Method for promoting plant to generate multiple buds and application thereof
By cloning and overexpressing genes that promote the growth of plant flower head count and expressing these genes on the buds of Arabidopsis, the problem of increasing plant flower head count in the prior art is solved, the effect of significantly increasing flower head count and improving plants growth and adaptability.
Patent Information
- Application Number
- CN202411761271.9
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2024-12-03
- Publication Date
- 2025-02-25
AI Technical Summary
The prior art has failed to effectively promote the increase in the number of plant flower heads through genetic modification.
By cloning and stably overexpressing genes that promote the growth of plant flower head count, Agrobacterium overexpression vector was constructed, and transferred to the buds of wild-type Arabidopsis to express specific genes to promote flower bud development.
It significantly increased the number of buds in Arabidopsis, improved the growth status and adaptability of plants, and has important agricultural and ecological significance.
Smart Images
Figure CN119506339A_ABST
Abstract
Description
Technical Field
[0001] The invention relates to the field of biotechnology, and in particular to a method for promoting plants to produce multiple flower buds and an application thereof. Background Art
[0002] In nature, Agrobacterium transfers its T-DNA into the plant genome through horizontal gene transfer, which may cause morphological changes such as tumors and hairy roots in plants. Agrobacterium is called a "natural genetic engineer" because of its ability to introduce T-DNA fragments into the plant genome. Scientists have transformed the Ti plasmid into a reliable and effective genetic engineering vector by studying the mechanism and characteristics of Agrobacterium. This modification allows Agrobacterium to be used in the laboratory to introduce foreign genes into plant cells to achieve the purpose of gene transformation and genetic improvement. This method of using Agrobacterium as a genetic engineering tool is widely used in agriculture, botany and biotechnology, and provides an important means for studying and improving plant genomes.
[0003] The number of flower buds plays an important role in the growth of plants. The following methods can be used to increase the number of flower heads:
[0004] Pruning: For succulent plants, such as Guanyin Lotus, Bear Paw, and Quiet Night, the growth of side buds can be promoted by pinching, thereby increasing the number of flower heads. Pinching usually means gently cutting off the tender buds at the top of the plant to promote the growth of new buds. Beheading method: For some succulent plants, such as Black Magic Master, the growth of new buds can be promoted by beheading. Cut off the tender buds at the heart of the flower head. After the wound heals, more new buds will grow from the original wound. Nutritional supplement: Apply 5% or 2% minoxidil to provide nutrition for the hair. Eat a balanced diet and supplement multiple vitamins and trace elements. Soil improvement: Use loose and fertile acidic soil, which is conducive to plant growth. Watering tips: Dry the water for two or three days before watering to evaporate the chlorine. Use pine leaves to pad the pot or spread on the soil surface to improve the air permeability of the soil and provide nutrients. Appropriate pruning: For plants such as roses, appropriate pruning can promote the growth of shoots, increase the number of branches, and thus promote flowering. Leaf picking propagation: By inserting the leaves of succulent plants into the soil, small seedlings can grow in groups. Use of drugs: After cutting the heart of the flower head, you can use fungicides such as carbendazim to apply to the wound to prevent infection. These methods can promote the increase of the number of flower heads of plants.
[0005] However, the method of obtaining transgenic plants with increased flower heads through genetic modification has not been studied. Therefore, technicians in this field are committed to developing a method for promoting the increase of plant flower heads by stably expressing specific genes. Summary of the invention
[0006] In view of the above-mentioned defects of the prior art, the technical problem to be solved by the present invention is how to express specific genes to promote the increase of the number of plant flower heads.
[0007] To achieve the above object, the present invention provides a method for promoting plants to produce multiple flower buds, comprising the following steps:
[0008] Step 1: Clone a gene that stably overexpresses and promotes the increase of plant flower head number;
[0009] Step 2, constructing an overexpression vector using the CDS sequence of the gene obtained in step 1 and the plant expression vector;
[0010] Step 3, transferring the overexpression vector constructed in step 2 into the Agrobacterium strain to prepare an Agrobacterium infection suspension;
[0011] Step 4, soaking the wild-type Arabidopsis thaliana in the Agrobacterium infection suspension prepared in step 3 to perform a flower soaking experiment;
[0012] Step 5: Obtain a homozygous mutant according to the flower dipping experiment results of step 4, and obtain its seeds.
[0013] Further, the genes in step 1 are: Agrobacterium tumefaciens LMG63 T-DNA genes acs, orf3n, orf8, orf10, orf11, orf12, orf13, orf14, TL-orf15-2, and / or TL-orf15-1 genes, camellia transarisanensis CaTH genes orf358, orf2, orf3n, orf8-1, rolB, rolC, orf13, orf8-2, and / or susL genes.
[0014] Furthermore, the plant expression vector in step 2 is a plasmid vector containing a CaMV 35S strong promoter and two restriction sites after restriction digestion.
[0015] Furthermore, step 2 also includes designing target gene-specific primers, obtaining the target gene by PCR amplification, selecting a plant expression vector (pCAMBIA1301 vector) containing a strong CaMV 35S promoter and having GFP and hygromycin, and using Novozymes' kits C112 and C115 to perform single-fragment ligation and multi-fragment ligation, respectively, to construct an overexpression vector.
[0016] Furthermore, step 3 further comprises: transferring the constructed overexpression vector into the Agrobacterium strain, and propagating the bacterial solution at 28°C in a shaking incubator to a concentration of OD 600 0.5-1.0; 25℃, collect the bacteria after centrifugation; add suspension MS to resuspend, 25℃, centrifuge, discard the supernatant; add MS to resuspend; measure OD 600is 0.5; add AS at a ratio of 1:10000 and expand in a shaking incubator at 28°C.
[0017] Further, step 3 also includes: transferring the constructed vector into the GV3101 Agrobacterium strain, and propagating the bacterial solution at a concentration of OD 600 0.5-1.0; 25℃, 5000rpm centrifuge for 10min to collect bacteria; add 5mL suspension MS to resuspend, 25℃, 5000rpm centrifuge for 10min, discard the supernatant; add 5mL MS to resuspend; measure OD 600 is 0.5; add AS at a ratio of 1:10000 and expand in a shaking incubator at 28°C for 2h.
[0018] Furthermore, it also includes picking a single colony of Agrobacterium on an LB plate containing antibiotics, inoculating it into LB containing antibiotics, culturing it at 28°C and 200 r / min for about 20 hours, and then inoculating it into LB containing antibiotics and As at a ratio of 1:1000, and culturing it overnight at 28°C and 200 r / min to the mid-logarithmic phase.
[0019] Furthermore, step 4 also includes the preparation of wild-type Arabidopsis: after the first stem of the wild-type Arabidopsis grows out, it is cut off from the lowest end with scissors, and its flower pods are cut off when it grows most luxuriantly, leaving only unopened flower buds, and the flower buds are immersed in the Agrobacterium infection suspension.
[0020] Furthermore, step 4 also includes adding Tween to the prepared Agrobacterium infection solution, adding 2 drops of Tween for every 200 mL; cutting off the flower pods of the wild-type Arabidopsis thaliana, leaving only the unopened flower buds, soaking the flower buds in the bacterial solution for 1 minute, and applying the bacterial solution to the flower buds that have not been soaked with a pipette (they can be soaked once after one week), and protecting from light for one day after soaking.
[0021] Furthermore, step 5 also includes: protecting the Arabidopsis thaliana that has undergone the flower immersion experiment from light, moving it into a culture room, and after growing for 1-2 months, the seeds mature and the seeds are collected. The seeds obtained at this time are T0 generation; sowing the T0 generation on a screening medium containing hygromycin, the screening medium is: 1 / 2MS, sucrose with a concentration of 20g / L, Agar with a concentration of 4g / L, plant gel with a concentration of 1.5g / L, hygromycin with a concentration of 30mg / L, and a pH of 5.8; when the T0 generation screens out positive seedlings, extracting DNA to detect whether it contains the target gene sequence; the seedlings containing the target gene sequence are continuously transplanted into the soil and cultured until the seeds mature and the seeds are collected. The seeds obtained at this time are T1 generation; then sowing the T1 generation into the screening medium, waiting for the seeds of the T1 generation to germinate for 7-10 days, counting the ratio of positive seedlings to negative seedlings, the positive: negative ratio is approximately 3:1, that is, the homozygous strain, the homozygous strain is moved into the soil again for culture until the seeds mature and the seeds are collected, and the homozygous T2 generation seeds are obtained at this time.
[0022] Further, the soil used for growing Arabidopsis thaliana includes soil: vermiculite:perlite in a volume ratio of 3:1:1.
[0023] The invention also provides an application of the method for promoting plants to produce multiple flower buds in promoting the increase of the number of flower buds in Arabidopsis thaliana.
[0024] In a preferred embodiment of the present invention, the process of stably overexpressing a gene that promotes the increase in the number of flower heads in plants is described in detail.
[0025] The beneficial technical effects of the present invention are as follows:
[0026] The present invention promotes the development of plant flower buds by stably overexpressing specific genes, thereby increasing the number of Arabidopsis flower buds. By constructing an Agrobacterium overexpression vector, one or more genes that can promote the increase of plant flower buds are expressed in the flower buds of wild-type Arabidopsis, thereby achieving the purpose of promoting the increase of plant flower heads. This helps to improve the growth condition and adaptability of plants, and is of great significance to agriculture and ecosystems. Specifically:
[0027] Enhance plant adaptability: Flower heads play an important role in the growth and adaptation of plants to the environment. By increasing the number of flower heads, plants can better adapt to various environmental conditions, such as drought, saline-alkali land, harsh climate, etc.
[0028] Increased biodiversity: A variety of flower species can attract different insects and birds, thereby increasing the diversity of the ecosystem.
[0029] Beautify the environment and purify the air: The increased number of flowers can beautify the environment and provide people with visual pleasure and relaxation; certain flowers can absorb harmful substances in the air, such as carbon dioxide, and release oxygen to help improve air quality.
[0030] Promotes plant health: An increase in the number of appropriate flower heads can be a sign of plant health, indicating that the plant is growing and developing well.
[0031] The concept, specific structure and technical effects of the present invention will be further described below in conjunction with the accompanying drawings to fully understand the purpose, characteristics and effects of the present invention. BRIEF DESCRIPTION OF THE DRAWINGS
[0032] Figure 1 It is a schematic diagram of the implementation process of a preferred embodiment of the present invention;
[0033] Figure 2 This is a result diagram of Arabidopsis thaliana multi-flowered head according to a preferred embodiment of the present invention;
[0034] Figure 3 This is a statistical diagram of the results of Arabidopsis thaliana multiple flower heads in a preferred embodiment of the present invention. DETAILED DESCRIPTION
[0035] The following describes several preferred embodiments of the present invention with reference to the drawings in the specification, so that the technical content is clearer and easier to understand. The present invention can be embodied in many different forms of embodiments, and the protection scope of the present invention is not limited to the embodiments mentioned in the text.
[0036] Example: Stable overexpression of a gene that promotes the increase in the number of flower heads in plants
[0037] The flowchart of the implementation of stable overexpression of genes that promote the increase of flower head number in plants is as follows Figure 1 As shown, the following steps are included:
[0038] 1. Gene cloning: Clone the CaTH gene orf3n gene of camellia transarisanensis, one or several CDS sequences; design gene-specific primers, and perform sequence amplification to obtain the target sequence.
[0039] Table 1 Genes that promote the increase of plant flower head number
[0040] Serial number Species Gene name 1 Agrobacterium rhizogenes LMG152 TL-orf15-2 2 Agrobacterium rhizogenes LMG152 TL-orf15-1 3 Agrobacterium rhizogenes LMG152 orf14 4 Agrobacterium rhizogenes LMG152 orf13 5 Agrobacterium rhizogenes LMG152 orf12 6 Agrobacterium rhizogenes LMG152 orf11 7 Agrobacterium rhizogenes LMG152 orf10 8 Agrobacterium rhizogenes LMG152 orf8 9 Agrobacterium rhizogenes LMG152 orf3n 10 Agrobacterium rhizogenes LMG152 acs 11 Camellia transarisanensis orf358 12 Camellia transarisanensis orf2 13 Camellia transarisanensis orf3n 14 Camellia transarisanensis orf8-1 15 Camellia transarisanensis rolB 16 Camellia transarisanensis rolC 17 Camellia transarisanensis orf13 18 Camellia transarisanensis orf8-2 19 Camellia transarisanensis susl
[0041] 2. Vector construction: Connect the gene CDS to pMD19-T using T4 ligase. Select a plant expression vector (pCAMBIA1301 vector, hereinafter referred to as 1301 vector) containing a strong CaMV35S promoter, and select two suitable restriction sites after the promoter to cut the plasmid vector. Using the pMD19-T vector containing the target gene as a template, design specific primers with homologous arms of the 1301 vector, amplify by PCR, and homologously recombine the target fragment into the linear 1301 vector. Transform the constructed 1301 gene overexpression vector into GV1301 Agrobacterium competent cells by quick freezing in liquid nitrogen.
[0042] 3. Preparation of wild-type Arabidopsis:
[0043] a) Cultivation of wild-type Arabidopsis seedlings: The wild-type Arabidopsis seedlings were sown in a culture medium (soil: vermiculite:perlite, volume ratio 3:1:1) and cultured. When the first stem of the Arabidopsis seedlings grew out, it was cut off from the root, and the floral dipping experiment was prepared when the Arabidopsis seedlings were in the most vigorous growth period.
[0044] b) Processing flower pods: Cut off the opened flowers and leave only the unopened buds.
[0045] 4. Preparation of Agrobacterium infection suspension:
[0046] Pick a single colony of Agrobacterium from an LB plate containing antibiotics, inoculate it into 1 mL of LB containing antibiotics, and culture it at 28°C and 200 r / min for about 20 hours. Then inoculate it into 50 mL of LB containing antibiotics and As at a ratio of 1:1000, and culture it at 28°C and 200 r / min overnight until the mid-logarithmic phase. The cultured Agrobacterium solution was transferred to a 50 mL centrifuge tube and propagated in a shaking incubator at 28°C to a concentration of OD 600 0.5-1.0; 25℃, 5000rpm centrifuge for 10min to collect bacteria; add 5mL suspension MS to resuspend, 25℃, 5000rpm centrifuge for 10min, discard the supernatant; add 5mL MS to resuspend; measure OD 600 is 0.5; add AS at a ratio of 1:10000 and expand in a shaking incubator at 28°C for 2h.
[0047] 5. Infection: Add Tween to the prepared Agrobacterium infection solution, add 2 drops of Tween for every 200 mL; cut off the flower pods of wild-type Arabidopsis, leaving only the unopened flower buds, and soak the flower buds in the bacterial solution for 1 minute. If the flower buds are not soaked, use a pipette to apply the bacterial solution (you can soak them once a week), and protect them from light for one day after soaking.
[0048] 6. Culture medium formula:
[0049] a) Seeding medium (antibiotic-free): 1 / 2MS + sucrose (20 g / L) + Agar (4 g / L) + plant gel (1.5 g / L); PH = 5.8.
[0050] b) Seeding medium (hygromycin resistance): 1 / 2MS + sucrose (20 g / L) + Agar (4 g / L) + plant gel (1.5 g / L) + hygromycin (30 g / L); PH = 5.8.
[0051] Tween stock solution: 50 ml infection solution + 50 drops of Tween, filter sterilize. Add 2 drops of Tween to 100 ml infection solution.
[0052] Experimental results: Flower bud pictures Figure 2 As shown, col-0 is a wild-type Arabidopsis plant, and the negative control group. Samples 1 and 2 are Arabidopsis plants treated by the method described in Example 1. The experimental statistical results are shown in Figure 3 As shown in the figure, the number of plants counted was 84 for col-0 plants, 85 for sample 1, and 85 for sample 2. The results showed that the number of flower buds of the two samples after treatment increased significantly. Sample 1 increased from an average of 4 flower buds to about 5, and sample 2 increased to about 6.
[0053] The preferred specific embodiments of the present invention are described in detail above. It should be understood that ordinary technicians in the field can make many modifications and changes based on the concept of the present invention without creative work. Therefore, all technical solutions that can be obtained by technicians in the technical field based on the concept of the present invention through logical analysis, reasoning or limited experiments on the basis of the prior art should be within the scope of protection determined by the claims.
Claims
1. A method for promoting plants to produce multiple flower buds, characterized in that: The method comprises the following steps: Step 1: Clone a gene that stably overexpresses and promotes the increase of plant flower head number; Step 2, constructing an overexpression vector using the CDS sequence of the gene obtained in step 1 and the plant expression vector; Step 3, transferring the overexpression vector constructed in step 2 into the Agrobacterium strain to prepare an Agrobacterium infection suspension; Step 4, soaking the wild-type Arabidopsis thaliana in the Agrobacterium infection suspension prepared in step 3 to perform a flower soaking experiment; Step 5: Obtain a homozygous mutant according to the flower dipping experiment results of step 4, and obtain its seeds.
2. The method according to claim 1, characterized in that The genes in step 1 are: Agrobacterium tumefaciens LMG63 T-DNA genes acs, orf3n, orf8, orf10, orf11, orf12, orf13, orf14, TL-orf15-2, and / or TL-orf15-1 genes, camellia transarisanensis CaTH genes orf358, orf2, orf3n, orf8-1, rolB, rolC, orf13, orf8-2, and / or susL genes.
3. The method according to claim 1, characterized in that The plant expression vector in step 2 is a plasmid vector containing a strong CaMV35S promoter and two restriction sites after being cut.
4. The method according to claim 1, characterized in that The step 3 further comprises: transferring the constructed overexpression vector into the Agrobacterium strain, and propagating the bacterial solution at 28°C in a shaking incubator to a concentration of OD 600 0.5-1.0; 25°C, centrifuge and collect the bacteria; add the suspension MS to resuspend, 25°C, centrifuge, discard the supernatant; add the MS to resuspend; measure OD 600 is 0.5; add AS at a ratio of 1:10000 and expand in a shaking incubator at 28°C.
5. The method according to claim 4, characterized in that The method also includes picking a single colony of Agrobacterium on an LB plate containing antibiotics, inoculating it into LB containing antibiotics, culturing it at 28°C and 200 r / min for about 20 hours, and then inoculating it into LB containing antibiotics and As at a ratio of 1:1000, and culturing it overnight at 28°C and 200 r / min to the mid-logarithmic phase.
6. The method according to claim 1, characterized in that The step 4 also includes the preparation of the wild-type Arabidopsis thaliana: after the first stem of the wild-type Arabidopsis thaliana grows out, the stem is cut off from the lowest end with scissors, and the flower pods are cut off when the stem grows most vigorously, leaving only unopened flower buds, and the flower buds are immersed in the Agrobacterium infection suspension.
7. The method according to claim 1, characterized in that The step 5 also includes: protecting the Arabidopsis thaliana that has undergone the flower dipping experiment from light, moving it into a culture room, and collecting the seeds after growing for 1-2 months until the seeds mature, and the seeds obtained at this time are T0 generation; sowing the T0 generation on a screening culture medium containing hygromycin; when positive seedlings are screened out of the T0 generation, extracting DNA to detect whether they contain the target gene sequence; continuing to transplant the seedlings containing the target gene sequence into the soil and culture them until the seeds mature and the seeds are collected, and the seeds obtained at this time are T1 generation; then sowing the T1 generation into the screening culture medium, waiting for the seeds of the T1 generation to germinate for 7-10 days, counting the ratio of positive seedlings to negative seedlings, and if the positive:negative ratio is approximately 3:1, it is a homozygous strain, and the homozygous strain is again moved into the soil for culture until the seeds mature and the seeds are collected, and the homozygous T2 generation seeds are obtained at this time.
8. The method according to claim 1, characterized in that The screening culture medium is: 1 / 2MS, sucrose with a concentration of 20g / L, Agar with a concentration of 4g / L, plant gel with a concentration of 1.5g / L, hygromycin with a concentration of 30mg / L, and a pH of 5.
8.
9. The method according to claim 7, characterized in that The soil used for growing Arabidopsis thaliana includes soil: vermiculite:perlite, with a volume ratio of 3:1:
1.
10. Use of the method according to claim 1 in promoting the increase of the number of flower buds in Arabidopsis thaliana.