Application of PavERF110 gene in regulating and controlling ripening direction of sweet cherry fruits

Abstract

The invention provides an application of a PavERF110 gene in regulating and controlling the ripening direction of sweet cherry fruits, and belongs to the technical field of molecular breeding. The nucleotide sequence of the PavERF110 gene is as shown in SEQ ID NO.1, and the amino acid sequence of the encoded protein of the PavERF110 gene is as shown in SEQ ID NO.2. The invention also provides an overexpression vector and a VIGS gene silencing vector constructed by the gene, a construction method of the overexpression vector and the VIGS gene silencing vector, and a recombinant microorganism containing the vectors. Engineering bacteria containing related carriers are injected and infected at the equator of the fruits 25 days after the sweet cherries blossom, and it is found that overexpression of the PavERF110 gene can accelerate fruit coloring and increase the expression level of the mature related genes. New technical support and theoretical basis are provided for sweet cherry fruit quality improvement and ripening regulation, market requirements can be met, and meanwhile understanding of a fruit ripening transcription regulation mechanism is deepened.

Description

Technical Field

[0001] The invention belongs to the technical field of molecular breeding, and in particular relates to application of the PavERF110 gene in regulating the ripening direction of sweet cherry fruits. Background Art

[0002] Fruits of appropriate maturity, with their excellent flavor, color and taste, not only meet consumers' demand for high-quality food, but also significantly enhance the market competitiveness and economic value of products. The process of promoting fruit ripening can timely supply ripe fruits in a specific season, meeting the urgent market demand for fresh fruits. In addition, this process supports an environmentally friendly and sustainable agricultural development model by reducing the use of chemical ripening agents. Sweet cherry, as a popular emerging fruit, is particularly favored by consumers because of its rich nutrients such as anthocyanins, vitamin C and carotenoids accumulated during the ripening process. However, the current understanding of the transcriptional regulatory mechanism during the ripening process of sweet cherry fruit is still insufficient. In-depth screening and research on the key transcription factors in the ripening process of sweet cherry can not only reveal their specific role in regulating fruit ripening, but also provide valuable technical support and strategies for improving the quality of sweet cherry fruit.

[0003] The AP2 / ERF family transcription factors play a vital role in plant growth and development and are one of the most critical members of the plant transcription factor family. These transcription factors finely regulate the expression of downstream target genes by specifically recognizing and binding to sequences in the promoter region of target genes. In the regulation of fruit ripening, ERF transcription factors play a systemic regulatory role by affecting key quality traits such as firmness, pigment accumulation, sugars and organic acids. Specifically, in tomatoes, overexpression of the LeERF1 gene causes the fruit to exhibit a constitutive ethylene response phenotype, thereby accelerating the ripening process; in bananas, MaERF9 acts as a transcriptional activator and participates in regulating the expression of genes related to ripening; in apples, the MdERF1B transcription factor interacts with the promoters of MdMYB9 and MdERF1101 to promote the accumulation of pigments during fruit ripening. These examples highlight the important role of ERF transcription factors in the ripening process of different fruits. Nevertheless, for sweet cherries, the specific role of ERF family transcription factors in ripening regulation remains to be elucidated. In-depth research on the regulatory mechanism of ERF transcription factors in sweet cherry fruit ripening will not only help to reveal the molecular basis for the formation and maintenance of its unique flavor, but also have important scientific significance and application prospects for improving fruit quality through biological means. Summary of the invention

[0004] In view of this, the purpose of the present invention is to provide an application of the PavERF110 gene in regulating the ripening direction of sweet cherry fruit, to reveal its mechanism of action in regulating the ripening of sweet cherry fruit, and to provide technical support for improving the quality of sweet cherry fruit.

[0005] In order to achieve the above-mentioned object of the invention, the present invention provides the following technical solutions:

[0006] Application of the PavERF110 gene in regulating the ripening direction of sweet cherry fruit, wherein the nucleotide sequence of the PavERF110 gene is shown in SEQ ID NO.1.

[0007] Preferably, the amino acid sequence of the protein encoded by the PavERF110 gene is shown in SEQ ID NO.2.

[0008] The present invention also provides an overexpression vector constructed by the PavERF110 gene.

[0009] Preferably, the construction method comprises the following steps: using sweet cherry cDNA as a template, amplifying the PavERF110 gene coding frame using the forward primer PavERF110-FO and the reverse primer PavERF110-RO, and connecting the PavERF110 gene coding frame to a vector by homologous recombination; the nucleotide sequence of the forward primer PavERF110-FO is shown in SEQ ID NO.3, and the nucleotide sequence of the reverse primer PavERF110-RO is shown in SEQ ID NO.4.

[0010] The present invention also provides a VIGS gene silencing vector constructed by the PavERF110 gene.

[0011] Preferably, the construction method comprises the following steps: using sweet cherry DNA as a template, amplifying the 3' non-conserved region fragment of the PavERF110 gene using the forward primer PavERF110-FV and the reverse primer PavERF110-RV, and connecting the amplified fragment to a vector by homologous recombination; the nucleotide sequence of the forward primer PavERF110-FV is shown in SEQ ID NO.5, and the nucleotide sequence of the reverse primer PavERF110-RV is shown in SEQ ID NO.6.

[0012] The present invention also provides a recombinant microorganism containing the above-mentioned overexpression vector or the above-mentioned VIGS gene silencing vector.

[0013] The present invention also provides the use of the above-mentioned overexpression vector, or VIGS gene silencing vector, or recombinant microorganism in regulating the ripening direction of sweet cherry fruit.

[0014] The present invention also provides a method for regulating the ripening direction of sweet cherry fruit, wherein the above-mentioned overexpression vector is transferred into DH5α Escherichia coli to prepare an engineered bacterium, or the above-mentioned VIGS gene silencing vector is transferred into GV3101 Agrobacterium competent state to prepare an engineered bacterium, and the bacterial liquid is injected at the equator of the sweet cherry fruit 25 days after flowering for infection.

[0015] The present invention also provides the use of the above-mentioned overexpression vector, or VIGS gene silencing vector, or recombinant microorganism in sweet cherry breeding.

[0016] Compared with the prior art, the present invention has the following beneficial effects:

[0017] (1) Promote fruit ripening. The present invention verifies the regulatory function of PavERF110 in the fruit ripening process by genetic and molecular means. Fruits overexpressing PavERF110 change color faster, and the expression levels of ripening-related genes CHS, NCED, PL1, and PL2 are significantly increased, thereby promoting the ripening of sweet cherry fruits and helping to meet the market demand for fresh fruits.

[0018] (2) Providing a basis for quality improvement. The cloned PavERF110 gene and the verification of its function provide a basis for the research and development of sweet cherry fruit quality improvement technology, which will help to cultivate sweet cherry varieties with better quality.

[0019] (3) Deepen the understanding of the mechanism. This invention helps to reveal the transcriptional regulatory mechanism of sweet cherry fruit ripening, provides a reference for further research on the molecular basis of fruit ripening, and is of great significance to the development of the sweet cherry industry. BRIEF DESCRIPTION OF THE DRAWINGS

[0020] Figure 1 The gene expression level of PavERF110 in sweet cherry fruit injected with PavERF110 gene overexpression bacterial solution;

[0021] Figure 2 is the gene expression level of PavERF110 in sweet cherry fruit injected with the PavERF110 gene VIGS gene silencing vector;

[0022] Figure 3 The color change of sweet cherry fruit with overexpression and silencing of PavERF110 gene;

[0023] Figure 4 The expression levels of CHS, NCED, PL1, and PL2 in sweet cherry fruits with overexpression and silencing of PavERF110 gene. DETAILED DESCRIPTION

[0024] The technical solutions provided by the present invention are described in detail below in conjunction with the embodiments, but they should not be construed as limiting the protection scope of the present invention.

[0025] In the embodiment of the present invention, the plant material is red light sweet cherry, and all sweet cherry fruit samples are collected from the Wenchuan sweet cherry research base of Sichuan Agricultural University. Strains: vector pBI121 is preserved by the laboratory of the College of Horticulture of Sichuan Agricultural University; vectors pTRV1 and pTRV2 are preserved by the laboratory of the College of Horticulture of Sichuan Agricultural University; DH5α Escherichia coli transformation strain and GV3101 Agrobacterium transformation strain are purchased from Qingke Xinye Biotechnology Co., Ltd.

[0026] Example 1

[0027] Cloning of PavERF110 gene from sweet cherry

[0028] Input the CDS sequence of the PavERF110 gene into the website (https: / / crm.vazyme.com / cetool / ) to design the PavERF110 gene cloning primers. The selected sequence needs to have an appropriate length (15-25bp), Tm value (50-60°C) and GC content (about 50%). The Tm values ​​of the primers on both chains need to be basically consistent. Add the KpnI restriction site sequence in front of the primer, select the KpnI restriction site on the website (https: / / crm.vazyme.com / cetool / singlefragment.html), and input the sequence of more than 20bp on the left and right of the restriction site on the vector, and finally generate cloning primers with restriction sites and recombination sequences.

[0029] The target gene was cloned by PCR, and the 2×PhantaFlashMasterMix high-fidelity enzyme purchased from Nanjing Novozyme was used for gene cloning. The red light sweet cherry fruit cDNA stored in this laboratory was used as an amplification template. The reaction system is as follows: 2×PhantaFlashMasterMix 25μL; PrimerF (10μM) 1μL; PrimerR (10μM) 1μL; cDNA 1μL; ddH2O 22μL. The amplification program is as follows: 98℃ pre-denaturation for 30s; 98℃ denaturation for 15s; 58℃ annealing for 15s; 72℃ extension for 10s; 35 cycles; 72℃ final extension for 5min; 4℃ storage. 50μL of PCR product was aspirated for 1% gel electrophoresis analysis and detection. After 120V electrophoresis for 20min, the size of the amplified band was observed using a gel imaging system. The product was purified using a PCR product gel recovery kit purchased from Qingke.

[0030] The results showed that the size of the electrophoresis band was consistent with the reference gene size. The PavERF110 gene sequence had a total of 1398 bases (SEQ ID NO.1) and the encoded protein contained 465 amino acids (SEQ ID NO.2).

[0031] Example 2

[0032] Construction and infection of PavERF110 gene overexpression and VIGS gene silencing vectors

[0033] This method uses homologous recombination method, using the purchased from Nanjing Novozymes Ⅱ One Step Cloning Kit, the purified PCR products were connected to the vector pBI121 and the vector pTRV2, respectively, to obtain the overexpression vector pBI121-PavERF110 and the VIGS gene silencing vector pTRV2-PavERF110.

[0034] Specific:

[0035] Construction of overexpression vector pBI121-PavERF110: Using sweet cherry cDNA as a template, the PavERF110 gene coding frame was amplified using forward primer PavERF110-FO (SEQ ID NO.3) and reverse primer PavERF110-RO (SEQ ID NO.4), and the coding frame of the PavERF110 gene was connected to the vector pBI121 by homologous recombination to obtain the overexpression vector pBI121-PavERF110.

[0036] Construction of VIGS gene silencing vector pTRV2-PavERF110: Using sweet cherry DNA as a template, the 3' non-conserved region fragment of the PavERF110 gene was amplified using the forward primer PavERF110-FV (SEQ ID NO.5) and the reverse primer PavERF110-RV (SEQ ID NO.6), and the amplified fragment was connected to the vector by homologous recombination to obtain the VIGS gene silencing vector pTRV2-PavERF110.

[0037] Then the constructed vectors pBI121-PavERF110 and pTRV2-PavERF110 were transformed into DH5α Escherichia coli and GV3101 Agrobacterium competent cells, respectively. Agrobacterium solution was slowly injected at the equator of the sweet cherry fruit 25 days after flowering. The PavERF110 gene overexpression test group included pBI121-PavERF110 solution, and the control group was pBI121 empty solution. The gene silencing test group included pTRV2-PavERF110 solution and pTRV1 solution, and the control group was pTRV2 empty solution and pTRV1 empty solution. Each treatment had 30 biological replicates. Sweet cherry fruits were collected 15 days after injection, frozen in liquid nitrogen, and stored in a -80℃ ultra-low temperature refrigerator for further analysis.

[0038] According to the instructions of the fruit RNA extraction kit, RNA was extracted from fruit samples of different experimental groups, the genome was removed from RNA, and reverse transcription was performed to obtain cDNA template, which was diluted to 100 ng / ml and stored at -20°C for later use. The fruit RNA extraction kit and reverse transcription kit were purchased from Tiangen Biochemical Technology Co., Ltd. qRT-PCR primers were designed using the website (https: / / crm.vazyme.com / cetool / ), and the gene expression level of PavERF110 was detected using a real-time fluorescence quantitative PCR instrument (Bio-Rad) according to the experimental steps provided by the qPCR kit. The Actin gene was used as an internal reference and three biological replicates were set. The qRT-PCR kit was purchased from Novozyme Biotech Co., Ltd.

[0039] Among them, the primers used for detecting the expression level of the sweet cherry PavERF110 gene based on the qRT-PCR method include the forward primer PavERF110-FQ (SEQ ID NO.7) and the reverse primer PavERF110-RQ (SEQ ID NO.8).

[0040] The results showed that the gene expression level of PavERF110 in sweet cherry fruits injected with PavERF110 gene overexpression solution was significantly increased compared with the control ( Figure 1 ), while the gene expression level of PavERF110 was significantly reduced in sweet cherry fruits injected with VIGS vector ( Figure 2 ). At the same time, the color change of fruits overexpressing PavERF110 was accelerated, while the color change of fruits silencing PavERF110 was slowed down ( Figure 3 ). These results indicate that the transcription factor PavERF110 can regulate the ripening of sweet cherry fruit.

[0041] Example 3

[0042] Detection of expression levels of genes related to sweet cherry fruit ripening

[0043] The qRT-PCR primers were designed using the website (https: / / crm.vazyme.com / cetool / ), and the experimental steps provided by the qPCR kit were followed. The cDNA obtained in Example 2 was used as a template, and the expression levels of the key genes CHS, NCED, PL1, and PL2 related to ripening in the fruit samples of different experimental groups were detected using a real-time fluorescence quantitative PCR instrument (Bio-Rad). The Actin gene was used as an internal reference and three biological replicates were set.

[0044] Among them, the primers for detecting the sweet cherry ripening-related gene CHS based on the qRT-PCR method include forward primer CHS-FQ (SEQ ID NO.9) and reverse primer CHS-RQ (SEQ ID NO.10); the primers for detecting the sweet cherry ripening-related gene NCED include forward primer NCED-FQ (SEQ ID NO.11) and reverse primer NCED-RQ (SEQ ID NO.12); the primers for detecting the sweet cherry ripening-related gene PL1 include forward primer PL1-FQ (SEQ ID NO.13) and reverse primer PL1-RQ (SEQ ID NO.14); the primers for detecting the sweet cherry ripening-related gene PL2 include forward primer PL2-FQ (SEQ ID NO.15) and reverse primer PL2-RQ (SEQ ID NO.16).

[0045] The results showed that compared with the control, the expression levels of ripening-related genes CHS, NCED, PL1, and PL2 in sweet cherry fruits overexpressing the PavERF110 gene were significantly increased, while the expression levels of ripening-related genes CHS, NCED, PL1, and PL2 in sweet cherry fruits silencing the PavERF110 gene were all decreased ( Figure 4 ). This indicates that PavERF110 can positively regulate the expression of key ripening genes, thereby promoting the ripening of sweet cherry fruit.

[0046] The above is only a preferred embodiment of the present invention. It should be pointed out that for ordinary technicians in this technical field, several improvements and modifications can be made without departing from the principle of the present invention. These improvements and modifications should also be regarded as the scope of protection of the present invention.

Claims

1. Application of the PavERF110 gene in regulating the ripening direction of sweet cherry fruit, the nucleotide sequence of the PavERF110 gene is shown in SEQ ID NO.

1.

2. The use according to claim 1, characterized in that: The amino acid sequence of the protein encoded by the PavERF110 gene is shown in SEQ ID NO.

2.

3. An overexpression vector constructed by the PavERF110 gene according to claim 1.

4. The overexpression vector according to claim 3, characterized in that The construction method includes the following steps: Using sweet cherry cDNA as template, the PavERF110 gene coding frame was amplified using forward primer PavERF110-FO and reverse primer PavERF110-RO, and the PavERF110 gene coding frame was connected to the vector by homologous recombination. The nucleotide sequence of the forward primer PavERF110-FO is shown in SEQ ID NO.3, and the nucleotide sequence of the reverse primer PavERF110-RO is shown in SEQ ID NO.

4.

5. A VIGS gene silencing vector constructed with the PavERF110 gene according to claim 1.

6. The VIGS gene silencing vector according to claim 5, characterized in that: The construction method includes the following steps: Using sweet cherry DNA as a template, the 3' non-conservative region fragment of the PavERF110 gene was amplified using the forward primer PavERF110-FV and the reverse primer PavERF110-RV, and the amplified fragment was connected to the vector by homologous recombination. The nucleotide sequence of the forward primer PavERF110-FV is shown in SEQ ID NO.5, and the nucleotide sequence of the reverse primer PavERF110-RV is shown in SEQ ID NO.

6.

7. A recombinant microorganism comprising the overexpression vector according to claim 3 or the VIGS gene silencing vector according to claim 5.

8. Use of the overexpression vector according to claim 3, or the VIGS gene silencing vector according to claim 5, or the recombinant microorganism according to claim 7 in regulating the ripening direction of sweet cherry fruit.

9. A method for regulating the ripening direction of sweet cherry fruit, characterized in that: The overexpression vector of claim 3 is transferred into DH5α Escherichia coli to prepare an engineered bacterium, or the VIGS gene silencing vector of claim 5 is transferred into GV3101 Agrobacterium competent cells to prepare an engineered bacterium, and the bacterial solution is injected at the equator of the sweet cherry fruit 25 days after flowering for infection.

10. Use of the overexpression vector according to claim 3, or the VIGS gene silencing vector according to claim 5, or the recombinant microorganism according to claim 7 in sweet cherry breeding.

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