Method for improving brightness of cucumber fruits, related biological material and identification method

Abstract

The invention relates to a method for improving the brightness of cucumber fruits, a related biological material and an identification method. According to the method for improving the brightness of the cucumber fruits, directed mutation is carried out through a gene editing technology, a CsZFP6 mutant containing mini ZFP6 protein is constructed, and the amino acid sequence of the mini ZFP6 protein is shown as SEQ ID NO: 1. A nucleic acid molecule comprises a nucleic acid molecule encoding the miniZFP6 protein in the method for improving the brightness of the cucumber fruit. The recombinant vector comprises the nucleic acid molecule. The recombinant microorganism comprises the nucleic acid molecule or the recombinant vector. On the basis of a 3D structure of the CsZFP6 protein, the CsZFP6 mutant containing the mini ZFP6 protein is created through a CRISPR / Cas9 technology, the brightness of cucumber fruits is effectively improved, and direction and convenience are provided for improvement of the brightness of cucumbers and breeding.

Description

Technical Field

[0001] The present application relates to the field of molecular biology technology, and specifically, to a method for improving the brightness of cucumber fruits, related biological materials and identification methods. Background Art

[0002] Cucumber (Cucumis sativus L.) is one of the most important vegetables. Cucumber is very popular among consumers for its unique flavor and taste. With the continuous development of social economy, people have higher and higher requirements for the quality of cucumber. The brightness of cucumber fruit is an important appearance quality, and shiny cucumbers are more attractive to consumers. Therefore, improving the brightness of the fruit has become one of the important goals of cucumber quality improvement.

[0003] At present, only one brightness-related gene (glossy fruit 1, glf1) has been precisely located in cucumber. It is located on Chr5 and encodes the C2H2 transcription factor CsZFP6. However, glf1 is not only involved in regulating the formation of fruit tumors, but also closely linked to the fruit color-related gene uniform immature fruit color (u) and the fruit thorn size-related gene small spines (ss), which limits the application of glf1 in breeding to a certain extent.

[0004] CRISPR / Cas9 is a simple and easy genome editing technology that can change plant traits by mutating or deleting target sequences. Therefore, directional control of target traits and related genes plays an important role in cucumber improvement. Summary of the invention

[0005] One purpose of the present application is to provide a method for improving the brightness of cucumber fruits, related biological materials and identification methods. The present application creates a CsZFP6 mutant, which contains the protein sequence miniZFP6, and can effectively improve the brightness of cucumber fruits.

[0006] In a first aspect, the present application claims a method for improving the brightness of cucumber fruits, wherein a CsZFP6 mutant containing a miniZFP6 protein is constructed by targeted mutagenesis using gene editing technology, and the amino acid sequence of the miniZFP6 protein is shown in SEQ ID NO: 1.

[0007] In a second aspect, the present application claims protection for a nucleic acid molecule comprising a nucleic acid molecule encoding the miniZFP6 protein in the method for improving the brightness of cucumber fruit as described above.

[0008] In a third aspect, the present application claims protection for a recombinant vector comprising the nucleic acid molecule as described above.

[0009] In a fourth aspect, the present application claims protection for a recombinant microorganism comprising the nucleic acid molecule or the recombinant vector as described above.

[0010] In a fifth aspect, the present application claims protection for a molecular marker for detecting the nucleic acid molecule as described above, which comprises a primer pair as shown in SEQ ID NO.2 and SEQ ID NO.3.

[0011] In a sixth aspect, the present application claims protection for the use of the above-mentioned nucleic acid molecules, or the above-mentioned recombinant vectors, or the above-mentioned recombinant microorganisms, or the above-mentioned cell lines, or the above-mentioned molecular markers in breeding high-quality cucumbers.

[0012] In a seventh aspect, the present application claims a method for identifying a nucleic acid encoding a miniZFP6 protein, using the molecular markers as described above to identify cucumbers.

[0013] Based on the 3D structure of CsZFP6 protein, this application created a CsZFP6 mutant containing miniZFP6 protein through CRISPR / Cas9 technology, which effectively improved the brightness of cucumber fruit and provided direction and convenience for the improvement of cucumber brightness and breeding.

[0014] The present application involves biological materials such as nucleic acid molecules, recombinant vectors, recombinant microorganisms, cell lines, and molecular markers. The present application uses molecular biological techniques to construct a CsZFP6 mutant containing miniZFP6 protein, which effectively improves the brightness of the fruit and has good application prospects in breeding high-quality cucumbers.

[0015] The method for identifying miniZFP6 protein of the present application can quickly identify miniZFP6 protein, including but not limited to identifying cucumber in the seedling stage. BRIEF DESCRIPTION OF THE DRAWINGS

[0016] Figure 1 :The protein 3D structure prediction of CsZFP6. The red one is the α-helix structure.

[0017] Figure 2 :pCUA-miniZFP6 vector map.

[0018] Figure 3 :The fruit brightness phenotype of cucumber containing miniZFP6 protein and its background type. The left one is the cucumber inbred line 'CCMC', and the right one is the cucumber containing miniZFP6 protein sequence in the 'CCMC' background.

[0019] Figure 4: Electrophoresis identification results of cucumbers containing miniZFP6 protein. Wherein, M: DNA marker; 1: 'CCMC'; 2: F1 plant of 'CCMC' and cucumber containing miniZFP6 protein; 3: cucumber containing miniZFP6 protein; 4: cucumber inbred line 'JY35'; 5: cucumber inbred line 'KP2'; 6: cucumber inbred line 'GY2'. DETAILED DESCRIPTION

[0020] The present application will be described in detail below with reference to the accompanying drawings and in combination with embodiments. The following specific implementations are only used to illustrate the present application and should not be considered to limit the scope of the present application. In the absence of contradiction, the embodiments in the present application and the features in the embodiments can be combined with each other.

[0021] A method for improving the brightness of cucumber fruits comprises the following steps: directing mutation through gene editing technology to construct a CsZFP6 mutant containing a miniZFP6 protein, wherein the amino acid sequence of the miniZFP6 protein is MAALENHYQTKQNNNNPATTRLKLFGFDVQEDLDQDDSTPTSSDSGAAVFRNPIVAAFAPPPHLLPSAAVTASSSWPVYIPRAPPQFQVSHGCVFTPSSSYGGGGDAPPPPSPPDFFTMGSRSSHGMVDAPMSLSRFSKVDGGTAFDDGPGLDLHLSLAPAAP (SEQ ID NO: 1).

[0022] Example 1 Creation of miniZFP6 protein in cultivated cucumber

[0023] In order to design the target more accurately and create the miniZFP6 protein sequence, Alphafold was first used to predict the 3D structure of CsZFP6 protein. The results showed that the CsZFP6 protein structure includes an α-helix, such as Figure 1As shown in Figure 3, α-helical structures are usually associated with protein-DNA interactions. Two 19 bp targets were designed at both ends of the α-helical structure, and the primer sequences were miniZFP6-CR-F: ATATATGGTCTCGATGGGGTTTCTAAAAACGGGTTTTAGAGCTAGAAATAGC (SEQ ID No. 4) and miniZFP6-CR-R: ATTATTGGTCTCGAAACCCGTTCCATCCTCCGGCGACAATCTCTTAGTCGACTCTAC (SEQ ID No. 5). Plasmid pCBC-DT1T2 (Addgene) was used as a template and 2×Phanta Max MasterMix (Vazyme) was used for PCR cloning. The PCR-purified fragments were ligated to the pCUA-miniZFP6 vector by the Golden Gate system. Positive clones were screened by colony PCR, and positive clones were subjected to Sanger sequencing. The sequencing primer was pBIN19-Seq-R: CAGCTGGCGAAAGGGGGAT (SEQ ID No. 6). After the sequencing was correct, the corresponding E. coli strain was added with glycerol and then quickly frozen with liquid nitrogen and stored at -80°C.

[0024] pCUA-miniZFP6( Figure 2 ) was transferred into Agrobacterium strain EHA105 (purchased from Weidi Biotechnology), and the positive colonies were used to vacuum infect the cotyledons of the cucumber inbred line 'CCMC'. After 2 days of co-cultivation at 25°C, the explants were transferred to the shoot regeneration medium, and 8 explants were placed in each culture dish. The light cycle in the culture room was adjusted to 16 hours of light / 8 hours of darkness, and the temperature was 25°C. After 20-25 days, positive shoots with GFP signals were found under a stereo microscope (Nikon). The positive shoots were cut off from the explants, transferred to the rooting medium, and continued to be placed in the culture room until positive tissue culture seedlings were obtained (generally 2 positive seedlings can be obtained from 100 explants). The GFP gene in the obtained regenerated plants was detected by PCR, and the primers were GFP-F: ATGGTGAGCAAGGGCGAG (SEQ ID No.7) and GFP-R: TTAAGATCTGTACAGCTCGTCCATGC (SEQ ID No.8). The positive plants were further subjected to Sanger sequencing of the CsZFP6 locus, translated into protein sequences (https: / / web.expasy.org / translate / ), and then screened for available miniZFP6 protein sequences. A total of 12 positive plants were obtained, of which 5 contained the miniZFP6 protein sequence shown in SEQ ID No.1, and its α-helical structure was completely missing. Figure 3 As shown, cucumber fruits containing miniZFP6 protein sequences were observed ( Figure 3 Right), which is significantly brighter than its background type 'CCMC' ( Figure 3 Left).

[0025] Example 2 Rapid identification of miniZFP6 protein

[0026] In order to improve the brightness of cultivated cucumbers, the DNA sequence encoding miniZFP6 can be transferred to other background types through backcrossing. In this case, molecular-assisted breeding tools are needed to accelerate the process. To this end, this embodiment provides a pair of primers miniZFP6-genotyping-F: TGGCAGCTCTAGAAAACCATTACCAAAC (SEQ ID No. 2) and miniZFP6-genotyping-R: TGGGGCGGCCGGAGCGAG (SEQ ID No. 3) for rapid identification of the DNA sequence encoding the miniZFP6 protein. The CTAB method was used to extract the genomic DNA of the cotyledons of the seedlings of the plants to be tested. The PCR reaction system: 10 μL 2×Taq PCRMasterMix, 1 μL gDNA, 1 μL of each primer, plus 4.5 μL ddH2O, the total reaction system is 15 μL. The PCR amplification program was: 98°C pre-denaturation for 5 min, 95°C denaturation for 30 s, 56°C annealing for 30 s, 72°C extension for 30 s, 31 cycles, 72°C extension for 10 min, and storage at 4°C. When the band size was 490 bp, the material contained the DNA sequence encoding miniZFP6, such as Figure 4 To verify the effectiveness of the primers, three other cultivated cucumber varieties ('JY35', 'KP2' and 'GY2') were randomly selected to extract their DNA for identification (lanes 4-6). It was found that the fragments were all larger than 490 bp, confirming that the primer pair can identify the DNA sequence encoding miniZFP6 from the cultivated cucumber population. In addition, heterozygous plants were also tested and found that the primers can also effectively identify them (lane 2).

[0027] Although the present application has been described in detail above with general instructions and specific implementation methods, those skilled in the art may make various changes or modifications to the present application based on the present application, and these equivalent forms also fall within the scope defined by the claims of the present application.

Claims

1. A method for improving the brightness of cucumber fruits, characterized in that: A CsZFP6 mutant containing a miniZFP6 protein was constructed by directed mutagenesis using gene editing technology. The amino acid sequence of the miniZFP6 protein is shown in SEQ ID NO:

1.

2. A nucleic acid molecule, characterized in that It comprises a nucleic acid molecule encoding the miniZFP6 protein in the method for improving the brightness of cucumber fruits as claimed in claim 1.

3. A recombinant vector, characterized in that: It comprises the nucleic acid molecule according to claim 2.

4. A recombinant microorganism, characterized in that It comprises the nucleic acid molecule according to claim 2 or the recombinant vector according to claim 3.

5. A molecular marker, characterized in that Used to detect the nucleic acid molecule as described in claim 2, it includes a primer pair as shown in SEQ ID NO.2 and SEQ ID NO.

3.

6. Use of the nucleic acid molecule according to claim 2, or the recombinant vector according to claim 3, or the recombinant microorganism according to claim 4, or the molecular marker according to claim 5 in breeding high-quality cucumbers.

7. A method for identifying a nucleic acid sequence encoding a miniZFP6 protein, characterized in that: Cucumber is identified using the molecular marker as described in claim 5.

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