Application of the SlZHD11 gene in regulating crop yield and flavor quality

Abstract

This invention discloses a SlZHD11 The application of genes in regulating crop yield and flavor quality falls under the field of genetic engineering technology. This invention utilizes CRISPR / Cas9 gene editing technology to insert endogenous genes from tomatoes... SlZHD11 Knockout in tomatoes significantly increases fruit size, yield, and sugar content. Without affecting the normal growth of the tomato plant, it improves both fruit yield and quality, providing genetic resources and strategies for tomato germplasm innovation and better meeting the needs of industry and consumers.

Description

TECHNICAL FIELD

[0001] The application belongs to the technical field of genetic engineering, and particularly relates to a kind of SlZHD11 Application of a gene in regulating crop yield and flavor quality. BACKGROUND

[0002] Tomato is an important economic crop, and is widely cultivated in the world as a fresh fruit vegetable due to its delicious fruit, rich nutrition and unique flavor. Fruit size is an important agronomic trait, which is closely related to the yield of horticultural crops. Sugar is a very important component in determining the formation of tomato fruit flavor quality, for example, about 50% of the dry matter in tomato fruit is sugar, mainly including sucrose, fructose and glucose, the three kinds of soluble sugar. Therefore, flavor quality and yield are the most important fruit quality characteristics of tomato, which directly affect the nutritional value and commodity value of the fruit.

[0003] However, in recent years, tomato breeding has paid too much attention to yield, appearance and other commodity traits. Although the yield of the fruit has been improved, the important gene locus controlling the flavor quality of the fruit has been lost, resulting in a significant decrease in the taste and flavor quality of the tomato, leading to the problem of poor flavor quality of high-yield tomatoes and low yield of good flavor quality tomatoes, which restricts the development of the industry. Therefore, in view of the industrial demand for changing from focusing on yield growth to paying attention to quality and quality of the fruit and vegetable industry in China, how to use genetic engineering technology to improve the flavor quality and yield of the fruit is an important goal of the breeding of horticultural crops in China. At present, traditional breeding methods are time-consuming and unstable, and often have great blindness and unpredictability. Therefore, using biological technology to improve the yield and quality of the fruit will be an important development direction of modern tomato breeding.

[0004] Some scholars have found that using overexpression technology, the cell proliferation can be inhibited by processing MIF gene, thereby affecting the development of flower meristem and ovule of tomato. In addition, some scholars have used RNA interference technology to down-regulate the expression of SlMIF2 Another name of SlZHD11 gene causes abnormal development of pistil in inflorescence, resulting in multi-chambered fruit. At the same time, using gene editing technology to edit the sequence of SlMIF2 promoter region causes some lines to also have the phenotype of multi-chambered fruit. The existing technology mainly focuses on analyzing the function of SlZHD11 gene in regulating inflorescence development, and does not explain other functions of SlZHD11 gene. Therefore, on the basis of avoiding off-target editing effect, how to edit SlZHD11 gene to regulate the size, yield and sugar content of tomato fruit is a technical problem to be solved at present. SUMMARY

[0005] The technical problem solved by the present application is to provide a kind of SlZHD11 The application of the gene in regulating the yield, total soluble solids, fructose and glucose content of tomato fruit solves the technical problem that the yield and flavor quality of tomato cannot be improved simultaneously.

[0006] To achieve the above-mentioned purpose, the technical scheme adopted by the present application is to provide a kind of SlZHD11 The application of the gene in regulating the yield, total soluble solids, fructose and glucose content of tomato fruit, SlZHD11 The nucleotide sequence of the gene is shown in SEQ ID NO: 1.

[0007] On the basis of the above-mentioned technical scheme, the present application can also be improved as follows:

[0008] Further, SlZHD11 The amino acid sequence of the protein encoded by the gene is shown in SEQ ID NO: 2.

[0009] Further, by inhibiting or knocking out SlZHD11 The gene, the yield, total soluble solids, fructose and glucose content of tomato fruit are regulated.

[0010] Further, by inhibiting or knocking out SlZHD11 The gene, the fruit size, single fruit weight and fruiting amount of tomato are improved, and the sugar content of tomato fruit is also improved.

[0011] Further, by inhibiting or knocking out SlZHD11 The gene, the total soluble solids, fructose and glucose content of tomato fruit are improved.

[0012] The beneficial effects of the present application are: by using CRISPR / cas9 gene editing technology, the endogenous gene SlZHD11 In tomato, the knockout of the gene significantly increases the size of the tomato fruit, the yield of the fruit, and the sugar content, thereby achieving the effect of simultaneous improvement of the yield and flavor quality of the tomato fruit, providing gene resources and strategies for innovation of tomato germplasm resources, and better meeting the needs of consumers. BRIEF DESCRIPTION OF DRAWINGS

[0013] Figure 1 The FastCas98 expression vector map is constructed;

[0014] Figure 2 The positive identification results of the CRISPR / cas9 gene knockout plant of the gene are shown in the following table: SlZHD11

[0015] Figure 3 The gene editing schematic diagram of the gene knockout line is shown in the following figure: SlZHD11 The gene knockout line of the gene​

[0016] Figure 4 Wild type tomato and SlZHD11 Statistical chart of fruit size of wild type tomato and

[0017] Figure 5 Wild type tomato and SlZHD11 Statistical chart of fruit diameter of wild type tomato and

[0018] Figure 6 Wild type tomato and SlZHD11 Statistical chart of fruit weight of wild type tomato and

[0019] Figure 7 Wild type tomato and SlZHD11 Statistical chart of fruit yield of wild type tomato and

[0020] Figure 8 Wild type tomato and SlZHD11 Statistical chart of total soluble solids content of wild type tomato and

[0021] Figure 9 Wild type tomato and SlZHD11 Statistical chart of fructose content of wild type tomato and

[0022] Figure 10 Wild type tomato and SlZHD11 Statistical chart of glucose content of wild type tomato and DETAILED DESCRIPTION

[0023] The specific embodiments of the present application are described below to enable those skilled in the art to understand the present application, and the specific conditions are not indicated in the examples, and the conventional conditions or the conditions suggested by the manufacturer are used, and the reagents or instruments are not indicated by the manufacturer, and are all conventional products that can be purchased in the market. However, it should be clear that the present application is not limited to the scope of the specific embodiments, and for those skilled in the art, it is obvious that various changes are within the spirit and scope of the present application defined and determined by the appended claims, and all the inventions using the concept of the present application are within the scope of protection.

[0024] SlZHD11 The nucleotide sequence of the gene is as follows:

[0025] ATGAAAAAAGTTTTGAGGAGAAATGATTACTCAAGAAATTCTACAAATTCATCCTTTACCATGAGGAGAGTGAGATATGTTGAGTGCCAGAGAAATCATGCTGCTAGTGTCGGTGGATACGTTATCGATGGGTGCCGGGAGTACATGCCCGAGGGCACTACTTCCGGCACCCTAAATTGTGCAGCCTGTGGCTGCCACCGCAATTTCCACAGAAGGGAAGTGGAAACTGATGTTGCTTCTGAATGTACTTCCGCTTCTTCTACTACTAAATGA (SEQ ID NO: 1).

[0026] SlZHD11 The amino acid sequence of the protein encoded by the gene is as follows:

[0027] MKKVLRRNDYSRNSTNSSFTMRRVRYVECQRNHAASVGGYVIDGCREYMPEGTTSGTLNCAACGCHRNFHRREVETDVASECTSASSTTK (SEQ ID NO: 2).

[0028] The primer sequences used in the following examples are shown in Table 1.

[0029] Table 1 Primer sequence table

[0030]

[0031] Example 1 Construction SlZHD11 CRISPR / cas9 gene knockout system of the gene

[0032] 1. Design SlZHD11 Target sequence of the gene

[0033] The target site design was performed using the website http: / / chopchop.cbu.uib.no / . In the results of the target site design, the sequence, position, GC content, potential off-target sites and other related information of all candidate target sites can be evaluated, and the target site with GC content in the range of 45% to 70%, position in the first 2 / 3 region of the CDS of the gene (after ATG, but not on the last exon), and at least three base mismatches with any other position in the genome was selected.

[0034] 2. Construction SlZHD11 CRISPR / cas9 vector of the gene

[0035] (1) Construction SlZHD11CRISPR / cas9 vector primer design of gene

[0036] ZHD11 Cas9 -F: TCTCACTCTCCTCATGGTAA (reverse complementary sequence of gRNA1) TTACCATGAGGAGAGTGAGA (SEQ ID NO: 5);

[0037] ZHD11 Cas9 -R: CAAATTCATCCTTTACCATG (gRNA2) CATGGTAAAGGATGAATTTG (SEQ ID NO: 6).

[0038] (2) Amplification of target fragment: the amplification system is shown in Table 2.

[0039] Table 2 PCR amplification system

[0040]

[0041] The amplification procedure is: 98℃ 3min; (98℃ 10s, 58℃ 20s, 72℃ 30s) 30 cycles; 72℃ 5min; 4℃ storage.

[0042] (3) FastCas9 vector enzyme digestion and fragment ligation: the ligation system is shown in Table 3.

[0043] Table 3 FastCas9 vector enzyme digestion and fragment ligation system

[0044]

[0045] The ligation procedure is: 37℃, 2min; 16℃, 5min. 6 cycles.

[0046] The FastCas98 expression vector map obtained is shown in Figure 1 .

[0047] Example 2 Obtain SlZHD11 Gene knockout line plant

[0048] 1. Screening of engineering bacteria

[0049] (1) Transformation of E. coli

[0050] Take CRISPR / cas9 terminal vector 10 μL into DH5α competent, ice bath 30 min, then 42℃ heat shock 45 s, then ice bath 2 min, add 500 μL of non-resistant LB culture solution, shake bacteria in 37℃ shaker for 1 h, then centrifuge at 6000 rpm for 1 min, discard supernatant, resuspend 50 μL of supernatant, plate with LB medium containing 50 μg / mL kanamycin, 37℃ inverted culture for 16 h. Select single colony strain, then perform PCR amplification identification on its colony, sequence positive strain, and extract plasmid from correct sequencing result.

[0051] (2) Transformation of Agrobacterium

[0052] Take 300 ng of plasmid containing the final transformation vector, mix with 50 μL of Agrobacterium competent GV3101, and gently mix on ice for 5 min, then sequentially stand in liquid nitrogen for 5 min, 37℃ water bath for 5 min, ice for 5 min, and finally add 500-600 μL of non-antibiotic liquid LB, incubate at 28℃ in a shaker for 2-3 h; centrifuge at 6000 rpm for 1 min. Resuspend 50 μL of supernatant, plate with LB medium containing 50 μg / mL kanamycin and 100 μg / mL rifampicin resistance, and incubate at 28℃ for 2-3 days to obtain Agrobacterium containing recombinant plasmid.

[0053] 2. Transform tomato explants with Agrobacterium containing recombinant plasmid by leaf disc method

[0054] (1) Seed disinfection

[0055] Put wild-type tomato seeds into a sterile container, immerse in 70% ethanol solution for 30 s to disinfect the surface of the seeds; pour out the ethanol solution and then rinse the seeds twice with sterile water, immerse in 5% sodium hypochlorite solution for 15 min, shake constantly during the period to ensure that each seed is disinfected sufficiently; after washing with sterile water for 3-4 times, pour out the water in the seeds, transfer the seeds to seed culture medium, and place in a light incubator, with culture conditions being: 25℃ light incubation for 14 h, 20℃ dark incubation for 10 h; light intensity being 250 μmol•m -2 •s -1 ; relative humidity being 80%; culture period being 8-10 days.

[0056] (2) Pre-culture of explants

[0057] After the seeds germinate for about 9-10 days, when the true leaves of the tomato seedlings are about to grow, it is the best time to obtain explants, place the seedlings on sterile filter paper, cut the cotyledon and hypocotyl with a sterilized scalpel, and then transfer to KCMS pre-culture medium for dark culture for one day.

[0058] (3) Agrobacterium infection of explants

[0059] In 20 mL LB medium (containing 20 μL, 50 μg / mL kanamycin and 40 μL, 100 μg / mL rifampicin), 100 μL of preserved Agrobacterium tumefaciens GV3101 was added, and cultured at 28°C, 230 rpm overnight until the optical density OD 600 = 0.8-1.0. After 1 mL of bacterial solution was taken and centrifuged at 5000 rpm for 5 min, the supernatant was discarded, the precipitate was washed once with KCMS liquid medium, and the bacterial solution was diluted to OD 600 = 0.1 with KCMS liquid medium. A drop of Agrobacterium dilution was added to the wound of the explant using a pipette, and the bacterial solution was not sucked out. After the plate was sealed and placed on KCMS medium for dark culture for 2 days.

[0060] (4) Differentiation and rooting of explants

[0061] After the explants were co-cultured with Agrobacterium for 2 days, the explants were transferred from KCMS medium to primary screening medium (2Z) added with 20 μM trans-zeatin riboside, and the primary screening medium was replaced every 15 days. When differentiated bud points grew out of the explants, the explants were transferred to subculture medium (1Z) added with 10 μM trans-zeatin riboside, and the medium was replaced every 15 days. After two times of culture in the subculture medium, the differentiated callus grew out of bud bodies with independent main stems, which were cut off and inserted into rooting medium (ENR medium), and transplanted after rooting.

[0062] (5) Identification of transgenic positive plants

[0063] The transgenic T0 generation seedlings were detected using the CRISPR / Cas9 carrier detection primers Cas9-F (SEQ ID NO: 3) / Cas-R (SEQ ID NO: 4) to confirm whether they were transgenic positive plants, and the detection results are shown in Figure 2 .

[0064] As shown in Figure 2 , PCR amplification was performed using the genomic DNA of T0 generation seedlings as a template, and a 500 bp fragment was detected by electrophoresis to determine that the plant was a transgenic positive plant.

[0065] Subsequently, the genomic DNA of T0 generation seedlings was used as a template, and detection primers containing target sites ZHD11 -Cas9check-F (SEQ ID NO: 7) / ZHD11-Cas9 check-R (SEQ ID NO: 8) was used for PCR amplification. After agarose gel electrophoresis, the target band was recovered, and the purified PCR product was constructed into the pEASY-Blunt-Zero cloning vector for single-clone sequencing. Twenty single clones were sequenced from each transgenic positive plant. The sequencing results were compared with the wild-type (WT) sequence to determine the editing status of the T0 generation plants. Figure 3 As shown, the selected SlZHD11 Gene knockout lines ( SlZHD11 -KO-#1 and SlZHD11 The mutation modes of -KO-#13 are deletion of 13 bases and insertion of 1 base.

[0066] Seeds from the T0 generation that were sequenced as heterozygous were selected and screened with kanamycin before being sown again. The editing status of the T1 generation plants was analyzed (using the same editing detection method as the T0 generation plants). Seeds from 2-3 homozygous lines with different editing forms were collected, screened with kanamycin, and sown into the T2 generation for subsequent research.

[0067] Example 3 Knockout SlZHD11 The effect of gene on plant yield

[0068] (1) Take 5 wild-type (WT) and 5 wild-type plants each. SlZHD11 Gene knockout lines ( SlZHD11 -KO-#1 and SlZHD11 -KO-#13), artificially pollinate it, and cultivate it until the fruits of the first three flower clusters are completely broken in color. Then, weigh and measure the fruits of the first three flower clusters to count the fruit yield.

[0069] (2) Take more than 10 fruits from each strain for tracking, and record the fruit diameter at each stage using vernier calipers, with a measurement interval of 5 days.

[0070] The results are as follows Figure 4 and Figure 5 As shown. From Figure 4 The comparison of the actual products revealed SlZHD11 Gene knockout lines ( SlZHD11 -KO-#1 and SlZHD11 The fruit size and diameter of the -KO-#13 were significantly larger than those of the wild type (WT). Figure 5 The statistical chart of fruit diameter data also verifies this conclusion. SlZHD11 The individual fruit weight of the gene knockout line was also significantly higher than that of the wild type. Figure 6 ),and SlZHD11 The fruit yield of the gene knockout line increased by approximately 18%. Figure 7 ).

[0071] Example 4 Knockout SlZHD11Effects of gene modification on sugar content in tomato fruit

[0072] (1) Detection of soluble solids content

[0073] Harvest wild-type (WT) and SlZHD11 Gene knockout lines ( SlZHD11 -KO-#1 and SlZHD11 The red-ripe fruit of -KO-#13 was washed and placed in gauze to squeeze out the juice. The juice was then transferred to a clean container, and the sugar and acidity of the crude juice extract were measured using a handheld digital saccharimeter (ATO).

[0074] (2) Determination of soluble sugar content in fruit

[0075] Collect wild-type (WT) and red-ripe wild-type fish. SlZHD11 Fruit tissue from the gene knockout line was flash-frozen in liquid nitrogen and ground into powder. 0.1 g of sample was weighed and mixed thoroughly with 1.4 mL of pre-chilled methanol to extract soluble sugars from the tomato fruit. After centrifugation, the supernatant was transferred to a new centrifuge tube, and pre-chilled distilled water was added to bring the volume to 3 mL. 750 μL of pre-chilled chloroform was added, and the mixture was vortexed thoroughly. After centrifugation for 10 min, the supernatant was transferred to a new centrifuge tube. The sample was derivatized using methoxyamine hydrochloride and pyridine, with 20 μL of ribitol (2 mg / mL) added as an internal standard. The derivatized sample was filtered through an organic phase filter membrane. The sample could be directly analyzed or stored at -80℃.

[0076] Soluble sugar content was determined using a Shimadzu GC2010pro gas chromatograph. The instrument parameters were as follows: SPL temperature 250℃, optimal split ratio 10:1; column initial temperature 130℃, increased to 185℃ at a rate of 5℃ / min, increased to 190℃ at a rate of 0.5℃ / min, increased to 300℃ at a rate of 8℃ / min, and held for 10 min; column flow rate 1 mL / min. Detector temperature was set to 320℃, and hydrogen, dry air, and nitrogen flow rates were 40 mL / min, 400 mL / min, and 40 mL / min, respectively.

[0077] The results are as follows Figures 8-10 As shown in the figure. It can be seen from the figure that... SlZHD11 Gene knockout fruit ( SlZHD11 -KO-#1 and SlZHD11 The total soluble solids content of -KO-#13 was significantly higher than that of the wild type (WT). Figure 8 (The red-ripe period) SlZHD11 The fructose and glucose content of the fruit of the gene knockout line was significantly increased. Figure 9 and Figure 10 The sugar content increases by about 25%.

[0078] In summary, this demonstrates that gene editing technology can be used to knock out the gene in tomatoes. SlZHD11 Genes can significantly increase tomato fruit size and yield. In addition, knocking out… SlZHD11 The gene also increased the sugar content of tomato fruit, thereby simultaneously improving fruit yield and flavor quality. This provides a valuable reference for innovation in tomato germplasm resources, aiming to better meet the needs of industry and consumers.

Claims

1. A kind SlZHD11 The application of genes in regulating tomato fruit yield, total soluble solids, fructose, and glucose content is characterized by, By inhibiting or knocking out SlZHD11 the gene, the fruit size and single fruit weight of tomato are increased, and the total soluble solids, fructose and glucose contents of tomato fruit are increased; the nucleotide sequence of the SlZHD11 gene is shown as SEQ ID NO: 1.

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