Gene csiaa4 for improving parthenocarpy of cucumber and application thereof

Abstract

The application discloses a gene CsIAA4 for improving cucumber parthenocarpy and application thereof, identifies a new cucumber parthenocarpic regulation gene CsIAA4, and the nucleotide sequence is as shown in SEQ ID NO. 1. The parthenocarpic capacity of the gene editing mutant of the application is weakened, and the parthenocarpic capacity of the overexpression mutant is significantly enhanced. The gene CsIAA4 of the application can be used for guiding the breeding and improvement of strong parthenocarpic germplasm resources of cucumber, and provides technical support for the breeding of cucumber parthenocarpic varieties. The application jointly explores the application of CsIAA4 in cucumber parthenocarpy through two kinds of transgenic technologies, provides new gene resources for the molecular genetic improvement of cucumber parthenocarpy, and provides the gene CsIAA4 for cucumber parthenocarpy, which has important application value in the research of improving the parthenocarpic capacity of cucumber.

Description

Technical Field

[0001] This invention belongs to the field of plant genetic engineering technology, specifically relating to a gene CsIAA4 that improves the parthenocarpy of cucumbers and its application. Background Technology

[0002] The cucumber (Cucumis sativus L.) is an important commercial organ, widely popular globally for its crisp texture, unique flavor, and nutritional value. As of 2022, the total planting area of ​​cucumbers reached 2.17 million hectares. Cucumber fruit development typically occurs through pollination and fertilization; however, unfavorable weather conditions can reduce pollen viability, leading to poor pollination and fertilization, and consequently, reduced fruit yield. Parthenocarpy, the development of fruit without pollination and fertilization, is the biological basis for increasing yield and saving labor costs, and is crucial for improving agricultural productivity. However, few cultivated cucumber varieties possess the ability to produce parthenocarpy. Therefore, identifying key genes regulating parthenocarpy formation in cucumbers is essential for improving their fruit-setting ability.

[0003] In plants, many developmental processes are finely regulated by auxin, such as vascular tissue formation, adventitious root development, tropism, apical dominance, and flower and fruit development. At the cellular level, auxin controls cell division, elongation, and differentiation. Dynamic spatiotemporal changes in auxin levels can precisely and rapidly trigger gene reprogramming, with early auxin response genes and the auxin / indole-3-acetic acid (Aux / IAA) family playing important roles. In maize, Zm-IAA14 increases root vigor and nutrient absorption under low nitrogen conditions; in Arabidopsis, At-IAA12 and At-IAA19 control lateral root development under abiotic stress. In rapeseed, the auxin signaling gene BnaA3.-IAA7 helps improve plant structure, yield, and heterosis. In Arabidopsis, At-IAA8 regulates seed germination. Research on genes and proteins that enhance parthenocarpy in cucumbers is limited. Summary of the Invention

[0004] Objective of the Invention: To address the shortcomings of existing technologies, this invention provides a gene, CsIAA4, to enhance the parthenocarpy ability of cucumbers. This invention identifies a novel parthenocarpy regulatory gene, CsIAA4, in cucumbers. Edited mutants exhibit weakened parthenocarpy ability, while overexpressed mutants show significantly enhanced parthenocarpy ability. The CsIAA4 gene of this invention can be used to guide the breeding and improvement of cucumber germplasm resources with strong parthenocarpy, providing technical support for the breeding of cucumber varieties with strong parthenocarpy.

[0005] The present invention also provides the application of the gene CsIAA4 that regulates parthenocarpy formation in cucumber.

[0006] Technical solution: In order to achieve the above objective, the present invention provides a gene CsIAA4 for improving the parthenocarpy of cucumbers, the nucleotide sequence of which is shown in SEQ ID NO.1.

[0007] The primer pair used to amplify the CsIAA4 gene is as follows:

[0008] CsIAA4-F:5'-ATGGCATTTCAAAATGGGTTA-3';

[0009] CsIAA4-R:5'-TTATGCAACACATCCTAATCCCT-3'.

[0010] The CDS gene of CsIAA4, which improves the parthenocarpy ability of cucumber, as described in this invention, has the CDS gene sequence shown in SEQ ID NO.2.

[0011] The primer pair used to amplify the CDS gene of the CsIAA4 gene is as follows:

[0012] CsIAA4-F-1:5'-ATGGCATTTCAAAATGGGTTAAA-3';

[0013] CsIAA4-R-1:5'-TGCAACACATCCTAATCCCTTTG-3'.

[0014] The gene knockout vector CRISPR-CsIAA4 for the gene CsIAA4 that improves the parthenocarpy ability of cucumber is described in this invention.

[0015] The gene knockout vector CRISPR-CsIAA4 is constructed by designing the target gene sequences sgRNA1 and sgRNA2 of CsIAA4.

[0016] sgRNA1:5'-CGAGGCCCGGTGGTCCAAGGCGG-3';

[0017] sgRNA2:5'-GACGGTGGCCGCGACAACGGCGG-3'.

[0018] Using pCBC-DT1T2(Cm) as a template, the dual targets were amplified, and the resulting double-stranded sgRNA1 / 2 was ligated into the pkSE402 vector. After transformation, the plasmid was extracted, and the gene knockout vector CRISPR-CsIAA4 was finally obtained.

[0019] The present invention contains the CDS gene overexpression vector OE-CsIAA4 containing the gene CsIAA4, which enhances the parthenocarpy of cucumbers.

[0020] The overexpression vector OE-CsIAA4 is characterized in that the overexpression vector OE-CsIAA4 is constructed by designing primers for the vector based on the CDS region of the CsIAA4 gene.

[0021] OE-CsIAA4-F:gagaacacgggggacggatccATGGCATTTCAAAATGGGTTAAA;

[0022] OE-CsIAA4-R:atggtctttgtagtcggatccTGCAACACATCCTAATCCCTTTG;

[0023] The gene was amplified, digested with enzymes, and ligated into the 1305.4 3*flag vector. After transformation, the plasmid was extracted, and the overexpression vector OE-CsIAA4 was finally obtained.

[0024] The application of the gene CsIAA4, or the CDS gene of the gene CsIAA4, or the gene knockout vector, or the overexpression vector, or a host bacterium containing the above-mentioned gene or vector, in regulating parthenocarpy in cucumber.

[0025] Specifically, editing the cucumber CsIAA4 gene with CRISPR / Cas9 reduced its parthenocarpy ability; overexpressing the cucumber CsIAA4 gene increased its parthenocarpy ability.

[0026] The present invention relates to the application of the gene CsIAA4, which regulates parthenocarpy in cucumber, or the CDS gene of CsIAA4, or the gene knockout vector, or the overexpression vector, or a host bacterium containing the above-mentioned gene or vector, in the cultivation of cucumber germplasm with strong parthenocarpy.

[0027] This invention uses the cucumber cultivar “CCMC” as material and constructs a dual-target CRISPR / Cas9 technology to obtain stable CRISPR-CsIAA4 gene-edited material. The parthenocarpy rate of the CRISPR-CsIAA4 transgenic material and the control material (WT) were statistically analyzed, and plant phenotypic changes were observed. The results showed that the parthenocarpy rate of the CsIAA4 gene knockout was significantly lower than that of the control. Using the cucumber cultivar “YN” as material, an OE-CsIAA4 overexpression vector was constructed using pCAMBIA1305.4 to obtain stable OE-CsIAA4 overexpression material. The parthenocarpy rate of the OE-CsIAA4 transgenic material and the control material (WT) were statistically analyzed, and plant phenotypic changes were observed. The results showed that the parthenocarpy rate of the CsIAA4 gene overexpression was significantly higher than that of the control. Through these two transgenic technologies, the application of CsIAA4 in the formation of parthenocarpy in cucumbers was explored, providing new gene resources for the molecular genetic improvement of strong parthenocarpy in cucumbers. This invention provides the gene CsIAA4, which regulates parthenocarpy in cucumber, and has important application value in studying how to improve the parthenocarpy ability of cucumber.

[0028] This invention relates to the application of a novel cucumber germplasm with strong parthenocarpy based on CsIAA4 overexpression. The transgenic plants obtained by overexpressing the CsIAA4 gene in this invention exhibit a significantly higher parthenocarpy rate and stronger parthenocarpy ability than the negative control plants. The novel cucumber germplasm with strong parthenocarpy created in this invention has significant application value in genetic breeding research on cucumber with strong parthenocarpy.

[0029] Beneficial effects: Compared with the prior art, the present invention has the following advantages:

[0030] This invention identified a novel gene regulating parthenocarpy, providing a new gene resource for the molecular genetic improvement of cucumber with enhanced parthenocarpy. Furthermore, this invention obtained new cucumber germplasm with enhanced parthenocarpy ability. Through genetic transformation, this invention obtained a cucumber CsIAA4 gene-edited mutant and an overexpression mutant. Compared with the wild type, the gene-edited mutant showed weakened parthenocarpy ability, while the overexpression mutant showed enhanced parthenocarpy ability.

[0031] This invention clones a cucumber auxin gene, CsIAA4, using molecular biology methods and demonstrates its function in regulating parthenocarpy. The gene mining and strong parthenocarpy acquisition method described in this invention has clear operational steps, high reproducibility, and can be applied to mature transgenic crops to obtain strong parthenocarpy germplasm, showing promising application prospects. Attached Figure Description

[0032] Figure 1Phenotypic results of transgenic cucumber plants after the CsIAA4 gene was knocked out using CRISPR / Cas9 technology. (a) Schematic diagram of CRISPR / Cas9-CsIAA4 target sites and genotyping; (b) Phenotypic observation of gene-edited plants (cr-1, cr-2, cr-3) and control plants; (c) Identification of gene expression levels in gene-edited plant cr-1 and control plants; WT represents the control plant, and cr-1, cr-2, and cr-3 represent the gene-edited plants. Variance is expressed as ±SDs, and three biological statistics were performed.

[0033] Figure 2 Phenotypic results of cucumber CsIAA4 gene overexpression plants obtained through transgenic technology. (a) Phenotypic observation of overexpressing plants (oe-1, oe-2) and control plants; (b) Identification of gene expression levels in overexpressing plant oe-1 and control plants. WT represents the control plant, and oe-1 and oe-2 represent the overexpressing plants. Variance is expressed as ±SDs, and three biological statistics were performed. Detailed Implementation

[0034] The present invention will be further described below with reference to the accompanying drawings and embodiments.

[0035] This invention uses the cultivated cucumber variety “ZK” for related experiments. This variety comes from the germplasm resource bank of the Cucumber Genetics and Breeding and Molecular Science Innovation Team of the College of Horticulture and Landscape Architecture, Yangzhou University, and was provided by Yangzhou University (Cytokinin and auxinmodulate cucumber parthenocarpy fruit development. Scientia Horticulturae, 2021, 282, 110026.).

[0036] The cucumber transformation receptor materials, “CCMC” and “YN”, are well-known receptor materials, provided by Yangzhou University. (A CsEIL3-CsARN6.1 module promotes waterlogging-triggered adventitious root formation in cucumber by activating the expression of CsPrx5, The Plant Journal, 2023, 114, 824-835: Characterization of Differences in the Composition and Content of Volatile Compounds in Cucumber Fruit. Foods. 2022 Apr 12; 11(8):1101.)

[0037] Example 1

[0038] The cloning method for the CsIAA4 gene includes the following steps:

[0039] (1) Cucumber tissue cDNA synthesis: RNA was extracted from cucumber “ZK” fruit tissue at the cotyledon stage and reverse transcribed to obtain first-strand cDNA; or RNA was extracted from the fruit tissue of cucumber transformation recipient materials “CCMC” and “YN” and reverse transcribed to obtain first-strand cDNA; (2) PCR amplification of CsIAA4 gene and its CDS sequence:

[0040] Primers were designed (CsIAA4-F: 5'-ATGGCATTTCAAAATGGGTTA-3'; CsIAA4-R: 5'-TTATGCAACACATCCTAATCCCT-3'), and cucumber tissue DNA was used as a template for PCR amplification. The PCR amplification products were recovered, purified, and sequenced.

[0041] Primers were designed (CsIAA4-F-1:5'-ATGGCATTTCAAAATGGGTTAAA-3'; CsIAA4-R-1:5'-TGCAACACATCCTAATCCCTTTG-3'), and cucumber tissue cDNA was used as a template for PCR amplification. The PCR amplification products were recovered, purified, and sequenced.

[0042] The nucleotide sequence encoding the cucumber CsIAA4 gene of this invention is shown in SEQ ID NO.1, and the CDS coding region sequence is shown in SEQ ID NO.2.

[0043] Example 2

[0044] (1) CRISPR / Cas9 vector was constructed using the cucumber CsIAA4 gene.

[0045] Construction of CsIAA4 gene knockout vector: Two target gene primer sequences for CsIAA4, sgRNA1 and sgRNA2, were designed. The designed gene sequences are as follows:

[0046] sgRNA1: 5'-CGAGGCCCGGTGGTCCAAGGCGG-3';

[0047] sgRNA2: 5'-GACGGTGGCCGCGACAACGGCGG-3';

[0048] Using pCBC-DT1T2(Cm) as a template, dual-target products were obtained by PCR amplification using PrimerSTAR high-fidelity enzyme. The reaction volume was 50 μL, containing 10 μL of 5×PrimeSTAR Buffer, 4 μL of dNTP Mixture, 1 μL of sgRNA1 primer, 1 μL of sgRNA2 primer, 32.5 μL of ddH2O, 1 μL of pCBC-DT1T2(Cm) plasmid, and 0.5 μL of PrimerSTAR high-fidelity enzyme. The reaction program was 95℃ for 5 min; 95℃ for 15 sec, 55℃ for 15 sec, 72℃ for 15 sec, 35 cycles; 72℃ for 5 min.

[0049] The amplified dual-target product sgRNA1 / 2 was ligated into the pkSE402 vector. The reaction mixture was 15 μL, containing 1.2 μL of pkSE402 vector, 2 μL of dual-target product, 1 μL of Bsal, 1 μL of T4 Liguse, 1.5 μL of 10×NEB T4 Buffer, and 8.3 μL of ddH2O. The reaction program was 37℃ for 5 min; 16℃ for 5 min, 60℃ for 5 min, and 12℃. 15 μL of the ligation product was added to 50 μL of competent E. coli cells. After mixing thoroughly, the mixture was frozen on ice for 30 min, heat-shocked at 42℃ for 30 sec, and then incubated on ice for 2 min. 500 μL of LB liquid medium was added, mixed well, and incubated at 37℃ for 1 h using a shaker. 100 μL of the bacterial cells were then spread onto Kan+LB solid medium and incubated at 37℃ for 12–16 h. Single colonies were picked for positive testing and sent to Qingke Biotechnology Co., Ltd. for sequencing. Correct single clones were selected for propagation. Plasmids were extracted using the Plasmid MiniKit plasmid DNA miniprep kit to obtain the gene knockout vector CRISPR-CsIAA4. The plasmid was transformed into Agrobacterium competent cells EHA105. The competent cells were thawed on ice. 50 μL of competent cells and 2 μL of plasmid were mixed, and the mixture was incubated sequentially on ice for 5 min, in liquid nitrogen for 5 min, at 37°C for 5 min, and on ice for 5 min. 700 μL of LB liquid medium was added, and the mixture was incubated at 28°C in a shaker for 2–3 h. 100 μL of the bacterial cells were spread on Kan+Rif LB solid medium and incubated at 28°C for 2–3 days. Single clones were picked, tested, and preserved to obtain Agrobacterium carrying the CRISPR-CsIAA4 gene for subsequent transgenic applications.

[0050] (2) Obtaining CRISPR-CsIAA4 transgenic plants and positive detection

[0051] Transgenic plants were obtained using Agrobacterium-mediated genetic transformation. The recipient material was "CCMC". The basic method was as follows: "CCMC" seeds were soaked in distilled water in a 55°C water bath for 30 min, the seed coat was removed, and the seeds were rinsed 3-4 times with sterile water. They were then soaked in 75% alcohol for 30 sec, soaked in 2% sodium hypochlorite solution for 10 min, and rinsed 4-5 times with sterile ddH2O. The seeds were then sown on SGM medium, wrapped in aluminum foil, and placed in a 28°C incubator for 36 h. Agrobacterium carrying the CRISPR-CsIAA4 gene was cultured in LB solid medium containing Kan and Rif antibiotics for 12 h. The bacterial cells were added to 40 mL of 1M liquid medium, mixed well, and the OD was measured. 600 , making OD 600 =0.2, placed in a 28℃ constant temperature incubator; remove the buds, cut off 1 / 3 of the seed and divide it in half, soak in IM liquid medium, after the seeds are treated, transfer them together to IM liquid medium containing bacterial solution, sonicate for 20 seconds with a KQ5200DE type CNC ultrasonic cleaner, vacuum penetrate for 90 seconds (twice in total), finally place the explants on IM solid medium with tweezers, wrap them with aluminum foil and place them in a 25℃ constant temperature incubator for 3 days; transfer the explants to SRM medium and culture them in a 26℃ light 16h / dark 8h environment for 2-3 weeks; observe with a fluorescence microscope and select explants with green fluorescence, cut off the putative transformant about 1cm in length, place the remaining part in RM medium to induce rooting, transfer the rooted tissue culture seedlings to the substrate for acclimatization culture, and finally transfer them to the greenhouse for cultivation.

[0052] CRISPR-CsIAA4 transgenic positive plants were screened by PCR, and the gene editing type of the knockout plants was further determined by sequencing.

[0053] Design specific primers near the CsIAA4 target gene sequence to detect gene knockout fragment sequences:

[0054] CRISPR-CsIAA4-F:5'-TCCCCCTTCCACCAAATCAA-3';

[0055] CRISPR-CsIAA4-R:5'-AAGTTTGGAGGAGTACAGGGTA-3';

[0056] Using CRISPR-CsIAA4 transgenic plant leaf DNA as a template, PCR amplification and sequencing were performed using PrimerSTAR high-fidelity enzyme to obtain the editing sites. The reaction system was 50 μL, containing 10 μL of 5×PrimeSTAR Buffer, 4 μL of dNTP Mixture, 1 μL of CRISPR-CsIAA4-F primer, 1 μL of CRISPR-CsIAA4-R primer, 32.5 μL of ddH2O, 1 μL of DNA template, and 0.5 μL of PrimerSTAR high-fidelity enzyme. The reaction program was 95℃ for 5 min; 95℃ for 15 sec, 55℃ for 15 sec, 72℃ for 15 sec, 35 cycles; 72℃ for 5 min. The results are as follows. Figure 1 (a) shows three types of editing. Sequencing analysis revealed that transgenic plants cr-1, cr-2 and cr-3 had an increase of 2 bp, a deletion of 14 bp and 13 bp, respectively.

[0057] Example 3

[0058] Gene phenotype observation, expression level detection, and auxin assay of CRISPR-CsIAA4 transgenic cucumber plants

[0059] (1) Phenotypic observation of CRISPR-CsIAA4 transgenic cucumber plants

[0060] To confirm the ability of the CsIAA4 gene to regulate parthenocarpy in cucumber, female flowers of control plants (WT: CCMC) and CRISPR-CsIAA4 transgenic plants (cr-1, cr-2, cr-3) were subjected to a flower-clamping treatment one day before flowering. After one week of continuous flower clamping, the parthenocarpy rate was calculated and phenotypic observations were performed. Figure 1 (b) and Table 1 show that the parthenocarpy rates of CRISPR-CsIAA4 transgenic plants were 67.74%, 62.22%, and 72.15%, respectively, which were significantly lower than the 95.70% of the control plants. The parthenocarpy capacity of the three types of transgenic plants was significantly lower than that of the control plants.

[0061] Table 1. Statistics on the parthenocarpy rate of control and edited plants.

[0062]

[0063] (2) Detection of gene expression levels in CRISPR-CsIAA4 transgenic cucumber plants

[0064] Fruits from control plants (WT) and CRISPR-CsIAA4 transgenic plants (cr-1, cr-2, cr-3) were subjected to flower-pinching treatment one day before flowering. RNA was extracted from fruits one day after flowering (1d) for reverse transcription and detected by real-time fluorescent PCR.

[0065] Cucumber actin (CsaV3_6G041900) was used as an internal reference gene:

[0066] actin-F:5'-GCTGGATTCTGGTGATGGTG-3';

[0067] actin-R:5'-AGCAAGGTCCAAACGGAGAA-3';

[0068] Primer sequences designed for the target gene IAA4:

[0069] IAA4-F:5'-CCGTAGGTGAATACTCTGAGAGAG-3';

[0070] IAA4-R:5'-TGGGACATCTCCAACCAACA-3'.

[0071] Expression levels of IAA4 in control plants and three types of edited transgenic plants. Figure 1 As shown in (c), one day after flowering (1d), the expression levels of transgenic plants with all three editing types were significantly lower than those of the control plants.

[0072] Example 4

[0073] The pCAMBIA1305.4 overexpression vector was constructed using the cucumber CsIAA4 gene.

[0074] (1) Primers were designed using the CDS region of the CsIAA4 gene (with the stop codon removed) to construct the vector. The primer sequences were as follows:

[0075] OE-CsIAA4-F-1:gagaacacgggggacggatccATGGCATTTCAAAATGGGTTAAA;

[0076] OE-CsIAA4-R-1:atggtctttgtagtcggatccTGCAACACATCCTAATCCCTTTG.

[0077] First, using the CsIAA4 gene CDS sequence as a template, PCR amplification was performed using PrimerSTAR high-fidelity enzyme to obtain the product. The reaction system was 50 μL, containing 10 μL of 5×PrimeSTAR Buffer, 4 μL of dNTP Mixture, 1 μL of OE-CsIAA4-F primer, 1 μL of OE-CsIAA4-R primer, 32.5 μL of ddH2O, 1 μL of template, and 0.5 μL of PrimerSTAR high-fidelity enzyme. The reaction program was 95℃ for 5 min; 95℃ for 15 sec, 55℃ for 15 sec, 72℃ for 15 sec, 35 cycles; 72℃ for 5 min. Homologous recombination kits were used... The purified PCR product was recombinantly ligated with a linear vector using the One Step Cloning Kit. 2 μL of 5×CE II Buffer, 1 μL of purified PCR product, 1 μL of pCAMBIA1305.4 vector fragment digested with BamHI, 1 μL of Exnase II, and 5 μL of sterile water were added to a 10 μL recombinant ligation system. After mixing, the mixture was ligated at 37℃ for 30 min. The mixture was then transformed into *E. coli* DH5α (TransGen, Beijing) using the heat shock method. Single clones were picked, amplified by PCR, and sequenced. Correct single clones were selected for propagation, and plasmids were extracted to obtain the overexpression vector OE-CsIAA4. The plasmid was transformed into *Agrobacterium* competent cells EHA105. Single clones were selected, tested, and preserved for subsequent transgenic applications (method as in Example 3(1)).

[0078] (2) Obtaining OE-CsIAA4 transgenic plants and positive detection

[0079] Transgenic plants were obtained using Agrobacterium-mediated genetic transformation, with “YN” as the transformation recipient material, and the method was the same as in Example 3(2).

[0080] Positive detection was performed on overexpressing transgenic plants. OE-CsIAA4 transgenic positive plants were screened by PCR, and primer sequences were detected.

[0081] OE-CsIAA4-F:5'-GTCACTTTATTGTGAAGATAGTGGA-3';

[0082] OE-CsIAA4-R:5'-atggtctttgtagtcggatTGCAACACATCCTAATCCCTTTG-3'.

[0083] Using leaf DNA from overexpressing transgenic plants as templates, PCR amplification was performed using Taq PCR MasterMix II. The reaction volume was 20 μL, containing 10 μL of 2×Taq PCR MasterMix II, 1 μL of OE-CsIAA4-F primer, 1 μL of OE-CsIAA4-R primer, 7 μL of ddH2O, and 1 μL of DNA template. The reaction program was 98℃ for 5 min; 98℃ for 30 sec, 55℃ for 30 sec, 72℃ for 3 min, for 35 cycles; 72℃ for 10 min. Positive OE-CsIAA4 transgenic plants (oe-1, oe-2) were screened after agarose gel electrophoresis.

[0084] Example 5

[0085] Gene phenotype observation, expression level detection, and auxin assay of OE-CsIAA4 transgenic cucumber plants

[0086] (1) Phenotypic observation of OE-CsIAA4 transgenic cucumber plants

[0087] To confirm the function of the CsIAA4 gene in regulating parthenocarpy in cucumber, female flowers of control plants (WT:YN) and OE-CsIAA4 transgenic plants (oe-1, oe-2) were subjected to a flower-pinching treatment one day before flowering. After one week of continuous flower-pinching, the parthenocarpy rate was calculated and phenotypic observations were performed. Figure 2 As shown in (a) and Table 2, the parthenocarpy rates of OE-CsIAA4 transgenic plants were 96.7% and 81.0%, respectively, which were significantly higher than the control plants by 20.5%. The parthenocarpy capacity of the two overexpressed transgenic plants was significantly higher than that of the control plants.

[0088] Table 2. Statistics on parthenocarpy rate of control plants and overexpression plants.

[0089]

[0090] (2) Detection of gene expression levels in OE-CsIAA4 transgenic cucumber plants

[0091] Fruits from control plants (WT) and OE-CsIAA4 transgenic plants (oe-1, oe-2) were subjected to flower-pinching treatment one day before flowering. RNA was extracted from fruits one day after flowering (1d) for reverse transcription and detected by real-time fluorescent PCR.

[0092] Cucumber actin (CsaV3_6G041900) was used as an internal reference gene:

[0093] actin-F:5'-GCTGGATTCTGGTGATGGTG-3';

[0094] actin-R:5'-AGCAAGGTCCAAACGGAGAA-3';

[0095] Primer sequences designed for the target gene IAA4:

[0096] IAA4-F:5'-CCGTAGGTGAATACTCTGAGAGAG-3';

[0097] IAA4-R:5'-TGGGACATCTCCAACCAACA-3'.

[0098] Expression levels of IAA4 in control plants and two overexpressing transgenic plants. Figure 2 As shown in (b), one day after flowering (1d), the expression levels of both overexpressing transgenic plants were significantly higher than those of the control plants.

Claims

1. A gene that enhances the parthenocarpy ability of cucumbers. CsIAA4 CRISPR gene knockout vector CsIAA4 The gene knockout vector CRISPR- CsIAA4 The construction method is based on design. CsIAA4 The target gene sequences sgRNA1 and sgRNA2; sgRNA1: 5'-CGAGGCCCGGTGGTCCAAGGCGG -3'; sgRNA2: 5'-GACGGTGGCCGCGACAACGGCGG -3'; Using pCBC-DT1T2(Cm) as a template, the dual targets were amplified, and the resulting double-stranded sgRNA1 / 2 was ligated into a vector. After transformation, the plasmid was extracted, and the gene knockout vector CRISPR-CsIAA4 was finally obtained.

2. A gene containing the aforementioned gene that enhances the parthenocarpy of cucumbers. CsIAA4 CDS gene overexpression vector OE- CsIAA4 The overexpression vector OE- CsIAA4 The construction method is based on CsIAA4 Primers for designing vectors from the CDS region of genes: OE- CsIAA4 -F: gagaacacgggggacggatccATGGCATTTCAAAATGGGTTAAA; OE- CsIAA4 -R: atggtctttgtagtcggatccTGCAACACATCCTAATCCCTTTG; The gene was amplified, digested with enzymes, and ligated into a vector. After transformation, the plasmid was extracted, ultimately yielding the overexpression vector OE-. CsIAA4 The gene CsIAA4 The nucleotide sequence of the CDS gene is shown in SEQ ID NO.

2.

3. Genes CsIAA4 Or genes CsIAA4 The application of the CDS gene, or the gene knockout vector of claim 1, or the overexpression vector of claim 2, or a host bacterium containing the above-mentioned gene or vector, in regulating parthenocarpy in cucumber, wherein the gene... CsIAA4 The nucleotide sequence of the gene is shown in SEQ ID NO.

1. CsIAA4 The nucleotide sequence of the CDS gene is shown in SEQ ID NO.

2.

4. The application according to claim 3, characterized in that, Editing cucumbers using CRISPR / Cas9 CsIAA4 Genes that reduce the parthenocarpy of cucumbers; by overexpressing these genes... CsIAA4 Genes that increase its ability to produce parthenocarpic fruit.

5. A gene regulating parthenocarpy in cucumbers CsIAA4 Or genes CsIAA4 The application of the CDS gene, or the gene knockout vector of claim 1, or the overexpression vector of claim 2, or a host bacterium containing the above-mentioned gene or vector, in the cultivation of strongly parthenocarpic cucumber germplasm, wherein the gene... CsIAA4 The nucleotide sequence of the gene is shown in SEQ ID NO.

1. CsIAA4 The nucleotide sequence of the CDS gene is shown in SEQ ID NO.2.

Images