WO2022052381A1

WO2022052381A1 - Application of gene in enlarging tomato fruits

Info

Publication number: WO2022052381A1
Application number: PCT/CN2020/141384
Authority: WO
Inventors: 马伯军; 陈析丰; 沈淑容; 许以灵; 李敏; 陈浩天
Original assignee: 浙江师范大学
Priority date: 2020-09-11
Filing date: 2020-12-30
Publication date: 2022-03-17
Also published as: JP2023526694A; CN112048510B; JP7313759B2; CN112048510A

Abstract

An application of a gene in enlarging tomato fruits. The size of tomato fruits can be increased by knocking out an IFW1 gene in tomato, so that the yield is increased; and a nucleotide sequence encoded by the gene IFW1 is as shown in SEQ ID NO: 1. Two knockout strains of the IFW1 gene are ifw1-1 and ifw1-2, and IFW1 gene mutation sequences in ifw1-1 and ifw1-2 plants are both SEQ ID NO: 2.

Description

Application of a gene in tomato fruit enlargement

technical field

The invention relates to a gene for promoting tomato fruit enlargement and its application, belonging to the field of crop molecular genetics.

Background technique

As an important horticultural plant and economic crop, tomato (Solanum lycopersicum) is widely planted around the world and has become a worldwide vegetable and fruit. Tomato fruit size and weight is an important agronomic trait that affects tomato appearance and yield. Increasing the diameter and weight of the fruit can not only improve tomato yield, but also enhance the visual aesthetics, increase the nutrient content in the fruit, and bring about a higher market and economy. value. Therefore, by mining new functional genes and using gene editing technology, it is a relatively quick way to breed new varieties of large-fruited tomato.

SUMMARY OF THE INVENTION

The technical problem to be solved by the present invention is how to effectively increase the size of tomato fruit, thereby increasing its fruit yield.

In order to solve the above technical problem, the present invention provides a tomato gene IFW1 (increase fruit size and w eight 1), the nucleotide sequence of which is shown in SEQ ID NO: 1.

The present invention also provides the use of the above-mentioned gene: knocking out the IFW1 gene in tomato can effectively increase (significantly increase) the size of its fruit, thereby increasing the yield.

As an improvement of the application of the IFW1 gene of the present invention in increasing tomato fruit: the two knockout lines of the IFW1 gene are ifw1-1 and ifw1-2; the IFW1 gene mutation sequences in the ifw1-1 and ifw1-2 plants are both SEQ ID NO: 2.

The present invention also provides a method for knocking out the IFW1 gene in tomato, comprising the following steps:

1) Using CRISPR/Cas9 technology to design the target sgRNA sequence for gene editing: 5'-GATAGAGGCAGAGGCAGAGG-3';

2) Synthesize primers using the sequences obtained in step 1), and construct them into the CRISPR/Cas9 vector;

3) Genetically transform the vector obtained in step 2) into a wild-type tomato variety MicroTom, thereby obtaining a corresponding transgenic plant; from the transgenic tomato plant, a plant with the IFW1 gene knocked out is identified.

The technical scheme of the present invention is as follows:

Using CRISPR/Cas9 gene editing technology, according to the nucleotide sequence of the IFW1 gene (SEQ ID NO: 1), a gRNA sequence that specifically targets the IFW1 gene was synthesized, the corresponding CRISPR/Cas9 vector was constructed, and genetically transformed into wild-type tomato In the variety MicroTom, the IFW1 gene in the genome was directionally edited to obtain transgenic plants. The IFW1 gene in the transgenic plants was PCR amplified and sequenced, and different knockout lines of the IFW1 gene were identified and obtained ifw1-1 and ifw1- 2 (Figure 1). The IFW1 gene mutation sequences in ifw1-1 and ifw1-2 plants are both SEQ ID NO: 2. By comparing the fruit types, the size (Figure 2, Figure 3) and weight of the mature fruit of the IFW1 gene knockout plants were significantly higher than those of the wild-type control variety (Figure 4), indicating that knocking out the IFW1 gene in tomato can effectively promote the increase of fruit. The synthesis of medium biomass has important breeding application value.

Description of drawings

The specific embodiments of the present invention will be further described in detail below with reference to the accompanying drawings.

Figure 1 shows the location and sequencing results of CRISPR/Cas9 target sites of tomato IFW1 gene knockout lines.

Figure 2 is a comparison diagram of the fruit type of the tomato IFW1 gene knockout line and the wild-type control MicroTom.

Figure 3 is the fruit size determination of the tomato IFW1 gene knockout line and the wild-type control MicroTom.

Figure 4 is the single fruit weight measurement of the tomato IFW1 knockout line and the wild-type control MicroTom.

In Figures 1 to 4, WT is the wild-type control variety MicroTom; ifw1-1 and ifw1-2 are two different knockout lines of the IFW1 gene. The values in Figure 3 and Figure 4 are the mean ± standard deviation, ** indicates that the t-test of the tomato IFW1 knockout line ifw1-1 (or ifw1-2) and the wild type (WT) control MicroTom is extremely significant ( P<0.01) difference.

detailed description

Step 1. Construction of tomato IFW1 gene knockout CRISPR/Cas9 vector

Using online professional software (http://crispr.mit.edu/), design the target sgRNA sequence edited by CRISPR/Cas9 in the coding sequence of IFW1 gene (SEQ ID NO: 1): 5'-GATAGAGGCAGAGGCAGAGG-3'; The corresponding primer sequences were synthesized in a biotechnology company: 5'-TGATTGATAGAGGCAGAGGCAGAGG-3' and 5'-AAACCCTCTGCCTCTGCCTCTATCA-3'. The corresponding CRISPR/Cas9 vector was constructed by CRISPR/Cas9 kit (Biogle, China), and the construction method was operated according to the product instructions.

Step 2. Genetic transformation of tomato constructed by CRISPR/Cas9 vector

The CRISPR/Cas9 vector constructed in step 1 was genetically transformed into the tomato variety MicroTom, and the transgenic method was based on the method of Kimura et al. (Kimura S et al, CHS Protoc, 2008) to obtain corresponding transgenic tomato plants.

Step 3. Sequencing analysis of IFW1 gene in transgenic tomato

Take 0.1 g of transgenic tomato leaves, grind them with liquid nitrogen, add 600 μl of extract (15.76 g Tris-cl, 29.22 g NaCl, 15.0 g SDS powder, add ultrapure water to 1 L, adjust pH=8.0), and incubate at 65°C for 60 min ; Add 200 μl 5M KAC, after mixing, ice bath for 10 min; add 500 μl chloroform, mix well, centrifuge at 10,000 rpm for 5 min; take the supernatant, add 500 μl isopropanol, mix, centrifuge at 12,000 rpm for 3 min, discard the supernatant; use 75 Wash the precipitate with 500 μl of % ethanol, centrifuge at 12,000 rpm for 3 min, and discard the supernatant; after inverting the DNA to dry for 15 min, add 30 μl of pure water to dissolve the DNA.

The upstream primer 5'-AACGTTCAACGGACAATC-3' and the downstream primer 5'-CAATAAAGTACACCACAT-3' were synthesized by PCR amplification of the IFW1 gene, using the genomic DNA of the transgenic tomato plant and its control variety MicroTom as the template, using 2 × Taq PCR Master Mix ( TIANGEN Company) carried out PCR amplification of the IFW1 gene. The PCR amplification system was 20 μl, containing 10 μl of 2×Taq PCR MasterMix, 1 μl of upstream and downstream primers (10 μM), 1 μl of template DNA (<1 μg), and 7 μl of sterile water; PCR amplification The incremental program was as follows: pre-denaturation at 94°C for 5 min; denaturation at 94°C for 30 sec, annealing at 55°C for 30 sec, extension at 72°C for 30 sec, 35 cycles; extension at 72°C for 10 min.

After sequencing and analysis of the PCR products, two lines, ifw1-1 and ifw1-2, successfully knocked out the IFW1 gene were identified. The coding region of the IFW1 gene was deleted by 2 bases in the plants of these two lines (Fig. 1). A frameshift mutation in the IFW1 gene results in loss of function of the gene. The nucleotide sequence of the IFW1 gene in the plants of the ifw1-1 and ifw1-2 lines is as described in SEQ ID NO:2.

Step 4. Determination of tomato fruit size

The IFW1 knockout lines ifw1-1 and ifw1-2 identified above and the wild-type control variety MicroTom were grown in a greenhouse at 25°C, 16h light, 8h dark. When the tomato fruits were mature, 3 plants of each variety were randomly selected, and 3 mature fruits were taken from each plant, and the stems were removed. Use vernier calipers to measure the longest diameter (cm) of each tomato fruit, calculate the average fruit diameter of each variety, and use t test to analyze the significance between the ifw1-1 (or ifw1-2) knockout line and the wild-type control. difference. The results obtained were that the fruit diameters of the IFW1 knockout lines ifw1-1 and ifw1-2 were significantly higher than those of the wild-type control (Figures 2 and 3), which were increased by 23.2% and 25.9%, respectively, compared with the control varieties.

Step 5. Determination of tomato fruit weight

Take the cultivated tomato fruit in step 4, weigh the weight of each tomato fruit (g) with an electronic balance, calculate the average fruit weight of each variety, and use the t test to analyze the results of the ifw1-1 (or ifw1-2) knockout line and wild. significant differences between controls. The results obtained were that the fruit weights of the IFW1 knockout lines ifw1-1 and ifw1-2 were significantly higher than those of the wild-type control (Fig. 4), which were 51.7% and 57.4% higher than the control varieties, respectively.

Finally, it should also be noted that the above enumeration is only a few specific embodiments of the present invention. Obviously, the present invention is not limited to the above embodiments, and many modifications are possible. All deformations that those of ordinary skill in the art can directly derive or associate from the disclosure of the present invention shall be considered as the protection scope of the present invention.

Claims

The application of a gene in tomato fruit enlargement, characterized in that: knocking out the IFW1 gene in tomato can increase the size of tomato fruit, thereby increasing yield;

The nucleotide sequence encoded by the gene IFW1 is shown in SEQ ID NO: 1.
The application of a gene according to claim 1 in increasing tomato fruit, wherein the two knockout lines of the IFW1 gene are ifw1-1 and ifw1-2; The IFW1 gene mutation sequences are all SEQ ID NO: 2.