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

Abstract

The invention discloses an application of a CsHT16 gene in control of cucumber plant type and fruit appearance. The invention belongs to the technical field of gene engineering, gene editing is carried out on a cucumber CsHT16 gene through a CRISPR / Cas9 technology, a mutant is obtained through further screening, and a homozygous editing cucumber dwarfing material is obtained through offspring screening. The obtained cucumber plant is dwarfed in plant type, space is fully utilized, short-stem thick and strong fruits are obtained, edible parts are increased, and the method has important application value.

Description

Technical Field

[0001] The present invention belongs to the technical field of cucumber genetic engineering, and specifically relates to the editing of cucumber CsHT16 gene by using the CRISPR / Cas9 system, so as to create cucumber germplasms with dwarf plant types and short fruit stalks. Background Art

[0002] Cucumber (Cucumis sativus L.) is a plant of the Cucurbitaceae family and is one of the important vegetable crops widely cultivated in the world. Plant height has an important impact on cultivation management and yield. Dwarf plant types grow compactly, are suitable for close planting, can make full use of land and light. Without affecting the cucumber yield, creating cucumber varieties with dwarf plant types plays an important role in cultivating strong seedlings and preventing excessive growth in production. Without changing the fruit yield and quality, shortening the fruit stalk of cucumber fruits and increasing the edible part meet the market and consumer demands. The CsHT16 mutant cucumber fruits are short and thick, and the fruit stalks are short, which is of great significance for cultivating cucumbers with different appearance qualities.

[0003] CsHT16 encodes a hexose transporter in cucumber. Hexose transporters are a subgroup of the plant monosaccharide transporter superfamily, which are abbreviated as STP, MST, and HT in Arabidopsis thaliana, rice, and grape respectively. Hexose transporters have 12 transmembrane domains, and the transmembrane domains interact with each other to form a central pore, so that soluble hexoses can pass through the hydrophobic membrane smoothly (Büttner and Sauer, 2000). There are a total of 14 STP subgroup members in Arabidopsis thaliana, and 6 of them (AtSTP2, AtSTP4, AtSTP6, AtSTP9, AtSTP10, AtSTP11) are mainly or specifically expressed and function at specific stages of anther or pollen development (Schneidereit ect. 2003, 2005). A similar situation was also found in rice. The hexose transporter subgroup OsMSTs in rice is homologous to STPs in Arabidopsis thaliana. OsMST8 is mainly expressed in the tapetum cells and microspore stage until mature pollen in the early stage of male gametophyte development (Oliver et al. 2007a). While OSMST7 is mainly expressed in pollen grains and anther walls in the later stage of development, but not in the tapetum (Oliver et al. 2007b). The functions of hexose transporters expressed in flowers are more related to pollen development or pollen tube elongation.

[0004] In addition to the large number of hexose transporters present in flowers, a large number of hexose transporters have also been found in fruits. It is known that the soluble sugar content in mature grapes accounts for 65% - 91% of their dry weight, and most of these sugars are transported to the grape pulp through the apoplast pathway during the late stage of grape ripening (Zhang et al., 2006). Some possible hexose transporters in the grape sink tissue were analyzed by gene chip technology, indicating that VvHT2, VvHT3, and VvHT11 in grapes are all members of the STP subfamily, and they have relatively high expression levels during the process from fruit set to maturity in grapes. Thus, it can be speculated that these transporters play a role in apoplast unloading in the fruit vascular tissue (Afoufa et al., 2010). Another study showed that 70% - 80% of the hexoses stored in mature tomato fruits are transported in through monosaccharide transporters (McCurdy et al., 2010). Although many hexose transporter genes have been identified and reported, mutations in their single genes are rarely found to cause obvious phenotypic changes in plants.

[0005] The applicant first discovered that the fruits of the CsHT16 mutant cucumber are shorter and thicker, and the fruit stalks are shorter. Therefore, the technical solution of the present invention is of great significance for cultivating cucumbers with different appearance qualities. Summary of the Invention

[0006] The object of the present invention is to provide the application of the CsHT16 gene in controlling the cucumber plant type and / or fruit shape, and the protein encoded by the CsHT16 gene is shown as SEQ ID NO.2.

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

[0008] The last object of the present invention is to provide the application of the CsHT16 gene in preparing cucumbers with shortened fruit stalks, and the protein encoded by the CsHT16 gene is shown as SEQ ID NO.2.

[0009] In order to achieve the above object, the present invention takes the following technical measures:

[0010] The protection scope of the present invention includes:

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

[0012] Application of CsHT16 gene in preparing cucumber with dwarf plant type and / or shortened pedicel of cucumber fruit, wherein the protein encoded by the CsHT16 gene is shown as SEQ ID NO.2.

[0013] For the above application, preferably, the gene is shown as SEQ ID NO.1.

[0014] The above application specifically is:

[0015] Knocking out, inhibiting or silencing the coding gene of CsHT16 protein in cucumber to dwarf the plant type of cucumber and / or shorten the pedicel of cucumber fruit.

[0016] In the above application, preferably, the knocking out adopts the CRISPR / Cas9 system, and the target site of gRNA in the system is GACACGTAAGTTCGGTCGT.

[0017] In the above application, preferably, the obtained edited cucumber contains the gene shown as SEQ ID NO.3.

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

[0019] (1) The applicant firstly discloses that the cucumber CsHT16 gene can be used to create germplasm of cucumber with dwarf plant type and short fruit pedicel.

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

[0021] (3) The present invention uses the CRISPR / Cas9 gene editing technology to create cucumber CsHT16 mutant materials, and obtains homozygous lines by self-crossing, which is more time-saving than traditional breeding. Description of the Drawings

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

[0023] Among them, the upper figure is: gene editing type of CsHT16 mutant, the insertion of one base causes frameshift mutation of the gene sequence; the lower figure is: detection result of whether Cas9 is still carried in the CsHT16 mutant.

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

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

[0026] The following figure shows the comparison of plant phenotypes of mutants at the seedling stage (one-leaf-one-heart stage and two-leaves-one-heart stage).

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

[0028] Among them, the left figure shows the comparison of adult plant phenotypes between CsHT16 knockout mutant and WT control

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

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

[0031] Among them, a is the fruit photos of wt and ht16 mutants (big fruit stage, 16 days after flowering);

[0032] b is the single fruit weight, yield per plant (3 fruits per plant), soluble solid content of fruit, fruit length, fruit diameter and peripheral length of wt and ht16 mutant fruits (16 days after flowering);

[0033] c is the dynamic data of fruit development of wt and ht16 mutant fruits (DAA, days after anthesis);

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

[0035] Example 1:

[0036] Construction of CRISPR / Cas9 vector and Agrobacterium transformation

[0037] (1) Cloning of cucumber CsHT16 gene

[0038] Using cucumber "Xintaimici" as a template, total RNA of leaves was extracted according to the operation method of the Trizol RNA extraction kit instructions, and cDNA was obtained by reverse transcription. The coding sequence of CsHT16 (SEQ ID NO1) was amplified using the following specific primers. The primers are:

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

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

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

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

[0043] The designed specific sgRNA fragment of CsHT16 is 5'-GACACGTAAGTTCGGTCGT-3'. The sgRNA of CsHT16 was constructed into the gene editing vector PKSE402 with Cas9. After sequencing, the knockout plasmid was obtained. This gene editing vector carries an EGFP fluorescent tag, and the seedlings transformed can be judged whether they are positive seedlings by observing the GFP fluorescence under ultraviolet light.

[0044] (3) Agrobacterium transformation

[0045] Take 2 μL of the sequenced knockout plasmid in a laminar flow hood, add it to 100 μL of Agrobacterium tumefaciens EHA105 competent cells and pipette to mix evenly. After mixing, ice-bath for 5 min, put it into liquid nitrogen for 1 min, and then quickly place it in a 37°C water bath for 5 min. Add 800 μL of LB medium, and culture it on a shaker at 28°C and 220 rpm for 2 h. Centrifuge at 12000 rpm for 1 min to concentrate the bacterial solution, add 200 μL of LB liquid medium to resuspend, mix evenly, and spread it evenly on the solid medium of LB+Kana, and culture it in the dark at 28°C for 2 days. Pick out the Agrobacterium monoclonal colonies, shake the bacteria in LB+kana+rif liquid for 12 h, and store the bacterial solution in a -80°C ultra-low temperature refrigerator for later use.

[0046] Example 2:

[0047] Genetic transformation of cucumber seedlings with CsHT16 gene editing

[0048] Using cucumber cotyledons as explants, mediated by Agrobacterium tumefaciens EHA105, T0 generation transgenic cucumbers were obtained.

[0049] The main steps are as follows:

[0050] (1) Take cucumber seeds of "Xintaimici", soak them in warm water at 55°C for more than half an hour to remove the seed coat. In a laminar flow hood, first wash with 75% alcohol for 30 s, then soak in 0.3% NaClO solution for 15 min, gently shake during this period, and finally wash with sterile water 5 times. Transfer the disinfected seeds to the pre-prepared seed germination medium and place them in a 28-degree oven for dark culture for 24 h.

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

[0052] (3) One day in advance, pick a single colony of positive 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 shake it overnight at 28°C and 200 rpm. When the bacterial solution grows to OD 600 When OD = 0.4-0.8, centrifuge at 6000 rpm for 8 min to collect Agrobacterium, resuspend the cells in IM liquid medium and dilute to OD 600 0.2, ultrasonic in a water bath with a power of 100W for 30s. Remove the piston core rod of the syringe, add the cotyledon explants to the syringe barrel of a 20mL syringe, draw more than 10mL of Agrobacterium liquid from the needle hole, and expel excess air until the piston is pushed to the 10mL scale position and stops. Seal the needle hole at the head of the syringe with a rubber plug, slowly pull the piston core rod backward with force, and gently shake the syringe to make the force on each explant uniform. When the piston stops at the 20mL scale, stay for 1.5min to achieve the purpose of applying vacuum negative pressure. Gently release the hand, and the piston slowly returns to the 10mL scale position, and repeat once more.

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

[0054] Example 3:

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

[0056] (1) DNA extraction and positive plant detection of transgenic cucumber plants

[0057] The CTAB method was used to extract the transgenic cucumber leaf DNA in Example 2, and the extracted DNA was used as a template, the knockout plasmid DNA constructed in Example 1 was used as a positive control, and the receptor material DNA was used as a negative control to detect transgenic positive plants. The detection primers were:

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

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

[0060] Using the DNA of the positive fluorescent seedlings grown in Example 1 as a template for PCR amplification, and sending the PCR products with bands identified by gel electrophoresis to the company for Hi-tom sequencing. The sequencing results showed that the editing types were as Figure 1 shown, and the mutated sequence was as shown in SEQ ID NO 3.

[0061] Detect Cas9 in the mutant lines of the T2 generation. Using the DNA of the CsHT16 mutant plants as a template for PCR amplification and observing the bands by gel electrophoresis. The length of the amplified fragment was approximately 500 bp. As Figure 1 shown in the gel diagram, the mutant plants without bands were those without the editing marker Cas9. The PCR amplification primers used were:

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

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

[0064] After 4 generations of self-crossing, a homozygous CsHT16 gene-edited line (without the editing marker Cas9) was obtained.

[0065] Example 4:

[0066] Observation of the phenotypic traits of homozygous mutant cucumber dwarf plant type and short and thick fruit

[0067] To further determine the effect of CsHT16 transgenic cucumbers on plant dwarfing and short fruits, compare the seedlings and mature fruits of the homozygous mutants in Example 3 with wild-type cucumber plants. It can be seen from Figure 2 that compared with the wild type, the homozygous mutants had a significant 15.11 cm shortening of the hypocotyl and a significant 4.36 SPAD increase in the relative chlorophyll content at the one-leaf-one-heart and two-leaf-one-heart stages of the seedling stage.

[0068] As Figure 3 shown, when comparing the plant heights of CsHT16 mutants and wild-type plants, the mutant plants were significantly dwarfed to different degrees compared with the wild-type plants from 3 d to 18 d after transplantation. Specifically, the wt plants were 11.84 cm taller than the ht16 plants at 3 d, 7.42 cm taller at 6 d, 11.00 cm taller at 9 d, 15.34 cm taller at 12 d, 22.36 cm taller at 15 d, and 23.10 cm taller at 18 d.

[0069] AsFigure 4 As shown, from pollination to 22 days of fruit growth, there is no significant difference in single fruit weight, yield (leaving 3 fruits per plant), and soluble solids between the mutant cucumber fruits and the wild type. However, during the growth process, the fruit length is significantly shortened. After 16 days of pollination, the fruit diameter of ht16 is significantly increased by 0.48 cm compared to wt, the fruit girth length is significantly increased by 1.68 cm, and the peduncle length is significantly shortened by 0.47 cm. As shown in Figure d, microscopic observation of the longitudinal section cells of the 16-day-old fruits shows that the shortening of the longitudinal cells of the ht16 fruits is the main factor leading to the shortening of the ht16 fruits, while the increase in the number of transverse cells of the fruits is the main factor leading to the thickening of the fruits.

[0070] The present invention provides a method for creating dwarf cucumber materials. According to the above method, cucumber plants with targeted gene editing are prepared, and then the gene-edited cucumber plants are self-crossed to create dwarf and thick-short fruit-shaped cucumber germplasms.

Claims

1. Application of CsHT16 gene in controlling cucumber plant type and / or fruit shape, the CsHT16 The protein encoded by the gene is shown in SEQ ID NO.

2.

2. Application of the CsHT16 gene in preparing dwarf cucumber plant type and / or cucumber fruit with shortened stems, the CsHT16 The protein encoded by the gene is shown in SEQ ID NO.

2.

3. The use according to claim 1 or 2, wherein the gene is shown as SEQ ID NO.

1.

4. The use according to claim 1 or 2, characterized in that: Knocking out, inhibiting or silencing the gene encoding the CsHT16 protein in cucumber can dwarf the plant shape of cucumber and / or shorten the handle of cucumber fruit.

5. The use according to claim 4, wherein the knockout is carried out using the CRISPR / Cas9 system.

6. The use according to claim 5, wherein the target site of gRNA in the system is GACACGTAAGTTCGGTCGT.

7. The use according to claim 6, wherein the cucumber obtained in the use process contains the gene shown in SEQ ID NO.3.

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