Application of Solyc11g071810 gene in regulation and control of tomato fruit size

Abstract

The invention discloses an application of a Solyc11g071810 gene in regulating and controlling the size of tomato fruits, and is characterized in that the nucleotide sequence of cDNA (complementary deoxyribonucleic acid) of the Solyc11g071810 gene is as shown in SEQ ID NO. 1; according to the invention, a CRISPR / Cas9 gene editing technology is adopted, the Solyc11g071810 gene is edited, and an amino acid sequence coded by the Solyc11g071810 gene is changed, so that the function of the Solyc11g071810 gene disappears or changes, and the size of tomato fruits can be obviously reduced, therefore, the Solyc11g071810 gene can regulate and control the size of the tomato fruits, in addition, the invention also provides a CRISPR / Cas9 vector of the targeted Solyc11g071810 gene, and the expression of the Solyc11g071810 gene is improved by limiting a target site. And a better editing effect is achieved.

Description

Technical Field

[0001] The invention relates to the technical field of agricultural planting, and in particular to an application of a Solyc11g071810 gene in regulating tomato fruit size. Background Art

[0002] The low yield per unit area of ​​tomatoes is still a key factor restricting tomato production. The yield per unit area of ​​tomatoes is closely related to the fruit size, which is mainly controlled by genetic genes. Therefore, it is extremely important for tomato production to deeply explore the genetic genes that regulate tomato fruit size and then regulate tomato fruit size.

[0003] The gene loci that have been cloned to control tomato fruit size include fw2.2 (Cell Number Regulator) (Frary et al., 2000; Cong et al., 2006), fw3.2 (SlKLUH) (Zhang et al., 2012; Chakrabarti et al., 2013; Li et al., 2021) and fw11.3 (Cell Size Regulator) (Chakrabarti et a., 2015; Mu et al., 2015; Mu et al., 2017).

[0004] As the research deepened, it was found that tomato fruit size is the result of the simultaneous control of multiple genes. To date, there are still many regulatory genes for tomato fruit size that have not been discovered, and the gene regulatory network for tomato fruit size is still incomplete. Therefore, the discovery of new genes that regulate tomato fruit size has important theoretical significance and practical application value in production, and is one of the important areas of tomato breeding research.

[0005] In view of this, the present invention is proposed. Summary of the invention

[0006] The object of the present invention is to provide an application of the Solyc11g071810 gene in regulating the size of tomato fruits. The present invention can adjust the size of tomato fruits by editing the Solyc11g071810 gene using CRISPR / Cas9 gene editing technology.

[0007] In order to achieve the above-mentioned purpose of the present invention, the following technical solutions are particularly adopted:

[0008] In a first aspect, the present invention provides an application of the Solyc11g071810 gene in regulating tomato fruit size. The nucleotide sequence of the Solyc11g071810 gene cDNA is shown in SEQ ID NO.1.

[0009] Preferably, the CRISPR / Cas9 vector targeting the Solyc11g071810 gene is used to regulate tomato fruit size.

[0010] Preferably, the CRISPR / Cas9 vector targeting the Solyc11g071810 gene comprises sgRNA1 and / or sgRNA2;

[0011] The nucleotide sequence of the sgRNA1 is shown in SEQ ID NO.2; the nucleotide sequence of the sgRNA2 is shown in SEQ ID NO.3.

[0012] The second aspect of the present invention provides a method for constructing a CRISPR / Cas9 vector targeting the Solyc11g071810 gene, the construction method comprising the following steps:

[0013] (a) Design and synthesize sgRNA for Solyc11g071810 gene;

[0014] (b) Inserting sgRNA into VK005-14 vector;

[0015] (c) The VK005-14 vector containing sgRNA is transformed into Escherichia coli, and then positive screening, expansion culture, and extraction are performed to obtain the CRISPR / Cas9 vector targeting the Solyc11g071810 gene.

[0016] Preferably, the sgRNA includes sgRNA1 and / or sgRNA2;

[0017] The nucleotide sequence of the sgRNA1 is shown in SEQ ID NO.2; the nucleotide sequence of the sgRNA2 is shown in SEQ ID NO.3.

[0018] The third aspect of the present invention provides a CRISPR / cas9 vector targeting the Solyc11g071810 gene constructed by the above construction method.

[0019] A fourth aspect of the present invention provides a method for reducing the size of a tomato fruit, the method for reducing the size of a tomato fruit comprising the following steps:

[0020] The CRISPR / Cas9 vector targeting the Solyc11g071810 gene constructed by the construction method is transferred into Agrobacterium, and then infects tomato explants for cultivation.

[0021] Preferably, the Agrobacterium is Agrobacterium LB4404.

[0022] Compared with the prior art, the beneficial effects of the present invention include at least:

[0023] The present invention adopts CRISPR / Cas9 gene editing technology, edits the Solyc11g071810 gene, changes the amino acid sequence encoded by the Solyc11g071810 gene, makes the function of the Solyc11g071810 gene disappear or change, and can significantly reduce the size of tomato fruit. Therefore, the Solyc11g071810 gene can regulate the size of tomato fruit. In addition, the present invention also provides a CRISPR / Cas9 vector targeting the Solyc11g071810 gene, and achieves a better editing effect by limiting the target site. BRIEF DESCRIPTION OF THE DRAWINGS

[0024] In order to more clearly illustrate the specific embodiments of the present invention or the technical solutions in the prior art, the following is a brief introduction to the drawings required for the specific embodiments or the description of the prior art. In all the drawings, similar elements or parts are generally identified by similar reference numerals. In the drawings, the elements or parts are not necessarily drawn according to the actual scale.

[0025] Figure 1 The detection of CAS9 gene in transgenic plants and the specific sequence comparison of Solyc11g071810 gene in transgenic plants in the embodiments of the present invention;

[0026] Figure 2 The fruit sizes of the wild-type and transgenic plants in the examples of the present invention are shown. DETAILED DESCRIPTION

[0027] The following embodiments of the technical solution of the present invention are described in detail in conjunction with the embodiments. The following embodiments are only used to more clearly illustrate the technical solution of the present invention, and are therefore only used as examples, and cannot be used to limit the protection scope of the present invention.

[0028] It should be noted that, unless otherwise specified, the technical terms or scientific terms used in this application should have the common meanings understood by those skilled in the art to which the present invention belongs.

[0029] The embodiment of the present invention provides an application of the Solyc11g071810 gene in regulating tomato fruit size. The nucleotide sequence of the Solyc11g071810 gene cDNA is shown in SEQ ID NO.1, and the specific sequence is.

[0030] The present invention adopts CRISPR / Cas9 gene editing technology, edits the Solyc11g071810 gene, changes the amino acid sequence encoded by the Solyc11g071810 gene, makes the function of the Solyc11g071810 gene disappear or change, and can significantly reduce the size of tomato fruit. Therefore, the Solyc11g071810 gene can regulate the size of tomato fruit. In addition, the present invention also provides a CRISPR / Cas9 vector targeting the Solyc11g071810 gene, and achieves a better editing effect by limiting the target site.

[0031] In one embodiment, a CRISPR / Cas9 vector targeting the Solyc11g071810 gene is used to regulate tomato size.

[0032] In one embodiment, the CRISPR / Cas9 vector targeting the Solyc11g071810 gene comprises sgRNA1 and / or sgRNA2;

[0033] The nucleotide sequence of the sgRNA1 is shown in SEQ ID NO.2, specifically UCCUCAGAACGCGUUUGUU; the nucleotide sequence of the sgRNA2 is shown in SEQ ID NO.3, specifically UAGUGACAGUAAGAUGUGGGC.

[0034] The present invention does not specifically limit the construction method of the CRISPR / Cas9 vector targeting the Solyc11g071810 gene. Those skilled in the art can use a conventional construction kit in the art to construct it. In one embodiment, the construction method of the CRISPR / Cas9 vector targeting the Solyc11g071810 gene includes the following steps:

[0035] (a) Design and synthesize sgRNA for Solyc11g071810 gene;

[0036] (b) Inserting sgRNA into VK005-14 vector;

[0037] (c) The VK005-14 vector containing sgRNA is transformed into Escherichia coli, and then positive screening, expansion culture, and extraction are performed to obtain the CRISPR / Cas9 vector targeting the Solyc11g071810 gene.

[0038] In one embodiment, the sgRNA includes sgRNA1 and / or sgRNA2;

[0039] The nucleotide sequence of the sgRNA1 is shown in SEQ ID NO.2; the nucleotide sequence of the sgRNA2 is shown in SEQ ID NO.3.

[0040] Another embodiment of the present invention provides a CRISPR / Cas9 vector targeting the Solyc11g071810 gene constructed by the above construction method.

[0041] Another embodiment of the present invention provides a method for reducing the size of a tomato fruit, the method for reducing the size of a tomato fruit comprising the following steps:

[0042] The CRISPR / Cas9 vector targeting the Solyc11g071810 gene constructed by the construction method is transferred into Agrobacterium, and then infects tomato explants for cultivation.

[0043] In one embodiment, the Agrobacterium is Agrobacterium LB4404.

[0044] The technical solution of the present invention is further described in detail below through specific embodiments.

[0045] Example

[0046] This example is a verification of the biological function of the Solyc11g071810 gene:

[0047] 1. Construction of CRISPR / Cas9 vector targeting Solyc11g071810 gene:

[0048] The website (http: / / crispr.mit.edu / ) was used to design sgRNA sequences for the first and second exons of the tomato Solyc11g071810 gene (Gene Bank accession number EU557674), sgRNA1: UCCUCAGAACGCGUUUGUU; sgRNA2: UAGUGACAGUAAGAUGUGGGC.

[0049] After the commissioned company synthesized the sgRNA sequence oligonucleotide fragment, it synthesized the oligo dimer and inserted the oligo dimer into the CRISPR / Cas9 vector to complete the vector construction (Plant Cas9 / gRNA Plasmid Construction Kit, Catalog.No.VK005-14);

[0050] In heat-shock transformation of E. coli DH5α, positive clones were screened using kanamycin, and single clones were positively detected using CAS9 primers;

[0051] The positive clones were expanded and cultured to extract plasmids. After sequencing, plasmids with sequencing results consistent with the reference sequence were selected for preservation and transferred into Agrobacterium tumefaciens LB4404 for explant infection.

[0052] 2. Tomato genetic transformation:

[0053] AC germplasm (Ailsa Craig, germplasm source TGRC) was genetically transformed by Agrobacterium-mediated method, and DNA was extracted from transformed seedlings for detection of target genes. The detection results are shown in Table 1.

[0054] Table 1

[0055]

[0056]

[0057] As shown in Table 1, the Solyc11g071810 gene of the three transgenic plants has mutated, and it is a mosaic mutation or a biallelic mutation, and all contain CRISPR / Cas9 T-DNA insertion. The target gene detection of the transgenic plant progeny is shown in the following figure. Figure 1 As shown in Figure A, Figure 1 From A, we can see that the Solyc11g071810 gene of the progeny of transgenic plants #2, #3, and #6 has mutated. Figure 1 As shown in Figure B, none of the offspring of the transgenic plants contained the CAS9 gene, indicating that the transgenic plants did not contain the CRISPR / Cas9 vector sequence and that the transgenic plants were stably inherited.

[0058] The fruit sizes of wild-type and transgenic plants were statistically analyzed. Figure 2 As shown;

[0059] Depend on Figure 2 It can be seen that compared with the wild type, the fruit diameter of the transgenic plants was significantly reduced, and the fruit diameters of the transgenic plants #2, #3 and #6 were reduced by 14.86%, 24.49% and 18.82%, respectively; the fruit weight of the transgenic plants was significantly reduced, and the fruit weight of the transgenic plants #2, #3 and #6 were reduced by 13.12%, 22.62% and 16.90%, respectively; further indicating that Solyc11g071810 can regulate the size of tomato fruit.

[0060] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention, rather than to limit it. Although the present invention has been described in detail with reference to the aforementioned embodiments, those skilled in the art should understand that they can still modify the technical solutions described in the aforementioned embodiments, or replace some or all of the technical features therein by equivalents. These modifications or replacements do not make the essence of the corresponding technical solutions deviate from the scope of the technical solutions of the embodiments of the present invention, and they should all be included in the scope of the claims and specification of the present invention.

Claims

1. Application of the Solyc11g071810 gene in regulating tomato fruit size, characterized in that: The nucleotide sequence of the Solyc11g071810 gene cDNA is shown in SEQ ID NO.

1.

2. The use according to claim 1, characterized in that: Application of CRISPR / cas9 vector targeting Solyc11g071810 gene in regulating tomato fruit size.

3. The use according to claim 1, characterized in that: The CRISPR / Cas9 vector targeting the Solyc11g071810 gene includes sgRNA1 and / or sgRNA2; The nucleotide sequence of the sgRNA1 is shown in SEQ ID NO.2; the nucleotide sequence of the sgRNA2 is shown in SEQ ID NO.

3.

4. A method for constructing a CRISPR / Cas9 vector targeting the Solyc11g071810 gene, characterized in that: The steps include: (a) Design and synthesize sgRNA for Solyc11g071810 gene; (b) Inserting sgRNA into VK005-14 vector; (c) The VK005-14 vector containing sgRNA is transformed into Escherichia coli, and then positive screening, expansion culture, and extraction are performed to obtain the CRISPR / Cas9 vector targeting the Solyc11g071810 gene.

5. The construction method according to claim 4, characterized in that: The sgRNA includes sgRNA1 and / or sgRNA2; The nucleotide sequence of the sgRNA1 is shown in SEQ ID NO.2; the nucleotide sequence of the sgRNA2 is shown in SEQ ID NO.

3.

6. The CRISPR / Cas9 vector targeting the Solyc11g071810 gene constructed by the construction method according to claim 4 or 5.

7. A method for reducing the size of tomato fruits, characterized in that: The steps include: The CRISPR / Cas9 vector targeting the Solyc11g071810 gene constructed by the construction method of claim 4 or 5 is transferred into Agrobacterium, and then infects tomato explants for cultivation.

8. The method of reducing tomato fruit size according to claim 7, characterized in that: The Agrobacterium is Agrobacterium LB4404.

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