Application of slhb40 gene in creating tomato dwarf material

Abstract

The application discloses application of a SlHB40 gene in creating a tomato dwarf material and relates to the fields of plant genetic engineering and molecular breeding. The CDS sequence of the SlHB40 gene is shown as SEQ ID NO. 1, and the tomato dwarf material is created by overexpressing the SlHB40 gene. The application research finds that the SlHB40 gene can affect the plant height of the tomato, a recombinant microbial strain carrying the SlHB40 CDS sequence is constructed, the recombinant microbial strain is used to mediate tomato transformation, and a tomato transgenic plant overexpressing the SlHB40 is constructed. It is proved by phenotype detection that the tomato transgenic plant overexpressing the SlHB40 is a tomato dwarf material. The tomato dwarf material obtained by the application has the advantages of high-density planting, full use of space and land, enhanced photosynthetic efficiency, low water consumption and lodging resistance.

Description

Technical Field

[0001] This invention relates to the fields of plant genetic engineering and molecular breeding, and in particular to the application of the SlHB40 gene in the creation of dwarfing materials for tomatoes. Background Technology

[0002] Tomatoes are one of the most widely cultivated vegetable crops in the world, with high commercial value and an important position in global agricultural production. The plant type of the tomato plant plays a crucial role in dense planting, space and light utilization, irrigation, lodging resistance, and yield. Summary of the Invention

[0003] The purpose of this invention is to provide the application of the SlHB40 gene in the creation of tomato dwarfing materials, so as to solve the problems existing in the prior art. This invention has found that the SlHB40 gene can affect the plant height of tomatoes. By constructing transgenic tomato lines overexpressing SlHB40, tomato dwarfing materials can be obtained, which has important application value.

[0004] To achieve the above objectives, the present invention provides the following solution:

[0005] This invention provides the application of the SlHB40 gene in the creation of tomato dwarfing materials. The tomato dwarfing materials are created by overexpressing the SlHB40 gene.

[0006] The CDS sequence of the SlHB40 gene is shown in SEQ ID NO.1.

[0007] The present invention also provides the application of a recombinant expression vector carrying the SlHB40 gene in the creation of tomato dwarfing materials, wherein the CDS sequence of the SlHB40 gene is shown in SEQ ID NO.1.

[0008] The present invention also provides the application of a recombinant microbial strain carrying the SlHB40 gene in the creation of tomato dwarfing materials, wherein the CDS sequence of the SlHB40 gene is shown in SEQ ID NO.1.

[0009] This invention also provides a method for creating tomato dwarfing materials through gene editing, comprising the steps of introducing the SlHB40 gene into tomato explants and creating SlHB40 gene overexpressing plants through tissue culture; the SlHB40 gene overexpressing plants are the tomato dwarfing materials.

[0010] The CDS sequence of the SlHB40 gene is shown in SEQ ID NO.1.

[0011] Furthermore, the tomato explant is the cotyledon of a tomato seedling.

[0012] Furthermore, the method for introducing the SlHB40 gene into the tomato explant includes:

[0013] The SlHB40 gene was ligated into an expression vector to construct a recombinant expression vector.

[0014] The recombinant expression vector was transformed into a microbial strain to obtain a recombinant microbial strain;

[0015] The recombinant microbial strain was transformed into the tomato explant.

[0016] Furthermore, the SlHB40 gene is first ligated into the entry vector pENTRTDirectional TOPO, and then subjected to an LR substitution reaction with the overexpression vector pK7FWG2.0 to obtain the recombinant expression vector.

[0017] Furthermore, the microbial strain is Agrobacterium GV1301.

[0018] Furthermore, the recombinant microbial strain was transformed into the tomato explants using an infection method.

[0019] Furthermore, the infection time for the aforementioned infection method is 8-10 minutes.

[0020] The present invention discloses the following technical effects:

[0021] This invention discovered that the SlHB40 gene can affect tomato plant height. A recombinant microbial strain carrying the SlHB40 CDS sequence was constructed. Then, using tomato cotyledons as explants, the recombinant microbial strain mediated tomato transformation, resulting in SlHB40-overexpressing transgenic tomato plants. Phenotypic analysis confirmed that these SlHB40-overexpressing transgenic tomato plants are dwarfing materials for tomatoes. The tomato dwarfing material obtained by this invention has advantages such as high-density planting, efficient use of space and land, enhanced photosynthetic efficiency, low water consumption, and lodging resistance, making it of significant application value for different tomato cultivation methods. Attached Figure Description

[0022] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the drawings used in the embodiments will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0023] Figure 1 The results of agarose gel electrophoresis of SlHB40 CDS PCR products;

[0024] Figure 2The results of qRT-PCR detection of SlHB40 expression levels in wild-type and SlHB40 overexpression lines; WT-1 and WT-2 are wild-type plants, and OX-1, OX-2 and OX-3 are SlHB40 overexpression lines;

[0025] Figure 3 The results show the phenotypic identification of SlHB40 overexpressing lines; where A is a height comparison diagram between wild-type and SlHB40 overexpressing lines; B is the average plant height of wild-type and SlHB40 overexpressing lines; in A, WT-1 and WT-2 represent wild-type plants, and OX-1, OX-2 and OX-3 represent SlHB40 overexpressing lines; in B, WT represents wild-type plants, and OX represents SlHB40 overexpressing lines. Detailed Implementation

[0026] Various exemplary embodiments of the present invention will now be described in detail. This detailed description should not be considered as a limitation of the present invention, but rather as a more detailed description of certain aspects, features, and embodiments of the present invention.

[0027] It should be understood that the terminology used in this invention is merely for describing particular embodiments and is not intended to limit the invention. Furthermore, with respect to numerical ranges in this invention, it should be understood that each intermediate value between the upper and lower limits of the range is also specifically disclosed. Any stated value or intermediate value within a stated range, as well as each smaller range between any other stated value or intermediate value within said range, is also included in this invention. The upper and lower limits of these smaller ranges may be independently included or excluded from the range.

[0028] Unless otherwise stated, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art. While only preferred methods and materials have been described herein, any methods and materials similar or equivalent to those described herein may be used in the implementation or testing of this invention. All references to this specification are incorporated by way of citation to disclose and describe methods and / or materials associated with those references. In the event of any conflict with any incorporated reference, the content of this specification shall prevail.

[0029] Various modifications and variations can be made to the specific embodiments described in this specification without departing from the scope or spirit of the invention, as will be apparent to those skilled in the art. Other embodiments derived from this specification will also be readily apparent to those skilled in the art. This specification and embodiments are merely exemplary.

[0030] The terms “include,” “including,” “have,” “contain,” etc., used in this article are all open-ended terms, meaning that they include but are not limited to.

[0031] Example 1

[0032] 1. Experimental materials

[0033] Tomato variety: MoneyMaker

[0034] 2. Experimental Methods

[0035] 2.1 PCR amplification of the SlHB40 CDS sequence

[0036] Total RNA was extracted from MoneyMaker tomato seedlings using Trizol reagent and an RNA miniprep kit (Invitrogen). cDNA was then synthesized using a cDNA reverse transcription kit (Applied Biosystems). The CDS sequence of SlHB40 was amplified using the high-fidelity enzyme Phusion (Thermo Scientific) with the cDNA as a template. The agarose gel electrophoresis results of the PCR products are shown below. Figure 1 The PCR product, namely the SlHB40 CDS sequence, is shown in SEQ ID NO.1.

[0037] The PCR primers were as follows: forward primer: 5'-CACCATGAACCATCAGGTTATAG-3' (SEQ ID NO.2); reverse primer: 5'-AATATATAAGTTCACCCAATCTAA-3' (SEQ ID NO.3); the PCR reaction system (20 μL) is shown in Table 1; the PCR reaction conditions were as follows: pre-denaturation 98℃ for 30 s; denaturation 98℃ for 10 s, annealing 58℃ for 30 s, extension 72℃ for 30 s, 35 cycles; final extension 72℃ for 5 min.

[0038] Table 1 PCR reaction system

[0039]

[0040]

[0041] SEQ ID NO.1:

[0042] .

[0043] 2.2 Construction of SlHB40 overexpression vector

[0044] The PCR-amplified SlHB40 CDS sequence was ligated into the entry vector pENTRT Directional TOPO using the pENTRT Directional TOPO Cloning Kit. The entry vector pENTRT Directional TOPO (Invitrogen) was then ligated into the entry vector using the LR Clonase™ Enzyme Mix Kit. TMThe fusion expression vector plasmid was obtained by performing an LR substitution reaction on the final overexpression vector pK7FWG2.0 (disclosed in the literature "Karimi M, Inzé D, Depicker A. 2002. GATEWAY vectors for Agrobacterium-mediated plant transformation. Trends in Plant Science 7, 193–195"). The constructed fusion expression vector plasmid was transformed into *E. coli* DH5α competent cells, and the transformed bacteria were plated on LB selection plates containing 50 mg / L spectinomycin. After colony growth, single colonies were picked for PCR verification and sent to the company for sequencing verification. The recombinant plasmid, after sequencing verification, was transformed into *Agrobacterium* GV1301 to obtain recombinant *Agrobacterium*.

[0045] 2.3 Construction of transgenic plants

[0046] (1) Preparation of sterile seedlings: Select plump mature tomato seeds, disinfect them with 75% alcohol for 1 min, then disinfect them with sodium hypochlorite solution for 10 min, rinse them with sterile water 4 times, and then inoculate them on 1 / 2 MS solid medium. After culturing in the dark at 25℃ for 3 days, they are then cultured in the light for one week.

[0047] (2) Preparation of explants: Take the cotyledons of sterile seedlings that have just unfolded, cut them from the middle part and place them in MS agar medium for 2 days.

[0048] (3) Agrobacterium propagation: The recombinant Agrobacterium obtained in 2.2 was streaked on LB solid medium (containing 50 mg / L spectinomycin + 50 mg / L gentamicin + 50 mg / L rifampin) and cultured at 28℃ for 2 days. Single colonies were picked and inoculated into LB liquid medium (containing 100 mg / L spectinomycin + 50 mg / L gentamicin + 50 mg / L rifampin) to prepare OD. 600 =0.8 resuspension of recombinant Agrobacterium.

[0049] (4) Agrobacterium infection and co-culture: The pre-cultured explants were placed in OD 600 Infect the bacteria in a recombinant Agrobacterium resuspension at 0.8 for 8 min (8-10 min can achieve the same effect), then place them on MS agar medium and incubate in the dark at 25°C for 3 days.

[0050] (5) Bud differentiation induction: After co-culture, the explants were transferred to MS agar medium containing 20 mg / L kanamycin to induce bud formation. The medium was changed every two weeks.

[0051] (6) Rooting and screening of resistant plants: The differentiated resistant buds were transferred to gel MS resistance medium containing 20 mg / L kanamycin and allowed to form complete small plants.

[0052] (7) Transplanting of transgenic plants: When the roots grow to 5cm, the seedlings are transplanted into square pots containing nutrient soil and vermiculite. After covering and keeping them moist for 7 days, the soil is gradually uncovered and irrigated with nutrient water to obtain T0 generation SlHB40 overexpression transgenic tomatoes.

[0053] (8) Screening of T0 generation SlHB40 overexpressing plants: Compared with wild type, the expression level of SlHB40 in the overexpressing lines was detected by qRT-PCR. The reaction system (5 μL) was as shown in Table 2 according to the SYBR (Applied Biosystems) kit instructions. The reaction program was as follows: 95℃ pre-denaturation for 5 min; 95℃ denaturation for 10 s, 60℃ annealing / extension for 30 s; 40 cycles; melting curve: 95℃ denaturation for 15 s; 60℃ annealing for 60 s; 95℃ termination for 15 s. The primer sequences are as follows: SlHB40 forward primer: 5'-ACCACAACAAGGAGAGTCAAAGCC-3' (SEQ ID NO.4); reverse primer: 5'-TGATCATTCCCACTCCCTTCTCC-3' (SEQ ID NO.5); internal reference gene SlGAPDH forward primer: 5'-ACTGGTGCTGCTAAGGCTGT-3' (SEQ ID NO.6); reverse primer: 5'-ACAAGGTCCACAACCGAGAC-3' (SEQ ID NO.7). qRT-PCR detection results are shown below. Figure 2 .

[0054] Table 2 qRT-PCR reaction system

[0055] SYBR Master Mix (2×) 2.5 forward primer 1 reverse primer 1 cDNA 0.5

[0056] (9) Phenotypic identification of SlHB40 overexpression lines: Three plants were randomly selected from the T0 generation SlHB40 overexpression plants, and T1 generation seeds were collected from each plant. Then, one plump mature T1 generation tomato seed was selected from each T0 plant and planted to obtain three T1 generation lines, which were denoted as OX-1, OX-2 and OX-3, respectively. The planting culture and phenotypic detection methods are as follows:

[0057] Mature tomato seeds were first disinfected with 75% alcohol for 1 minute, then with sodium hypochlorite solution for 10 minutes, rinsed four times with sterile water, and then sown on MS solid medium containing 20 mg / L kanamycin. After incubation in the dark at 4°C for 3 days, they were then cultured under long-day conditions (16 hours light / 8 hours dark) for 10 days. When the roots reached 5 cm, the seedlings were transplanted into square pots containing nutrient soil and vermiculite, covered and kept moist for 7 days, then gradually uncovered and irrigated with nutrient water. Fifty days after sowing, plant height was measured and photographed (wild-type tomatoes with the same culture method and growth cycle were used as a control). Phenotypic results are shown below. Figure 3 As shown in Table 3, the results indicate that overexpression of SlHB40 can dwarf tomato plants.

[0058] Table 3. Plant height of wild-type and SlHB40-overexpressing plants

[0059] WT 72；70；79；76；71 OX-1 58；56；48 OX-2 55；47；53 OX-3 47；44；51

[0060] Note: WT is wild type, and OX-1, OX-2 and OX-3 are SlHB40 overexpression lines.

[0061] The embodiments described above are merely preferred embodiments of the present invention and are not intended to limit the scope of the present invention. Various modifications and improvements made by those skilled in the art to the technical solutions of the present invention without departing from the spirit of the present invention should fall within the protection scope defined by the claims of the present invention.

Claims

1. SlHB40 The application of genes in the creation of tomato dwarfing materials is characterized by, By making the SlHB40 Gene overexpression was used to create the tomato dwarfing material described above; The SlHB40 The CDS sequence of the gene is shown in SEQ ID NO.

1.

2. A type of carrier SlHB40 The application of recombinant gene expression vectors in the creation of tomato dwarfing materials is characterized by, The SlHB40 The CDS sequence of the gene is shown in SEQ ID NO.

1.

3. A type of carrier SlHB40 The application of recombinant microbial strains in the creation of tomato dwarfing materials is characterized by, The SlHB40 The CDS sequence of the gene is shown in SEQ ID NO.

1.

4. A method for creating a tomato dwarfing material, characterized in that, Including SlHB40 Genes were introduced into tomato explants and created through tissue culture. SlHB40 Steps for establishing gene overexpression in plants; The SlHB40 The overexpressing gene plants are the tomato dwarfing materials mentioned above; The SlHB40 The CDS sequence of the gene is shown in SEQ ID NO.

1.

5. The method according to claim 4, characterized in that, The tomato explants are the cotyledons of tomato seedlings.

6. The method according to claim 4, characterized in that, The SlHB40 Methods for introducing genes into the tomato explants include: The SlHB40 Genes are linked to expression vectors to construct recombinant expression vectors; The recombinant expression vector was transformed into a microbial strain to obtain a recombinant microbial strain; The recombinant microbial strain was transformed into the tomato explant.

7. The method according to claim 6, characterized in that, The SlHB40 The gene was first ligated into the entry vector pENTRDirectional TOPO, and then subjected to an LR substitution reaction with the overexpression vector pK7FWG2.0 to obtain the recombinant expression vector.

8. The method according to claim 6, characterized in that, The microbial strain is Agrobacterium GV1301.

9. The method according to claim 8, characterized in that, The recombinant microbial strain was transformed into the tomato explants using an infection method.

10. The method according to claim 9, characterized in that, The infiltration time for the aforementioned infiltration method is 8-10 minutes.

Images