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

[0001] The present application is a divisional application of a patent application with the application date of October 9, 2024, the application number of CN202411403437.X, and the patent name of a method for establishing a hybrid Liriodendron hybrid somatic embryogenesis system based on LhAHL10 / 15 genes. TECHNICAL FIELD

[0002] The present application belongs to the technical field of plant genetic engineering, more specifically, relates to a method for establishing a hybrid Liriodendron hybrid somatic embryogenesis system based on LhAHL10 / 15 genes. BACKGROUND

[0003] Hybrid Liriodendron (Liriodendron sino-americanum P.C.Yieh ex Shang et Z.R.Wang) is also known as hybrid tulip tree or hybrid yellowwood. It is a plant of Magnoliaceae Liriodendron. It is obtained by artificial hybridization with Liriodendron as the female parent and Liriodendron tulipifera as the male parent. The leaf of hybrid Liriodendron is similar to a military coat, so it is also called hybrid yellowwood. Its flowers are large and yellow, with a sweet fragrance, and are solitary on the branch tips, resembling tulips, so it is also called "wooden tulip". It grows fast and has good wood quality, and is a valuable commercial tree species. The leaf type is similar to a military coat, and the flowers are like golden saucers, making it an excellent landscaping tree species. It is an ancient relic plant that was widely distributed in the temperate regions of the northern hemisphere, and is also a national second-class key rare and endangered protected plant. Liriodendron tulipifera is native to the southeastern United States, and also has distribution in Qingdao, Lushan, Nanjing, Guangzhou and other places in China. The female and male flower periods of Liriodendron do not coincide, resulting in low natural reproduction efficiency. Currently, the propagation method of Liriodendron tree species generally uses asexual reproduction, including cutting, grafting, tissue culture, etc. However, the artificial propagation methods of cutting and grafting are difficult and have low survival rates.

[0004] 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 factors, such as transcription factors, hormones, epigenetic modifications, etc. The ectopic expression of specific transcription factors or the absence of certain chromatin modification proteins can lead to the acquisition of totipotency by somatic cells and their transformation into embryonic cells. In previous studies, many single genes have been reported to directly induce somatic embryogenesis, such as BBM (Baby Boom), WUS (WUSCHEL), LEC 1 (LEAFY COTYLEDON 1), WOX 2 (WUSCHEL-RELATED HOMEOBOX 2), etc. AHL15( AT-HOOK MOTIF NUCLEAR LOCALED 15 )

[0005] ​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.

[0006] 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 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

[0007] 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.

[0008] In order to solve the above technical problems, the technical scheme adopted by the present application is as follows:

[0009] 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.

[0010] The regulation of LhAHL15 gene expression is to promote the expression of LhAHL15 gene.

[0011] The hybrid Liriodendron somatic embryogenesis system establishment method based on LhAHL15 gene comprises:

[0012] 1) Constructing an overexpression vector of LhAHL15 gene of hybrid Liriodendron;

[0013] 2) transforming the constructed hybrid Liriodendron tulipifera LhAHL15 gene overexpression vector into hybrid Liriodendron tulipifera;

[0014] 3) cultivating, screening and obtaining transgenic hybrid Liriodendron tulipifera plants with significantly improved somatic embryogenesis efficiency.

[0015] A hybrid Liriodendron tulipifera somatic embryogenesis system establishment method based on LhAHL10 gene, by regulating the expression of LhAHL10 gene, so as to achieve the purpose of regulating the somatic embryogenesis of hybrid Liriodendron tulipifera; the nucleotide sequence of the LhAHL10 gene is shown in SEQ ID NO. 1

[0016] The LhAHL10 gene expression is inhibited.

[0017] The hybrid Liriodendron tulipifera somatic embryogenesis system establishment method based on LhAHL10 gene comprises:

[0018] 1) constructing an editing vector of the LhAHL10 gene of hybrid Liriodendron tulipifera;

[0019] 2) transforming the constructed editing vector of the LhAHL10 gene of hybrid Liriodendron tulipifera into hybrid Liriodendron tulipifera;

[0020] 3) cultivating, screening and obtaining transgenic hybrid Liriodendron tulipifera plants with significantly improved somatic embryogenesis efficiency.

[0021] Application of the LhAHL15 gene of hybrid Liriodendron tulipifera in regulating the expression of somatic embryogenesis related genes of hybrid Liriodendron tulipifera.

[0022] The somatic embryogenesis related genes are BBM, LEC1, PIN1 and PLT2.

[0023] Application of the LhAHL10 gene of hybrid Liriodendron tulipifera in regulating the expression of somatic embryogenesis related genes of hybrid Liriodendron tulipifera.

[0024] The somatic embryogenesis related genes are LEC1, PIN1 and PLT2.

[0025] Compared with the prior art, the beneficial effects of the present application are:

[0026] 1) The present application promotes the expression of LhAHL15 gene, so as to achieve the purpose of promoting the somatic embryogenesis of hybrid Liriodendron tulipifera, the nucleotide sequence of the LhAHL15 gene is shown in SEQ ID NO. 3; an overexpression vector of the LhAHL15 gene of hybrid Liriodendron tulipifera is constructed; the constructed overexpression vector of the LhAHL15 gene of hybrid Liriodendron tulipifera is transformed into hybrid Liriodendron tulipifera; and transgenic hybrid Liriodendron tulipifera plants with significantly improved somatic embryogenesis efficiency are cultivated, screened and obtained.

[0027] 2) The application promotes somatic embryogenesis of hybrid Liriodendron by inhibiting the expression of LhAHL10 gene, the nucleotide sequence of which is shown in 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.

[0028] 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.

[0029] 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. Cotyledon embryos overexpressing LhAHL15 have a more robust hypocotyl and cotyledon flesh compared with wild type and empty load. At the seedling stage, overexpression of LhAHL15 has a thick stem 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 zero, but the plant transformation rate of ahl10-ko has no significant difference from that of the control group.

[0030] 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. 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 amount of PLT2 gene. Knocking out LhAHL15 significantly reduces the gene expression amount of PLT2. However, knocking out LhAHL10 significantly increases the gene expression amount of PLT2. BRIEF DESCRIPTION OF DRAWINGS

[0031] Figure 1 Figure is a positive identification diagram of LhAHL10 / 15 transgenic callus;

[0032] Figure 2 Figure is a qRT-PCR result diagram of LhAHL10 / 15 transgenic callus;

[0033] Figure 3 Figure is a positive identification and first generation sequencing result diagram of ahl10 / 15-ko transgenic callus (a is ahl10-ko transgenic positive callus and second generation sequencing result and peak diagram; b is ahl10-ko gene editing protein sequence result, the stop codon is indicated in the red box; c is ahl15-ko transgenic positive callus and second generation sequencing result and peak diagram; d is ahl15-ko gene editing protein sequence result, the stop codon is indicated in the red box);

[0034] Figure 4 Figure is a somatic embryogenesis induction diagram (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 result);

[0035] Figure 5 Figure is a somatic embryogenesis induction diagram (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 result;

[0036] 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 overexpressing LhAHL15; d is the plantlet of overexpressing LhAHL15; e is the plant transformation rate of different transgenic lines; f is a statistical diagram of the plant transformation rate);

[0037] 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;

[0038] 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 vector, ahl15-ko, ahl0-ko single embryo; b is the callus induced by pBI121 empty vector, 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);

[0039] Figure 9 Figure 11 is a diagram of the effect of LhAHL10 / 15 on the expression amount of somatic embryogenesis related genes. DETAILED DESCRIPTION

[0040] In order to make the purpose, technical scheme and advantages of the present application more clear, the present application will be further described below in combination with specific examples. In the following examples, if no detailed description is given, the technical means used are all conventional means well known to those skilled in the art.

[0041] The material selected in the present application is embryogenic callus induced by immature embryos, and the genotype is 154102 (subcultured for about 15 days). The embryogenic callus is cultured in a constant temperature incubator at 22°C in dark.

[0042] The pBI121, pYLsgRNA-AtU3b, pYLsgRNA-AtU3d, pYLsgRNA-AtU6-1, pYLCRISPR_Cas9P35S-N selected by the application are preserved by the Molecular Laboratory of Nanjing Forestry University, and the pClone007 Blunt is purchased from Beijing Genki Biological Company; the E. coli strain is Trelief™ 5α Chemically Competent Cell purchased from Beijing Genki Biological Company, and the Agrobacterium strain is EHA105 purchased from Shanghai Weidi Biological Technology Co., Ltd.

[0043] The RNA extraction kit (Vazyme) selected by the application; the RNA reverse transcription kit (Vazyme); the 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 (Genki); plasmid small extraction kit (TIANGEN); AceQ qPCR SYBR Green Master Mix (Vazyme). Antibiotics: Geneticin (G418); Cefpiramide (Cef); Kanamycin (Kan); Ampicillin (Amp).

[0044] The culture medium used by the application is as follows:

[0045] M13 callus subculture medium: 3 / 4MS + 2,4-D 1 mg / L + 6-BA 0.2 mg / L + Vc 5 mg / L + CH 0.5 g / L + sucrose 30 g / L + agar 2.5 g / L; the medium pH value is 5.72-5.74.

[0046] Z36 suspension culture medium: 3 / 4MS + Vc 5 mg / L + KT 0.5 mg / L + BA 0.2 mg / L + NAA 0.2 mg / L + CH 0.5 g / L + sucrose 50 g / L + agar 2.4 g / L; the medium pH value is 5.72-5.74.

[0047] Z14 somatic embryo induction medium: 3 / 4MS + Vc 5 mg / L + LH 0.2 g / L + sucrose 40 g / L + agar 2.4 g / L; the medium pH value is 5.72-5.74.

[0048] Embryogenic callus induction medium: 3 / 4MS + 2,4-D 2 mg / L + 6-BA 0.2 mg / L + Vc 5 mg / L + CH 1 g / L + sucrose 40 g / L + agar 3.6 g / L; medium pH value is 5.72-5.74.

[0049] LB liquid medium: NaCl (10 g / L) + tryptone 10 g / L + yeast extract 5 g / L.

[0050] Example 1

[0051] 1. Total RNA extraction and cDNA acquisition

[0052] RNA of L. hypohaloides embryogenic callus was extracted using an RNA extraction kit (Vazyme). 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°C to reduce the number of repeated freeze-thaw cycles.

[0053] 2. LhAHL10 / 15 gene cloning

[0054] Using LhAHL10 / 15 gene CDS sequence as reference sequence, LhAHL10 / 15 gene cloning primer sequence was designed using Oligo7 website. Sequence information is as follows:

[0055] pLhAHL10-clone-F: 5'-ACTACCCACTCACTCCCTTG-3',

[0056] pLhAHL10-clone-R: 5'-TTACATGTGGGCCTTGACCT-3';

[0057] pLhAHL15-clone-F: 5'-TCCTCCTCACAAAACACCAGA-3',

[0058] pLhAHL15-clone-R: 5'-TATGCACAGCCACAACATCG-3'.

[0059] Using L. hypohaloides cDNA as a template, a premix reaction system was configured, and the mixture was placed in a 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.

[0060] 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, cDNA 2 μL.

[0061] PCR reaction program: 95 °C pre-denaturation 3 min; 95 °C denaturation 15 sec, 60 °C annealing 15 sec, 72 °C extension 3 min, 35 cycles; 72 °C thorough extension 5 min.

[0062] The PCR amplification product band is clear and bright, and the target fragment is recovered using the DNA gel recovery kit (TSP602-200) of TSINGKE Company. The gel cutting recovery fragment is connected to the pClone007 intermediate vector.

[0063] 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 the pipette is gently blown and mixed.

[0064] The connection reaction program is: 22-30 °C room temperature reaction, and after the reaction is completed, do not place on ice to prevent reducing the connection efficiency.

[0065] The intermediate vector connected with the target fragment is transformed into DH5α E. coli, and the bacterial solution is verified by PCR. The correct bacterial solution 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 °C refrigerator.

[0066] The CDS sequence of the finally cloned LhAHL10 gene 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 the 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.

[0067] Example 2

[0068] 1. Construction of overexpression vector

[0069] The homologous arm of the target sequence is added, and the homologous arm primer sequence of the vector is as follows:

[0070] OL-LhAHL10-F:

[0071] 5'-tggagagaacacgggggactATGTCGGGTAGAGAGTCCTTTGG-3',

[0072] OL-LhAHL10-R:

[0073] 5'-cgatcggggaaattcgagctTTATTTCCATGGCATGTTCGCCAAG-3';

[0074] OL-LhAHL15-F:

[0075] 5'-tggagagaacacgggggactATGGGAGGAGTCGATCTGTCG-3',

[0076] OL-LhAHL15-R:

[0077] 5'-cgatcggggaaattcgagctTTAGTAAGATGGTGGTGGCCTAGT-3'.

[0078] 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.

[0079] The enzyme reaction system was 10x cutsmart 5 μL, Xba I 1 μL, Sac I 1 μL, plasmid 7 μL, ddH2O 36 μL.

[0080] The enzyme reaction program was 37°C for 30 min, 80°C for 20 min, and 4°C for infinity.

[0081] 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.

[0082] The homologous recombination connection reaction system was 5x CE II Buffer 4 μL, Exnase® II 2 μL, linearized vector 4 μL, target fragment 2 μL, ddH2O 8 μL.

[0083] The homologous recombination connection reaction program was 37°C for 60 min, and 4°C for infinity.

[0084] The homologous recombination connection reaction products of the two genes were transformed into DH5a E. coli, respectively, and the bacterial liquid was verified by PCR. The correct bacterial liquid was sent to the company for sequencing. After the sequencing of the correct DH5a E. coli was completed, the plasmid was extracted using the Zhen Gene Plasmid Extraction Kit (DP106), and then the agrobacterium was transformed. The specific steps are as follows.

[0085] The agrobacterium prepared in the ultra-low temperature refrigerator was placed at room temperature for melting. After melting, it was placed in an ice box to keep low temperature; 100 μL of competent cells were added to about 1 μg of plasmid DNA, and the plasmid and bacterial liquid were mixed by gently stirring the bottom of the centrifuge tube with hands, and then 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 in ice for 5 min; about 700 μL of LB liquid medium without antibiotics was added to the tube, and placed in a 28°C (150 rpm) shaking incubator for 3 h; the bacteria were collected by centrifugation 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 inverted and placed in a 28°C incubator for 3 days. Single colonies were picked and subjected to the next experiment.

[0086] 2. Transformation and screening of hybrid liriodendron positive callus

[0087] 1) Pre-culture of hybrid liriodendron embryogenic callus

[0088] Pre-culture for 2-3 weeks on 3 / 4 medium with AS concentration of 1 mg / mL, the purpose is to make the embryogenic callus reach the most vigorous state, which is conducive to the infection of agrobacterium.

[0089] 2) Preparation of agrobacterium liquid

[0090] Pick the agrobacterium single colony that grew on the medium containing kan. Inoculate into 700 μL of liquid LB medium, and incubate at 28°C, 220 rpm shaking incubator for 4-6 h. After the positive bacterial liquid is verified by PCR, 20 μL of bacterial liquid is added to 2 mL of LB medium and incubated at 28°C, 220 rpm shaking incubator for 12 h. Then the bacterial liquid is expanded by 1:25 for 4 h. The OD value of the bacterial liquid is detected by ultraviolet spectrophotometer, and the OD value of the bacterial liquid is controlled between 0.6-0.8. Centrifuge at 5000 rpm for 10 min at room temperature, remove the supernatant, and collect the bacterial body. Resuspend the bacterial body with a certain volume of 3 / 4 MS liquid medium (AS concentration of 1 mg / mL) to prepare for use.

[0091] 3) Infection and co-culture

[0092] The pre-cultured 18d hybrid Liriodendron callus was placed in a 250mL conical flask, and gently crushed with sterilized forceps, then the callus was slowly washed to the bottom of the conical flask with the bacteria used in the previous step. The conical flask was placed on a low-speed shaker, 90rpm for 10 minutes, so that the bacteria solution and the 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.

[0093] 4) De-sterilization and screening of positive callus

[0094] The hybrid Liriodendron callus after co-culture was washed at least 3 times (about 5min each time) with MS liquid medium added with Cef 1000mg / L, and then washed 3 times with sterilized ddH2O, and then the water was absorbed with sterilized cotton and filter paper. The callus was placed on recovery medium added with Cef (400mg / L) for culture. After 7d, the medium was replaced with basic medium added with Cef (400mg / L) and G418 (90mg / L). The medium was replaced every 21 days until yellowish-white callus grew on the callus, and then the callus was multiplied.

[0095] 5) Identification of transgenic positive callus

[0096] DNA was extracted from 3 randomly selected overexpression transgenic lines, and PCR reaction was performed for positive identification of the transgenic callus, 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).

[0097] 3, Induction of transgenic positive somatic embryo

[0098] 1) Induction of somatic embryogenesis

[0099] 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 that has been suspended 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 back flush into the conical flask, cultivate on a 90 rpm shaker for 2 d; use a 1 mL pipette to take 1 mL of the suspended cultivated 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. Dilute the suspended material using Z36 medium to ensure that there are 8000-10000 cells on each filter paper.

[0100] 2) Direct induction of somatic embryos from callus

[0101] 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 lines for each gene, and repeat 5 dishes for each line.

[0102] Example 3

[0103] 1. Construction of sgRNA expression cassette

[0104] 1) One round of PCR

[0105] 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:

[0106] U-F:

[0107] 5'-CTCCGTTTTACCTGTGGAATCG-3',

[0108] 3d-R:

[0109] 5'- ATCGCAACCATCGATGCATATgaccaatggtgctttg-3',

[0110] grT1:

[0111] 5'- TATGCATCGATGGTTGCGATgttttagagctagaaat-3';

[0112] GR-R:

[0113] 5'-CGGAGGAAAATTCCATCCAC-3',

[0114] 3b-R:

[0115] 5'- GTGGGCGGCCCCTTGCTTTTgaccaatgttgctcc-3',

[0116] grT2:

[0117] 5'- AAAGCAAGGGGCCGCCCACgttttagagctagaaat-3';

[0118] 6-1-R:

[0119] 5'- TGTTGGACGACCGGTGTGGCaatcactacttcgtct-3',

[0120] grT3:

[0121] 5'- CCACACCGGTCGTCCAACAgttttagagctagaaat-3'.

[0122] PCR reaction system: ddH2O 8.5 μL, 2x 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.

[0123] PCR reaction program: 95°C pre-denaturation 3 min; 95°C denaturation 15 sec, 56°C annealing 15 sec, 72°C extension 30 sec, 35 cycles; 72°C thorough extension 5 min.

[0124] 2) 2 rounds of PCR

[0125] 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:

[0126] U-F:

[0127] 5'-CTCCGTTTTACCTGTGGAATCG-3',

[0128] GR-R:

[0129] 5'-CGGAGGAAAATTCCATCCAC-3'.

[0130] 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.

[0131] PCR reaction program: 95°C pre-denaturation 3 min; 95°C denaturation 15 sec, 56°C annealing 15 sec, 72°C extension 36 sec, 35 cycles; 72°C thorough extension 5 min.

[0132] Electrophoresis detection was performed using 2% agarose gel, and the fragments were recovered by gel cutting. AtU3d, AtU3b and AtU6-1 with the target gene were obtained, and were named AtU-t1-3d, AtU-t2-3b and AtU-t3-6-1, respectively.

[0133] 3) 3 rounds of PCR

[0134] 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:

[0135] U-GAL:

[0136] 5'-ACCGGTAAGGCGCGCCGTAGTGCTCGACTAGTATGGAATCGGCAGCAAAGG-3',

[0137] pgs-GA2:

[0138] 5'-CAGGGAGCGGATAACAATTTCACACAGGCACATCCACTCCAAGCTCTTG-3',

[0139] U-GA2:

[0140] 5'-GTGCCTGTGTGAAATTGTTATCCGCTCCCTGGAATCGGCAGCAAAGG-3',

[0141] pgs-GA3:

[0142] 5'-CCACGCATACGATTTAGGTGACACTATAGCGCATCCACTCCAAGCTCTTG-3',

[0143] U-GA3:

[0144] 5'-CGCTATAGTGTCACCTAAATCGTATGCGTGGTGGAATCGGCAGCAAAGG-3'

[0145] pgs-GAR:

[0146] 5'-TAGCTCGAGAGGCGCGCCAATGATACCGACGCGTATCCATCCACTCCAAGCTCTTG-3'.

[0147] 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 product of the last round of gel recovery 2 μL.

[0148] 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 thorough extension 5 min.

[0149] Electrophoresis detection was performed using 2% agarose gel, and the fragments were gel recovered and bidirectionally detected. At this time, AtU-t1-3d, AtU-t2-3b and AtU-t3-6-1 were named oT1, oT2 and oT3 respectively.

[0150] 2. Connection of sgRNA expression cassette and CRISPR / Cas9 to construct gene editing vector

[0151] Enzymatic digestion of plasmid (Cas9 plasmid concentration = 373.9 ng / μL).

[0152] Enzymatic digestion reaction system: 10 × cutsmart 5 μL, Bsa1-HFV2 1 μL, plasmid 7 μL, ddH2O 37 μL.

[0153] Enzymatic digestion reaction program: 37°C 30 min, 80°C 20 min, 4°C ∞.

[0154] Linearized vector and target gene fragment recovered by gel recovery were subjected to homologous recombination using ClonExpress II One Step Cloning Kit.

[0155] The homologous recombination connection reaction system is: oT11 1 μL, oT2 1 μL, oT3 1 μL, linearized vector 100 ng 3 μL, Gibson Assembly Master Mix 7 μL, ddH2O 7 μL.

[0156] The homologous recombination connection reaction procedure is: 50°C for 30 min, 4°C for ∞.

[0157] The homologous recombination connection reaction product (gene editing vector) is respectively transformed into DH5α E. coli, and the bacterial liquid is verified by PCR. The bacterial liquid with correct PCR verification is sent to the company for sequencing. After the DH5α E. coli with completed and correct sequencing is extracted by using the Zhen Gene Plasmid Miniprep Kit (DP106), the plasmid is transformed into Agrobacterium. The gene editing vector is transformed into hybrid Liriodendron callus, and positive callus identification is performed.

[0158] 3、Transgenic positive callus identification

[0159] Yellow and delicate callus grows on the black and brown callus. Randomly select 3 transgenic callus tissues after recovery to extract DNA. Then, transgenic positive identification is performed on different strains by PCR reaction. The fragment cloning primer is designed on the genome, and the primer sequence is as follows:

[0160] Target-F:

[0161] 5'- ATGTCGGGTAGAGAGTCCTTT-3',

[0162] Target-R:

[0163] 5'- CCAAAGCAACCATCTGCTTC-3',

[0164] The sequences of the 3 target points are amplified by taking DNA as a template. Then, the PCR product is connected to the intermediate vector 007B, and 5 single clones are selected from each strain for first-generation sequencing.

[0165] The results are shown in Figure 3 The ahl10-ko has a single base insertion and deletion site, and the ahl15-ko has a small fragment deletion and a single base insertion site. Protein sequence analysis results show that the sequencing peak chart and the protein sequence table show that the protein sequences of ahl10-ko and ahl15-ko both appear the phenomenon of premature termination. In summary, the gene editing events of LhAHL10 and LhAHL15 in hybrid Liriodendron are successful.

[0166] Example 4

[0167] 1、LhAHL10 / 15 on the effect of somatic embryo induction efficiency

[0168] 1) Using LhAHL15-OE, ahl15-ko and control calli as experimental materials, the effect of LhAHL15 on somatic embryogenesis was further explored by inducing somatic embryogenesis.

[0169] The results are shown in Figure 4 that overexpression of LhAHL15 promoted somatic embryogenesis on the medium without exogenous hormones, while the number of somatic embryos of the gene edited strain (ahl15-ko) was reduced, and most of them were arrested at the globular embryo stage, and a small amount of somatic embryos could continue to develop. Overexpression could form complete cotyledon embryos, while gene editing would cause somatic embryo development to be deformed and unable to form normal plants.

[0170] 2) Using LhAHL10-OE, ahl10-ko and control calli as experimental materials, the effect of LhAHL10 on somatic embryogenesis was further explored by inducing somatic embryogenesis.

[0171] The results are shown in Figure 5 that 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 the control. The number of somatic embryos induced by ahl10-ko increased significantly compared with the control and was similar to that of overexpression.

[0172] 2, the effect of LhAHL10 / 15 on somatic morphology

[0173] 1) The results are shown in Figure 6 that the hypocotyl base of ahl15-ko mutant callus induced somatic embryos was swollen and showed the phenomenon of radicle absence. At the small plant stage, the root end development was arrested and callus accumulation occurred. Cotyledon embryos overexpressing LhAHL15 had a thicker hypocotyl and cotyledon flesh compared to wild type and empty vector. At the seedling stage, overexpression of LhAHL15 had thick stems and fleshy leaves.

[0174] 2) The results are shown in Figure 7 that the volume of somatic embryos induced by ahl10-ko mutant callus was significantly smaller than that of the control, but the embryo development was complete and had no obvious defects. Cotyledon embryos overexpressing LhAHL10 developed disorderly, had no obvious embryo structure, and could not develop into small plants at the later stage. The plant transformation rate of LhAHL10-OE tended to zero, but the plant transformation rate of ahl10-ko had no significant difference from the control.

[0175] 3, the effect of LhAHL10 / 15 on embryogenic callus induction

[0176] The results are shown in Figure 8As shown, AHL genes promote cotyledon embryo to induce embryogenic callus. LhAHL10-OE has an induction efficiency of embryogenic callus of up to 90% or more, while ahl10 has an inhibitory effect on the induction of embryogenic callus, and the induction efficiency of embryogenic callus is 10% to 20%. LhAHL15-OE promotes cotyledon embryo to induce callus, and ahl15 inhibits the induction of embryogenic callus.

[0177] 4. Effects of LhAHL10 / 15 on somatic embryogenesis related genes

[0178] Ectopic overexpression of BBM can promote the formation of callus and adventitious buds, change the morphology of leaves, and hormone-free regeneration of explants; PLT2 controls cell division and starch formation to regulate root cap development and gravitropism; in embryos, LEC1 regulates seed development by interacting with other transcription factors, and LEC1 is essential for embryo maturation; PIN1 is important for auxin transport during somatic embryogenesis.

[0179] Using transgenic positive callus overexpressing and gene editing as experimental materials, the expression of BBM, LEC1, PIN1 and PLT2 genes was detected by using fluorescent quantitative PCR technology after extracting RNA and reversing it into cDNA.

[0180] As shown in the results, Figure 9 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 has no significant effect on the expression level of BBM, 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, and knocking out LhAHL15 significantly reduces the gene expression level of PLT2. However, knocking out LhAHL10 significantly increases the gene expression level of PLT2.

[0181] The above description is only illustrative in nature and is not limiting, and those skilled in the art understand that many modifications, changes or equivalents can be made without departing from the spirit and scope defined by the appended claims, but all will fall within the protection scope of the present application.

Claims

1. A method for establishing a hybrid Liriodendron tulipifera embryogenesis system based on LhAHL10 gene, characterized by, The application discloses a method for regulating somatic embryogenesis of Liriodendron hybridum by regulating expression of LhAHL10 gene, and the nucleotide sequence of the LhAHL10 gene is shown as SEQ ID NO.

1.

2. The method of hybrid Liriodendron body embryogenesis system establishment according to claim 1, characterized in that, The regulation of the LhAHL10 gene expression is inhibition of the LhAHL10 gene expression.

3. The method of hybrid Liriodendron body embryogenesis system establishment according to claim 2, characterized in that, The method comprises the following steps: 1) constructing an editing vector of the LhAHL10 gene of Liriodendron hybridum; 2) transforming the constructed editing vector of the LhAHL10 gene of Liriodendron hybridum into Liriodendron hybridum; 3) cultivating, screening and obtaining transgenic Liriodendron hybridum plants with significantly improved somatic embryogenesis efficiency.

4. Application of the LhAHL10 gene of Liriodendron hybridum in regulation of expression of somatic embryogenesis related genes of Liriodendron hybridum.

5. Use according to claim 4, characterized in that, The somatic embryogenesis related genes are LEC1, PIN1 and PLT2.

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