Application of tomato HD-Zip transcription factor SlHZ07 in regulating tomato plant height

Abstract

This invention discloses the application of the tomato HD-Zip transcription factor SlHZ07 in regulating tomato plant height, belonging to the fields of plant genetic engineering and molecular breeding technology. This invention provides an SlHZ07 gene for regulating plant height. The nucleotide sequence of the SlHZ07 gene is shown in SEQ ID NO.1, and the expressed SlHZ07 protein belongs to the tomato HD-Zip transcription factor family. Experimental results show that overexpression of the SlHZ07 gene in tomato plants can significantly increase tomato plant height, which can be used for targeted genetic improvement of tomato plant height and the breeding of superior germplasm. This invention successfully creates new tomato germplasm, providing important experimental materials for subsequent research, and also providing gene resources and breeding parents for further regulation of tomato plant height, contributing to the development of the tomato breeding industry.

Description

Technical Field

[0001] This invention relates to the fields of plant genetic engineering and molecular breeding technology, and in particular to the application of the tomato HD-Zip transcription factor SlHZ07 in regulating the growth rate of tomato plants. Background Technology

[0002] Tomato (Solanum lycopersicum L.) is one of the most widely cultivated vegetable crops, with high commercial value and rich in nutrients such as folic acid, vitamin C, potassium, and lycopene. At the same time, the tomato is also a model plant for plant science research, playing an important role in agricultural production and plant science research.

[0003] In traditional cultivation, tomato plant height is jointly regulated by genetic factors (such as gibberellin and brassinolide signaling pathways) and environmental factors (light, temperature, water, and fertilizer). In recent years, with the development of greenhouse agriculture and molecular breeding technology, optimizing plant height has become a research hotspot, aiming to balance yield potential and cultivation adaptability. Indeterminate tomatoes can have longer fruiting branches and increased fruit set per plant by increasing plant height. Tall varieties can fully utilize vertical space in greenhouse cultivation, increasing yield per unit area. Moderately increasing height can improve canopy light penetration, reduce shading from lower leaves, and enhance overall photosynthetic efficiency; it also reduces fruit-soil contact, lowering the risk of disease. Increasing tomato plant height has significant value in high-yield cultivation and greenhouse agriculture.

[0004] However, there are currently few successfully created tall tomato materials with stable traits. There is an urgent need to provide a method for creating tall tomato materials through gene overexpression and its application, to alleviate the pressure on tomato production against the backdrop of population growth and reduced arable land, to balance yield potential and cultivation adaptability, to increase tomato plant height, to improve the economic benefits of tomato cultivation, and to promote the development of the tomato industry. Summary of the Invention

[0005] The purpose of this invention is to provide the application of the tomato HD-Zip transcription factor SlHZ07 in regulating tomato plant height, in order to solve the problems existing in the prior art. This invention overexpresses the tomato SlHZ07 gene to increase tomato plant height, providing new technical support for tomato breeding and industrial development.

[0006] To achieve the above objectives, the present invention provides the following solution:

[0007] This invention provides an application of the tomato HD-Zip transcription factor SlHZ07 protein in any of the following:

[0008] (1) Application in regulating the growth rate of tomato plants;

[0009] (2) Application in cultivating tomato varieties with increased plant height;

[0010] The nucleotide sequence of the gene encoding the tomato HD-Zip transcription factor SlHZ07 protein is shown in SEQ ID NO.1.

[0011] This invention also provides an application of the SlHZ07 gene expressing the tomato HD-Zip transcription factor in any of the following:

[0012] (1) Application in regulating the growth rate of tomato plants;

[0013] (2) Application in cultivating tomato varieties with increased plant height;

[0014] The nucleotide sequence of the SlHZ07 gene is shown in SEQ ID NO.1.

[0015] Preferably, the SlHZ07 gene is overexpressed in tomatoes to increase the plant height of the tomatoes.

[0016] The present invention also provides the use of a recombinant vector containing the above-described SlHZ07 gene in any of the following:

[0017] (1) Application in regulating the growth rate of tomato plants;

[0018] (2) Application in cultivating tomato varieties with increased plant height.

[0019] Preferably, the recombinant vector is an overexpression vector.

[0020] The present invention also provides the use of engineered bacteria comprising the above-described recombinant vector in any of the following:

[0021] (1) Application in regulating the growth rate of tomato plants;

[0022] (2) Application in cultivating tomato varieties with increased plant height.

[0023] Preferably, the originating bacteria of the engineered bacteria are Agrobacterium.

[0024] The present invention also provides a method for increasing the height of tomato plants, including the step of overexpressing the SlHZ07 gene in tomatoes to increase the height of the tomato plants;

[0025] The nucleotide sequence of the SlHZ07 gene is shown in SEQ ID NO.1.

[0026] Preferably, the overexpression of the SlHZ07 gene in tomato includes the following steps:

[0027] The nucleotide sequence of the SlHZ07 gene was ligated to the PCG3301 vector to construct an overexpression vector;

[0028] The overexpression vector was transferred into tomatoes using Agrobacterium-mediated transformation, cultured, and tomato plants with elevated SlHZ07 gene expression levels were screened, thus completing the overexpression of the SlHZ07 gene in tomatoes.

[0029] This invention also provides a method for cultivating transgenic tomatoes with increased plant height, comprising the following steps:

[0030] The SlHZ07 gene was overexpressed in tomato cells, the tomato cells were then cultured, and the tomato cells were used to regenerate tomatoes, thus obtaining the transgenic tomato with increased plant height.

[0031] The nucleotide sequence of the SlHZ07 gene is shown in SEQ ID NO.1.

[0032] The present invention discloses the following technical effects:

[0033] This invention, through overexpression of the SlHZ07 gene, yielded tomato plants with increased height. Compared to wild-type plants, the resulting tomato plants exhibited significantly increased height and more vigorous growth. In greenhouse tomato cultivation, this invention allows for more efficient use of space, with layered canopy distribution reducing leaf shading and improving overall photosynthetic efficiency. It also fosters multi-layered fruiting branches, increasing yield per unit area. The tall canopy provides excellent ventilation and light penetration, reducing sunburn and deformed fruit rates. Furthermore, it extends the harvest period, making it suitable for year-round greenhouse cultivation. This invention provides crucial tomato germplasm for improving the economic benefits of the tomato industry and promoting its development. Attached Figure Description

[0034] 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.

[0035] Figure 1 The graph shows the relative expression levels of the SlHZ07 gene in the wild-type line (WT) and the SlHZ07 overexpression lines (OX-1 and OX-2); where ** indicates P < 0.01 compared to the wild-type line.

[0036] Figure 2 Phenotypic comparison of wild-type lines (WT) and SlHZ07 overexpression lines (OX-1 and OX-2);

[0037] Figure 3 The plant height of wild-type lines (WT) and SlHZ07 overexpression lines (OX-1 and OX-2) 30 days after sowing is statistically shown; where ** indicates P<0.01 compared with wild-type lines. Detailed Implementation

[0038] 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.

[0039] 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.

[0040] 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.

[0041] 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.

[0042] 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.

[0043] The SlHZ07 gene targeted in this invention belongs to the Homeodomain-Leucine Zipper (HD-Zip) transcription factor. HD-Zip is a type of transcription factor unique to plants, which plays a certain regulatory role in regulating plant growth and development, organ and tissue morphology, and responses to biotic and abiotic stresses.

[0044] Example 1

[0045] 1. Experimental Materials

[0046] Tomato variety: MicroTom, published in the literature "Aoki, Koh et al. Large-scale analysis of full-length cDNAs from the tomato (Solanum lycopersicum) cultivar Micro-Tom, a reference system for the Solanaceae genomics. BMC genomics vol. 11210. 30 Mar. 2010".

[0047] Vector: The overexpression vector PCG3301 has been published in the literature “Li JT, Yu G, Sun XH, Jia CG, Du Q, LiQY, Pan HY. Modification of vectors for functional genomic analysis in plants. Genet Mol Res. 2014 Sep 26; 13(3):7815-25.”. The URL for this literature is: https: / / sci-hub.se / 10.4238 / 2014.september.26.20.

[0048] The applicant promises to release the above-mentioned biological materials to the public within 20 years from the date of application of this invention.

[0049] 2. Experimental Methods

[0050] 2.1 Construction of SlHZ07 overexpression vector

[0051] The CDS sequence of the SlHZ07 gene (as shown in SEQ ID NO.1) was synthesized, and a KpnI restriction site was added to its 5' end and an XbaI restriction site was added to its 3' end. The overexpression vector PCG3301 was ligated to the SlHZ07 gene CDS after double digestion with KpnI and XbaI. The constructed fusion expression vector plasmid was transformed into *E. coli* Top10 competent cells, and the transformed bacteria were plated on LB selection plates containing 50 mg / L kanamycin. After colony growth, single colonies were picked and inoculated into LB liquid medium (containing 50 mg / L kanamycin), cultured overnight, and the plasmid was extracted and verified by double digestion with KpnI and XbaI, and then sent to the company for sequencing verification. The recombinant plasmid, after sequencing verification, was transformed into *Agrobacterium* GV1301.

[0052] SEQ ID NO.1:

[0053] .

[0054] 2.2 Preparation of sterile vaccines

[0055] Select plump, mature tomato seeds, disinfect them with 75% alcohol for 1 minute, then with sodium hypochlorite solution for 10-20 minutes, rinse four times with sterile water, and inoculate them onto 1 / 2 MS solid medium. Incubate in the dark at 25°C for 3 days, then incubate under light for one week.

[0056] 2.3 Preparation of explants

[0057] Take newly unfolded cotyledons from sterile seedlings, cut them in the middle, and incubate them in MS agar medium for 2 days.

[0058] 2.4 Agrobacterium propagation

[0059] Glycerin bacteria were streaked onto LB solid medium (containing 100 mg / L spectinomycin or kanamycin + 50 mg / L gentamicin + 50 mg / L rifampin) and incubated at 28°C for 2 days. Single colonies were then picked and inoculated onto LB liquid medium (containing 100 mg / L kanamycin + 50 mg / L gentamicin + 50 mg / L rifampin) to prepare OD. 600 Agrobacterium resuspension at a concentration of 0.4-1.2.

[0060] 2.5 Agrobacterium infection and co-culture

[0061] After pre-culture, the explants were placed in a resuspended Agrobacterium solution for 8-10 min to infect them, and then placed on MS agar medium and incubated in the dark at 25°C for 2-4 days.

[0062] 2.6 Bud differentiation induction

[0063] After co-culturing, the explants were transferred to MS agar medium containing 10 mg / L hygromycin to induce shoot growth, and the medium was changed every two weeks.

[0064] 2.7 Screening of Rooted and Resistant Plants

[0065] The differentiated resistant shoots were transferred to gel MS resistance medium containing 10 mg / L hygromycin and allowed to develop into complete plantlets.

[0066] 2.8 Screening of T0 generation plants overexpressing SlHZ07

[0067] Compared with the wild type, the expression level of the SlHZ07 gene in the overexpression lines was detected by qRT-PCR. The reaction system is shown in Table 1. The qRT-PCR amplification program followed the instructions of the SYBR (Applied Biosystems) kit. The primer sequences are as follows:

[0068] Forward primer (SlHZ07-F): 5'-AAGCTTAACGTAAGGCCTCGTC-3' (SEQ ID NO.2);

[0069] Reverse primer (SlHZ07-R): 5'-GCTTCAACTTCGTTCTTGCTCTTC-3' (SEQ ID NO.3).

[0070] Forward primer for internal reference gene SlGAPDH: 5'-ACTGGTGCTGCTAAGGCTGT-3' (SEQ ID NO.4);

[0071] Reverse primer: 5'-ACAAGGTCCACAACCGAGAC-3' (SEQ ID NO.5).

[0072] Table 1 qRT-PCR reaction system

[0073] reagents Amount added (μL) SYBRMasterMix (2×) 2.5 forward primer 1 reverse primer 1 cDNA 0.5

[0074] 3. Experimental Results

[0075] The qRT-PCR test results are shown below. Figure 1 It can be seen that the expression level of SlHZ07 gene in the SlHZ07 overexpression lines (OX-1 and OX-2) is significantly higher than that in the wild-type line (WT), P<0.01.

[0076] Phenotypic comparison observation results are as follows: Figure 2 As shown, the SlHZ07 overexpression lines (OX-1 and OX-2) exhibit significantly more vigorous growth than the wild-type lines (WT).

[0077] The statistical results of plant height measurement 30 days after sowing are as follows: Figure 3 As shown, the average plant height of the SlHZ07 overexpression lines (OX-1 and OX-2) was 10.24 cm and 10.93 cm, respectively, which was significantly higher than the average plant height of the wild-type line (WT) of 7.82 cm (P<0.01).

[0078] 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. The application of a tomato HD-Zip transcription factor SlHZ07 protein in any of the following: (1) Application in improving the height of tomato plants; (2) Application in cultivating tomato varieties with increased plant height; The nucleotide sequence of the gene encoding the tomato HD-Zip transcription factor SlHZ07 protein is shown in SEQ ID NO.1; The encoded gene was overexpressed in tomatoes.

2. A method for overexpressing the tomato HD-Zip transcription factor SlHZ07 The application of genes in any of the following: (1) Application in improving the height of tomato plants; (2) Application in cultivating tomato varieties with increased plant height; The SlHZ07 The nucleotide sequence of the gene is shown in SEQ ID NO.1; Overexpression of the above in tomatoes SlHZ07 Gene.

3. A method for increasing tomato plant height, characterized in that, Including overexpression in tomatoes SlHZ07 The steps of using genes to increase the height of the tomato plant; The SlHZ07 The nucleotide sequence of the gene is shown in SEQ ID NO.

1.

4. The method as described in claim 3, characterized in that, The overexpression in tomatoes SlHZ07 Genes include the following steps: The SlHZ07 The nucleotide sequence of the gene was linked to the PCG3301 vector to construct an overexpression vector; The overexpression vector was transferred into tomatoes using Agrobacterium-mediated transformation, followed by culture and screening. SlHZ07 Tomato plants with elevated gene expression levels, i.e., those that have achieved the overexpression in tomatoes, are examples of plants that have successfully completed the process described above. SlHZ07 Gene.

5. A method for cultivating transgenic tomatoes with increased plant height, characterized in that, Includes the following steps: Overexpression in tomato cells SlHZ07 Genes are then used to cultivate tomato cells, and the tomato cells are used to regenerate tomatoes, thus obtaining the transgenic tomatoes with increased plant height. The SlHZ07 The nucleotide sequence of the gene is shown in SEQ ID NO.1.

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