Application of PavERF110 gene in regulating ripening direction of sweet cherry fruit

Abstract

The application provides application of a PavERF110 gene in regulation of ripening direction of sweet cherry fruits, and belongs to the technical field of molecular breeding.The nucleotide sequence of the PavERF110 gene is shown as SEQ ID NO.1, and the amino acid sequence of the encoded protein is shown as SEQ ID NO.2.The application also provides an overexpression vector and a VIGS gene silencing vector constructed from the gene and a construction method of the vectors, and recombinant microorganisms containing the vectors.It is found that overexpression of the PavERF110 gene can accelerate fruit color change and increase expression levels of mature-related genes by injecting and infecting engineering bacteria containing relevant vectors at the equator of fruits 25 days after flowering of the sweet cherry.The application provides new technical support and theoretical basis for improvement of fruit quality and regulation of ripening of the sweet cherry, helps to meet market demand, and deepens understanding of the transcriptional regulation mechanism of fruit ripening.

Description

Technical Field

[0001] This invention belongs to the field of molecular breeding technology, and in particular relates to the application of the PavERF110 gene in regulating the ripening direction of sweet cherry fruit. Background Technology

[0002] Properly ripened fruit, with its superior flavor, color, and texture, not only satisfies consumers' demand for high-quality food but also significantly enhances the product's market competitiveness and economic value. Promoting fruit ripening allows for the timely supply of ripe fruit during specific seasons, meeting the market's urgent need for fresh fruit. Furthermore, this process supports an environmentally friendly and sustainable agricultural development model by reducing the use of chemical ripening agents. Sweet cherries, as a popular emerging fruit, are particularly favored by consumers due to their rich accumulation of anthocyanins, vitamin C, and carotenoids during ripening. However, current understanding of the transcriptional regulatory mechanisms during sweet cherry ripening is still insufficient. In-depth screening and research of key transcription factors in the sweet cherry ripening process can not only reveal their specific roles in regulating fruit ripening but also provide valuable technical support and strategies for improving the quality of sweet cherry fruit.

[0003] AP2 / ERF family transcription factors play a crucial role in plant growth and development, and are among the most critical members of the plant transcription factor family. These transcription factors precisely regulate the expression of downstream target genes by specifically recognizing and binding to the sequences of the promoter regions of target genes. In the regulation of fruit ripening, ERF transcription factors exert a systemic regulatory effect by influencing key quality traits such as firmness, pigment accumulation, sugar content, and organic acids. Specifically, in tomatoes, overexpression of the LeERF1 gene induces a constitutive ethylene response phenotype in the fruit, thereby accelerating the ripening process; in bananas, MaERF9, as a transcriptional activator, participates in regulating the expression of ripening-related genes; in apples, the MdERF1B transcription factor interacts with the promoters of MdMYB9 and MdERF1101, promoting pigment accumulation during fruit ripening. These examples highlight the important role of ERF transcription factors in different fruit ripening processes. Nevertheless, the specific role of ERF family transcription factors in ripening regulation in sweet cherries remains to be elucidated. In-depth research into the regulatory mechanism of ERF transcription factors in the ripening of sweet cherries will not only help to reveal the molecular basis for the formation and maintenance of their unique flavor, but also has 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 this invention is to provide the 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] To achieve the above-mentioned objectives, the present invention provides the following technical solution:

[0006] The 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.

[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 from the above-mentioned PavERF110 gene.

[0009] Preferably, the construction method includes the following steps: using sweet cherry cDNA as a template, the PavERF110 gene coding frame is amplified using the forward primer PavERF110-FO and the reverse primer PavERF110-RO, and the PavERF110 gene coding frame is ligated 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.

[0010] The present invention also provides a VIGS gene silencing vector constructed from the above-mentioned PavERF110 gene.

[0011] Preferably, the construction method includes the following steps: using sweet cherry DNA as a template, the 3' non-conserved region fragment of the PavERF110 gene is amplified using the forward primer PavERF110-FV and the reverse primer PavERF110-RV, and the amplified fragment is ligated 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 application 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 engineered bacteria, or the above-mentioned VIGS gene silencing vector is transferred into GV3101 Agrobacterium competent cells to prepare engineered bacteria, and the bacterial solution is injected into the equator of the fruit 25 days after flowering to infect it.

[0015] The present invention also provides the application 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) Promoting fruit ripening. This invention verified the regulatory function of PavERF110 in the fruit ripening process through genetic and molecular methods. Overexpression of PavERF110 accelerated fruit color change and significantly increased the expression levels of ripening-related genes CHS, NCED, PL1, and PL2, thereby promoting the ripening of sweet cherries and helping to meet the market demand for fresh fruit.

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

[0019] (3) Deepening 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. Attached Figure Description

[0020] Figure 1 The gene expression level of PavERF110 in sweet cherry fruits injected with bacteria that overexpress the PavERF110 gene;

[0021] Figure 2 The gene expression level of PavERF110 in sweet cherry fruits injected with the PavERF110 gene VIGS gene silencing vector.

[0022] Figure 3 The color change of sweet cherry fruits with overexpression and silence of the PavERF110 gene;

[0023] Figure 4 The expression levels of CHS, NCED, PL1, and PL2 in sweet cherry fruits with overexpression and silencing of the PavERF110 gene were determined. Detailed Implementation

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

[0025] In this embodiment of the invention, the plant material was the Red Lantern sweet cherry, and all sweet cherry fruit samples were collected from the Wenchuan Sweet Cherry Research Base of Sichuan Agricultural University. Strains: Vector pBI121 was preserved by the laboratory of the College of Horticulture, Sichuan Agricultural University; vectors pTRV1 and pTRV2 were preserved by the laboratory of the College of Horticulture, Sichuan Agricultural University; DH5α Escherichia coli transformation strain and GV3101 Agrobacterium transformation strain were purchased from Qingke Xinyue Biotechnology Co., Ltd.

[0026] Example 1

[0027] Cloning of the PavERF110 gene in sweet cherry

[0028] Input the PavERF110 gene CDS sequence into the website (https: / / crm.vazyme.com / cetool / ) to design PavERF110 gene cloning primers. The selected sequences need to have appropriate length (15-25 bp), Tm value (50-60℃), and GC content (around 50%). The Tm values ​​of the two primer strands should be basically consistent. Add the KpnI restriction site sequence before the primers. Select the KpnI restriction site on the website (https: / / crm.vazyme.com / cetool / singlefragment.html) and input sequences of at least 20 bp to the left and right of the restriction site on the vector. Finally, cloning primers with restriction sites and recombinant sequences are generated.

[0029] The target gene was cloned using PCR. A 2×PhantaFlashMasterMix high-fidelity enzyme (purchased from Nanjing Novizan) was used for gene cloning, and cDNA from red-light sweet cherry fruit preserved in our laboratory was used as the amplification template. The reaction system was as follows: 2×PhantaFlashMasterMix 25 μL; Primer F (10 μM) 1 μL; Primer R (10 μM) 1 μL; cDNA 1 μL; ddH2O 22 μL. The amplification program was as follows: 98℃ pre-denaturation for 30 s; 98℃ denaturation for 15 s; 58℃ annealing for 15 s; 72℃ extension for 10 s; 35 cycles; 72℃ final extension for 5 min; storage at 4℃. 50 μL of the PCR product was analyzed by 1% gel electrophoresis. After electrophoresis at 120V for 20 min, the size of the amplified bands was observed using a gel imaging system. The product was purified using a PCR product gel extraction kit purchased from Qingke.

[0030] The results showed that the size of the electrophoretic bands was consistent with the size of the reference gene. The PavERF110 gene sequence has a total of 1398 bases (SEQ ID NO.1) and encodes a protein containing 465 amino acids (SEQ ID NO.2).

[0031] Example 2

[0032] PavERF110 gene overexpression and VIGS gene silencing vector construction and infection

[0033] This method uses homologous recombination, employing reagents purchased from Nanjing Novizan. II. The One Step Cloning Kit was used to ligate the purified PCR products to vectors pBI121 and pTRV2, respectively, to obtain the overexpression vector pBI121-PavERF110 and the VIGS gene silencing vector pTRV2-PavERF110.

[0034] Specifically:

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

[0036] Construction of the 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). The amplified fragment was then ligated into the vector using homologous recombination to obtain the VIGS gene silencing vector pTRV2-PavERF110.

[0037] The constructed vectors pBI121-PavERF110 and pTRV2-PavERF110 were then transformed into DH5α *E. coli* and GV3101 *Agrobacterium* competent cells, respectively. Twenty-five days after flowering, *Agrobacterium* bacterial suspension was slowly injected into the equatorial region of the sweet cherry fruit. The PavERF110 gene overexpression group included pBI121-PavERF110 bacterial suspension, while the control group consisted of pBI121 empty vector bacterial suspension. The gene silencing group included pTRV2-PavERF110 and pTRV1 bacterial suspensions, while the control group consisted of pTRV2 and pTRV1 empty vector bacterial suspensions. Each treatment had 30 biological replicates. Sweet cherry fruits were harvested 15 days after injection, frozen in liquid nitrogen, and stored at -80°C for further analysis.

[0038] RNA was extracted from fruit samples of different experimental groups according to the instructions of the fruit RNA extraction kit. The genomic DNA was removed from the RNA, and the cDNA template was obtained by reverse transcription. The cDNA template was diluted to 100 ng / ml and stored at -20℃ until use. Both the fruit RNA extraction kit and the reverse transcription kit were purchased from Tiangen Biotech 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 quantitative PCR instrument (Bio-Rad) following the experimental steps provided by the qRT-PCR kit. The Actin gene was used as an internal control, and three biological replicates were set up. The qRT-PCR kit was purchased from Novizan Biosciences Co., Ltd.

[0039] The primers used for detecting the expression level of the PavERF110 gene in sweet cherry using 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, compared with the control, the gene expression level of PavERF110 in sweet cherry fruits injected with PavERF110 gene overexpression bacterial solution was significantly increased. Figure 1 In contrast, the gene expression level of PavERF110 was significantly reduced in sweet cherry fruits injected with the VIGS vector. Figure 2 Meanwhile, overexpression of PavERF110 accelerated fruit color change, while silencing PavERF110 slowed down fruit color change. Figure 3 These results all indicate that the transcription factor PavERF110 can regulate the ripening of sweet cherry fruits.

[0041] Example 3

[0042] Detection of expression levels of genes related to ripening in sweet cherries

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

[0044] The primers for detecting the sweet cherry ripening-related gene CHS using the qRT-PCR method include the forward primer CHS-FQ (SEQ ID NO. 9) and the reverse primer CHS-RQ (SEQ ID NO. 10); the primers for detecting the sweet cherry ripening-related gene NCED include the forward primer NCED-FQ (SEQ ID NO. 11) and the reverse primer NCED-RQ (SEQ ID NO. 12); the primers for detecting the sweet cherry ripening-related gene PL1 include the forward primer PL1-FQ (SEQ ID NO. 13) and the reverse primer PL1-RQ (SEQ ID NO. 14); and the primers for detecting the sweet cherry ripening-related gene PL2 include the forward primer PL2-FQ (SEQ ID NO. 15) and the 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 were significantly increased in sweet cherry fruits overexpressing the PavERF110 gene, while the expression levels of ripening-related genes CHS, NCED, PL1, and PL2 were decreased in sweet cherry fruits with silenced PavERF110 gene. Figure 4 This indicates that PavERF110 can positively regulate the expression of key ripening genes, thereby promoting the ripening of sweet cherry fruits.

[0046] The above description is only a preferred embodiment of the present invention. It should be noted that for those skilled in the art, several improvements and modifications can be made without departing from the principle of the present invention, and these improvements and modifications should also be considered within the scope of protection of the present invention.

Claims

1. Use of the PavERF110 gene in regulating the direction of fruit ripening in sweet cherry, characterized in that, The nucleotide sequence of the PavERF110 gene is shown as SEQ ID NO. 1, and overexpression of the PavERF110 gene can accelerate the color change of sweet cherry fruits and promote the maturation of sweet cherry fruits.

2. The use of the overexpression vector containing the PavERF110 gene construct of claim 1 or the recombinant GV3101 Agrobacterium containing the overexpression vector in accelerating the color change of sweet cherry fruits and promoting the maturation of sweet cherry fruits.

3. A method of accelerating the color break and ripening of sweet cherry fruit, characterized by, The overexpression vector of claim 2 is transformed into GV3101 Agrobacterium to prepare an engineering bacterium, and the bacterium liquid is injected into the equator of the fruits 25 days after the flowering of sweet cherry.

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