A method for establishing hybrid liriodendron body embryo system based on LhAHL10 / 15 gene

Abstract

The application discloses a hybrid Liriodendron hybridum somatic embryogenesis system establishment method based on LhAHL10 / 15 genes, and relates to the technical field of plant genetic engineering. The hybrid Liriodendron hybridum somatic embryogenesis system establishment method based on LhAHL10 / 15 genes disclosed by the application promotes the expression of LhAHL15 genes or inhibits the expression of LhAHL10 genes, so that the purpose of promoting the somatic embryogenesis of the hybrid Liriodendron hybridum is achieved; the nucleotide sequences of the LhAHL15 genes and the LhAHL10 genes are respectively shown in SEQ ID NO. 3 and SEQ ID NO. 1. The results show that overexpression of LhAHL15 in the hybrid Liriodendron hybridum is beneficial to improving the indirect somatic embryogenesis efficiency of the hybrid Liriodendron hybridum. Overexpression of LhAHL10 in the hybrid Liriodendron hybridum can also improve the somatic embryogenesis efficiency, but can affect the patterning development process of the somatic embryos, so that the somatic embryos are developed to stagnate at the globular embryo stage.

Description

TECHNICAL FIELD

[0001] The application belongs to the technical field of plant genetic engineering, and more particularly relates to a hybrid Liriodendron chinense somatic embryogenesis system establishment method based on LhAHL10 / 15 genes. BACKGROUND

[0002] Hybrid Liriodendron sino-americanum P.C.Yieh ex Shang et Z.R.Wang, also known as hybrid Liriodendron or hybrid Liriodendron tulipifera, is a plant of Magnoliaceae Liriodendron. It is obtained by artificial hybridization, taking Liriodendron chinense as the female parent and Liriodendron tulipifera as the male parent. The leaf of the hybrid Liriodendron tulipifera is similar to a frock, so it is also called hybrid Liriodendron tulipifera. Its flowers are large, yellow, fragrant, single on the branch top, and similar to tulips, so it is also called "wooden tulip". It grows fast and has good wood quality, and is a precious commodity tree species. The leaf type is similar to a frock, and the flowers are like golden saucers, so it is an excellent landscaping tree species. It is an ancient relic plant and was widely distributed in the temperate regions of the northern hemisphere. Liriodendron tulipifera is originally from the southeastern United States, and there are also distributions in Qingdao, Lushan, Nanjing, Guangzhou and other places in China. The female and male flower periods of Liriodendron do not meet, resulting in low natural reproduction efficiency. At present, the breeding methods of Liriodendron tree species generally adopt asexual reproduction, including cutting, grafting, tissue culture and the like. However, the artificial breeding means of cutting and grafting is difficult and has low survival rate.

[0003] Somatic embryogenesis is a manifestation of plant cell totipotency, and somatic cells or other cells develop into embryos without fertilization. Somatic embryogenesis is regulated by many aspects, such as transcription factors, hormones, epigenetic modifications and the like. The ectopic expression of specific transcription factors or the deletion of certain chromatin modification proteins can lead to the acquisition of totipotency by somatic cells and the transformation into cells with embryonic properties. In previous studies, it has been reported that the ectopic expression of many single genes can directly induce somatic embryogenesis, such as BBM (Baby Boom), WUS (WUSCHEL), LEC 1 (LEAFY COTYLEDON 1), AHL15 (AT-HOOK MOTIF NUCLEAR LOCATED 15) and the like.

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

[0005] At present, most of the researches on somatic embryogenesis of forest trees are still in the optimization and improvement of somatic embryogenesis conditions, and the research on the molecular mechanism of somatic embryogenesis is still not much. Strengthening the research on the regulation mechanism of somatic embryogenesis is conducive to further providing reference genes for plants with low somatic embryogenesis ability and improving the somatic embryogenesis ability and efficiency. Hybrid Liriodendron as a plant with great potential economic value, has the problems of low natural seed setting rate, poor reproductive ability and insufficient supply of hybrid species. The research on improving the efficiency of somatic embryogenesis and plant regeneration of hybrid Liriodendron has important significance for the preservation, genetic improvement and rapid propagation of its germplasm resources. SUMMARY

[0006] In view of the above problems existing in the prior art, the technical problem to be solved by the present application is to provide a hybrid Liriodendron somatic embryogenesis system establishment method based on LhAHL10 / 15 gene, which is used for regulating the formation and development of hybrid Liriodendron somatic embryo.

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

[0008] A hybrid Liriodendron somatic embryogenesis system establishment method based on LhAHL15 gene, by regulating the expression of LhAHL15 gene, so as to achieve the purpose of regulating the somatic embryogenesis of hybrid Liriodendron. The nucleotide sequence of LhAHL15 gene is shown in SEQ ID NO. 3.

[0009] The regulation of LhAHL15 gene expression is to promote the expression of LhAHL15 gene.

[0010] The hybrid Liriodendron somatic embryogenesis system establishment method based on LhAHL15 gene comprises:

[0011] 1) Constructing an overexpression vector of LhAHL15 gene of hybrid Liriodendron;

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

[0013] 3) cultivating, screening and obtaining the transgenic hybrid Liriodendron tulipifera plant with significantly improved somatic embryogenesis efficiency.

[0014] The application discloses a hybrid Liriodendron tulipifera somatic embryogenesis system establishment method based on a LhAHL10 gene.

[0015] The LhAHL10 gene expression is inhibited.

[0016] The application discloses a hybrid Liriodendron tulipifera somatic embryogenesis system establishment method based on a LhAHL10 gene.

[0017] 1) constructing an editing vector of a hybrid Liriodendron tulipifera LhAHL10 gene;

[0018] 2) transforming the constructed editing vector of the hybrid Liriodendron tulipifera LhAHL10 gene into the hybrid Liriodendron tulipifera;

[0019] 3) cultivating, screening and obtaining the transgenic hybrid Liriodendron tulipifera plant with significantly improved somatic embryogenesis efficiency.

[0020] Application of the hybrid Liriodendron tulipifera LhAHL15 gene in regulating expression of a somatic embryogenesis related gene of the hybrid Liriodendron tulipifera.

[0021] The somatic embryogenesis related gene is BBM, LEC1, PIN1 or PLT2.

[0022] Application of the hybrid Liriodendron tulipifera LhAHL10 gene in regulating expression of a somatic embryogenesis related gene of the hybrid Liriodendron tulipifera.

[0023] The somatic embryogenesis related gene is LEC1, PIN1 or PLT2.

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

[0025] 1) The application promotes expression of the LhAHL15 gene, thereby achieving the purpose of promoting somatic embryogenesis of the hybrid Liriodendron tulipifera, the nucleotide sequence of the LhAHL15 gene is shown as SEQ ID NO. 3, an overexpression vector of the hybrid Liriodendron tulipifera LhAHL15 gene is constructed, the constructed overexpression vector of the hybrid Liriodendron tulipifera LhAHL15 gene is transformed into the hybrid Liriodendron tulipifera, and the transgenic hybrid Liriodendron tulipifera plant with significantly improved somatic embryogenesis efficiency is cultivated and screened.

[0026] 2) The application promotes somatic embryogenesis of hybrid Liriodendron by inhibiting the expression of LhAHL10 gene, the nucleotide sequence of LhAHL10 gene is shown as SEQ ID NO. 1; an editing vector of hybrid Liriodendron LhAHL10 gene is constructed; the constructed editing vector of hybrid Liriodendron LhAHL10 gene is transformed into hybrid Liriodendron; and transgenic hybrid Liriodendron plants with significantly improved somatic embryogenesis efficiency are obtained through cultivation and screening.

[0027] 3) The results of the application show that overexpression of LhAHL15 promotes somatic embryogenesis on the medium without exogenous hormones, the number of somatic embryos of the gene edited strain (ahl15-ko) is reduced, and most of them are stalled at the globular embryo stage, and a small amount of somatic embryos can continue to develop. Overexpression can form complete cotyledon embryos, while gene editing will cause somatic embryo development to be deformed and unable to form normal plants. Compared with the control group, the number of somatic embryos of LhAHL10-OE significantly increases, but the development of somatic embryos is stalled at the globular embryo stage, and the volume of globular embryos is significantly larger than that of the control group. The number of somatic embryos induced by ahl10-ko is significantly increased compared with the control and is close to the number of somatic embryos induced by overexpression.

[0028] 4) The results of the application show that the hypocotyl base of somatic embryos induced by ahl15-ko mutant callus is swollen and the phenomenon of loss of radicle occurs. At the small plant stage, the root end development is stalled and callus accumulation occurs. Compared with wild type and empty vector, cotyledon embryos overexpressing LhAHL15 have thicker hypocotyls and cotyledon flesh. At the seedling stage, overexpression of LhAHL15 has thick stems and fleshy leaves. The volume of somatic embryos induced by ahl10-ko mutant positive callus is significantly smaller than that of the control group, but the embryo development is complete and has no obvious defects. Cotyledon embryos overexpressing LhAHL10 develop disorderly, have no obvious embryo structure, and cannot develop into small plants at the later stage. The plant transformation rate of LhAHL10-OE tends to be zero, but the plant transformation rate of ahl10-ko has no significant difference from that of the control group.

[0029] 5) The results of the present application 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 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 PLT2 gene. Knocking out LhAHL15 significantly reduces the expression level of PLT2 gene. However, knocking out LhAHL10 significantly increases the expression level of PLT2 gene. BRIEF DESCRIPTION OF DRAWINGS

[0030] Figure 1 Figure for positive identification of LhAHL10 / 15 transgenic callus;

[0031] Figure 2 Figure for qRT-PCR results of LhAHL10 / 15 transgenic callus;

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

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

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

[0035] Figure 6 Figure 8 is a phenotype diagram of ahl15-ko knockout mutants and LhAHL15-OE at the cotyledon embryo stage and the 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 of overexpression of LhAHL15; d is the plantlet of overexpression of LhAHL15; e is the plant transformation rate of different transgenic lines; f is a statistical diagram of the plant transformation rate);

[0036] Figure 7 Figure 9 is a cotyledon embryo picture and a statistical data diagram of the plant transformation rate of ahl10-ko knockout mutants and LhAHL10-OE; a is a cotyledon embryo picture of LhAHL10-OE and ahl10-ko; b is a statistical diagram of the cotyledon embryo size; c is a statistical diagram of the effect of LhAHL10 gene on the plant transformation rate;

[0037] Figure 8 Figure 10 is a diagram of the effect of LhAHL10 / 15 on the induction of embryogenic callus (a is the callus induced by CRISPR / Cas9 empty, ahl15-ko, ahl0-ko single embryo; b is the callus induced by pBI121 empty, LhAHL15-OE, LhAHL10-OE single embryo; c-d are schematic diagrams of non-embryogenic callus; e is a schematic diagram of embryogenic callus; f is a statistical diagram of the induction efficiency of LhAHL10 on embryogenic callus; g is a statistical diagram of the induction efficiency of LhAHL15 on embryogenic callus);

[0038] Figure 9 Figure 11 is a diagram of the effect of LhAHL10 / 15 on the expression amount of somatic embryogenesis related genes. DETAILED DESCRIPTION

[0039] In order to make the purpose, technical scheme and advantages of the present application clearer, the present application will be further described below in combination with specific examples. If no detailed description is given in the following examples, the technical means used are all conventional means well known to those skilled in the art.

[0040] The material selected in the present application is embryogenic callus induced by 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 selected in the present application are preserved by the Molecular Laboratory of Nanjing Forestry University, and pClone007 Blunt is purchased from Beijing Genki Biological Company; the E. coli strain is Trelief TM5αChemically Competent Cell purchased from Beijing Genesee Biotech Co., Ltd., Agrobacterium strain EHA105 purchased from Shanghai Weidi Biological Technology Co., Ltd.

[0042] The RNA extraction kit (Vazyme) selected in the application; RNA reverse transcription kit (Vazyme); restriction endonuclease EcoR I, Kpn I, Hind III, Xba I (NEB); ClonExpress II One Step Cloning Kit (Vazyme); Phanta Max Super-Fidelity DNA Polymerase (Vazyme); gel recovery kit (Genesee); plasmid 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 the 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; medium pH value is 5.72-5.74.

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

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

[0049] Example 1

[0050] 1. Total RNA extraction and cDNA acquisition

[0051] The RNA of the hybrid Liriodendron chinense embryonic callus was extracted using an RNA extraction kit (Vazyme). The extracted total RNA was used as a template for reverse transcription experiment using an 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] The LhAHL10 / 15 gene cloning primer sequence was designed using the Oligo7 website with the LhAHL10 / 15 gene CDS sequence as the reference sequence. 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] The hybrid Liriodendron chinense cDNA was used as a template to configure a premix reaction system, and the mixture was placed in a PCR instrument for reaction. All operations must be performed on ice, and each component must be thoroughly mixed and centrifuged to the bottom of the tube.

[0059] The PCR reaction system was: ddH2O 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: 95℃ pre-denaturation for 3min; 95℃ denaturation for 15sec, 60℃ annealing for 15sec, 72℃ extension for 3min, 35 cycles; 72℃ thorough extension for 5min.

[0061] PCR amplification product band is clear and bright, the DNA gel recovery kit (TSP602-200) of TSINGKE company is used to recover the target fragment. The cut gel recovery fragment is connected to pClone007 intermediate vector.

[0062] The connection reaction system is: target fragment 3 μL, pClone007 Blunt Vector 1 μL, 10×Topo Mix 1 μL, ddH2O 5 μL. All solutions are directly added to the bottom of the PCR tube, and gently blown and mixed with a pipette.

[0063] The connection reaction program is: 22-30℃ room temperature reaction, after the reaction is completed, do not put on ice, prevent reducing the connection efficiency.

[0064] The intermediate vector connected with the target fragment is transformed into DH5α E. coli, and the bacterial liquid is verified by PCR. The correct bacterial liquid is sent to the company for sequencing, and the DH5α E. coli with correct sequencing is extracted by using the small amount of plasmid extraction kit (DP106) of Tian Gen. The prepared intermediate vector plasmid containing the target fragment is placed in the-20℃ refrigerator.

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

[0066] Example 2

[0067] 1, construction of overexpression vector

[0068] The homologous arm of the target sequence is added, and the homologous arm primer sequence of 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 Xba I and Sac I as enzyme sites. The vector digestion product was recovered using a DNA recovery kit.

[0078] The enzyme reaction system was: 10x cutsmart 5 μL, Xba I 1 μL, Sac I 1 μL, plasmid 7 μL, ddH2O 36 μL

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

[0080] The linearized vector and the target gene fragment recovered after cutting the gel were subjected to homologous recombination using ClonExpress II One Step Cloning Kit.

[0081] The homologous recombination ligation reaction system was: 5x CE II Buffer 4 μL, II 2 μL, linearized vector 4 μL, target fragment 2 μL, ddH2O 8 μL.

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

[0083] The homologous recombination ligation reaction products of the two genes were transformed into DH5α E. coli, respectively, and the bacterial liquid was verified by PCR. The bacterial liquid with correct PCR verification was sent to the company for sequencing. After the DH5α E. coli with completed and correct sequencing was extracted using the Zhen Gene Plasmid Miniprep Kit (DP106), the agrobacterium was transformed, and the specific steps were as follows.

[0084] The agrobacterium competent cells stored in the ultra-low temperature refrigerator were thawed at room temperature, and then placed in an ice box to keep low temperature. 100 μL of the competent cells were added to about 1 μg of plasmid DNA, and the plasmid and the bacterial solution were mixed by gently stirring the bottom of the centrifuge tube with hands. Then, the centrifuge tube was sequentially placed on ice for 5 min, then placed in liquid nitrogen for 5 min, immediately placed in a 37°C water bath for 5 min, and then placed on ice for 5 min. About 700 μL of LB liquid medium without antibiotics was added to the tube, and the tube was placed in a 28°C (150 rpm) shaking incubator for 3 h. The bacteria were centrifuged in a centrifuge at 5000 rpm for about 1 min, and then about 100 μL of supernatant was taken and resuspended by gently blowing the bacterial body. The bacterial body was coated on a plate containing LB solid medium containing the corresponding antibiotic using a sterile coating rod, and the plate was placed in a 28°C incubator for 3 days. Single colonies were picked and used for the next experiment.

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

[0086] 1) Pre-culture of hybrid Liriodendron embryogenic callus

[0087] The embryogenic callus was pre-cultured on 3 / 4 medium with an AS concentration of 1 mg / mL for 2-3 weeks, so that the embryogenic callus reached the most vigorous growth state, which was beneficial to agrobacterium infection.

[0088] 2) Preparation of agrobacterium solution

[0089] Agrobacterium single colonies growing on the medium containing kan were picked and inoculated into 700 μL of liquid LB medium, which was cultured at 28°C, 220 rpm on a shaking table for 4-6 h. After the positive bacteria solution was verified by PCR, 20 μL of the bacteria solution was added to 2 mL of LB medium, which was cultured at 28°C, 220 rpm on a shaking table for 12 h. Then, the bacteria solution was expanded at a ratio of 1:25 for 4 h, and the OD value of the bacteria solution was detected by a UV spectrophotometer. The OD value of the bacteria solution was controlled between 0.6 and 0.8. The supernatant was removed by centrifugation at 5000 rpm for 10 min at room temperature, and the bacterial body was collected. The bacterial body was resuspended in a certain volume of 3 / 4 MS liquid medium (AS concentration of 1 mg / mL) for standby.

[0090] 3) Infection and co-culture

[0091] The pre-cultured 18d hybrid Liriodendron callus was placed in a 250mL conical flask, and gently pressed with sterilized forceps, then the callus was slowly washed to the bottom of the conical flask with the bacteria from the previous step. The conical flask was placed on a low-speed shaker, 90rpm for 10 minutes, so that the bacteria solution and callus were in full contact. Then, the callus was filtered with a 400-mesh cell sieve, and the bacteria solution on the surface of the callus was absorbed with sterilized dry filter paper and cotton. The callus was placed on 3 / 4 medium with AS concentration of 1mg / mL for co-culture for 2 days.

[0092] 4) De-sterilization and screening of positive callus

[0093] The hybrid Liriodendron callus after co-culture was washed with MS liquid medium added with Cef 1000mg / L for at least 3 times (about 5min each time), and then washed with sterilized ddH2O for 3 times, and then the water was absorbed with sterilized cotton and filter paper. The callus was placed on recovery medium added with Cef (400mg / L). After 7 days, the medium was replaced with basic medium added with Cef (400mg / L) and G418 (90mg / L). The medium was replaced every 21 days until the callus grew yellowish white callus, and then the callus was multiplied.

[0094] 5) Identification of transgenic positive callus

[0095] DNA was extracted from 3 randomly selected overexpression transgenic lines, and the transgenic callus was positively identified by PCR reaction, with pBI121 empty transgenic and WT as control group. Figure 1 Then, RNA was extracted from the above positive callus lines, and qRT-PCR was performed after reverse transcription to cDNA to detect the expression amount. As shown in Figure 2 , the expression amount of LhAHL10 or LhAHL15 gene in the transgenic lines was significantly higher than that in the wild type and empty transgenic callus (control group).

[0096] 3、Transgenic positive somatic embryo induction

[0097] 1) Induction of somatic embryogenesis

[0098] Take 1 g of positive callus in a 250 mL conical flask, add 50 mL of M13 liquid medium, cultivate on a 90 rpm shaker for 14 d, replace the medium once during the cultivation; filter the callus cultivated for 14 d using a 150 mesh upper layer and a 400 mesh lower layer cell sieve; retain the single cells on the 400 mesh cell sieve, use 50 mL of Z36 medium to backflush into the conical flask, cultivate on a 90 rpm shaker for 2 d; use a 1 mL pipette to take 1 mL of the suspension cultivation material into a centrifuge tube. Mix thoroughly by inverting, then take 10 μL onto a glass 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. Dilute the suspension material using Z36 medium to ensure that there are 8000-10000 cells on each filter paper.

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

[0100] Take 0.1 g of callus in a 10 mL centrifuge tube, add 3 mL of M13 liquid medium, mix thoroughly. Take 70 μL of the mixed solution onto filter paper padded with cotton, and after the cotton has absorbed the water on the filter paper, use tweezers to transfer the filter paper to Z14 medium for cultivation for 28 d. Select 3 strains for each gene, and repeat 5 dishes for each strain.

[0101] Example 3

[0102] 1. Construction of sgRNA expression cassette

[0103] 1) One round of PCR

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

[0105] U-F:

[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: ddH2O 8.5 μL, 2 × Phanta Max Buffer 12.5 μL, dNTP Mix 0.5 μL, Primer-F 1 μL, Primer-R 1 μL, Phanta Max Super-Fidelity DNA Polimerase 0.5 μL, DNA 1 μL.

[0122] PCR reaction program: 95 ℃ pre-denaturation 3 min; 95 ℃ denaturation 15 sec, 56 ℃ annealing 15 sec, 72 ℃ extension 30 sec, 35 cycles; 72 ℃ complete extension 5 min.

[0123] 2) 2 rounds of PCR

[0124] The primers of the 3 target sites are all U-F / GR-R, and the template is the 10-fold dilution of the PCR product of the previous step. The primer sequences are as follows:

[0125] U-F:

[0126] 5'-CTCCGTTTTACCTGTGGAATCG-3',

[0127] GR-R:

[0128] 5'-CGGAGGAAAATTCCATCCAC-3'.

[0129] PCR reaction system: ddH2O 17 μL, 2 × Phanta Max Buffer 25 μL, dNTP Mix 1 μL, Primer-F 2 μL, Primer-R 2 μL, Phanta Max Super-Fidelity DNA Polimerase 1 μL, 1 μL of the PCR product of the previous round.

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

[0131] Electrophoresis detection was performed using 2% agarose gel, and the fragments were recovered by gel cutting. Thus, AtU3d, AtU3b and AtU6-1 to which the target genes were added were obtained, and were named AtU-t1-3d, AtU-t2-3b and AtU-t3-6-1, respectively.

[0132] 3) 3 rounds of PCR

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

[0134] U-GAL:

[0135] 5'-ACCGGTAAGGCGCGCCGTAGTGCTCGACTAGTATGGAATCGGCAGCAAAGG-3',

[0136] pgs-GA2:

[0137] 5'-CAGGGAGCGGATAACAATTTCACACAGGCACATCCACTCCAAGCTCTTG-3',

[0138] U-GA2:

[0139] 5'-GTGCCTGTGTGAAATTGTTATCCGCTCCCTGGAATCGGCAGCAAAGG-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: ddH2O 17 μL, 2 × Phanta Max Buffer 25 μL, dNTP Mix 1 μL, Primer-F 2 μL, Primer-R 2 μL, Phanta Max Super-Fidelity DNA Polimerase 1 μL, the recovered product of the last round of gel 2 μL.

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

[0148] Electrophoresis detection was performed using 2% agarose gel, the fragments were recovered by gel cutting, and bidirectional detection was performed. At this time, AtU-t1-3d, AtU-t2-3b, and AtU-t3-6-1 were named oT1, oT2, and oT3, respectively.

[0149] 2. Connection of sgRNA expression cassette and CRISPR / Cas9 to construct gene editing vector

[0150] Enzymatic digestion of plasmid (Cas9 plasmid concentration = 373.9 ng / μL).

[0151] Enzymatic reaction system: 10 × cutsmart 5 μL, Bsa1-HFV2 1 μL, plasmid 7 μL, ddH2O 37 μL.

[0152] Enzymatic reaction program: 37 °C for 30 min, 80 °C for 20 min, 4 °C for ∞.

[0153] Linearized vector and target gene fragment recovered by gel cutting were subjected to homologous recombination using ClonExpress II One Step Cloning Kit.

[0154] The homologous recombination ligation reaction system consisted of: 1 μL oT1, 1 μL oT2, 1 μL oT3, 3 μL linearized carrier (100 ng), 7 μL Gibson Assembly Master Mix, and 7 μL ddH2O.

[0155] The homologous recombination ligation reaction program was: 50℃ for 30 min, then 4℃ for ∞.

[0156] The homologous recombination ligation product (gene-editing vector) was transformed into DH5α *E. coli*, and the bacterial culture was verified by PCR. Cultures with correct PCR verification were sent to the company for sequencing. The sequenced DH5α *E. coli* were then used to extract plasmids using the Tiangen Plasmid Mini-Prep Kit (DP106) and transformed into *Agrobacterium*. The gene-editing vector was then transformed into hybrid *Liriodendron tulipifera* callus tissue, and positive callus identification was performed.

[0157] 3. Identification of transgenic positive callus

[0158] Yellow, fine callus tissue grew on the dark brown callus. DNA was extracted from three randomly selected transgenic callus tissues after recovery. Transgenic positivity was then identified for different strains using PCR. Fragment cloning primers were designed on the genome; the primer sequences are shown below:

[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 target sites were amplified. The PCR products were then ligated into the intermediate vector 007B, and five single clones from each line were selected for first-generation sequencing.

[0164] The results are as follows Figure 3 As shown, ahl10-ko contains single-base insertion and deletion sites, while ahl15-ko contains small-fragment deletions and single-base insertion sites. Protein sequence analysis results, including sequencing peak diagrams and protein sequences, indicate that both ahl10-ko and ahl15-ko protein sequences exhibit premature termination. In conclusion, the gene editing events on LhAHL10 and LhAHL15 in hybrid tulip trees were successful.

[0165] Example 4

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

[0167] 1) Using LhAHL15-OE, ahl15-ko and control group callus tissue 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, on a culture medium without exogenous hormones, overexpression of LhAHL15 promotes somatic embryogenesis, while the gene-edited line (ahl15-ko) exhibits reduced somatic embryogenesis, with most embryos arresting at the globular embryo stage, and only a small number continuing to develop. Overexpression can lead to the formation of complete cotyledonary embryos, while gene editing results in abnormal somatic embryo development, failing to form normal plants.

[0169] 2) Using LhAHL10-OE, ahl10-ko and control group callus tissue 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, compared with the control, the number of somatic embryos in LhAHL10-OE was significantly increased, but the development of somatic embryos was arrested at the globular embryo stage, and the volume of the globular embryos was significantly larger than that of the control somatic embryos. The number of somatic embryos induced by ahl10-ko was significantly increased compared with the control and was similar to the number of somatic embryos induced by overexpression.

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

[0172] 1) Results are as follows Figure 6 As shown, somatic embryos induced by callus from the ahl15-ko mutant exhibit hypocotyl base swelling and radicle loss. During the plantlet stage, root tip development is arrested, and callus accumulation occurs. Cotyledonary embryos overexpressing LhAHL15 show thicker hypocotyls and fleshy cotyledons compared to wild-type and unexpressed embryos. In the seedling stage, LhAHL15 overexpression results in robust stems and fleshy leaves.

[0173] 2) Results are as follows Figure 7 As shown, the somatic embryos induced by ahl10-ko mutant positive callus were significantly smaller than those in the control group, but their embryonic development was complete and without obvious defects. Cotyledonary embryos overexpressing LhAHL10 exhibited disordered development, lacked obvious embryonic structure, and failed to develop into small plantlets in later stages. The transformation rate of LhAHL10-OE plants approached zero, but the transformation rate of ahl10-ko plants was not significantly different from that of the control group.

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

[0175] The results are as follows Figure 8As shown, the AHL gene promotes the induction of embryogenic callus from cotyledonous embryos. LhAHL10-OE achieves an induction efficiency of over 90% for embryogenic callus, while ahl10 inhibits it, with an induction efficiency of only 10%–20%. LhAHL15-OE promotes callus induction from cotyledonous embryos, while ahl15 inhibits it.

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

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

[0178] Using transgenic positive callus tissue that was overexpressed and gene-edited as experimental material, RNA was extracted and reversed into cDNA, and the expression of BBM, LEC1, PIN1, and PLT2 genes was detected by real-time PCR.

[0179] The results are as follows Figure 9 As shown, overexpression of LhAHL15 significantly increased BBM expression levels, while overexpression of LhAHL10 had no significant effect on BBM expression levels. Overexpression of LhAHL15 significantly increased LEC1 expression levels, while overexpression of LhAHL10 had no significant effect on BBM expression levels; knockout of LhAHL10 significantly increased LEC1 expression levels. Overexpression of LhAHL15 significantly increased PIN1 expression levels, gene editing with LhAHL15 significantly decreased PIN1 expression levels, while knockout of LhAHL10 significantly increased PIN1 expression levels. Overexpression of LhAHL15 significantly increased PLT2 expression levels, while knockout of LhAHL15 significantly decreased PLT2 gene expression levels. However, knockout of LhAHL10 significantly increased PLT2 gene expression levels.

[0180] The above description is illustrative only and not restrictive of the present invention. Those skilled in the art will understand that many modifications, variations or equivalents can be made without departing from the spirit and scope defined by the appended claims, and all such modifications, variations or equivalents will fall within the protection scope of the present invention.

Claims

1. A method based on LhAHL15 A method for establishing a hybrid embryogenetic system of tulip tree (Liriodendron tulipifera) is characterized by, By promoting LhAHL15 Gene expression, thereby achieving the goal of improving somatic embryogenesis in hybrid tulip trees; the aforementioned LhAHL15 The nucleotide sequence of the gene is shown in SEQ ID NO.

3.

2. The method for establishing a hybrid tulip tree somatic embryogenesis system according to claim 1, characterized in that, include: 1) Constructing a hybrid tulip tree LhAHL15 Gene overexpression vectors; 2) Construct hybrid tulip trees LhAHL15 The gene overexpression vector was transformed into hybrid tulip trees; 3) Cultivate, screen and obtain transgenic hybrid tulip trees with significantly improved somatic embryogenesis efficiency.

3. Hybrid tulip tree LhAHL15 Application of gene in improving the expression of somatic embryogenesis-related genes during the induction of hybrid tulip tree somatic embryos; the somatic embryogenesis-related genes are... BBM , LEC1 , PIN1 , PLT2 .

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