Transcription factors regulating tomato growth, research methods, and preparation methods of tomato mutant plants

Abstract

The present invention discloses a transcription factor for regulating tomato growth, a research method, and a method for preparing a tomato mutant plant. The gene accession number of the transcription factor for regulating tomato growth is Solyc01g106030, its protein sequence is shown in SEQ ID NO.1, and its encoding DNA sequence is shown in SEQ ID NO.2. The transcription factor has a regulatory effect on tomato growth, specifically for regulating tomato fruit development, plant height, and / or the number of lateral branches. Therefore, by changing the expression specificity of the transcription factor or mutating the transcription factor, the downstream gene regulated by the transcription factor is changed, thereby regulating tomato fruit development, plant height, and / or the number of lateral branches.

Description

Technical Field

[0001] The present invention relates to the technical field of plant genetic engineering, and in particular to a transcription factor for regulating tomato growth, an application thereof, and a method for preparing a tomato mutant plant. Background Art

[0002] Tomato is a widely cultivated fruit and vegetable crop worldwide. Regulation of tomato fruit development and plant architecture are crucial for tomato production. The TIFY transcription factor family is a class of zinc-finger protein domain-containing transcription factors in plants, possessing a conserved TIFY domain. Currently, the functions of some TIFY transcription factors have been identified in crops such as Arabidopsis, cotton, rice, maize, and wheat. These transcription factors are involved in a variety of biological processes, including plant morphogenesis, floral organ development, leaf development, cotton fiber initiation, biosynthesis of tanshinones, and plant defense. This suggests that TIFY transcription factors play an important role in regulating the development of various plant tissues and organs and in their ability to adapt to the environment. Genome sequencing analysis has identified 26 TIFY transcription factors in tomato, with limited reports on their gene expression characteristics and phylogenetic trees. However, limited research has examined the specific biological functions of TIFY transcription factors in tomato, limiting their development and utilization. Summary of the Invention

[0003] The purpose of the present invention is to provide a transcription factor for regulating tomato growth, its application and a method for preparing tomato mutant plants, so as to solve the problem that the specific biological function of TIFY transcription factor in tomato is rarely studied in the prior art, which limits its development and utilization.

[0004] In a first aspect, the present invention discloses a transcription factor for regulating tomato growth. Its gene accession number is Solyc01g106030, its protein sequence is shown in SEQ ID NO. 1, and its encoding DNA sequence is shown in SEQ ID NO. 2. This factor regulates tomato growth, specifically fruit development, plant height, and / or the number of lateral branches. Therefore, by altering the expression specificity of the transcription factor or mutating the transcription factor to alter the downstream genes it regulates, tomato fruit development, plant height, and / or the number of lateral branches can be regulated.

[0005] In a second aspect, a method for preparing a tomato mutant plant comprises:

[0006] Designing a sgRNA target site in the first exon of a transcription factor, synthesizing the double-stranded DNA molecule required for the sgRNA target site, introducing the sgRNA target site into an editing vector using DNA ligase, and then introducing the editing vector into host bacteria for culture. Tomato plants are then genetically transformed to obtain tomato mutant plants.

[0007] Preferably, the nucleotide sequence of the sgRNA target site is shown as SEQ ID NO.3.

[0008] Using this technical approach, the recombinant plasmid is introduced into normal tomato plants via host bacteria, causing them to mutate, resulting in mutant plants. Cultivating these mutant plants reveals which aspects of the plant are altered by altering the transcription factor, thereby revealing the specific regulatory mechanisms of the transcription factor. For a desired outcome, this preparation method can be used to refine the target site, double-stranded DNA molecule, primers, and other factors, resulting in mutant plants that meet the requirements.

[0009] As a possible design, the nucleotide sequence of the sense strand in the double-stranded DNA molecule is shown as SEQ ID NO.4, and the nucleotide sequence of the antisense strand is shown as SEQ ID NO.5.

[0010] As a possible design, primers are designed for the sgRNA target site and PCR amplification and screening are performed to obtain tomato mutant plants; wherein: the nucleotide sequence of the forward primer is shown in SEQ ID NO.6, and the nucleotide sequence of the reverse primer is shown in SEQ ID NO.7.

[0011] As a possible design, the host bacterium is Agrobacterium LBA4404.

[0012] In a third aspect, the present invention provides a method for studying transcription factor regulation of tomato growth, comprising:

[0013] In the process of mutating a transcription factor, a sgRNA target site is designed for the first exon of the transcription factor, a double-stranded DNA molecule required for the sgRNA target site is synthesized, the sgRNA target site is introduced into an editing vector using DNA ligase, the editing vector is then introduced into host bacteria, cultured, and genetically transformed into tomato plants to screen for tomato mutant plants;

[0014] The nucleotide sequence of the sgRNA target site is shown in SEQ ID NO.3.

[0015] When the above technical solution is adopted, for the desired results set in advance, the above research methods can be referred to to improve the corresponding targets, double-stranded DNA molecules, primers, etc., so as to obtain mutant plants that meet the conditions.

[0016] As a possible design, the nucleotide sequence of the sense strand in the double-stranded DNA molecule is shown as SEQ ID NO.4, and the nucleotide sequence of the antisense strand is shown as SEQ ID NO.5.

[0017] As a possible design, primers are designed for the sgRNA target site and PCR amplification and screening are performed to obtain tomato mutant plants; wherein: the nucleotide sequence of the forward primer is shown in SEQ ID NO.6, and the nucleotide sequence of the reverse primer is shown in SEQ ID NO.7. BRIEF DESCRIPTION OF THE DRAWINGS

[0018] Figure 1 This is a comparison diagram of the phenotype of the Solyc01g106030 gene knockout mutant and normal plants in Example 2 of the present invention;

[0019] Figure 2 This is the expression of the Solyc01g106030 gene in different tissues and organs in Example 3 of the present invention. DETAILED DESCRIPTION

[0020] In order to make the technical problems, technical solutions and beneficial effects to be solved by the present invention more clearly understood, the present invention is further described in detail below. It should be understood that the specific embodiments described herein are only used to explain the present invention and are not intended to limit the present invention.

[0021] Example 1

[0022] Obtaining the tomato Solyc01g106030 gene mutant

[0023] (1) Construction of tomato Solyc01g106030 gene editing vector and genetic transformation

[0024] S1-1. Using tomato breeding materials as recipients, an sgRNA target site was designed in the first exon of the Solyc01g106030 gene. Its nucleotide sequence is shown in Table 1.

[0025] S1-2. Artificially synthesize the sense single-stranded DNA and antisense single-stranded DNA containing linkers at the sgRNA target site of the tomato Solyc01g106030 gene. Take 1uM of the sense single-stranded DNA and antisense single-stranded DNA respectively, mix them, denature at 95°C, and then cool naturally to form a double-stranded sgRNA target site DNA molecule with a sticky-end linker.

[0026] S1-3. Use T4 DNA ligase to introduce the sgRNA target site into the prepared CRISPR / Cas9 plant gene editing vector to obtain a recombinant plasmid.

[0027] S1-4. The recombinant plasmid was introduced into Agrobacterium LBA4404, and tomato breeding materials were genetically transformed, and 5 transgenic positive plants were obtained by screening.

[0028] (2) Screening of tomato Solyc01g106030 gene mutants

[0029] Primers were designed at either end of the sgRNA target site in the Solyc01g106030 gene to detect target site mutations in the five transgenic plants obtained. PCR amplification was performed using genomic DNA from the transgenic plants as a template, and the amplified products were sequenced to identify target site mutations. The sequencing results, shown in Table 2, indicate that three Solyc01g106030 knockout mutants were obtained. Although all three mutants were heterozygous, frameshift mutations occurred in both homologous Solyc01g106030 genes in each strain, preventing the translation of a functional protein.

[0030] The protein sequence of the Solyc01g106030 gene in the above-mentioned non-mutated tomato breeding material is shown in SEQ ID NO. 1, and is specifically as follows:

[0031] MAEANRRANMYGRETMNAALHQDRHQQTQIDDDDDDDVVAAVGGGGSGGGGIESMDNPTPHIRYDQHHHSSHALHNGGAGGSMEMNGVEGVSHNALYGPPSEIVPTAGSGASDQLTLSFQGEVYVFDAVSPEKVQAVLLLLGGYEVPPGIPAVNVVPQSQRA SGDFPGRLNQPERAASLNRFREKRKERCFDKKIRYTVRKEVAMRMQRKKGQFTSAKSIPDEVGSSADWNEGSGQEEQETSCRHCNISSKSTPMMRRGPAGPRSLCNACGLKWANKGILRDLSKVPAPGTQDQTAKPGEQSHGEPNGSDDMAAIITPDDNNPVG

[0032] The DNA sequence of the Solyc01g106030 gene in the above-mentioned non-mutated tomato breeding material is shown in SEQ ID NO. 2, which is as follows:

[0033] ATGGCAGAAGCAAATCGCAGAGCTAACATGTACGGACGGGAGACGATGAACGCTGCACTTCACCAAGATAGACACCAGCAAACTCAGATCGACGACGACGATGATGACGACGTCGTCGCTGCTGTCGGTGGCGGTGGCAGTGGTGGGGGAGGAATAGAGTCTATGGACAACCCTACTCCTCACATTCGCTACGACCAACATCATCACTCTCATTCTCACGCGCTTCACAACGGCGGCGCCGGCGGTTCTATGGAGATGAATGGTGTGGAAGGTGTTTCTCATAACGCCTTGTATGGTCCTCCTTCCGAAATTGTTCCTACTGCTGGTAGTGGAGCTTCCGATCAGCTTACGCTGTCGTTTCAAGGCGAAGTGTACGTTTTTGATGCCGTTTCACCTGAAAAGGTTCAGGCGGTGCTGTTACTGTTGGGGGGATACGAAGTCCCTCCTGGTATCCCTGCTGTAAATGTGGTTCCCCAAAGTCAGAGGGCTTCAGGTGACTTTCCTGGAAGATTAAATCAACCGGAAAGAGCTGCTTCTTTAAATCGTTTTAGGGAAAAGAGGAAAGAACGGTGTTTTGATAAAAAGATCCGCTATACTGTGCGGAAGGAAGTTGCGATGAGGATGCAGCGCAAGAAAGGTCAGTTTACATCTGCCAAGTCAATACCTGACGAAGTAGGTTCTTCTGCAGATTGGAATGAAGGCTCTGGTCAAGAAGAGCAGGAAACATCATGTAGACATTGCAATATTAGTTCGAAATCCACTCCTATGATGCGTCGGGGACCAGCTGGCCCAAGGTCTCTTTGTAATGCATGTGGACTCAAGTGGGCCAATAAGGGAATTTTAAGAGATCTTTCTAAAGTTCCAGCTCCTGGAACTCAGGACCAAACTGCGAAACCTGGTGAACAGAGCCATGGTGAACCTAATGGCTCGGATGACATGGCTGCTATCATCACTCCGGATGACAACAACCCAGTGGGGTGA。

[0034] Table 1

[0035]

[0036]

[0037] Table 2

[0038]

[0039] As shown in Table 2, the Solyc01g106030 gene of the three mutants obtained in this example all had frameshift mutations and could not translate proteins with normal functions.

[0040] Example 2

[0041] Phenotypic Analysis of Tomato Solyc01g106030 Mutants

[0042] The three tomato Solyc01g106030 gene knockout mutants prepared in Example 1 (as a mutant group) and normal tomato plants without mutations (as a control group) were planted in a greenhouse and grown and managed normally to observe their phenotypic traits. The tomato Solyc01g106030 gene knockout mutants all showed phenotypes such as dwarfing, shortened internode length, and increased axillary buds. Figure 2 As shown, Figure 2 The left side is the mutant, and the right side is the non-mutated plant.

[0043] After entering reproductive growth, the mutant group had fewer flower buds and lower fertility compared with the control group. The statistical data are shown in Table 3.

[0044] Table 3

[0045]

[0046]

[0047] As shown in Table 3, the Solyc01g106030 mutant plants had a plant height of approximately 63 cm, 49.1% of the control plant. The average internode length was 3.87 cm, 48.1% of the control plant. The number of axillary buds increased 2.09 times, while the number of fruit set decreased by 2-3 per plant. This indicates that the Solyc01g106030 gene is a key regulator of tomato plant morphogenesis and reproductive development. The average longitudinal and transverse diameters of the Solyc01g106030 mutant plants were 3.4 cm and 5.2 cm, respectively, 70.8% and 85.2% of the control plant. The average fruit weight was 46.3% of the control plant. This suggests that the Solyc01g106030 mutation affects fruit development and is a key transcription factor in regulating tomato fruit development.

[0048] Example 3

[0049] Transcriptome data analysis of Solyc01g106030 gene in different tissues of tomato

[0050] The transcriptome data of different tissue parts of tomato were analyzed, and the results were as follows Figure 2 As shown by Figure 2 The Solyc01g106030 gene was expressed in leaves, hypocotyls, cotyledons, stem apex, flowers, and fruits, with relatively high expression in roots and stem apex, and lowest expression in mature leaves. This suggests that the Solyc01g106030 gene is stably expressed in different tissues and organs of tomato plants and is involved in regulating tissue and organ development. In fruits, Solyc01g106030 expression gradually increases with fruit development, indicating its involvement in regulating tomato fruit development.

[0051] The specific implementation methods described above further illustrate the objectives, technical solutions and beneficial effects of the present invention in detail. It should be understood that the above description is only a specific implementation method of the present invention and is not intended to limit the scope of protection of the present invention. Any modifications, equivalent substitutions, improvements, etc. made within the spirit and principles of the present invention should be included in the scope of protection of the present invention.

Claims

1. An application of a transcription factor in regulating tomato growth, characterized in that: By knocking out the transcription factor, the longitudinal and transverse diameters and single fruit weight of tomato fruits are reduced, the height of tomato plants is reduced, and / or the number of tomato side branches is increased; The gene accession number of the transcription factor is Solyc01g106030, its protein sequence is shown in SEQ ID NO.1, and its encoding DNA sequence is shown in SEQ ID NO.

2.

2. The use of a transcription factor in regulating tomato growth according to claim 1, characterized in that: The transcription factor is knocked out by designing an sgRNA target site for the first exon of the transcription factor, synthesizing a double-stranded DNA molecule required for the sgRNA target site, introducing the sgRNA target site into an editing vector using DNA ligase, and then introducing the editing vector into host bacteria for cultivation, and genetically transforming tomato plants to obtain tomato mutant plants.

3. The use of the transcription factor according to claim 2 in regulating tomato growth, characterized in that: The nucleotide sequence of the sgRNA target site is shown in SEQ ID NO.

3.

4. The use of the transcription factor according to claim 2 in regulating tomato growth, characterized in that: The nucleotide sequence of the sense strand in the double-stranded DNA molecule is shown in SEQ ID NO.4, and the nucleotide sequence of the antisense strand is shown in SEQ ID NO.

5.

5. Use of the transcription factor according to claim 3 or 4 in regulating tomato growth, characterized in that: Primers were designed for the sgRNA target site and PCR amplification and screening were performed to obtain tomato mutant plants; wherein: the nucleotide sequence of the forward primer is shown in SEQ ID NO.6, and the nucleotide sequence of the reverse primer is shown in SEQ ID NO.7.

Images