Application of LhAHL15 gene in inducing larch (larix gmelinii) polyploid

Abstract

The application discloses application of LhAHL15 gene in induction of a hybrid Liriodendron tulipifera polyploid, and relates to the technical field of plant genetic engineering. The hybrid Liriodendron tulipifera LhAHL15 gene is disclosed for the first time, the nucleotide sequence of which is shown as SEQ ID NO. 3, and the amino acid sequence of which is shown as SEQ ID NO. 4. The application constructs an expression vector of the hybrid Liriodendron tulipifera LhAHL15 gene, transforms the constructed expression vector of the hybrid Liriodendron tulipifera LhAHL15 gene into the hybrid Liriodendron tulipifera, and cultivates, screens and obtains a polyploid transgenic hybrid Liriodendron tulipifera plant. The transgenic somatic embryo obtained by construction has the characteristics of deepened leaf blade color, thickened leaf blade, thickened stem, dwarfed plant, increased total leaf area, increased contents of chlorophyll a, chlorophyll b and carotenoid, and increased expression amounts of a cyclin gene and a cell wall expansion related gene.

Description

Technical Field

[0001] The invention belongs to the technical field of plant genetic engineering, and more particularly relates to the application of LhAHL15 gene in inducing polyploidy in hybrid Liriodendron tulipifera. Background Art

[0002] Hybrid tulip tree (Liriodendron sino-americanum PC Yieh ex Shang et Z.R. Wang), also known as hybrid tulip tree or hybrid magnolia, is a species of plant in the genus Liriodendron in the Magnoliaceae family. It is the result of artificial hybridization, with Liriodendron sino-americanus as the female parent and Liriodendron sino-americanus as the male parent. Its leaves resemble magnolias, hence the name hybrid magnolia. Its large, yellow, fragrant flowers, solitary at the tops of branches, resemble tulips, earning it the nickname "woody tulip." Its rapid growth and high-quality wood make it a valuable commercial tree. Its magnolia-like leaves and magnolia-like flowers make it an excellent landscape tree. It is an ancient relict plant, once widely distributed in temperate regions of the Northern Hemisphere, and is a nationally designated Class II rare and endangered plant. Native to southeastern North America, it is also found in Qingdao, Lushan, Nanjing, and Guangzhou in my country. The inconsistent flowering periods of male and female tulip trees result in low natural reproduction efficiency. Currently, asexual propagation, including cuttings, grafting, and tissue culture, is common in the genus Tulipa. However, artificial propagation methods such as cuttings and grafting are difficult and have low survival rates.

[0003] In nature, most plants exist in the form of diploids, but there are many naturally formed polyploids that have thick stems, strong growth, enhanced stress resistance, etc., and are ideal breeding materials. Compared with diploids, tetraploids have the characteristics of gigantic organs, low fertility, strong stress resistance, and increased secondary metabolites. The first natural tetraploid discovered due to its gigantic organs was the giant evening primrose (Oenothera gigas), which caused a huge sensation as soon as it was discovered. The gigantic organs of tetraploid plants are not caused by an increase in the number of cells but by an increase in cell volume. Polyploid plants may produce aneuploid gametes during meiosis, which can lead to reduced fertility or even infertility of the plant.

[0004] So far, among flowers, tetraploid morning glories have shown phenotypes with thicker leaves, sturdier stems, and enlarged floral organs. Among vegetables, researchers have successfully induced tetraploid chili peppers, providing new germplasm resources for pepper breeding. Among fruits, researchers have successfully induced tetraploid plants in Actinidia chinensis and found that tetraploid kiwifruit has enhanced drought tolerance.

[0005] Polyploidy induction is an important way of plant breeding improvement. The common breeding methods for tetraploid induction are chemical induction and physical induction. Colchicine is a common chemical mutagen in plant polyploid induction. It can inhibit the formation of spindle in cell division process, so that the cell cannot be divided into two daughter cells, thus leading to the doubling of the number of chromosomes. However, colchicine itself has certain toxicity and low induction efficiency. There are some problems in forest polyploidy, such as slow separation and purification of chimera, urgent need of new inducer, long evaluation period of forest polyploidy, and insufficient research on molecular mechanism of homologous polyploidy. Physical induction of tetraploid includes ray treatment, low temperature induction, high temperature induction, etc. However, physical induction of tetraploid may have problems such as high proportion of chimera, complex experimental operation, and low induction rate. Molecular breeding has shown great advantages in recent years, but there are few reports on breeding tetraploid by using molecular methods. Only in Arabidopsis thaliana, overexpression of AtAHL15 successfully induced tetraploid plants, and the proportion of tetraploid was large. 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 application of LhAHL15 gene in inducing hybrid Liriodendron chinense polyploidy, for efficiently inducing hybrid Liriodendron chinense polyploidy.

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

[0008] A hybrid Liriodendron chinense LhAHL15 gene, the nucleotide sequence of which is shown in SEQ ID NO. 3.

[0009] The expression protein of the hybrid Liriodendron chinense LhAHL15 gene, the amino acid sequence of which is shown in SEQ ID NO. 4.

[0010] A vector, a recombinant bacterium or a host cell containing the hybrid Liriodendron chinense LhAHL15 gene.

[0011] Application of the hybrid Liriodendron chinense LhAHL15 gene in inducing hybrid Liriodendron chinense polyploidy, comprising:

[0012] 1) constructing an expression vector of the hybrid Liriodendron chinense LhAHL15 gene;

[0013] 2) transforming the constructed expression vector of the hybrid Liriodendron chinense LhAHL15 gene into hybrid Liriodendron chinense;

[0014] 3) cultivating, screening and obtaining polyploid transgenic hybrid Liriodendron chinense plants.

[0015] Application of the hybrid Liriodendron chinense LhAHL15 gene in deepening the color of somatic embryo seedling leaves, thickening the leaves, thickening the stems, dwarfing the plants and increasing the total leaf area.

[0016] The application of the LhAHL15 gene of hybrid tulipwood in increasing the width of leaf epidermal cells, the length of leaf epidermal cells, the area of ​​leaf epidermal cells, the density of stomata, the length of stomata and the area of ​​stomata in somatic seedlings.

[0017] Application of the LhAHL15 gene from hybrid Liriodendron chinense to increase the contents of chlorophyll a, chlorophyll b and carotenoids in somatic embryo seedlings.

[0018] The invention relates to an application of the hybrid tulipwood LhAHL15 gene in promoting the expression of cyclin genes and cell wall expansion-related genes. The cyclin genes are cyclin-p3, CDKF-4, and ribosome-recycling factor; and the cell wall expansion-related gene is Expansin-A8.

[0019] Compared with the prior art, the present invention has the following beneficial effects:

[0020] 1) The hybrid tulipwood LhAHL15 gene disclosed for the first time in this application has a nucleotide sequence as shown in SEQ ID NO. 3 and an amino acid sequence as shown in SEQ ID NO. 4. The present invention constructs an expression vector for the hybrid tulipwood LhAHL15 gene; transforms the constructed hybrid tulipwood LhAHL15 gene expression vector into hybrid tulipwood; and cultivates, screens, and obtains polyploid transgenic hybrid tulipwood plants.

[0021] 2) The chromosome number of the hybrid Liriodendron chinense diploid is 2n=2x=38, and the chromosome number of LhAHL15-OE is 4n=4x=76. The results showed that the chromosome number (76) of the LhAHL15-OE positive callus constructed in the present invention was twice that of the wild type and the empty vector (38), and no chimeras were found in all the division phases.

[0022] 3) The present invention uses embryonic callus of LhAHL15-OE as material to induce somatic embryogenesis. Somatic embryo seedlings of diploid LhAHL15-OE have light green, thin leaves, slender stems, and slender plants. Tetraploid somatic embryo seedlings have dark green, thick leaves, sturdy stems, and dwarf plants. The number of leaves in tetraploid plants is not significantly different from that in diploid plants, but the total leaf area of ​​tetraploid plants is greater than that of diploid plants.

[0023] 4) In the leaves of transgenic plants, the stomata and epidermal cells of LhAHL15-OE transgenic plants were larger than those of the wild-type and untransfected plants. The width of leaf epidermal cells of LhAHL15 transgenic plants was over 400 μm, twice that of the wild-type and untransfected plants.

[0024] 5) The leaf color of LhAHL15-OE transgenic plants was significantly darker than that of the control group; the statistical analysis results showed that the chlorophyll a, chlorophyll b and carotenoid contents of LhAHL15-OE transgenic plants were significantly higher than those of the control group.

[0025] 6) Overexpression of LhAHL10 / 15 significantly increased the expression of cyclin genes and cell wall expansion-related genes. Cyclin gene expression was differentially expressed in the ahl10 mutant, and the cell wall expansion-related gene Expansin-A8 showed significant differences. In the ahl15 mutant, the expression of cyclin genes and cell wall expansion-related genes was significantly reduced. BRIEF DESCRIPTION OF THE DRAWINGS

[0026] Figure 1 This is a positive identification image of LhAHL10 / 15 transgenic callus;

[0027] Figure 2 This is the qRT-PCR result of LhAHL10 / 15 transgenic callus;

[0028] Figure 3 Figure 2 shows the positive identification and first-generation sequencing results of ahl10 / 15-ko transgenic callus (a is the ahl10-ko transgenic positive callus and the second-generation sequencing results and peak diagram; b is the ahl10-ko gene editing protein sequence result, and the terminator is indicated in the red box; c is the ahl15-ko transgenic positive callus and the second-generation sequencing results and peak diagram; d is the ahl15-ko gene editing protein sequence result, and the terminator is indicated in the red box);

[0029] Figure 4 The figures are the chromosome preparation and flow cytometric analysis results of callus tissue (a is the chromosome preparation and flow cytometric analysis results of wild-type callus tissue; b is the chromosome preparation and flow cytometric analysis results of transgenic empty callus tissue; c is the chromosome preparation and flow cytometric analysis results of LhAHL10 transgenic callus tissue);

[0030] Figure 5 The figures are the chromosome preparation and flow cytometric analysis results of callus tissue (a is the chromosome preparation and flow cytometric analysis results of wild-type callus tissue; b is the chromosome preparation and flow cytometric analysis results of transgenic empty callus tissue; c is the chromosome preparation and flow cytometric analysis results of LhAHL15 transgenic callus tissue);

[0031] Figure 6 Phenotypic observation diagram of LhAHL10-OE and control group during cotyledon embryo stage (scale bar is 1 mm);

[0032] Figure 7Graphs showing the total leaf area phenotypes of LhAHL15-OE transgenic plants and the control group cultured under light for 60 days (a is a graph showing the total leaf area of ​​wild-type plants; b is a graph showing the total leaf area of ​​pBI121 empty-vector transgenic plants; c is a graph showing the total leaf area of ​​LhAHL15-OE transgenic plants; d is a statistical graph showing the difference in leaf number; e is a statistical graph showing the difference in total leaf area);

[0033] Figure 8 The following are pictures and statistical graphs of plant leaf stomata (a is a picture of the stomata of the wild type and LhAHL15-OE transgenic plants; b is a statistical graph of the length, width and area of ​​leaf epidermal cells and the length, width and area of ​​stomata);

[0034] Figure 9 Statistical graphs of chlorophyll and carotenoid contents in leaves of LhAHL15-OE transgenic plants (a is leaf phenotype observation; b is statistical graphs of chlorophyll and carotenoid contents);

[0035] Figure 10 The expression analysis diagram of LhAHL15 gene in wild type, colchicine-induced tetraploid and tetraploid embryos produced by overexpressing LhAHL15;

[0036] Figure 11 This is an analysis chart of the expression levels of cell cycle proteins and cell wall expansion-related genes affected by LhAHL10 / 15 genes. DETAILED DESCRIPTION

[0037] In order to make the purpose, technical solutions 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.

[0038] The material used in this application is embryonic callus induced from immature embryos, which was cultured in a constant temperature incubator at 22° C. in the dark.

[0039] 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 TM 5αChemically Competent Cell was purchased from Beijing Qingke Biological Company, and Agrobacterium strain EHA105 was purchased from Shanghai Weidi Biotechnology Co., Ltd.

[0040] The following instruments were used in this application: RNA extraction kit (Vazyme); RNA reverse transcription kit (Vazyme); restriction endonucleases Sac I (NEB) and Xba I (NEB); ClonExpress II One Step Cloning Kit (Vazyme); Phanta Max Super-Fidelity DNA Polymerase (Vazyme); Gel Extraction Kit (Qingke); Plasmid Miniprep Kit (TIANGEN); AceQ qPCR SYBR Green Master Mix (Vazyme). Antibiotics included Geneticin (G418); Cefpiramide (Cef); Kanamycin (Kan); and Ampicillin (Amp).

[0041] The culture medium used in this application is as follows:

[0042] 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 pH value of the culture medium is 5.72-5.74.

[0043] 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 culture medium pH is 5.72-5.74.

[0044] Z14 somatic embryo induction medium: 3 / 4MS + Vc 5mg / L + LH 0.2g / L + sucrose 40g / L + agar 2.4g / L; the medium pH is 5.72-5.74.

[0045] 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; the pH value of the culture medium is 5.72-5.74.

[0046] LB liquid medium: NaCl (10 g / L) + tryptone 10 g / L + yeast extract 5 g / L.

[0047] Example 1

[0048] 1. Total RNA extraction and cDNA acquisition

[0049] RNA was extracted from embryonic calli of hybrid Liriodendron tulipifera using an RNA extraction kit (Vazyme). Reverse transcription was performed using the extracted total RNA as a template using an RNA reverse transcription kit (Vazyme) to generate cDNA. The product was stored at -20°C to minimize freeze-thaw cycles.

[0050] 2. LhAHL10 / 15 gene cloning

[0051] Using the LhAHL10 / 15 gene CDS sequence as a reference, the LhAHL10 / 15 gene cloning primer sequences were designed using the Oligo7 website. The sequence information is shown below:

[0052] LhAHL10-clone-F: 5'-ATGTCGGGTAGAGAGTCCTTT-3',

[0053] LhAHL10-clone-R: 5'-TTATTTCCATGGCATGTTCGCC-3';

[0054] LhAHL15-clone-F: 5'-ATGGGAGGAGTCGATCTGTCGG-3',

[0055] LhAHL15-clone-R: 5'-TTAGTAAGATGGGTGGTGGCCT-3'.

[0056] Prepare a premixed reaction system using hybrid Liriodendron chinense cDNA as a template. Place the mixture in a PCR instrument for reaction. All operations must be performed on ice. All components must be thoroughly mixed and centrifuged to the bottom of the tube.

[0057] The PCR reaction system was: 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, and cDNA 2 μL.

[0058] The PCR reaction program was as follows: pre-denaturation at 95°C for 3 min; 35 cycles of denaturation at 95°C for 15 sec, annealing at 60°C for 15 sec, and extension at 72°C for 3 min; and complete extension at 72°C for 5 min.

[0059] The PCR amplification product band was clear and bright, and the target fragment was recovered using the DNA gel recovery kit (TSP602-200) from TSINGKE. The gel-extracted fragment was ligated into the pClone007 intermediate vector.

[0060] The ligation reaction system is: 3 μL of target fragment, 1 μL of pClone007 Blunt Vector, 1 μL of 10× Topo Mix, and 5 μL of ddH2O. Add all the solutions directly to the bottom of the PCR tube and mix thoroughly by gently pipetting.

[0061] The ligation reaction procedure is: room temperature reaction at 22-30℃. Do not place on ice after the reaction is completed to prevent reducing the ligation efficiency.

[0062] The intermediate vector containing the target fragment was transformed into DH5α E. coli and the bacterial solution was verified by PCR. The correct bacterial solution was sent to the company for sequencing. The plasmid was extracted from the DH5α E. coli with the sequenced and correct results using the Tiangen Plasmid Mini-Prep Kit (DP106). The prepared intermediate vector plasmid containing the target fragment was stored in a -20℃ refrigerator.

[0063] 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.

[0064] Example 2

[0065] 1. Construction of overexpression vector

[0066] The homology arms are added to the target sequence, and the homology arm primer sequences for constructing the vector are as follows:

[0067] OL-LhAHL10-F:

[0068] 5'-tggagagaacacgggggactATGTCGGGTAGAGAGTCCTTTGG-3',

[0069] OL-LhAHL10-R:

[0070] 5'-cgatcggggaaattcgagctTTATTTCCATGGCATGTTCGCCAAG-3';

[0071] OL-LhAHL15-F:

[0072] 5'-tggagagaacacgggggactATGGGAGGAGTCGATCTGTCG-3',

[0073] OL-LhAHL15-R:

[0074] 5'-cgatcggggaaattcgagctTTAGTAAGATGGTGGTGGCCTAGT-3'.

[0075] 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.

[0076] The enzyme digestion reaction system was 10x cutsmart 5 μL, Xba I 1 μL, Sac I 1 μL, plasmid 7 μL, and ddH2O 36 μL.

[0077] The enzyme digestion reaction program was 37°C for 30 min, 80°C for 20 min, and 4°C for ∞.

[0078] 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.

[0079] The homologous recombination ligation reaction system was 5x CE II Buffer 4 μL, ClonExpress II 2 μL, linearized vector 4 μL, target fragment 2 μL, and ddH2O 8 μL.

[0080] The homologous recombination ligation reaction program was 37°C for 60 min and 4°C for ∞.

[0081] The homologous recombination ligation reaction products of the two genes were transformed into DH5α E. coli, respectively, and the bacterial liquid was subjected to PCR verification. 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 a small amount of plasmid extraction kit (DP106) of Tiangen, the agrobacterium was transformed, and the specific steps were as follows.

[0082] The agrobacterium competent preserved in the ultra-low temperature refrigerator was thawed at room temperature, and after thawing, it was placed in an ice box to keep low temperature; 100 μL of competent cells were added with about 1 μg of plasmid DNA, and the plasmid and bacterial liquid were mixed thoroughly by gently stirring the bottom of the centrifuge tube. Then, it 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 in ice for 5 min; about 700 μL of LB liquid medium without antibiotics was added in the tube, and it was placed in a 28°C (150 rpm) shaking incubator for shaking culture 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 to resuspend the bacterial body by gently blowing, and the bacterial body was coated on a plate made of LB solid medium containing the corresponding antibiotic using a sterile coating rod, and then inverted and placed in a 28°C incubator for culture for 72 h. Single colonies were picked and subjected to the next experiment.

[0083] ​2. Transformation and screening of positive calli of hybrid Liriodendron chinense

[0084] 1) Pre-culture of hybrid tulipwood embryonic callus

[0085] 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.

[0086] 2) Preparation of Agrobacterium culture solution

[0087] 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 to be between 0.6-0.8. Centrifuge at room temperature for 10 minutes at 5000 rpm, remove the supernatant, and collect the bacteria. Resuspend the bacteria in a certain volume of 3 / 4 MS liquid culture medium (AS concentration is 1 mg / mL) and prepare it for use.

[0088] 3) Infection and co-culture

[0089] Place the hybrid tulipwood callus tissue that has been pre-cultured for 18 days into a 250mL Erlenmeyer flask and gently crush it with sterilized tweezers. Then, slowly flush the callus tissue to the bottom of the Erlenmeyer flask using the bacteria cultured in the previous step. Place the Erlenmeyer flask on a low-speed shaker at 90 rpm for 10 minutes to ensure full contact between the bacterial solution and the callus tissue. Subsequently, filter the callus tissue using a 400-mesh cell sieve and use sterilized dry filter paper and cotton to absorb the bacterial solution on the callus surface. The callus tissue is placed on 3 / 4 medium with an AS concentration of 1 mg / mL and co-cultured for 2 days.

[0090] 4) Degerming and screening positive callus

[0091] After co-cultivation, the hybrid tulipwood callus was washed at least three times (approximately 5 minutes each time) with MS liquid medium supplemented with 1000 mg / L of cephalosporin, then rinsed three times with sterilized ddH2O. The water was then blotted dry with sterilized cotton and filter paper, and the callus was cultured on recovery medium supplemented with Cef (400 mg / L). After 7 days, the medium was replaced with minimal medium supplemented with Cef (400 mg / L) and G418 (90 mg / L). The medium was changed every 21 days until yellow-white callus formed on the callus, at which time the callus was propagated.

[0092] 5) Identification of transgenic positive callus

[0093] Yellow, fine callus grew on the black-brown callus. Randomly selected 3 transgenic recovered calli, WT and pBI121 transgenic empty load to extract DNA. Then the different strains were respectively transgenic positive identification by PCR reaction( Figure 1 ). With pBI121 transgenic empty load and wild type as control group, the above callus strains were extracted RNA, reversed to cDNA and then qRT-PCR was used to detect the expression amount( Figure 2 ).

[0094] 3、Transgenic somatic embryo induction

[0095] 1) Somatic embryogenesis induction

[0096] Take 1g positive callus in 250mL conical flask, add 50mL M13 liquid medium, culture on 90rpm shaker for 14d, replace the medium during the period; the callus suspended for 14d was filtered with 150 mesh upper layer and 400 mesh lower layer cell screen; retain the single cells on 400 mesh cell screen, use 50mL Z36 medium to back flush into the conical flask, culture on 90rpm shaker for 2d; use 1mL pipette to take 1mL suspended culture material in centrifuge tube. Mix thoroughly by turning over, then take 10μL on 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 suspended material with Z36 medium to ensure 8000-10000 cells on each filter paper.

[0097] 2) Direct induction of somatic embryo from callus

[0098] Take 0.1g callus in 10mL centrifuge tube, add 3mL M13 liquid medium, mix thoroughly. Take 70μL mixed solution to the filter paper padded with cotton, and then transfer the filter paper to Z14 medium with tweezers for culture for 28d. Select 3 strains for each gene, and repeat 5 dishes for each strain.

[0099] Example 3

[0100] 1、Construction of sgRNA expression cassette

[0101] 1) One round of PCR

[0102] Dilute AtU3d, AtU3b, AtU6-1 plasmids to 2-5ng / μL, use 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 3d, 3b, 6-1 plasmids. The primer sequences are as follows:

[0103] U-F:

[0104] 5'-CTCCGTTTTACCTGTGGAATCG-3',

[0105] 3d-R:

[0106] 5'-ATCGCAACCATCGATGCATATgaccaatggtgctttg-3',

[0107] grT1:

[0108] 5'-TATGCATCGATGGTTGCGATgttttagagctagaaat-3';

[0109] GR-R:

[0110] 5'-CGGAGGAAAATTCCATCCAC-3',

[0111] 3b-R:

[0112] 5'-GTGGGCGGCCCCTTGCTTTTgaccaatgttgctcc-3',

[0113] grT2:

[0114] 5'-AAAGCAAGGGGCCGCCCACgttttagagctagaaat-3';

[0115] 6-1-R:

[0116] 5'-TGTTGGACGACCGGTGTGGCaatcactacttcgtct-3',

[0117] grT3:

[0118] 5'-CCACACCGGTCGTCCAACAgttttagagctagaaat-3'.

[0119] 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.

[0120] The PCR reaction program was as follows: pre-denaturation at 95°C for 3 min; 35 cycles of denaturation at 95°C for 15 sec, annealing at 56°C for 15 sec, and extension at 72°C for 30 sec; and complete extension at 72°C for 5 min.

[0121] 2) 2 rounds of PCR

[0122] The primers for the three target sites are all UF / GR-R, and the template is the PCR product from the previous step diluted 10 times. The primer sequences are as follows:

[0123] UF:

[0124] 5'-CTCCGTTTTACCTGTGGAATCG-3',

[0125] GR-R:

[0126] 5′-CGGAGGAAAATTCCATCCAC-3′.

[0127] The PCR reaction system was as follows: 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, and 1 μL of each of the previous round of PCR products.

[0128] The PCR reaction program was as follows: pre-denaturation at 95°C for 3 min; 35 cycles of denaturation at 95°C for 15 sec, annealing at 56°C for 15 sec, and extension at 72°C for 36 sec; and complete extension at 72°C for 5 min.

[0129] Electrophoresis was performed on a 2% agarose gel, and the fragments were excised and recovered to obtain AtU3d, AtU3b, and AtU6-1 containing the target gene, which were named AtU-t1-3d, AtU-t2-3b, and AtU-t3-6-1, respectively.

[0130] 3) 3 rounds of PCR

[0131] 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:

[0132] U-GAL:

[0133] 5'-ACCGGTAAGGCGCGCCGTAGTGCTCGACTAGTATGGAATCGGCAGC AAAGG-3',

[0134] pgs-GA2:

[0135] 5'-CAGGGAGCGGATAACAATTTCACACAGGCACATCCACTCCAAGCTCTTG-3',

[0136] U-GA2:

[0137] 5'-GTGCCTGTGTGAAATTGTTATCCGCTCCCTGGAATCGGCAGCAAAG G-3',

[0138] pgs-GA3:

[0139] 5'-CCACGCATACGATTTAGGTGACACTATAGCGCATCCACTCCAAGCTCTTG-3',

[0140] U-GA3:

[0141] 5'-CGCTATAGTGTCACCTAAATCGTATGCGTGGTGGAATCGGCAGCAAA GG-3'

[0142] pgs-GAR:

[0143] 5'-TAGCTCGAGAGGCGCGCCAATGATACCGACGCGTATCCATCCACTCC AAGCTCTTG-3'.

[0144] The PCR reaction system was as follows: ddH2O 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.

[0145] The PCR reaction program was as follows: pre-denaturation at 95°C for 3 min; 35 cycles of denaturation at 95°C for 15 sec, annealing at 56°C for 15 sec, and extension at 72°C for 42 sec; and complete extension at 72°C for 5 min.

[0146] Electrophoresis was performed on a 2% agarose gel, and the fragments were excised, recovered, and sent for bidirectional testing. At this time, AtU-t1-3d, AtU-t2-3b, and AtU-t3-6-1 were named oT1, oT2, and oT3, respectively.

[0147] 2. Connecting the sgRNA expression cassette with CRISPR / Cas9 to construct a gene editing vector

[0148] The plasmid was digested with enzymes (Cas9 plasmid concentration = 373.9 ng / μL).

[0149] The enzyme digestion reaction system was: 10×cutsmart 5 μL, Bsa1-HFV2 1 μL, plasmid 7 μL, and ddH2O 37 μL.

[0150] The enzyme digestion reaction program was: 37°C for 30 min, 80°C for 20 min, and 4°C for ∞.

[0151] The linearized vector and the target gene fragment recovered after gel excision were homologously recombined using the ClonExpress II One Step Cloning Kit.

[0152] The homologous recombination ligation reaction system is: oT1 1μL, oT2 1μL, oT3 1μL, linearized vector 100ng3μL, Gibson Assembly Master Mix 7μL, ddH2O 7μL.

[0153] The homologous recombination ligation reaction program was: 50°C for 30 min, 4°C for ∞.

[0154] The homologous recombination ligation reaction products (gene editing vectors) were transformed into DH5α Escherichia coli, and the bacterial cultures were verified by PCR. The positive PCR-verified cultures were sent to the company for sequencing. The plasmids from the sequenced and correct DH5α E. coli were extracted using the Tiangen Plasmid Miniprep Kit (DP106) and then transformed into Agrobacterium. The gene editing vectors were then transformed into hybrid Liriodendron chinense calli, and positive calli were identified.

[0155] 3. Identification of transgenic positive callus

[0156] Yellow, delicate callus tissue grew from the dark brown callus. Three randomly selected transgenic calli were used for DNA extraction. PCR was then used to identify the transgenic lines. Genome-wide cloning primers were designed. The primer sequences are shown below:

[0157] Target-F:

[0158] 5'-ATGTCGGGTAGAGAGTCCTTT-3',

[0159] Target-R:

[0160] 5'-CCAAAGCAACCATCTGCTTC-3',

[0161] Using DNA as a template, the sequences of the three targets were amplified. The PCR products were then ligated into the intermediate vector 007B, and five single clones from each strain were selected for first-generation sequencing.

[0162] The results are as follows Figure 3 As shown, ahl10-ko has single-base insertion and deletion sites, while ahl15-ko has small deletions and single-base insertion sites. Protein sequence analysis revealed that both ahl10-ko and ahl15-ko exhibited premature termination. In summary, the gene editing of LhAHL10 and LhAHL15 in hybrid Liriodendron chinense was successful.

[0163] Example 4

[0164] 1. Chromosome preparation of hybrid Liriodendron chinense

[0165] Place 0.1 g of callus tissue in a 1.5 mL centrifuge tube. Pretreat with saturated paradichlorobenzene for 2-3 hours, fix with Carnoy's fixative for 3-4 hours, dissociate with hydrochloric acid at 60°C for 5-7 minutes, and stain with Carbofuchsin for at least 20 minutes. Place a portion of the stained callus tissue on a slide, cover with a coverslip, and add one drop of Carbofuchsin dye. Use an unused 2B pencil to tap the coverslip evenly and rhythmically, ensuring that individual chromosomes are clearly distributed in the same plane under the microscope. Look for clear mitotic phases. Repeat three times for each sample and count the chromosomes.

[0166] 2. Identification of tetraploidy by flow cytometry

[0167] Place 0.1 g of callus tissue, approximately 18 days after subculture, into a 6 cm diameter disposable Petri dish. Add 200 μL of pre-chilled nuclear lysis buffer and quickly mix the callus using a sharp double-edged blade. Then, add 800 μL of staining buffer and incubate for 20 seconds. Add 1 mL of 40 g / L PVP solution and shake thoroughly. Filter the sample through a 48 μm filter into a sample tube and analyze it using a Cyflow Plody Analyser flow cytometer. Collect at least 3,000 nuclei per sample.

[0168] The results are as follows Figure 4 As shown, the chromosome number of the hybrid Liriodendron chinense diploid is 2n=2x=38, and the chromosome number of LhAHL10-OE is 38-154. The results show that the chromosome number of LhAHL10-OE positive callus is 2-4 times that of the wild type and empty vector (38), and there is a high proportion of chimeras and a small amount of octoploids in the mitotic phase.

[0169] The results are as follows Figure 5As shown, the chromosome number of the hybrid Liriodendron chinense diploid is 2n=2x=38, and the chromosome number of LhAHL15-OE is 4n=4x=76. The results showed that the chromosome number of LhAHL15-OE positive callus (76) was twice that of the wild type and empty vector (38), and no chimeras were found in all division phases.

[0170] 3. Comparison of morphological characteristics

[0171] Somatic embryogenesis was induced using embryonic callus of LhAHL10-OE.

[0172] The results are as follows Figure 6 As shown, LhAHL10-OE exhibits developmental disorders during somatic embryogenesis, and normal cotyledonary embryos cannot be obtained at the cotyledonary embryo stage, resulting in the inability to form normal plantlets in the later stages of embryonic development.

[0173] Somatic embryogenesis was induced using embryogenic callus of LhAHL15-OE. Morphological observation was performed on somatic embryos of wild-type, empty-loaded, and LhAHL15-OE plants that had the same culture time and good growth.

[0174] The results are as follows Figure 7 As shown, diploid seedlings have light green, thin leaves, slender stems, and slender plants. Tetraploid seedlings have dark green, thick leaves, sturdy stems, and dwarf plants. The number of leaves in tetraploids is not significantly different from that in diploids, but the total leaf area of ​​tetraploid plants is larger than that of diploids.

[0175] 4. Stomatal Analysis of Tetraploid Leaves

[0176] Select 3 transgenic lines, repeat three times for each line, apply a thin layer of transparent nail polish on the surface of the leaf, let it air dry for about 5 minutes, and then tear it off from the edge with tweezers. The film size is about 1 cm 2 Next, the torn leaf epidermis was flattened on the center of a glass slide dripping with clean water to prepare a temporary mount of plant leaf stomata. Stomata were observed under a microscope and photographed. Stomatal size, length, height, and area, as well as leaf epidermal cell length, width, and area, were measured using ImageJ software. Histograms were plotted using GraphPad, and data were analyzed using SPSS.

[0177] The results are as follows Figure 8 As shown in the figure, in the leaves of transgenic plants, the stomata and epidermal cells of LhAHL15-OE transgenic plants were larger than those of wild-type and empty-type plants. The width of leaf epidermal cells of LhAHL15 transgenic plants was over 400 μm, which was twice that of wild-type and empty-type plants.

[0178] 5. Chlorophyll content determination

[0179] Take 0.15g of leaves in a mortar, add a small amount of quartz sand, calcium carbonate and 1ml of 96% ethanol, and grind into a homogenate. Then add 5ml of ethanol and let it stand for 3 minutes. Fold the filter paper into a cone shape and place it in a funnel, and place the funnel in a 25ml volumetric flask. Transfer the tissue homogenate through the filter paper and funnel to a 25ml volumetric flask and make up the volume with alcohol. Rinse the tissue residue in the mortar three times. Use a spectrophotometer to measure the absorbance of different tissue samples. The maximum absorption peak of carotenoids is 470nm, the maximum absorption peak of chlorophyll a is 665, and the maximum absorption peak of chlorophyll b is 649. Repeat three times for different strains.

[0180] Ca=13.95A 665 -6.88A 649 ; Cb=24.96A 649 -7.32A 665 ;

[0181] Chlorophyll content (mg / g) = [chlorophyll concentration × extract volume × dilution factor] / sample fresh weight;

[0182] Carotenoid content (mg / L) = (1000D 470 -2.05Ca-114.8Cb) / 245

[0183] The results are as follows Figure 9 As shown, the leaf color of LhAHL15-OE transgenic plants was significantly darker than that of the control group; the statistical analysis results showed that the chlorophyll a, chlorophyll b and carotenoid contents of LhAHL15-OE transgenic plants were significantly higher than those of the control group.

[0184] 6. LhAHL15 gene expression in tetraploids

[0185] The gene expression differences in tetraploid somatic seedlings of LhAHL15-OE were compared with those in tetraploid seedlings induced by colchicine and wild-type seedlings of 154102.

[0186] The results are as follows Figure 10 As shown, the expression level of LhAHL15 in tetraploids induced by colchicine was not significantly different from that in the wild type, while the expression level of LhAHL15 in the LhAHL15-OE strain was significantly different from that in the wild type and tetraploids induced by chemical reagents.

[0187] 7. Gene Expression Difference Analysis

[0188] qRT-PCR was used to identify the expression levels of cyclin genes and cell wall expansion-related genes. The primer sequences are as follows:

[0189] cyclin-p3-F: 5'-ACAGATTGGAGCTGAAATTCCT-3',

[0190] cyclin-p3-R: 5'-GATGTGCTCCTCCAGTAGCTT-3';

[0191] CDKF-4-F: 5'-CCTACGAATACAGGCATGTCGTT-3',

[0192] CDKF-4-R: 5'-GATCGCATAGTGGACCCAACC-3';

[0193] ribosome-recyclingfactor-F: 5'-CAAATCCTGCCATGCTTGACC-3',

[0194] ribosome-recyclingfactor-R: 5'-ACTATGGCCTTCTCGATAGCTT-3';

[0195] Expansin-A8-F: 5'-CAGAACTGGCAGAGCAACTCC-3',

[0196] Expansin-A8-R: 5'-TGGCTCCCTTCGAACGTCT-3';

[0197] Expansin-A15-F: 5'-TTCTGCCCACCAAACAATGCC-3',

[0198] Expansin-A15-R: 5'-TACCAGCCCTGTACTGAGCAA-3'.

[0199] The results are shown in Table 1. Figure 11 As shown in Table 1, overexpression of LhAHL10 / 15 significantly increased the expression of the above genes, cyclin genes had expression differences in ahl10 mutant, and cell wall expansion-related gene Expansin-A8 had significant differences. In ahl15 mutant, the expression of the above genes was significantly reduced.

[0200] The above description is only illustrative and not restrictive, 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 hybrid Liriodendron chinense LhAHL15 gene, the nucleotide sequence of which is shown in SEQ ID NO.

3.

2. The expressed protein of the hybrid Liriodendron chinense LhAHL15 gene according to claim 1, whose amino acid sequence is shown in SEQ ID NO.

4.

3. A vector and recombinant bacteria containing the hybrid Liriodendron chinense LhAHL15 gene according to claim 1.

4. Use of the hybrid tulipwood LhAHL15 gene of claim 1 in inducing tetraploidy in hybrid tulipwood.

5. The use according to claim 4, characterized in that include: 1) Construction of an expression vector for the hybrid Liriodendron chinense LhAHL15 gene; 2) Transforming the constructed hybrid tulipwood LhAHL15 gene expression vector into hybrid tulipwood; 3) Cultivate, screen and obtain tetraploid transgenic hybrid Liriodendron chinense plants.

6. Use of the hybrid tulipwood LhAHL15 gene of claim 1 in darkening the leaf color, thickening the leaf blades, thickening the stems, dwarfing the plant, and increasing the total leaf area of ​​hybrid tulipwood tetraploid somatic seedlings by overexpression.

7. Use of the hybrid tulipwood LhAHL15 gene of claim 1 in increasing the width, length, area, stomatal density, length, and area of ​​leaf epidermal cells of hybrid tulipwood tetraploid somatic seedlings.

8. Use of the hybrid tulipwood LhAHL15 gene of claim 1 in increasing the contents of chlorophyll a, chlorophyll b, and carotenoids in hybrid tulipwood tetraploid somatic seedlings.

9. Use of the hybrid tulipwood LhAHL15 gene of claim 1 in promoting the expression of cyclin genes and cell wall expansion-related genes in hybrid tulipwood tetraploid embryos; the cyclin genes are cyclin-p3, CDKF-4, and ribosome-recycling factor; and the cell wall expansion-related gene is Expansin-A8.

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