Application of CsHT16 gene in controlling cucumber plant type and fruit shape

Abstract

This invention discloses the application of the CsHT16 gene in controlling cucumber plant architecture and fruit morphology. This invention belongs to the field of genetic engineering technology, through... CRISPR / Cas9 Technology for cucumbers CsHT16 Gene editing was performed, and mutants were further screened to obtain homozygous dwarf cucumber materials. The cucumber plants obtained by this invention have a dwarfed plant type, making full use of space, producing short-stemmed and robust fruits, increasing the edible portion, and have important application value.

Description

Technical Field

[0001] This invention belongs to the field of cucumber genetic engineering technology, specifically involving the use of the CRISPR / Cas9 system to edit the cucumber CsHT16 gene, thereby creating dwarf plant type and short fruit stem cucumber germplasm. Background Technology

[0002] Cucumber (Cucumis sativus L.) is a plant of the Cucurbitaceae family and one of the most widely cultivated vegetable crops in the world. Plant height has a significant impact on cultivation management and yield. Dwarf plant types have compact growth, adapt to dense planting, and can make full use of land and sunlight. Creating dwarf cucumber varieties without affecting yield is important for cultivating strong seedlings and preventing excessive vegetative growth. Shortening the cucumber stem without changing fruit yield and quality increases the edible portion, meeting market and consumer demands. The CsHT16 mutant cucumber has short and thick fruits with a shorter stem, which is of great significance for cultivating cucumbers with different appearance qualities.

[0003] CsHT16 encodes a cucumber hexose transporter. Hexose transporters are a subfamily of the plant monosaccharide transporter family, abbreviated as STP, MST, and HT in Arabidopsis, rice, and grape, respectively. Hexose transporters possess 12 transmembrane domains, which interact to form a central pore, allowing soluble hexoses to pass smoothly through the hydrophobic membrane (Büttner and Sauer, 2000). In Arabidopsis, there are 14 members of the STP subfamily, six of which (AtSTP2, AtSTP4, AtSTP6, AtSTP9, AtSTP10, AtSTP11) are primarily or specifically expressed and function at specific stages of anther or pollen development (Schneidereit et al., 2003, 2005). A similar finding has been observed in rice, where the OsMST subfamily of hexose transporters is homologous to the STPs in Arabidopsis. OsMST8 is mainly expressed in the early stages of male gametophyte development, from the villous mucilage cells and microspores to mature pollen (Oliver et al. 2007a). OSMST7, on the other hand, is mainly expressed in the pollen grains and anther walls during later development, but not in the villous mucilage (Oliver et al. 2007b). The functions of hexose transporters expressed in flowers are primarily related to pollen development or pollen tube elongation.

[0004] Besides the abundance of hexose transporters in flowers, a large number of hexose transporters have also been found in fruits. It is known that soluble sugars account for 65%–91% of the dry weight of ripe grapes, and most of these sugars are transported to the grape pulp via the apoplastic pathway during the later stages of ripening (Zhang et al., 2006). Studies using gene chip technology have analyzed some possible hexose transporters in grape storage tissues, showing that VvHT2, VvHT3, and VvHT11 are all members of the STP subfamily and have high expression levels during grape fruit set and ripening. This suggests that these transporters function as apoplastic unloading agents in the vascular bundles of the fruit (Afoufa et al., 2010). Other studies have shown that 70%–80% of the hexoses stored in ripe tomato fruits are transported via monosaccharide transporters (McCurdy et al., 2010). Although many hexose transporter genes have been identified and reported, mutations in a single gene are rarely found to cause significant phenotypic changes in the plant.

[0005] The applicant has discovered for the first time that the CsHT16 mutant cucumber has shorter and thicker fruits and a shorter stem. Therefore, the technical solution of this invention is of great significance for cultivating cucumbers with different appearance qualities. Summary of the Invention

[0006] The purpose of this invention is to provide the application of the CsHT16 gene in controlling cucumber plant architecture and / or fruit morphology, wherein the protein encoded by the CsHT16 gene is shown in SEQ ID NO.2.

[0007] Another object of the present invention is to provide the application of the CsHT16 gene in the preparation of dwarf cucumbers, wherein the protein encoded by the CsHT16 gene is shown in SEQ ID NO.2.

[0008] The final objective of this invention is to provide the application of the CsHT16 gene in the preparation of cucumbers with shortened stems, wherein the protein encoded by the CsHT16 gene is shown in SEQ ID NO.2.

[0009] To achieve the above objectives, the present invention adopts the following technical measures:

[0010] The scope of protection of this invention includes:

[0011] The application of the CsHT16 gene in controlling cucumber plant architecture and / or fruit morphology, wherein the protein encoded by the CsHT16 gene is shown in SEQ ID NO.2.

[0012] The application of the CsHT16 gene in the preparation of dwarf cucumber plants and / or cucumbers with shortened stems, wherein the protein encoded by the CsHT16 gene is shown in SEQ ID NO.2.

[0013] In the above-described applications, preferably, the gene is shown in SEQ ID NO.1.

[0014] The applications described above are specifically as follows:

[0015] Knockout, suppression, or silence of the gene encoding the CsHT16 protein in cucumber can dwarf the plant type and / or shorten the stem of cucumber fruit.

[0016] In the above-described applications, preferably, the knockout is performed using the CRISPR / Cas9 system, where the target site of the gRNA is GACACGTAAGTTCGGTCGT.

[0017] In the above-described applications, preferably, the edited cucumber contains the gene shown in SEQ ID NO.3.

[0018] Compared with the prior art, the present invention has the following advantages:

[0019] (1) The applicant disclosed for the first time that the cucumber CsHT16 gene can be used to create dwarf plant type and short fruit stem cucumber germplasm.

[0020] (2) Compared with traditional breeding methods, the creation method provided by this invention can quickly obtain CsHT16 mutant cucumber plants.

[0021] (3) This invention uses CRISPR / Cas9 gene editing technology to create cucumber CsHT16 mutant materials and obtains homozygous lines through self-pollination, which is more time-saving than traditional breeding. Attached Figure Description

[0022] Figure 1 Gene editing status of cucumber CsHT16 mutant;

[0023] The top image shows the gene editing type of the CsHT16 mutant, where the insertion of one base leads to a frameshift mutation in the gene sequence; the bottom image shows the detection results for whether the CsHT16 mutant still carries Cas9.

[0024] Figure 2 Seedling phenotypes of cucumber CsHT16 knockout mutant and wild type;

[0025] The figure above shows the statistical data on the relative chlorophyll content of the mutant leaves and the plant height (ht16 is the CsHT16 gene-edited mutant, and WT is the non-transgenic wild-type control).

[0026] The figure below shows a comparison of the phenotypes of mutant seedlings at the one-leaf-one-heart stage and the two-leaf-one-heart stage.

[0027] Figure 3 Plant height phenotype of adult cucumber CsHT16 knockout mutant;

[0028] The left figure shows a comparison of the phenotypes of the CsHT16 knockout mutant and the WT control adult plants.

[0029] The right figure shows the statistical data on the change in plant height from 3 to 18 days after transplanting (ht16 is the CsHT16 gene editing mutant, and WT is the non-transgenic wild-type control).

[0030] Figure 4 Fruit phenotypes of cucumber CsHT16 knockout mutant and wild type;

[0031] Where a is a photo of the fruit of the wt and ht16 mutants (large fruit stage, 16 days after flowering);

[0032] b represents the single fruit weight, single plant yield (3 fruits per plant), soluble solids content, fruit length, fruit diameter, and periphery length (16 days after flowering) of the wt and ht16 mutant fruits.

[0033] c represents the dynamic data of fruit development (DAA, days after flowering) for the wt and ht16 mutants;

[0034] d represents the cell state and size in the longitudinal section of the fruit (16 days after flowering). Detailed Implementation

[0035] Example 1:

[0036] CRISPR / Cas9 vector construction and Agrobacterium-mediated transformation

[0037] (1) Cloning of cucumber CsHT16 gene

[0038] Using cucumber 'Xintai Mici' as a template, total RNA was extracted from the leaves according to the instructions of the Trizol RNA Extraction Kit. cDNA was obtained by reverse transcription, and the coding sequence of CsHT16 (SEQ ID NO1) was amplified using the following specific primers:

[0039] CsHT16-F: 5'-ATGCCTGCTGTCGCCGCAATCGTCC-3'

[0040] CsHT16-R: 5'-TTAAACTGTCTTAATGATTTGATCTCC-3';

[0041] PCR reaction conditions: 94℃ pre-denaturation for 3 min, 94℃ denaturation for 30 s, 56℃ annealing for 30 s, 72℃ extension for 2 min, for a total of 35 cycles, and a final extension at 72℃ for 5 min.

[0042] (2) Construction of CsHT16-CRISP / Cas9 vector

[0043] The designed specific sgRNA fragment for CsHT16 is 5'-GACACGTAAGTTCGGTCGT-3'. The CsHT16 sgRNA was constructed into the gene-editing vector PKSE402 containing Cas9, and after sequencing, the knockout plasmid was obtained. This gene-editing vector carries an EGFP fluorescent tag, allowing for observation of GFP fluorescence under UV light to determine whether the transformed seedlings are positive.

[0044] (3) Agrobacterium-mediated transformation

[0045] In a clean bench, take 2 μL of the sequenced knockout plasmid and add it to 100 μL of EHA105 Agrobacterium competent cells, then mix thoroughly. After mixing, incubate on ice for 5 min, freeze in liquid nitrogen for 1 min, and then quickly place in a 37°C water bath for 5 min. Add 800 μL of LB medium and incubate at 28°C and 220 rpm on a shaker for 2 h. Centrifuge at 12000 rpm for 1 min to concentrate the bacterial culture, resuspend in 200 μL of LB liquid medium, mix well, and spread evenly on LB+Kana solid medium. Incubate in the dark at 28°C for 2 days. Pick out Agrobacterium single-clone plaques and shake in LB+Kana+RIF liquid medium for 12 h. Store the bacterial culture at -80°C for later use.

[0046] Example 2:

[0047] Genetic transformation of CsHT16 gene-edited cucumber seedlings

[0048] Using cucumber cotyledons as explants, cucumbers were transformed using Agrobacterium EHA105 to obtain T0 generation transgenic cucumbers.

[0049] The main steps are as follows:

[0050] (1) Take “Xintai Mici” cucumber seeds and soak them in 55℃ warm water for more than half an hour to remove the seed coat. In a clean bench, first wash with 75% alcohol for 30 seconds, then soak in 0.3% NaClO solution for 15 minutes, gently shaking during the process, and finally rinse 5 times with sterile water. Transfer the sterilized seeds to the prepared seed germination medium and incubate in a 28℃ oven in the dark for 24 hours.

[0051] (2) Take the germinated seeds, cut off about 1 / 3 of the cotyledons at the far end in a clean bench, remove the hypocotyl, separate the two cotyledons, and each cotyledon will form a U-shaped wound at the near end. Make a small dot at the U-shaped opening with a knife to obtain the explant.

[0052] (3) One day in advance, pick a single colony of Agrobacterium EHA105 carrying the CsHT16 knockout plasmid and place it in 10 mL of LB medium containing 50 mg / L Kana and 50 mg / L Rif, and incubate overnight at 28°C and 200 rpm with a shaker. When the bacterial culture grows to OD... 600 When the concentration of bacteria is 0.4-0.8, centrifuge at 6000 rpm for 8 min, collect Agrobacterium cells, resuspend the cells in 1M liquid medium, and dilute to OD200. 600 0.2, sonicate in a 100W water bath for 30 seconds. Remove the plunger from the syringe, add the cotyledonary explant to a 20mL syringe barrel, and draw more than 10mL of Agrobacterium tumefaciens solution through the needle hole to expel excess air until the plunger stops at the 10mL mark. Seal the needle hole at the syringe tip with a rubber stopper, and slowly pull the plunger backward while gently shaking the syringe to distribute the pressure evenly on each explant. Hold the plunger at the 20mL mark for 1.5 minutes to apply a vacuum. Release the pressure gently; the plunger will slowly return to the 10mL mark. Repeat once more.

[0053] (4) After infection, the plants were cultured at 24°C in the dark for 4 days and then transferred to differentiation medium to induce bud growth. After multiple screenings, several fluorescent buds were obtained (the fluorescent buds can be preliminarily identified as positive buds). However, only one fluorescent bud successfully rooted, grew and bore fruit after tissue culture.

[0054] Example 3:

[0055] Identification of gene editing type in positive cucumber plants

[0056] (1) Extraction of DNA from transgenic cucumber plants and detection of positive single plants

[0057] DNA was extracted from the transgenic cucumber leaves in Example 2 using the CTAB method. The extracted DNA was used as a template, the knockout plasmid DNA constructed in Example 1 was used as a positive control, and the recipient material DNA was used as a negative control. Transgenic positive single plants were detected using the following primers:

[0058] Hi-HT16-F 5'-ggagtgagtacggtgtgcCGATGTTCACGTCGTCGTTGTA-3'

[0059] Hi-HT16-R 5'-gagttggatgctggatggTTAGCAAAGCCATTAATGAT-3' (Note: lowercase letters are primer adapters for Hi-tom sequencing).

[0060] Using the DNA from the positive fluorescent seedlings grown in Example 1 as a template, PCR amplification was performed, and the PCR products with bands identified by gel electrophoresis were sent to the company for Hi-tom sequencing. The sequencing results revealed the editing type as follows: Figure 1 As shown, the mutated sequence is shown in SEQ ID NO 3.

[0061] Cas9 detection was performed on the T2 generation mutant lines. PCR amplification was performed using DNA from the CsHT16 mutant plant as a template, and the bands were observed by gel electrophoresis. The amplified fragment length was approximately 500 bp. Figure 1 As shown in the gel image, plants without bands are mutant plants lacking the Cas9 editing marker. The PCR amplification primers used were:

[0062] Cas9-F 5'-CTCCCTAAGCACTCGCTCCTG-3'

[0063] Cas9-R 5'-TTGTCCCGAATGCCGTTGAT-3'

[0064] After four generations of self-pollination, homozygous CsHT16 gene-editing lines (excluding the editing marker Cas9) were obtained.

[0065] Example 4:

[0066] Phenotypic observation of dwarfing plant type and short and thick fruit traits in homozygous mutant cucumbers

[0067] To further determine the effects of CsHT16 transgenic cucumber on plant dwarfing and short fruit, the homozygous mutants in Example 3 were compared with wild-type cucumber plants at the seedling and mature stages. Figure 2 It can be seen that, compared with the wild type, the hypocotyl of the homozygous mutant was significantly shortened by 15.11 cm at the one-leaf-one-heart and two-leaf-one-heart stages of the seedling stage, and the relative chlorophyll content was significantly increased by 4.36 SPAD.

[0068] like Figure 3 As shown, comparing the plant height of the CsHT16 mutant and the wild-type plant, the mutant plants were significantly dwarfed to varying degrees compared to the wild-type plants from 3 to 18 days after transplanting. Specifically, the wt plants were 11.84 cm taller than the ht16 plants at 3 days, 7.42 cm taller at 6 days, 11.00 cm taller at 9 days, 15.34 cm taller at 12 days, 22.36 cm taller at 15 days, and 23.10 cm taller at 18 days.

[0069] like Figure 4 As shown in Figure d, from pollination to fruit growth (22 days), the mutant cucumber showed no significant difference in single fruit weight, yield (3 fruits per plant), and soluble solids compared to the wild type. However, the fruit length was significantly shortened during the growth process. 16 days after pollination, the diameter of the HT16 fruit was significantly increased by 0.48 cm compared to the WT fruit, the circumference was significantly increased by 1.68 cm, and the stem length was significantly shortened by 0.47 cm. As shown in Figure d, microscopic observation of longitudinal sections of the fruit at 16 days showed that the shortening of longitudinal cells in the HT16 fruit was the main factor leading to the shortening of the fruit, while the increase in the number of transverse cells was the main factor leading to the thickening of the fruit.

[0070] This invention provides a method for creating dwarf cucumber materials. The method involves preparing cucumber plants with the target gene edited according to the above method, and then performing self-pollination on the gene-edited cucumber plants to create dwarf cucumber germplasm with short and thick fruit shape.

Claims

1. Use of a gene encoding a protein of CsHT16 in cucumber for inhibiting the plant type of a dwarf cucumber and / or shortening the peduncle of a cucumber fruit, wherein the protein encoded by the gene is as shown in SEQ ID NO.

2. CsHT16 ​ 2. The use according to claim 1, wherein the gene is as shown in SEQ ID NO.

1.

3. The use according to claim 1, wherein the inhibition is in the form of knock-out, and the CRISPR / Cas9 system is used.

4. The use according to claim 3, wherein the target site of gRNA in the system is GACACGTAAGTTCGGTCGT.

5. The use according to claim 4, wherein the cucumber obtained in the process contains the gene as shown in SEQ ID NO. 3.

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