Application of LasSDE4425 gene of Asian species of phloem bacillus to doubling plant chromosomes

Abstract

The invention relates to the technical field of agricultural biology, in particular to application of a phloem bacillus Asian species LasSDE4425 gene in doubling plant chromosomes. The LasSDE4425 is stably expressed in eight plants of different families such as solanaceae, leguminosae, cruciferae, gramineae, cucurbitaceae and compositae, so that chromosomes of receptor plants are doubled, and the LasSDE4425 can be widely applied to creation of plant polyploid germplasm resources.

Description

Technical Field

[0001] The present invention relates to the field of agricultural biotechnology, and in particular to application of a LasSDE4425 gene of Bacillus subtilis asiaticus in doubling plant chromosomes. Background Art

[0002] Plant polyploid breeding refers to the selection of new plant varieties or new germplasm through chromosome / genome doubling. Compared with diploid plants, polyploid plants usually show morphological characteristics such as tall plants, strong growth, lush roots and thick stems, large and thick leaves, and enlarged floral organs, fruits, and seeds. In addition, polyploid plants contain significantly more organic substances such as fats, sugars, proteins, as well as vitamins and minerals than diploid varieties, and their ability to resist diseases and adverse environments (drought, high temperature, cold) is significantly enhanced. Therefore, the cultivation of polyploid plants helps to improve the yield, stress resistance, and nutritional quality of crops and cash crops, and has attracted widespread attention from countries around the world.

[0003] Natural doubling and artificial induction are currently the commonly used methods for plant polyploid breeding, but the probability of chromosome doubling in the former is extremely low. Artificial induction is divided into physical induction methods such as high temperature treatment, low temperature treatment, and radiation mutagenesis, chemical induction methods such as colchicine, oryzalin, trifluralin, and biological methods such as sexual hybridization and cell fusion. To date, there is no method for "promoting plant chromosome doubling by expressing exogenous genes." Colchicine treatment is currently the most commonly used artificial induction method, but the mutation efficiency is still low, and there are problems such as high mosaicism rate, time-consuming and labor-intensive screening and identification processes, which limit the development and application of plant polyploid breeding. Therefore, it is urgent to develop new polyploidy induction methods or technologies to overcome the above difficulties, so as to quickly and efficiently create excellent plant polyploid varieties and germplasm resources, and promote the leapfrog development of agriculture, animal husbandry, and forestry.

[0004] The LasSDE4425 gene from Candidatus Liberibacter asiaticus is 309 base pairs long, encoding 102 amino acids and annotated as a hypothetical protein. According to existing databases, LasSDE4425 and its homologous genes have not been found in other species. Literature reports indicate that LasSDE4425 can stimulate reactive oxygen species (ROS) production and cell death in plants, interfere with the yeast cell cycle, and inhibit cell division in yeast and tobacco. To date, there have been no reports of LasSDE4425 expression leading to chromosome doubling in plants. Summary of the Invention

[0005] The present invention aims to provide an application of the LasSDE4425 gene of Bacillus subtilis asiaticus for doubling plant chromosomes.

[0006] The amino acid sequence of the Bacillus spp. asiaticus LasSDE4425 gene is shown in SEQ ID NO: 1. MRRFPTYGSEERIATCAKPGYSSRLAQLCAENEKRLKEFDKITRELNTLSENEKKA FFEHEKKVTSNLNYNARDRKHNINQFYEARGKYRYGNGYYRNYRSQ

[0007] The nucleotide sequence of the Bacillus asiaticus LasSDE4425 gene is shown in SEQ ID NO: 2. ATGAGAAGATTCCCAACCTATGGATCTGAAGAACGTATAGCAACGTGCGCAAAACCGGGCTATTCTTCACGATTAGCACAACTATGTGCAGAAAACGAAAAAAGACTTAAAGAATTCGACAAAATAACAAGAGAATTGAATACGTTATCAGAAAACGAAAAAAAAGCATTCTTTGAACACGAGAAAAAAGTAACGAGCAATCTAAACTACAACGCAAGAGACAGAAAGCATAATATAAATCAATTCTACGAAGCGAGAGGAAAGTACCGCTACGGAAATGGATATTATCGAAACTACCGATCCCAGTAA.

[0008] Given the high conservation of plant cell cycle regulation mechanisms, combined with the present invention, transgenic expression of LasSDE4425 can trigger chromosome doubling in one tetraploid plant (common tobacco) and seven diploid plants (tomato, soybean, rapeseed, corn, rice, cucumber, and rubber grass). The recipient plants involved six families and eight genera, namely Solanaceae, Leguminosae, Cruciferae, Poaceae, Cucurbitaceae, and Asteraceae. Therefore, it is inferred that LasSDE4425 can be widely used for chromosome doubling and polyploid germplasm resource creation in higher plants.

[0009] The cell cycle is a complex and delicate basic life process. Plants use a precise and conserved molecular network composed of cell cycle proteins, cyclin-dependent kinases, cyclin-dependent kinase inhibitors, cell cycle checkpoint kinases, etc. to accurately regulate the sequential transition of cells from the four phases of G1, S, G2, and M. The present invention confirms that the expression of the Asian species LasSDE4425 gene of Bacillus subtilis promotes plant chromosome doubling. According to a specific embodiment of the present invention, flow cytometric analysis showed that the chromosome ploidy of common tobacco, tomato, soybean, rapeseed, corn, rice, cucumber, and rubber grass expressing LasSDE4425 increased to 2 times that of wild-type plants. This shows that LasSDE4425 can widely promote chromosome doubling in plants of different families and genera, which is speculated to be related to its function of interfering with the cell cycle. BRIEF DESCRIPTION OF THE DRAWINGS

[0010] Figure 1 Chromosome doubling of tobacco expressing LasSDE4425 was shown;

[0011] Figure 2 Chromosome doubling in tomatoes expressing LasSDE4425 was shown;

[0012] Figure 3 Chromosome doubling of soybean expressing LasSDE4425 was shown;

[0013] Figure 4 Chromosome doubling of rapeseed expressing LasSDE4425 was shown;

[0014] Figure 5 Chromosome doubling of maize expressing LasSDE4425 was shown;

[0015] Figure 6 Chromosome doubling of rice expressing LasSDE4425 was shown;

[0016] Figure 7 Chromosome doubling of cucumber expressing LasSDE4425 was shown;

[0017] Figure 8 Chromosome doubling of rubber grass expressing LasSDE4425 was shown. DETAILED DESCRIPTION

[0018] Example 1: Preparation and ploidy analysis of common tobacco expressing LasSDE4425

[0019] Pick EHA105 Agrobacterium containing SDE4425 gene, inoculate into 50mL YEB liquid medium (50mg / L Kan, 50mg / L Rif), culture overnight at 28℃, 220rpm; collect bacteria, resuspend bacteria in 1 / 2MS liquid medium, adjust OD600 To around 0.6.

[0020] Cut the young leaves of sterile common tobacco K326 into 1-1.5cm 2 Immerse the leaf discs in the prepared Agrobacterium solution for 5 minutes. Remove the leaf discs, drain off excess bacterial solution, and place them on co-cultivation medium plates (4.43 g / L MS, 30 g / L sucrose, 1 mg / L 6-BA, 4 g / L Agar, pH 5.8) for 2 days in the dark at 25°C. Transfer the leaf discs to differentiation medium (4.43 g / L MS, 30 g / L sucrose, 1 mg / L 6-BA, 0.1 mg / L α-NAA, 50 mg / L Kan, pH 5.8) and incubate at 25°C in the light for 3-4 weeks. Once differentiated shoots reach ~3-5 cm in length, transfer them to rooting medium (4.43 g / L MS, 30 g / L sucrose, 50 mg / L Kan, pH 5.8). After 3-4 weeks, regenerated plants will fully root. Harden them for 2-3 days, transplant them to nutrient soil, and continue incubation in a greenhouse (25°C, 16 h light / 8 h dark). RNA was extracted from leaves and RT-PCR was performed to confirm the transcriptional expression of LasSDE4425 in transgenic plants.

[0021] 0.1 g of mature leaves were collected from wild-type (wt) tobacco and transgenic plants expressing LasSDE4425 (4425-Nt). 2 mL of cell lysis buffer (0.1764 g sodium citrate, 0.3506 g sodium chloride, 100 μL Tween-20, pH 7.0, and water was added to 40 mL) and finely minced with a sharp blade. The filtrate was filtered through an 80 μm pore size filter, collected, and centrifuged at 1000 rpm for 5 minutes at 4°C. The supernatant was discarded, and the pelleted nuclei were collected. Propidium iodide (PI) staining solution (400 μg PI, 0.1764 g sodium citrate, 0.3506 g sodium chloride, pH 7.0, and water was added to 40 mL) was added and stained in the dark for 20 minutes to fluorescently label nuclear DNA. 4425-Nt nuclei were analyzed by flow cytometry using wt nuclei as a control. Ploidy was determined based on the fluorescence intensity of the control sample. The results showed that the chromosome ploidy of 4425-Nt was twice that of wt, achieving chromosome doubling ( Figure 1 ).

[0022] Example 2: Preparation and ploidy analysis of tomatoes expressing LasSDE4425

[0023] Select plump Micro-Tom tomato seeds, disinfect them with 75% alcohol and 8% sodium hypochlorite for 5 minutes and 8 minutes respectively, wash them with sterile H2O, dry them, transfer them to T0 medium (2.25g / L MS, 15g / L sucrose, 6.675g / L Agar, pH 5.8), culture them in the dark until they turn white, and continue to culture them in the light for 2-3 days; take the cotyledons, cut them into 5×5mm squares, transfer them to pre-culture medium (4.44g / LMS, 30g / L sucrose, 1mg / L 6-BA, 0.1mg / L IAA, pH 5.8), and treat them in the dark for 2 days.

[0024] Pick up EHA105 Agrobacterium containing SDE4425 gene, inoculate into YEB liquid medium (50mg / L Kan, 50mg / LRif), culture overnight at 28℃ and 220rpm; collect the bacteria, resuspend in ddH2O (OD 600 =0.1-0.15). The explants pre-cultured for 2 days were immersed in Agrobacterium solution, taken out after 5 minutes, the bacterial solution was aspirated, and placed on T1 medium for dark culture; after 2 days, the explants were transferred to bud induction medium (4.44g / LMS, 3g / L sucrose, 0.1mg / L IAAA, 1mg / L ZT, 200mg / L Temeiting, 50mg / L Hyg, pH 5.8), cultured at 25°C with 16h light, and T1 was replaced every 2 weeks. 21 Culture medium was added until the explants fully germinated. When the buds reached approximately 2-3 cm in length, they were transferred to bud elongation medium (4.44 g / L MS, 3 g / L sucrose, 1 mg / L GA3, 0.5 mg / L ZT, 200 mg / L Temei Ting, 50 mg / L Hyg, pH 5.8), with the medium changed every two weeks. When the buds reached 4-5 cm in length, they were transferred to rooting medium (4.44 g / L MS, 3 g / L sucrose, 1.5 mg / L TMT, 2 mg / L IBA, pH 5.8) and cultured until the root system was fully developed. After hardening the seedlings for 5-7 days, the seedlings were transferred to sterile soil and cultured at 25°C under a 16 h light / 8 h dark cycle. Leaf RNA was extracted and RT-PCR was performed to confirm the transcriptional expression of LasSDE4425 in the transgenic plants.

[0025] 0.1 g of mature leaves were collected from wild-type (wt) tomatoes and transgenic tomatoes expressing LasSDE4425 (4425-Sl), 2 mL of cell lysis buffer (0.1764 g of sodium citrate, 0.3506 g of sodium chloride, 100 μL of Tween-20, pH 7.0, and water was added to 40 mL), and chopped with a sharp blade. Filter with an 80 μm pore size filter membrane, collect the filtrate, centrifuge at 4°C, 1000 rpm for 5 minutes, discard the supernatant, and collect the precipitated cell nuclei. Add propidium iodide staining solution, place in the dark for staining for 20 minutes, and fluorescently label the nuclear DNA. Using the wt cell nucleus as a control, the cell nucleus of 4425-Sl was analyzed by flow cytometry, and its ploidy was identified based on the fluorescence intensity of the control sample. The results showed that the chromosome ploidy of 4425-Sl was twice that of wt, and the chromosomes were doubled ( Figure 2 ).

[0026] Example 3: Preparation and ploidy analysis of soybeans expressing LasSDE4425

[0027] Pick up LBA4404 Agrobacterium containing SDE4425 gene, inoculate into 50mL YEB liquid medium (50mg / L Kan, 50mg / L R Fif), and culture at 28℃ and 220rpm until OD 600 =0.6-0.8; collect the bacteria and resuspend them in 50 mL of MS liquid culture medium (100 μM AS, pH 5.4) to prepare a bacterial suspension.

[0028] Soybean pods of the Zhonghuang 35 soybean variety, 10-15 days after flowering, were removed. Immature cotyledons and hypocotyls were peeled and soaked in 70% ethanol and 2% sodium hypochlorite for 1 minute and 15 minutes, respectively, followed by rinsing with sterile water 3-5 times. The cotyledons and hypocotyls were cut into 5-8 mm segments, immersed in the bacterial suspension, and gently shaken for 10-15 minutes. The bacterial suspension was then removed, blotted dry, and placed on a co-culture medium (4.44 g / L MS, 1.0 mg / L 6-BA, 100 μM AS, 30 g / L sucrose, pH 5.8) for incubation in the dark at 22°C for 2-3 days. The explants were rinsed three times with sterile water, the surface moisture was blotted, and the cells were placed on screening medium (4.44 g / L MS, 1.0 mg / L 6-BA, 0.5 mg / L IBA, 30 g / L sucrose, 500 mg / L CTX, 50 mg / L Kan, pH 5.8) and cultured at 25°C with 16 h light / 8 h dark alternating cycles, subcultured every 2 weeks for 4-6 weeks. Actively growing calli were selected and transferred to regeneration medium (4.44 g / L MS, 0.5 mg / L 6-BA, 0.1 mg / L GA3, 50 mg / L Kan, pH 5.8) and cultured at 25°C with 16 h light / 8 h dark alternating cycles, subcultured every 2 weeks until the shoots were 2-3 cm long. Buds were transferred to rooting medium (2.22 g / L MS, 0.5 mg / L IBA, 15 g / L sucrose, pH 5.8) and cultured until root development was complete. After hardening for 5-7 days, the seedlings were transplanted to sterilized soil and incubated for one week in a moisturizing environment before normal management. Leaf RNA was extracted and RT-PCR was performed to confirm the transcriptional expression of LasSDE4425 in the transgenic plants.

[0029] 0.1 g of mature leaves were collected from wild-type (wt) soybeans and transgenic soybeans expressing LasSDE4425 (4425-Gm), 2 mL of cell lysis buffer (0.1764 g of sodium citrate, 0.3506 g of sodium chloride, 100 μL of Tween-20, pH 7.0, and water was added to 40 mL), and chopped with a sharp blade. Filter with an 80 μm pore size filter membrane, collect the filtrate, centrifuge at 4°C, 1000 rpm for 5 minutes, discard the supernatant, and collect the precipitated cell nuclei. Add propidium iodide staining solution, place in the dark for staining for 20 minutes, and fluorescently label the nuclear DNA. Using the wt cell nucleus as a control, the cell nucleus of 4425-Gm was analyzed by flow cytometry, and its ploidy was identified based on the fluorescence intensity of the control sample. The results showed that the chromosome ploidy of 4425-Gm was twice that of wt, and the chromosomes were doubled ( Figure 3 ).

[0030] Example 4: Preparation and ploidy analysis of rapeseed expressing LasSDE4425

[0031] Select plump rapeseed varieties Zhongshuang 11 seeds, disinfect them with 70% ethanol for 1 minute and 2% sodium hypochlorite for 15 minutes, and finally wash them with sterile water 5-7 times. Use sterile filter paper to absorb the moisture, sow them on MS solid culture medium, and culture them at 25℃ for 5-7 days.

[0032] Pick up EHA105 Agrobacterium containing SDE4425 gene, inoculate into 50mL YEB liquid medium (50mg / L Kan, 50mg / L R Fif), and culture at 28℃ and 220rpm until OD 600 =0.6-0.8; collect the bacteria, resuspend them in 50 mL MS liquid medium (100 μM AS), and let them stand at room temperature for 1 hour.

[0033] Hypocotyls (cut into 5-8 mm stem segments) and cotyledons (removing leaf tips and bases) from sterile seedlings were immersed in Agrobacterium culture medium and gently shaken for 10-15 minutes. The culture medium was removed, blotted dry, and transferred to co-cultivation medium (4.4 g / L MS, 1 mg / L 6-BA, 0.1 mg / L NAA, 100 μM AS) and incubated in the dark at 25°C. After 2-3 days, the explants were removed, rinsed three times with sterile water, and then soaked in MS liquid medium (300 mg / L Temetin) for 10 minutes. The explants were then transferred to screening medium (4.4 g / L MS, 1 mg / L 6-BA, 0.1 mg / L NAA, 50 mg / L Kan, 300 mg / L Temetin) and incubated at 25°C under alternating light and dark conditions (16 h light / 8 h dark). The medium was changed every 10 days until resistant shoots appeared and grew to 1-2 cm. Resistant buds were cut and transferred to rooting medium (1 / 2 MS, 0.1 mg / L NAA, 50 mg / L Kan) and cultured until the root system was fully developed. After hardening the seedlings for 3 days, they were transferred to sterilized soil, kept moist, and maintained as normal after 1 week.

[0034] 0.1 g of mature leaves were collected from wild-type (wt) rapeseed and transgenic rapeseed expressing LasSDE4425 (4425-Bc), 2 mL of cell lysis solution (0.1764 g of sodium citrate, 0.3506 g of sodium chloride, 100 μL of Tween-20, pH 7.0, and water was added to 40 mL), and chopped with a sharp blade. Filter with an 80 μm pore size filter membrane, collect the filtrate, centrifuge at 4°C, 1000 rpm for 5 minutes, discard the supernatant, and collect the precipitated cell nuclei. Add propidium iodide staining solution, place in the dark for staining for 20 minutes, and fluorescently label the nuclear DNA. Using the wt cell nucleus as a control, the cell nucleus of 4425-Bc was analyzed by flow cytometry, and its ploidy was identified based on the fluorescence intensity of the control sample. The results showed that the chromosome ploidy of 4425-Bc was twice that of wt, and the chromosomes were doubled ( Figure 4 ).

[0035] Example 5: Preparation and ploidy analysis of maize expressing LasSDE4425

[0036] Pick up LBA4404 Agrobacterium containing SDE4425 gene, inoculate into 50mL YEB liquid medium (50mg / L Kan, 50mg / L Rif), and culture at 28℃ and 220rpm until OD 600 =0.6-0.8; collect the bacteria and resuspend them in 50 mL N6 liquid culture medium (200 μM AS, pH 5.4) to prepare a bacterial suspension.

[0037] Maize ears from B104, 10-14 days after pollination, were selected. 1.0-1.5 mm immature embryos were removed and sterilized in 70% ethanol and 2% sodium hypochlorite for 1 minute and 15 minutes, respectively. The embryos were rinsed 3-5 times with sterile water, blotted dry, and immersed in Agrobacterium culture medium with gentle shaking for 30 minutes. The embryos were removed, the excess culture medium was blotted with sterile filter paper, and the embryos were transferred to co-cultivation medium (N6, 30 g / L sucrose, 10 g / L glucose, 200 μM AS, pH 5.8) and incubated at 23°C in the dark for 2-3 days. The embryos were then transferred to recovery medium (N6, 2 mg / L 2,4-D, 30 g / L sucrose, 200 μM AS, 500 mg / L CTX, pH 5.8) and incubated at 25°C in the dark. After 5-7 days, transfer to screening medium (N6, 2 mg / L 2,4-D, 30 g / L sucrose, 5 mg / L glufosinate), subculture once every 2 weeks for 6-8 weeks; select vigorously growing callus tissue and transfer to differentiation medium (MS, 0.5 mg / L 2,4-D, 1.0 mg / L 6-BA, 0.5 mg / L NAA, 30 g / L sucrose, 5 mg / L glufosinate, pH 5.8), 25 ° C photoperiod (16 h light / 8 h dark), and culture until green shoots appear; when the shoots are 3-5 cm long, transfer to rooting medium (1 / 2 MS, 0.1 mg / L NAA, 1.0 g / L activated carbon, 20 g / L sucrose, 5 mg / L glufosinate, pH 5.8) and culture until the root system is fully developed; harden the seedlings for 3-5 days, transplant to the greenhouse, moisturize and culture for 1 week, and then manage normally. RNA was extracted from leaves and RT-PCR was performed to confirm the transcriptional expression of LasSDE4425 in transgenic plants.

[0038] 0.1 g of mature leaves were collected from wild-type (wt) corn and transgenic corn (4425-Zm) expressing LasSDE4425, 2 mL of cell lysis solution (0.1764 g of sodium citrate, 0.3506 g of sodium chloride, 100 μL of Tween-20, pH 7.0, and water was added to 40 mL), and chopped with a sharp blade. Filter with an 80 μm pore size filter membrane, collect the filtrate, centrifuge at 4°C, 1000 rpm for 5 minutes, discard the supernatant, and collect the precipitated cell nuclei. Add propidium iodide staining solution, place in the dark for staining for 20 minutes, and fluorescently label the nuclear DNA. Using the wt cell nucleus as a control, the cell nucleus of 4425-Zm was analyzed by flow cytometry, and its ploidy was identified based on the fluorescence intensity of the control sample. The results showed that the chromosome ploidy of 4425-Zm was twice that of wt, and the chromosomes were doubled ( Figure 5 ).

[0039] Example 6: Preparation and ploidy analysis of rice expressing LasSDE4425

[0040] Mature and plump Zhonghua 11 rice seeds (about 150 seeds) were selected for shelling, rinsed with sterile water 1-2 times, disinfected with 70% ethanol for 1 min and 2% sodium hypochlorite for 9 min, and finally washed with sterile water 5-7 times. The water was absorbed with sterile filter paper and placed on callus induction medium (4.44 g / L MS, 4 mg / L 2,4-D, 100 mg / L inositol, 30 g / L sucrose, 0.3 g / L acid hydrolyzed casein, 2.8 g / L proline, pH 5.8), and cultured in the dark at 28°C until round, dense, milky yellow embryonic calli grew. The calli were then transferred to MS medium and cultured in the dark at 28°C for 7 days.

[0041] LBA4404 Agrobacterium containing the SDE4425 gene was picked, spread on an LB plate (50 mg / L Kan, 50 mg / L R-Rif), and inverted at 28° for 1 day. The bacterial moss was scraped and placed in AAM liquid medium containing 40 mg / L LAS, and cultured at 28°C and 200 rpm for 1 hour. The bacteria were collected and resuspended in the same medium, and the OD was adjusted to 0.12-0.15.

[0042] Embryogenic calli that had been pre-cultured for 7 days were taken, immersed in Agrobacterium solution, and gently shaken for 30 minutes; the calli were removed, dried with filter paper, and transferred to a co-culture medium containing 20 mg / LAS (N6, 2 mg / L 2,4-D, 100 mg / L inositol, 30 g / L sucrose, 10 g / L glucose, pH 5.2), and cultured in the dark at 22°C for 3-4 days; the callus tissue was rinsed with sterile water 5-7 times, placed in a washing solution containing 200 mg / L Temei Ting (N6, 2 mg / L 2,4-D, 100 mg / L inositol, 30 g / L sucrose, 0.3 g / L acid hydrolyzed casein, 0.5 g / L proline, pH 5.72), and gently shaken for 1 hour; the calli were removed, dried with filter paper, and transferred to a selection medium (N6, 2 mg / L The culture medium was cultured in the dark at 28°C with subculture every 2 weeks until yellow resistant calli the size of millet grains appeared. Resistant calli were transferred to differentiation medium (4.44 g / L MS, 0.02 mg / L NAA, 2 mg / L KT, 100 mg / L myo-inositol, 30 g / L sorbitol, 30 g / L sucrose, 0.3 g / L acid-hydrolyzed lobrafen, 40 mg / L Basta, 4.6 g / L Hytagel, pH 5.8) at 28°C with alternating light and dark cycles (16 h light / 8 h dark). The medium was changed every two weeks until shoots appeared. Strong shoots were excised and transferred to seedling medium (2.22 g / L MS, 100 mg / L myo-inositol, 30 g / L sucrose, 6.0 g / L Agar, pH 5.8) at 28°C with alternating light and dark cycles until root development was complete. Seedlings were hardened for 3-5 days before being transplanted to a greenhouse, incubated in a moisturizing chamber for 1 week, and then maintained normally. Leaf RNA was extracted and RT-PCR performed to confirm the transcriptional expression of LasSDE4425 in the transgenic plants.

[0043] 0.1 g of mature leaves were collected from wild-type (wt) rice and transgenic rice expressing LasSDE4425 (4425-Os), 2 mL of cell lysis buffer (0.1764 g of sodium citrate, 0.3506 g of sodium chloride, 100 μL of Tween-20, pH 7.0, and water was added to 40 mL), and chopped with a sharp blade. Filter with an 80 μm pore size filter membrane, collect the filtrate, centrifuge at 4°C, 1000 rpm for 5 minutes, discard the supernatant, and collect the precipitated cell nuclei. Add propidium iodide staining solution, place in the dark for staining for 20 minutes, and fluorescently label the nuclear DNA. Using the wt cell nucleus as a control, the cell nucleus of 4425-Os was analyzed by flow cytometry, and its ploidy was identified based on the fluorescence intensity of the control sample. The results showed that the chromosome ploidy of 4425-Os was twice that of wt, and the chromosomes were doubled ( Figure 6).

[0044] Example 7: Preparation and ploidy analysis of cucumbers expressing LasSDE4425

[0045] Select plump Jinyan No. 4 cucumber seeds, soak them in 70% ethanol for 1 minute, rinse them once with sterile water, soak them in 20% sodium hypochlorite (containing 0.1% Tween-20) for 15-20 minutes, and rinse them five times with sterile water. Inoculate them onto MS medium and culture them at 25°C under a 16-hour light / 8-hour dark regime for 5-7 days. Cut the cotyledonary nodes (5×5 mm pieces) into pre-culture medium (4.44 g / L MS, 1 mg / L 6-BA, 0.1 mg / L NAA) and culture them in the dark at 25°C for 1-2 days.

[0046] Pick Agrobacterium and inoculate it into 5 mL of YEP liquid medium (containing the same antibiotics) and culture at 28°C with shaking at 200 rpm until the OD600 is 0.6-0.8. Collect the cells by centrifugation and resuspend them in 1 / 2 MS liquid medium containing 200 μM AS. Adjust the OD600 to 0.3-0.5 and set aside.

[0047] The pre-cultured explants were immersed in Agrobacterium culture medium and shaken gently. After 10-15 minutes, the explants were taken out and the excess culture medium was aspirated. The explants were transferred to a co-culture medium (4.44 g / L MS, 1 mg / L 6-BA, 200 μM AS) and cultured in the dark at 25°C for 2-3 days. The explants were rinsed 3-5 times with sterile water and transferred to a screening medium (4.44 g / L MS, 1 mg / L 6-BA, 0.1 mg / L NAA, 50 mg / L Kan, 300 mg / LCTX) and cultured at 25°C with alternating light and dark (16 h light / 8 h dark). The culture medium was changed every 10 days. The screening was continued for 4-6 weeks, and the surviving calli were transferred to a regeneration medium (4.44 g / L MS, 2 mg / L TDZ, 0.05mg / L NAA, 50mg / L Kan, 300mg / L CTX) were used to induce bud differentiation; 2-3cm regenerated buds were cut and inserted into rooting medium (2.22g / L MS + 0.2mg / L IBA) and cultured until the root system was fully developed; seedlings were hardened for 3-5 days, transplanted to sterilized nutrient soil, moisturized for 1 week, and then transferred to normal greenhouse conditions.

[0048] 0.1 g of mature leaves were collected from wild-type (wt) cucumbers and transgenic cucumbers expressing LasSDE4425 (4425-Cs), 2 mL of cell lysis solution (0.1764 g of sodium citrate, 0.3506 g of sodium chloride, 100 μL of Tween-20, pH 7.0, and water was added to 40 mL), and chopped with a sharp blade. Filter with an 80 μm pore size filter membrane, collect the filtrate, centrifuge at 4°C, 1000 rpm for 5 minutes, discard the supernatant, and collect the precipitated cell nuclei. Add propidium iodide staining solution, place in the dark for staining for 20 minutes, and fluorescently label the nuclear DNA. Using the wt cell nucleus as a control, the cell nucleus of 4425-Cs was analyzed by flow cytometry, and its ploidy was identified based on the fluorescence intensity of the control sample. The results showed that the chromosome ploidy of 4425-Cs was twice that of wt, and the chromosomes were doubled ( Figure 7 ).

[0049] Example 8: Preparation and ploidy analysis of rubber grass expressing LasSDE4425

[0050] Cut the leaves of the sterile rubber grass seedlings into 0.5-1cm pieces 2 The small pieces were placed in pre-culture medium (4.4 g / L MS, 30 g / L sucrose, 2.5 g / L phytagel, 0.4 mg / L IAA, 1 mg / L KT) and cultured in the dark at 26°C for 48 h.

[0051] Agrobacterium LBA4404 containing the SDE4425 gene was picked and inoculated into 50 mL YEB liquid medium (50 mg / L Kan, 50 mg / L Rif), and cultured at 28°C and 220 rpm to an OD600 of 0.6-0.8. The bacteria were collected and resuspended in infection liquid medium (4.4 g / L MS, 30 g / L sucrose, 100 μM AS) to prepare a bacterial suspension with an OD600 of 0.3-0.5.

[0052] The pre-cultured leaves were immersed in Agrobacterium liquid and cultured at 28°C and 100 rpm for 30 min. The leaves were removed, the liquid was dried with filter paper, and placed in co-culture medium (4.4 g / L MS, 30 g / L sucrose, 2.5 g / L phytagel, 0.4 mg / L IAA, 1 mg / L KT, 100 μM AS) and cultured in the dark at 28°C for 2 d. The leaves were transferred to recovery medium (4.4 g / L MS, 30 g / L sucrose, 2.5 g / L phytagel, 0.4 mg / L IAA, 1 mg / L KT, 300 mg / L Temeiting) and cultured at 28°C with alternating light and dark (16 h light / 8 h dark). After one week, the leaves were transferred to screening medium (4.4 g / L MS, 30 g / L sucrose, 2.5 g / L phytagel, 0.4 mg / L IAA, 1 mg / L KT, 300 mg / L Temeiting). KT, 300 mg / L Temetin, 10 mg / L Hyg); replace the culture medium every 10-14 days until buds differentiate; transfer the buds to rooting medium (4.4 g / L MS, 2.5 g / L Phytogel, 300 mg / L Temetin, 10 mg / L Hyg) until rooting; when the root system is fully developed, harden the seedlings for 3-5 days, move them to sterilized nutrient soil, keep them moist for 1 week, and then transfer them to normal greenhouse conditions.

[0053] 0.1 g of mature leaves were collected from wild-type (wt) rubber grass and transgenic rubber grass expressing LasSDE4425 (4425-Tk), 2 mL of cell lysis solution (0.1764 g of sodium citrate, 0.3506 g of sodium chloride, 100 μL of Tween-20, pH 7.0, and water was added to 40 mL), and chopped with a sharp blade. Filter with an 80 μm pore size filter membrane, collect the filtrate, centrifuge at 4°C, 1000 rpm for 5 minutes, discard the supernatant, and collect the precipitated cell nuclei. Add propidium iodide staining solution, place in the dark for staining for 20 minutes, and fluorescently label the nuclear DNA. Using the wt cell nucleus as a control, the cell nucleus of 4425-Tk was analyzed by flow cytometry, and its ploidy was identified based on the fluorescence intensity of the control sample. The results showed that the chromosome ploidy of 4425-Tk was twice that of wt, and the chromosomes were doubled ( Figure 8 ).

[0054] The above embodiments are only used to explain the technical solutions of the present application and do not limit the protection scope of the present application.

Claims

1. A method for doubling plant chromosomes, characterized in that: The method comprises the step of expressing the Bacillus asiaticus LasSDE4425 gene in a plant, wherein the Bacillus asiaticus LasSDE4425 gene encodes a protein with an amino acid sequence as shown in SEQ ID NO:

1.

2. The method for doubling plant chromosomes according to claim 1, wherein The nucleotide sequence of the Bacillus subtilis asiaticus LasSDE4425 gene is shown in SEQ ID NO:

2.

3. The method for doubling plant chromosomes according to claim 1, wherein The plant is a plant selected from the family Solanaceae, Leguminosae, Cruciferae, Poaceae, Cucurbitaceae or Asteraceae.

4. The method for doubling plant chromosomes according to claim 1, wherein The plant is common tobacco, tomato, soybean, rape, corn, rice, cucumber or rubber grass.

Images