CN116023456A - Transcription factor regulating tomato growth and research method and preparation method of tomato mutant plant - Google Patents

Abstract

The invention discloses a transcription factor regulating tomato growth and a research method and a method for preparing tomato mutant plants. The gene accession number of the transcription factor regulating tomato growth is Solyc01g106030, and its protein sequence is shown in SEQ ID NO.1, and its coding DNA The sequence is shown in SEQ ID NO.2; it has a regulatory effect on tomato growth, and is specifically used to regulate tomato fruit development, plant height and/or the number of side branches. Therefore, by changing the expression specificity of the transcription factor or making mutations to the transcription factor, The downstream genes regulated by it are changed to regulate tomato fruit development, plant height and/or lateral branch quantity.

Description

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

Technical Field

The 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

Tomato is a fruit and vegetable crop widely grown worldwide. Tomato fruit development regulation and plant type control are of great significance to tomato production. The TIFY transcription factor family is a class of transcription factors containing zinc finger protein domains in plants, with a conserved TIFY domain. At present, the functions of some TIFY transcription factors have been identified in crops such as Arabidopsis, cotton, rice, corn, and wheat. These transcription factors are involved in a variety of biological processes such as plant morphology, floral organ development, leaf development, cotton fiber initiation, biosynthesis of tanshinone, and plant defense, indicating that TIFY transcription factors play an important role in the developmental regulation of different tissues and organs of plants and their ability to adapt to the environment. Through genome sequencing analysis, 26 TIFY transcription factors were found in tomatoes, and there are a few reports on the expression characteristics and phylogenetic trees of the genes, but the specific biological functions of TIFY transcription factors in tomatoes have been less studied, which limits their development and utilization.

Summary of the invention

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

In the first aspect, the present invention discloses a transcription factor for regulating tomato growth, whose gene accession number is Solyc01g106030, whose protein sequence is shown in SEQ ID NO.1, and whose coding DNA sequence is shown in SEQ ID NO.2. It has a regulating effect on tomato growth, and is specifically used to regulate tomato fruit development, plant height and/or the number of lateral branches, so the tomato fruit development, plant height and/or the number of lateral branches can be regulated by changing the expression specificity of the transcription factor or mutating the transcription factor to change the downstream gene regulated by it.

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

Designing sgRNA target sites in the first exon of the transcription factor, synthesizing double-stranded DNA molecules required for the sgRNA target sites, introducing the sgRNA target sites into editing vectors through DNA ligase, and then introducing the editing vectors into host bacteria for culture, and genetically transforming tomato plants to screen for tomato mutant plants;

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

In the case of the above technical solution, the recombinant plasmid is introduced into the normal tomato plant by the host bacteria, causing it to mutate, thereby obtaining a mutant plant. By cultivating the mutant plant, it can be shown which aspects of the plant can be changed by changing the transcription factor, and then the specific situation of the transcription factor being regulated can be obtained. For the desired results set in advance, the corresponding target, DNA double-stranded molecule, primer, etc. can be improved with reference to the preparation method, so as to obtain a mutant plant that meets the conditions.

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.

As a possible design, primers are designed for the sgRNA target site and PCR amplification screening is used 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.

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

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

In mutating the 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 by DNA ligase, the editing vector is then introduced into host bacteria for culture, and tomato plants are genetically transformed to obtain tomato mutant plants;

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

When the above technical solution is adopted, for the desired results to be 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.

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.

As a possible design, primers are designed for the sgRNA target site and PCR amplification screening is used 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

FIG1 is a comparison diagram of the phenotype of a Solyc01g106030 gene knockout mutant and a normal plant in Example 2 of the present invention;

FIG. 2 shows the expression of the Solyc01g106030 gene in different tissues and organs in Example 3 of the present invention.

DETAILED DESCRIPTION

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 used to limit the present invention.

Example 1

Obtaining the tomato Solyc01g106030 gene mutant

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

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

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 the antisense single-stranded DNA respectively, mix them, denature at 95℃ and then cool naturally to form a double-stranded sgRNA target site DNA molecule with a sticky-end linker.

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.

S1-4. The recombinant plasmid was introduced into Agrobacterium LBA4404, the tomato breeding materials were genetically transformed, and 5 transgenic positive plants were screened.

(2) Screening of tomato Solyc01g106030 gene mutants

Primers were designed at both ends of the sgRNA target site of the Solyc01g106030 gene to detect the target site mutations of the 5 transgenic positive plants obtained. PCR amplification was performed using the genomic DNA of the transgenic positive plants as a template, and the amplified products were sequenced to identify the target site mutations. The sequencing results are shown in Table 2, indicating that 3 mutants with the Solyc01g106030 gene knocked out were obtained. Although the 3 mutants are all heterozygous strains, the two homologous chromosome Solyc01g106030 genes in each strain have frameshift mutations and cannot translate normal functional proteins.

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

MAEANRRANMYGRETMNAALHQDRHQQTQIDDDDDDDVVAAVGGGGSGGGGIESMDNPTPHIRYDQHHHSSHALHNGGAGGSMEMNGVEGVSHNALYGPPSEIVPTAGSGASDQLTLSFQGEVYVFDAVSPEKVQAVLLLLGGYEVPPGIPAVNVVPQSQRASGDFPGRLNQPERAASLNRFREKRKERCFDKKIRYTVRKEVAMRMQRKKGQ FTSAKSIPDEVGSSADWNEGSGQEEQETSCRHCNISSKSTPMMRRGPAGPRSLCNACGLKWANKGILRDLSKVPAPGTQDQTAKPGEQSHGEPNGSDDMAAIITPDDNNPVG

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

ATGGCAGAAGCAAATCGCAGAGCTAACATGTACGGACGGGAGACGATGAACGCTGCACTTCACCAAGATAGACACCAGCAAACTCAGATCGACGACGACGATGATGACGACGTCGTCGCTGCTGTCGGTGGCGGTGGCAGTGGTGGGGGAGGAATAGAGTCTATGGACAACCCTACTCCTCACATTCGCTACGACCAACATCATCACTCTCATTCTCACGCGCTTCACAACGGCGGCGCCGGCGG TTCTATGGAGATGAATGTGTGGAAGGTGTTTCTCATAACGCCTTGTATGGTCCTCCTTCCGAAATTGTTCCTACTGCTGGTAGTGGAGCTTCCGATCAGCTTACGCTTGTCGTTTCAAGGCGAAGTGTACGTTTTTGATGCCGTTTCACCTGAAAAGGTTCAGGCGGTGCTGTTACTGTTGGGGGGATACGAAGTCCCTCCTGGTATCCCTGCTGTAAATGTGGTTCCCCAAAGTCAGAGGGCTTC AGGTGACTTTCCTGGAAGATTAAATCAACCGGAAAGAGCTGCTTCTTTAAATCGTTTTAGGGAAAAGAGGAAAGAACGGTGTTTTGATAAAAAGATCCGCTATACTGTGCGGAAGGAAGTTGCGATGAGGATGCAGCGCAAGAAAGGTCAGTTTACATCTGCCAAGTCAATACCTGACGAAGTAGGTTCTTCTGCAGATTGGAATGAAGGCTCTGGTCAAGAAGAGCAGGAAACATCATGTAGAC ATTGCAATATTAGTTCGAAATCCACTCCTATGATGCGTCGGGGACCAGCTGGCCCAAGGTCTCTTTGTAATGCATGTGGACTCAAGTGGGCCAATAAGGGAATTTTAAGAGATCTTTCTAAAGTTCCAGCTCCTGGAACTCAGGACCAAACTGCGAAACCTGGTGAACAGAGCCATGGTGAACCTAATGGCTCGGATGACATGGCTGCTATCATCACTCCGGATGACAACAACCCAGTGGGGTGA.

Table 1

Table 2

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

Example 2

Phenotypic Analysis of Tomato Solyc01g106030 Mutant

The three tomato Solyc01g106030 gene knockout mutants (as a mutant group) and non-mutated normal tomato plants (as a control group) prepared in Example 1 were planted in a greenhouse, grown and managed normally, and the phenotypic traits were observed. The tomato Solyc01g106030 gene knockout mutants all showed phenotypes such as dwarfing, shortened internode length, and increased axillary buds, as shown in Figure 2, where the mutants are on the left and the non-mutated plants are on the right.

After entering reproductive growth, the mutant group had very few flower buds and reduced fertility compared with the control group. The statistical data are shown in Table 3.

Table 3

As shown in Table 3, the plant height of the mutant plant of Solyc01g106030 gene is about 63 cm, which is 49.1% of the control group, the average internode length is 3.87 cm, which is 48.1% of the control group, the number of axillary buds increases by 2.09 times that of the control group, and the number of fruit sets decreases by 2-3 per plant, indicating that Solyc01g106030 gene is an important regulatory factor in the morphological construction and reproductive development of tomato plants. The average longitudinal diameter and average transverse diameter of the mutant plant of Solyc01g106030 gene are 3.4 cm and 5.2 cm, which are 70.8% and 85.2% of the control group, respectively; the average single fruit weight is 46.3% of the control group, indicating that the mutation of Solyc01g106030 gene affects fruit development and is an important transcription factor regulating tomato fruit development.

Example 3

Transcriptome data analysis of Solyc01g106030 gene in different tissues of tomato

The transcriptome data of different tissues of tomato were analyzed, and the results are shown in Figure 2. As shown in Figure 2, the Solyc01g106030 gene is expressed in leaves, hypocotyls, cotyledons, stem tips, flowers, fruits and other tissues, especially in roots and stem tips, where the expression level is relatively high, and the expression level is lowest in mature leaves, indicating that the Solyc01g106030 gene is stably expressed in different tissues and organs of tomato plants and participates in the regulation of tissue and organ development. In the fruit, the expression level of the Solyc01g106030 gene gradually increases with the development of the fruit, indicating that it is involved in the regulation of tomato fruit development.

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 (10)

1. A transcription factor for regulating tomato growth, characterized in that 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 transcription factor according to claim 1, characterized in that the transcription factor is used to regulate tomato fruit development, plant height and/or the number of lateral branches. 3. Use of the transcription factor according to claim 1 or 2 in regulating tomato growth, characterized in that tomato fruit development, plant height and/or the number of lateral branches are regulated by changing the expression specificity of the transcription factor; or; By mutating transcription factors, the downstream genes they regulate change, thereby regulating tomato fruit development, plant height and/or the number of lateral branches. 4. A research method for regulating tomato growth by transcription factors according to claim 1 or 2, characterized in that the research method comprises: In mutating the transcription factor, an 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 by DNA ligase, the editing vector is then introduced into host bacteria for culture, and tomato plants are genetically transformed to obtain tomato mutant plants; preferably, the nucleotide sequence of the sgRNA target site is as shown in SEQ ID NO.3. 5. The research method according to claim 4 is characterized in that the nucleotide sequence of the sense strand in the double-stranded DNA molecule is as shown in SEQ ID NO.4, and the nucleotide sequence of the antisense strand is as shown in SEQ ID NO.5. 6. The research method according to claim 4 or 5, characterized in that primers are designed for the sgRNA target site and PCR amplification screening is used 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. 7. A method for preparing a tomato mutant plant, characterized in that the preparation method comprises: Designing an sgRNA target site in the first exon of the transcription factor according to claim 1 or 2, synthesizing a double-stranded DNA molecule required for the sgRNA target site, introducing the sgRNA target site into an editing vector by DNA ligase, then introducing the editing vector into host bacteria for cultivation, and genetically transforming tomato plants to obtain tomato mutant plants; The nucleotide sequence of the sgRNA target site is shown in SEQ ID NO.3. 8. The method for preparing a tomato mutant plant according to claim 7, characterized in that the nucleotide sequence of the sense strand in the double-stranded DNA molecule is as shown in SEQ ID NO.4, and the nucleotide sequence of the antisense strand is as shown in SEQ ID NO.5. 9. The method for preparing a tomato mutant plant according to claim 7 or 8, characterized in that primers are designed for the sgRNA target site and PCR amplification screening is used to obtain a tomato mutant plant; 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. 10 . The method for preparing a tomato mutant plant according to claim 7 , wherein the host bacteria is Agrobacterium LBA4404.

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