Application of PavMYB1 gene in regulating anthocyanin synthesis in sweet cherry

Abstract

The application provides application of PavMYB1 gene in regulation of anthocyanin synthesis of sweet cherry. The nucleotide sequence of the PavMYB1 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 provides an overexpression vector and a VIGS gene silencing vector constructed by related genes and a construction method thereof, and a recombinant microorganism containing the vectors. The application proves through experiments that overexpression of PavMYB1 can increase pigment accumulation and expression of key anthocyanin synthesis genes of fruits, provides technical support and theoretical basis for improvement of fruit quality of sweet cherry, and helps to reveal the regulation mechanism of anthocyanin synthesis of sweet cherry.

Description

Technical Field

[0001] The invention belongs to the technical field of molecular breeding, and in particular relates to application of the PavMYB1 gene in regulating the synthesis of anthocyanins in sweet cherry. Background Art

[0002] Sweet cherry, an emerging fruit variety in my country, plays an important role in human health because it is rich in anthocyanins, vitamin C, carotenoids, potassium, magnesium and other nutrients. Anthocyanins are water-soluble flavonoid pigments that are mainly stored in the cell sap of plants and can give plants a variety of colors. They are also important plant secondary metabolites with significant antioxidant, anti-inflammatory, and anti-cancer pharmacological activities, and play a key role in maintaining human health. Currently, the anthocyanin biosynthesis pathway has been extensively studied in plants such as Arabidopsis, apple, and grape, but the biosynthesis mechanism of anthocyanins in sweet cherries is still unclear.

[0003] Transcription factors are an important class of proteins that regulate gene expression in organisms. Among these transcription factors, the R2R3-MYB family is one of the largest in plants and is widely involved in regulating biological processes such as plant growth and development, pigment accumulation, fruit ripening, and responses to biotic and abiotic stresses. In fruit trees such as strawberry, apple, and pear, MYB transcription factors play a crucial role in regulating anthocyanin biosynthesis. These MYB transcription factors can directly bind to the promoter regions of anthocyanin biosynthesis genes, activating or repressing their expression, thereby affecting anthocyanin production. However, the regulation of anthocyanin synthesis in sweet cherry fruit by MYB transcription factors remains unclear. In summary, understanding the regulatory mechanisms of anthocyanin biosynthesis by MYB transcription factors during sweet cherry fruit growth and development is crucial for maintaining the distinctive flavor and improving fruit quality through biological approaches. Summary of the Invention

[0004] In view of this, the purpose of the present invention is to provide an application of the PavMYB1 gene in regulating the synthesis of anthocyanins in sweet cherry, reveal its regulatory mechanism, and 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] The invention relates to an application of the PavMYB1 gene in regulating the synthesis of anthocyanins in sweet cherry. The nucleotide sequence of the PavDREB1 gene is shown in SEQ ID NO.1.

[0007] Preferably, the amino acid sequence of the protein encoded by the PavMYB1 gene is shown as SEQ ID NO.2.

[0008] The present invention also provides an overexpression vector constructed by the PavMYB1 gene.

[0009] Preferably, the construction method includes the following steps: using sweet cherry cDNA as a template, amplifying the PavMYB1 gene coding frame using the forward primer PavMYB1-FO and the reverse primer PavMYB1-RO, and connecting the PavMYB1 gene coding frame to a vector by homologous recombination; the nucleotide sequence of the forward primer PavMYB1-FO is shown in SEQ ID NO.3, and the nucleotide sequence of the reverse primer PavMYB1-RO is shown in SEQ ID NO.4.

[0010] The present invention also provides a VIGS gene silencing vector constructed with the PavMYB1 gene.

[0011] Preferably, the construction method includes the following steps: using sweet cherry DNA as a template, using the forward primer PavMYB1-FV and the reverse primer PavMYB1-RV to amplify the 3' non-conserved region fragment of the PavMYB1 gene, and connecting the amplified fragment to the vector by homologous recombination; the nucleotide sequence of the forward primer PavMYB1-FV is shown in SEQ ID NO.5, and the nucleotide sequence of the reverse primer PavMYB1-RV is shown in SEQ ID NO.6.

[0012] The present invention also provides a recombinant microorganism containing the above-mentioned overexpression vector or 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 synthesis of sweet cherry anthocyanins.

[0014] The present invention also provides a method for regulating the synthesis of anthocyanins in sweet cherries, comprising the steps of transferring the above-mentioned overexpression vector into DH5α Escherichia coli to prepare an engineered bacterium, or transferring the above-mentioned VIGS gene silencing vector into GV3101 competent Agrobacterium to prepare an engineered bacterium, and injecting the bacterial liquid 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 anthocyanin synthesis. The present invention verified the regulatory function of PavMYB1 in the anthocyanin metabolic pathway through genetic and molecular means. The pigment accumulation of fruits overexpressing PavMYB1 increased significantly, and the expression levels of the key anthocyanin biosynthesis genes CHS, ANS, DFR, and UFGT increased significantly, thereby promoting the synthesis of anthocyanins in sweet cherries and helping to improve fruit quality.

[0018] (2) Providing a basis for quality improvement. The cloned PavMYB1 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 higher anthocyanin content.

[0019] (3) Deepen understanding of the mechanism. This invention helps to reveal the regulatory mechanism of sweet cherry anthocyanin synthesis, provides a reference for further research on the molecular basis of plant anthocyanin synthesis, 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 PavMYB1 in sweet cherry fruit injected with PavMYB1 gene overexpression bacteria solution;

[0021] Figure 2 The gene expression level of PavMYB1 in sweet cherry fruit injected with the PavMYB1 gene VIGS gene silencing vector;

[0022] Figure 3 Pigment accumulation in sweet cherry fruit with overexpression and silencing of PavMYB1 gene;

[0023] Figure 4 The expression levels of CHS, ANS, DFR, and UFGT in sweet cherry fruits with overexpression of PavMYB1 gene and silencing of PavMYB1 gene. DETAILED DESCRIPTION

[0024] The technical solutions provided by the present invention are 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 the examples herein, the plant material was red sweet cherry. All sweet cherry fruit samples were collected from the Wenchuan Sweet Cherry Research Base of Sichuan Agricultural University. Strains: Vector pBI121 was maintained in the laboratory of the College of Horticulture at Sichuan Agricultural University; vectors pTRV1 and pTRV2 were also maintained in the laboratory of the College of Horticulture at Sichuan Agricultural University; DH5α Escherichia coli transformation strain and GV3101 Agrobacterium transformation strain were purchased from Qingke Xinye Biotechnology Co., Ltd.

[0026] Example 1

[0027] Cloning of the PavMYB1 gene from sweet cherry

[0028] Enter the PavMYB1 gene CDS sequence into the website (https: / / crm.vazyme.com / cetool / ) to design PavMYB1 gene cloning primers. The selected sequence should have an appropriate length (15-25 bp), Tm value (50-60°C), and GC content (around 50%). The Tm values ​​of the primers on both strands should be roughly consistent. Add the KpnI restriction site sequence to the primers. Select the KpnI restriction site on the website (https: / / crm.vazyme.com / cetool / singlefragment.html) and enter at least 20 bp of sequence to the left and right of the restriction site on the vector. This will generate cloning primers with the restriction site and recombination sequence.

[0029] The target gene was cloned by PCR using 2× PhantaFlash MasterMix high-fidelity enzyme purchased from Nanjing Novozymes. Red sweet cherry fruit cDNA stored in our laboratory was used as the amplification template. The reaction system was as follows: 2× PhantaFlash MasterMix 25 μL; Primer F (10 μM) 1 μL; Primer R (10 μM) 1 μL; cDNA 1 μL; ddH2O 22 μL. The amplification procedure was as follows: 98°C initial denaturation for 30 s; 98°C denaturation for 15 s; 58°C annealing for 15 s; 72°C extension for 10 s; 35 cycles; final extension at 72°C for 5 min; and storage at 4°C. 50 μL of the PCR product was analyzed by 1% gel electrophoresis at 120 V for 20 min. The amplified bands were visualized using a gel imaging system. The product was then 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 PavMYB1 gene sequence had a total of 225 bases (SEQ ID NO.1) and the encoded protein contained 74 amino acids (SEQ ID NO.2).

[0031] Example 2

[0032] Construction and infection of PavMYB1 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 was used to ligate the purified PCR products into vector pBI121 and vector pTRV2, respectively, to obtain the overexpression vector pBI121-PavMYB1 and the VIGS gene silencing vector pTRV2-PavMYB1.

[0034] Specifically:

[0035] Construction of the overexpression vector pBI121-PavMYB1: Using sweet cherry cDNA as a template, the forward primer PavMYB1-FO (SEQ ID NO. 3) and the reverse primer PavMYB1-RO (SEQ ID NO. 4) were used to amplify the PavMYB1 gene coding frame. The PavMYB1 gene coding frame was ligated into the vector pBI121 by homologous recombination to obtain the overexpression vector pBI121-PavMYB1.

[0036] Construction of the VIGS gene silencing vector pTRV2-PavMYB1: Using sweet cherry DNA as a template, the 3' non-conserved region of the PavMYB1 gene was amplified using the forward primer PavMYB1-FV (SEQ ID NO. 5) and the reverse primer PavMYB1-RV (SEQ ID NO. 6). The amplified fragment was ligated into the vector by homologous recombination to obtain the VIGS gene silencing vector pTRV2-PavMYB1.

[0037] The constructed vectors pBI121-PavMYB1 and pTRV2-PavMYB1 were then transformed into competent DH5α Escherichia coli and GV3101 Agrobacterium tumefaciens, respectively. Agrobacterium was slowly injected into the equator of sweet cherry fruits 25 days after anthesis. The PavMYB1 gene overexpression group consisted of pBI121-PavMYB1 culture, while the control group consisted of pBI121 empty vector culture. The gene silencing group consisted of pTRV2-PavMYB1 and pTRV1 culture, while the control group consisted of pTRV2 empty vector culture and pTRV1 empty vector culture. Thirty biological replicates were used for each treatment. Sweet cherry fruits were harvested 15 days after injection, frozen in liquid nitrogen, and stored in a -80°C freezer until further analysis.

[0038] RNA was extracted from fruit samples in different experimental groups according to the instructions of the fruit RNA extraction kit. The RNA was degenomic and reverse transcribed to obtain cDNA templates. The cDNA templates were diluted to 100 ng / ml and stored at -20°C until use. Both the fruit RNA extraction kit and the reverse transcription kit were purchased from Tiangen Biochemical Technology Co., Ltd. qRT-PCR primers were designed using the website (https: / / crm.vazyme.com / cetool / ). Following the protocol provided in the qPCR kit, PavMYB1 gene expression levels were measured using a real-time fluorescence quantitative PCR instrument (Bio-Rad). Actin was used as an internal control, and three biological replicates were performed. The qRT-PCR kit was purchased from Novozyme Biotech Co., Ltd.

[0039] The results showed that the gene expression level of PavMYB1 in sweet cherry fruits injected with PavMYB1 gene overexpression solution was significantly increased compared with the control ( Figure 1 ), while the gene expression level of PavMYB1 was significantly reduced in sweet cherry fruits injected with VIGS gene silencing vector ( Figure 2 ). At the same time, the pigment accumulation of fruits overexpressing PavMYB1 was significantly increased, while the pigment accumulation of fruits silencing PavMYB1 was significantly reduced ( Figure 3 These results indicate that the transcription factor PavMYB1 can regulate the synthesis of pigment substances in sweet cherry fruit.

[0040] Example 3

[0041] Detection of expression levels of genes related to anthocyanin synthesis in sweet cherry

[0042] qRT-PCR primers were designed using the website (https: / / crm.vazyme.com / cetool / ). Following the protocol provided in the qPCR kit, the cDNA obtained in Example 2 was used as a template. Real-time fluorescence quantitative PCR (Bio-Rad) was used to detect the expression levels of the key anthocyanin biosynthesis genes CHS, ANS, DFR, and UFGT in fruit samples from different experimental groups. The Actin gene was used as an internal reference, and three biological replicates were set up.

[0043] Among them, the primers for detecting the key anthocyanin biosynthesis gene CHS in sweet cherry fruit 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 key anthocyanin biosynthesis gene ANS in sweet cherry fruit include forward primer ANS-FQ (SEQ ID NO.11) and reverse primer ANS-RQ (SEQ ID NO.12); the primers for detecting the key anthocyanin biosynthesis gene DFR in sweet cherry fruit include forward primer DFR-FQ (SEQ ID NO.13) and reverse primer DFR-RQ (SEQ ID NO.14); the primers for detecting the key anthocyanin biosynthesis gene UFGT in sweet cherry fruit include forward primer UFGT-FQ (SEQ ID NO.15) and reverse primer UFGT-RQ (SEQID NO.16).

[0044] The results showed that compared with the control, the expression levels of the key anthocyanin biosynthesis genes CHS, ANS, DFR, and UFGT in the sweet cherry fruits with overexpression of the PavMYB1 gene were significantly increased, while the expression levels of the key anthocyanin biosynthesis genes CHS, ANS, DFR, and UFGT in the sweet cherry fruits with silenced PavMYB1 gene showed a downward trend, especially CHS and ANS, which were significantly reduced ( Figure 4 ). This indicates that PavMYB1 can positively regulate the expression of key anthocyanin biosynthesis genes such as CHS and ANS, thereby promoting anthocyanin biosynthesis.

[0045] 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 principles of the present invention. These improvements and modifications should also be regarded as within the scope of protection of the present invention.

Claims

1. PavMYB 1 gene in regulating the synthesis of sweet cherry anthocyanins, the PavDREB1 The nucleotide sequence of the gene is shown in SEQ ID NO.

1.

2. Containing the claim 1 PavDREB1 Application of gene overexpression vectors or recombinant microorganisms in regulating the synthesis of sweet cherry anthocyanins.

3. A method for regulating the synthesis of sweet cherry anthocyanins, characterized in that: The overexpression vector according to claim 2 is transformed into DH5α Escherichia coli 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.

4. Use of the overexpression vector or recombinant microorganism according to claim 2 in sweet cherry breeding.

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