Gene CsIAA4 for improving parthenocarpy capacity of cucumber and application of gene CsIAA4

Abstract

The invention discloses a gene CsIAA4 for improving parthenocarpy capacity of cucumbers and application of the gene CsIAA4, and identifies a novel parthenocarpy regulating gene CsIAA4 of the cucumbers, and the nucleotide sequence of the gene CsIAA4 is as shown in SEQ ID NO.1. The parthenocarpy ability of the gene editing mutant is weakened, and the parthenocarpy ability of the overexpression mutant is remarkably enhanced. The gene CsIAA4 can be used for guiding breeding and improvement of cucumber strong parthenocarpy germplasm resources, and technical support is provided for breeding of cucumber parthenocarpy varieties. The application of the CsIAA4 in cucumber parthenocarpy is jointly explored through two transgenic technologies, a new gene resource is provided for genetic improvement of cucumber parthenocarpy molecules, the cucumber parthenocarpy gene CsIAA4 is provided, and the cucumber parthenocarpy gene CsIAA4 has important application value in the aspect of research and improvement of cucumber parthenocarpy capacity.

Description

Technical Field

[0001] The invention belongs to the technical field of plant genetic engineering, and in particular relates to a gene CsIAA4 for improving the parthenocarpy ability of cucumber and an application thereof. Background Art

[0002] Cucumber (Cucumis sativus L.) fruit is an important commercial organ, which is popular all over the world for its crisp texture, unique flavor and nutritional value. As of 2022, the total cultivated area of ​​cucumber reached 2.17 million hectares. Cucumber fruit development is usually carried out through pollination and fertilization, however, adverse weather conditions can reduce pollen vitality, resulting in poor pollination and fertilization, and reducing fruit yield. Parthenocarpy refers to the development of fruit without pollination and fertilization. It is the biological basis for increasing yield and saving labor costs, and is crucial to improving agricultural productivity. However, few cultivated cucumber varieties have the ability to produce parthenocarpy. Therefore, exploring the key genes that regulate the formation of cucumber parthenocarpy is the genetic basis for improving the fruiting ability of cucumber.

[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 accurately and rapidly trigger gene reprogramming, among which the auxin early response genes, the auxin / indole-3-acetic acid (Aux / IAA) family, play an important role. Maize Zm-IAA14 can increase root activity and absorb nutrients under low nitrogen conditions, and Arabidopsis At-IAA12 and At-IAA19 control lateral root development under abiotic stress. The auxin signaling gene BnaA3.-IAA7 in rapeseed helps improve plant architecture and yield heterosis. Arabidopsis At-IAA8 regulates seed germination. Few studies have been conducted on genes and proteins that improve cucumber parthenocarpy. Summary of the invention

[0004] Purpose of the invention: In view of the deficiencies in the prior art, the present invention provides a gene CsIAA4 that improves the parthenocarpy ability of cucumber. The present invention identifies a new cucumber parthenocarpy regulatory gene CsIAA4, whose edited mutant has weakened parthenocarpy ability, while the overexpressed mutant has significantly enhanced parthenocarpy ability. The gene CsIAA4 of the present invention can be used to guide the breeding and improvement of cucumber germplasm resources with strong parthenocarpy, and provide technical support for the breeding of cucumber varieties with strong parthenocarpy.

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

[0006] Technical solution: In order to achieve the above-mentioned purpose, the present invention provides a gene CsIAA4 for improving the parthenocarpy ability of cucumber, and the nucleotide sequence of the gene CsIAA4 is shown in SEQ ID NO.1.

[0007] Among them, the primer pair used to amplify the gene CsIAA4 is:

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

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

[0010] The CDS gene of the gene CsIAA4 for improving the parthenocarpy ability of cucumber according to the present invention has a CDS gene sequence as shown in SEQ ID NO.2.

[0011] Among them, the primer pair used to amplify the CDS gene of the gene CsIAA4 is:

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

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

[0014] The gene knockout vector CRISPR-CsIAA4 of the gene CsIAA4 for improving the parthenocarpy ability of cucumber according to the present 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] pCBC-DT1T2 (Cm) was used as a template to amplify dual targets, and the formed double-stranded sgRNA1 / 2 was connected to the pkSE402 vector. After transformation and extraction of the plasmid, the gene knockout vector CRISPR-CsIAA4 was finally obtained.

[0019] The invention discloses a CDS gene overexpression vector OE-CsIAA4 containing the gene CsIAA4 for improving the parthenocarpy ability of cucumber.

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

[0021] OE-CsIAA4-F:gagaacacgggggacggatccATGGCATTTCAAAATGGGTTAAA;

[0022] OE-CsIAA4-R:atggtctttgtagtcggatccTGCAACACATCCTAATCCCTTTG;

[0023] The gene was amplified, restriction enzyme-cut and connected to the 1305.4 3*flag vector, and then transformed and the plasmid was extracted to finally obtain the overexpression vector OE-CsIAA4.

[0024] The gene CsIAA4 or the CDS gene of the gene CsIAA4 or the gene knockout vector or the overexpression vector or the host bacteria containing the above gene or vector described in the present invention are used in regulating cucumber parthenocarpy.

[0025] Among them, by editing the cucumber CsIAA4 gene through CRISPR / Cas9, its parthenocarpy ability was reduced; by overexpressing the cucumber CsIAA4 gene, its parthenocarpy ability was increased.

[0026] The invention discloses an application of the gene CsIAA4 for regulating cucumber parthenocarpy, or the CDS gene of the gene CsIAA4, or the gene knockout vector, or the overexpression vector, or a host bacteria containing the gene or the vector in cultivating strongly parthenocarpy cucumber germplasm.

[0027] The present invention uses the cucumber "CCMC" strain as material, constructs dual targets through CRISPR / Cas9 technology, obtains stable CRISPR-CsIAA4 gene editing materials, and performs fruit parthenocarpy statistics on CRISPR-CsIAA4 transgenic materials and control materials (WT), observes plant phenotypic changes, and the results show that after knocking out the CsIAA4 gene, its parthenocarpy rate is significantly lower than that of the control. Using the cucumber "YN" strain as material, pCAMBIA1305.4 is used as a vector to construct an OE-CsIAA4 overexpression vector, obtains stable OE-CsIAA4 overexpression materials, and performs fruit parthenocarpy statistics on OE-CsIAA4 transgenic materials and control materials (WT), observes plant phenotypic changes, and the results show that after overexpressing the CsIAA4 gene, its parthenocarpy rate is significantly higher than that of the control. Through two transgenic technologies, the application of CsIAA4 in the formation of cucumber parthenocarpy is jointly explored, providing new gene resources for molecular genetic improvement of strong parthenocarpy in cucumber. The invention provides a gene CsIAA4 for regulating cucumber parthenocarpy, which has important application value in research on improving the parthenocarpy ability of cucumber.

[0028] The application of the new cucumber germplasm with strong parthenocarpy created based on overexpression of CsIAA4 described in the present invention. The transgenic plants obtained by overexpressing the CsIAA4 gene in the present invention have a significantly higher parthenocarpy rate than the negative control plants, and their parthenocarpy ability is significantly stronger than that of the control plants. The new cucumber germplasm with strong parthenocarpy created by the present invention has important application value in the genetic breeding research of strong parthenocarpy of cucumber.

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

[0030] The present invention identifies a new gene that regulates parthenocarpy, providing a new gene resource for molecular genetic improvement of strong parthenocarpy in cucumber. Further, the present invention obtains a new cucumber germplasm with enhanced parthenocarpy ability. The present invention obtains cucumber CsIAA4 gene editing mutants and overexpression mutants through genetic transformation. Compared with the wild type, the gene editing mutant has weakened parthenocarpy ability, while the overexpression mutant has enhanced parthenocarpy ability.

[0031] The present invention clones a cucumber auxin gene CsIAA4 using molecular biological methods and proves its function of regulating parthenocarpy. The gene mining and strong parthenocarpy acquisition method of the present invention has clear operation steps and strong replicability. The gene can be applied to other crops with mature transgenic systems to obtain strong parthenocarpy germplasm, which has good application prospects. BRIEF DESCRIPTION OF THE DRAWINGS

[0032] Figure 1The phenotype of transgenic plants after the cucumber CsIAA4 gene was knocked out by CRISPR / Cas9 technology. (a) Schematic diagram of CRISPR / Cas9-CsIAA4 targeting site 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 plants cr-1 and control plants; WT is the control plant, cr-1, cr-2, cr-3 are gene-edited plants. Variance is expressed as ±SDs, and biological statistics were repeated three times.

[0033] Figure 2 The phenotype of the overexpressed cucumber CsIAA4 gene was obtained by transgenic technology. (a) Phenotypic observation of overexpressed plants (oe-1, oe-2) and control plants; (b) Identification of gene expression levels in overexpressed plants oe-1 and control plants. WT is the control plant, oe-1 and oe-2 are overexpressed plants. Variance is expressed as ±SDs, and biological statistics were performed three times. DETAILED DESCRIPTION

[0034] The present invention is further described below in conjunction with the accompanying drawings and embodiments.

[0035] The present invention uses the cultivated cucumber variety "ZK" for relevant experiments. This variety comes from the germplasm resource bank of the cucumber genetic breeding and molecular innovation team of the College of Horticulture and Landscape Architecture of Yangzhou University, and is provided by Yangzhou University (Cytokinin and auxin modulate 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. 2022Apr 12; 11(8): 1101.)

[0037] Example 1

[0038] The cloning method of CsIAA4 gene comprises the following steps:

[0039] (1) Cucumber tissue cDNA synthesis: Extract RNA from cucumber "ZK" fruit tissue at the cotyledon stage, and reverse transcribe to obtain the first-chain cDNA; or use the fruit tissue RNA of cucumber transformation recipient materials "CCMC" and "YN" to reverse transcribe to obtain the first-chain cDNA; (2) PCR amplification of CsIAA4 gene and its CDS sequence:

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

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

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

[0043] Example 2

[0044] (1) Construction of CRISPR / Cas9 vector using cucumber CsIAA4 gene.

[0045] Construction of CsIAA4 gene knockout vector: Design two CsIAA4 target gene primer sequences sgRNA1 and sgRNA2. The designed target gene sequences are:

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

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

[0048] Using pCBC-DT1T2 (Cm) as a template, PrimerSTAR high-fidelity enzyme was used for PCR amplification to obtain dual-target products. The reaction system was 50 μL, including 5× PrimeSTAR Buffer 10 μL, dNTP Mixture 4 μL, sgRNA1 primer 1 μL, sgRNA2 primer 1 μL, ddH 2 O 32.5 μL, pCBC-DT1T2 (Cm) plasmid 1 μL, PrimerSTAR high-fidelity enzyme 0.5 μL. The reaction program was 95°C for 5 min; 95°C for 15 sec, 55°C for 15 sec, 72°C for 15 sec, 35 cycles; 72°C for 5 min.

[0049] The amplified dual-target product sgRNA1 / 2 was connected to the pkSE402 vector. The reaction system was 15 μL, including 1.2 μL pkSE402 vector, 2 μL dual-target product, 1 μL Bsal, 1 μL T4 Liguse, 1.5 μL 10×NEB T4 Buffer, and ddH 2 O 8.3μL. The reaction procedure is 37℃5min; 16℃5min, 60℃5min, 12℃ insulation. Add 15μL of the ligation product to 50μL of E. coli competent cells, mix them evenly, freeze them on ice for 30min, heat shock them at 42℃ for 30sec, let them stand on ice for 2min, add 500μL of LB liquid culture medium, mix them evenly, and culture them in a 37℃ constant temperature oscillator for 1h. Take 100μL of the bacteria and spread them on Kan+LB solid culture medium. The culture medium is cultured in a 37℃ incubator for 12-16h. Pick a single colony for positive detection and send it to Qingke Biotechnology Co., Ltd. for sequencing. Select the correct single clone for expansion and use Plasmid MiniKit plasmid DNA extraction kit was used to extract plasmids, and gene knockout vector CRISPR-CsIAA4 was obtained by extracting plasmids. The plasmid was transferred into Agrobacterium competent EHA105, and competent EHA105 was placed on ice to melt. 50 μL competent and 2 μL plasmid were aspirated, mixed, and placed on ice for 5 minutes, liquid nitrogen for 5 minutes, 37°C for 5 minutes, and ice for 5 minutes in sequence. 700 μL LB liquid culture medium was added, mixed, and cultured in a 28°C shaker for 2-3 hours. 100 μL of bacteria was spread on Kan+Rif LB solid culture medium, and cultured in a 28°C constant temperature incubator for 2 to 3 days. The single clone strain was selected for detection and preservation, and Agrobacterium carrying CRISPR-CsIAA4 gene was obtained for subsequent transgenic.

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

[0051] Transgenic plants were obtained by Agrobacterium-mediated genetic transformation, and the transformation receptor material was "CCMC". The basic method was as follows: "CCMC" seeds were soaked in distilled water and placed in a 55°C water bath for 30 minutes, the seed coat was peeled off, washed with sterile water 3 to 4 times, soaked in 75% alcohol for 30 seconds, soaked in 2% sodium hypochlorite solution for 10 minutes, and sterilized with ddH 2 O flush 4-5 times, sow the seeds on SGM medium, wrap them in tin foil and place them in a constant temperature incubator at 28℃ for 36h; culture Agrobacterium carrying CRISPR-CsIAA4 gene in LB solid medium containing Kan and Rif antibiotics for 12h, add the bacteria to 40mL IM liquid medium, mix well and measure OD 600 , so that OD 600 =0.2, and placed in a constant temperature incubator at 28°C; skim off the buds, cut off 1 / 3 of the seeds and divide them into two, soak them in IM liquid culture medium, and after the seeds are processed, transfer them together to IM liquid culture medium containing bacterial solution, use KQ5200DE CNC ultrasonic cleaner to ultrasonicate for 20sec, vacuum infiltrate for 90sec (twice in total), and finally place the explants on IM solid culture medium with tweezers, wrap them with tin foil and place them in a constant temperature incubator at 25°C for 3d; transfer the explants to SRM culture medium and culture them in an environment of 26°C with 16h light / 8h darkness for 2-3 weeks; observe and select explants with green fluorescence under a fluorescence microscope, cut off the putative transformants with a length of about 1cm, and place the remaining parts in RM culture medium to induce rooting, transfer the rooted tissue culture seedlings to the substrate for acclimatization and culture, and finally move 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 the gene knockout fragment sequence:

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

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

[0056] Using the DNA of leaves of CRISPR-CsIAA4 transgenic plants as templates, PrimerSTAR high-fidelity enzyme was used for PCR amplification and sequencing to obtain the editing site. The reaction system was 50 μL, including 10 μL of 5× PrimeSTAR Buffer, 4 μL of dNTPMixture, 1 μL of CRISPR-CsIAA4-F primer, 1 μL of CRISPR-CsIAA4-R primer, and 1 μL of ddH 2 O 32.5μL, DNA template 1μL, PrimerSTAR high-fidelity enzyme 0.5μL. The reaction program was 95℃5min; 95℃15sec, 55℃15sec, 72℃15sec, 35 cycles; 72℃5min. The results were as follows Figure 1 (a) There are three types of editing. Sequencing analysis revealed that the transgenic plants cr-1, cr-2 and cr-3 had 2bp increase, 14bp and 13bp loss, respectively.

[0057] Example 3

[0058] Observation of gene phenotype, expression level detection and auxin determination in CRISPR-CsIAA4 transgenic cucumber plants

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

[0060] In order to confirm the ability of CsIAA4 gene to regulate cucumber parthenocarpy, the female flowers of control plants (WT: CCMC) and CRISPR-CsIAA4 transgenic plants (cr-1, cr-2, cr-3) were clipped one day before flowering. After one week of continuous clipping, the parthenocarpy rate was calculated and the phenotype was observed. 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 95.70% of the control plants. The parthenocarpy ability of the three edited types of transgenic plants was significantly lower than that of the control plants.

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

[0062]

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

[0064] The fruits of the control plants (WT) and CRISPR-CsIAA4 transgenic plants (cr-1, cr-2, cr-3) were pinched one day before flowering, and RNA was extracted from the fruits 1 day (1d) after flowering for reverse transcription and detection by real-time fluorescence PCR.

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

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

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

[0068] Primer sequences designed for target gene IAA4:

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

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

[0071] The expression level of IAA4 in the control plants and three types of edited transgenic plants. Figure 1 As shown in (c), one day after flowering (1d), the expression levels of the three edited types of transgenic plants 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 for vector construction were designed based on the CDS region of the CsIAA4 gene (excluding the stop codon). The designed primer sequences were:

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

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

[0077] First, the CDS sequence of the CsIAA4 gene was used as a template and PrimerSTAR high-fidelity enzyme was used for PCR amplification to obtain the product. The reaction system was 50 μL, including 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, and ddH 2 O 32.5μL, template 1μL, PrimerSTAR high-fidelity enzyme 0.5μL. The reaction program was 95℃5min; 95℃15sec, 55℃15sec, 72℃15sec, 35 cycles; 72℃5min. Homologous recombination kit ( ⅡOne Step Cloning Kit) was used to recombinantly connect the purified PCR product with the linear vector. 5×CE II Buffer 2μL, purified PCR product 1μL, BamHⅠ single enzyme digested pCAMBIA1305.4 vector fragment 1μL, recombinase Exnase II 1μL, and 5μL sterile water were added to 10μL of the recombination connection system. After mixing, the mixture was connected at 37°C for 30min. The heat shock method was used to transform Escherichia coli DH5a (Quanshijin, Beijing), and a single clone was picked. PCR amplification detection and sequencing were performed. The correct single clone was selected for propagation and plasmid extraction to obtain the overexpression vector OE-CsIAA4. The plasmid was transformed into Agrobacterium competent EHA105, and the single clone strain was picked for detection and preservation for subsequent transgenic (the method is the same as that in Example 3 (1)).

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

[0079] The transgenic plants were obtained by Agrobacterium-mediated genetic transformation, with the transformation recipient material being "YN" and the method being the same as in Example 3(2).

[0080] Positive detection of overexpressed transgenic plants was performed, and OE-CsIAA4 transgenic positive plants were screened by PCR. The detection primer sequences were:

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

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

[0083] Using the leaf DNA of the overexpressed transgenic plant as a template, PCR amplification was performed using Taq PCR MasterMixⅡ. The reaction system was 20 μL, including 2×Taq PCR MasterMixⅡ10 μL, OE-CsIAA4-F primer 1 μL, OE-CsIAA4-R primer 1 μL, ddH 2 O 7μL, DNA template 1μL. The reaction procedure was 98℃5min; 98℃30sec, 55℃30sec, 72℃3min, 35 cycles; 72℃10min; positive strains OE-CsIAA4 transgenic plants (oe-1, oe-2) were screened out by agarose gel electrophoresis.

[0084] Example 5

[0085] Observation of gene phenotype, expression level detection and auxin determination in OE-CsIAA4 transgenic cucumber plants

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

[0087] In order to confirm the function of CsIAA4 gene in regulating cucumber parthenocarpy, the female flowers of control plants (WT:YN) and OE-CsIAA4 transgenic plants (oe-1, oe-2) were clipped one day before flowering. After clipping for one week, the parthenocarpy rate was calculated and the phenotype was observed. 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 that of the control plants by 20.5%. The parthenocarpy ability of the two overexpressed transgenic plants was significantly higher than that of the control plants.

[0088] Table 2 Statistics of parthenocarpy rates of control plants and overexpression plants

[0089]

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

[0091] The fruits of the control plants (WT) and OE-CsIAA4 transgenic plants (oe-1, oe-2) were pinched one day before flowering, and RNA was extracted from the fruits one day after flowering for reverse transcription and detection by real-time fluorescence PCR.

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

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

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

[0095] Primer sequences designed for target gene IAA4:

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

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

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

Claims

1. A gene CsIAA4 for improving the parthenocarpy ability of cucumber, characterized in that: The nucleotide sequence of the gene CsIAA4 is shown in SEQ ID NO.

1.

2. The gene CsIAA4 for regulating cucumber parthenocarpy according to claim 1, characterized in that: The primer pair used to amplify the gene CsIAA4 is: CsIAA4-F:5'-ATGGCATTTCAAAATGGGTTA-3'; CsIAA4-R:5'-TTATGCAACACATCCTAATCCCT-3'.

3. A CDS gene of gene CsIAA4 for improving the parthenocarpy ability of cucumber, characterized in that: The CDS gene sequence is shown in SEQ ID NO.

2.

4. The CDS gene of the gene CsIAA4 for improving the parthenocarpy ability of cucumber according to claim 3, characterized in that: The primer pair used to amplify the CDS gene of the gene CsIAA4 is preferably: CsIAA4-F-1:5'-ATGGCATTTCAAAATGGGTTAAA-3'; CsIAA4-R-1:5'-TGCAACACATCCTAATCCCTTTG-3'.

5. A gene knockout vector CRISPR-CsIAA4 based on the gene CsIAA4 for improving the parthenocarpy ability of cucumber according to claim 1.

6. The gene knockout vector CRISPR-CsIAA4 according to claim 5, characterized in that The gene knockout vector CRISPR-CsIAA4 is constructed by designing the target gene sequences sgRNA1 and sgRNA2 of CsIAA4; sgRNA1:5'-CGAGGCCCGGTGGTCCAAGGCGG-3'; sgRNA2:5'-GACGGTGGCCGCGACAACGGCGG-3'. pCBC-DT1T2 (Cm) was used as a template to amplify dual targets, and the formed double-stranded sgRNA1 / 2 was connected to the vector. After transformation and extraction of the plasmid, the gene knockout vector CRISPR-CsIAA4 was finally obtained.

7. A CDS gene overexpression vector OE-CsIAA4 containing the gene CsIAA4 for improving the parthenocarpy ability of cucumber according to claim 3.

8. The overexpression vector OE-CsIAA4 according to claim 7, characterized in that: The overexpression vector OE-CsIAA4 is constructed by designing primers for vector construction based on the CDS region of the CsIAA4 gene: OE-CsIAA4-F:gagaacacgggggacggatccATGGCATTTCAAAATGGGTTAAA; OE-CsIAA4-R:atggtctttgtagtcggatccTGCAACACATCCTAATCCCTTTG; The gene was amplified, enzyme-cut and connected to the vector, and then transformed and the plasmid was extracted to finally obtain the overexpression vector OE-CsIAA4.

9. Use of the gene CsIAA4 according to claim 1 or the CDS gene of the gene CsIAA4 according to claim 3 or the gene knockout vector according to claim 5 or the overexpression vector according to claim 7 or a host bacteria containing the above genes or vectors in regulating cucumber parthenocarpy.

10. The use according to claim 9, characterized in that: By editing the cucumber CsIAA4 gene through CRISPR / Cas9, its parthenocarpy ability was reduced; by overexpressing the cucumber CsIAA4 gene, its parthenocarpy ability was increased.

11. Use of the gene CsIAA4 for regulating cucumber parthenocarpy according to claim 1, or the CDS gene of the gene CsIAA4 according to claim 3, or the gene knockout vector according to claim 5, or the overexpression vector according to claim 7, or a host bacteria containing the above genes or vectors in cultivating strong parthenocarpy cucumber germplasm.

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