhAHL15 gene expression is to promote LhAHL15 gene expression.

3. The method for establishing a somatic embryogenesis system of hybrid Liriodendron chinense according to claim 2, characterized in that: include: 1) constructing an overexpression vector of the hybrid Liriodendron chinense LhAHL15 gene; 2) transforming the constructed overexpression vector of hybrid tulipwood LhAHL15 gene into hybrid tulipwood; 3) Cultivate, screen and obtain transgenic hybrid tulipwood plants with significantly improved somatic embryogenesis efficiency.

4. A method for establishing a somatic embryogenesis system of hybrid Liriodendron chinense based on LhAHL10 gene, characterized in that: By regulating the expression of the LhAHL10 gene, the purpose of regulating the somatic embryogenesis of hybrid Liriodendron chinense is achieved; the nucleotide sequence of the LhAHL10 gene is shown in SEQ ID NO.

1.

5. The method for establishing a somatic embryogenesis system of hybrid Liriodendron chinense according to claim 4, characterized in that: The regulating LhAHL10 gene expression is inhibiting LhAHL10 gene expression.

6. The method for establishing a somatic embryogenesis system of hybrid Liriodendron chinense according to claim 5, characterized in that: include: 1) Construction of editing vector for hybrid Liriodendron chinense LhAHL10 gene; 2) Transforming the constructed editing vector of hybrid tulipwood LhAHL10 gene into hybrid tulipwood; 3) Cultivate, screen and obtain transgenic hybrid tulipwood plants with significantly improved somatic embryogenesis efficiency.

7. Application of the hybrid tulip tree LhAHL15 gene in regulating the expression of genes related to somatic embryogenesis in hybrid tulip tree.

8. The use according to claim 7, characterized in that: The somatic embryogenesis-related genes are BBM, LEC1, PIN1, and PLT2.

9. Application of the hybrid tulip tree LhAHL10 gene in regulating the expression of genes related to somatic embryogenesis in hybrid tulip tree.

10. The use according to claim 9, characterized in that: The somatic embryogenesis-related genes are LEC1, PIN1 and PLT2.

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