Application of PavMYB1 gene in regulation and control of sweet cherry anthocyanin synthesis

Abstract

The invention provides an application of a PavMYB1 gene in regulation and control of sweet cherry anthocyanin synthesis. The nucleotide sequence of the PavMYB1 gene is as shown in SEQ ID NO. 1, and the amino acid sequence of the encoded protein of the PavMYB1 gene is as shown in SEQ ID NO. 2. The invention provides an overexpression vector and a VIGS gene silencing vector constructed by related genes, a construction method of the overexpression vector and the VIGS gene silencing vector, and a recombinant microorganism containing the vectors. Experiments prove that overexpression of PavMYB1 can increase fruit pigment accumulation and anthocyanin synthesis key gene expression, technical support and theoretical basis are provided for improvement of sweet cherry fruit quality, and revealing of a regulation mechanism of sweet cherry anthocyanin synthesis is facilitated.

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 is an emerging fruit variety in my country. It is rich in anthocyanins, vitamin C, carotenoids, potassium, magnesium and other nutrients, which play an important role in human health. Anthocyanin is a water-soluble flavonoid pigment, which is mainly stored in the cell sap of plants and can give plants a variety of colors. It is also an important plant secondary metabolite with significant antioxidant, anti-inflammatory, anti-cancer and other pharmacological activities, and plays a key role in maintaining human health. At present, the anthocyanin biosynthesis pathway has been studied in plants such as Arabidopsis, apples, and grapes, but the biosynthesis mechanism of anthocyanins in sweet cherries is still unclear.

[0003] Transcription factors are an important class of proteins that can regulate gene expression in organisms. Among many transcription factors, R2R3-MYB is one of the largest transcription factor families in plants, and is widely involved in regulating biological processes such as plant growth and development, pigment accumulation, fruit ripening, and response to biotic and abiotic stresses. In fruit trees such as strawberries, apples, and pears, MYB transcription factors play a vital role in regulating the biosynthesis of anthocyanins. These MYB transcription factors can directly bind to the promoter region of anthocyanin biosynthesis genes and affect the production of anthocyanins by activating or inhibiting the expression of these genes. However, the regulation of anthocyanin synthesis in sweet cherries by MYB transcription factors is still unclear. In summary, studying the regulatory mechanism of MYB transcription factors on anthocyanin biosynthesis during the growth and development of sweet cherry fruits is of great significance for maintaining its unique flavor and improving its 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 PavMYB1 gene in regulating the synthesis of anthocyanins in sweet cherries, to reveal its regulatory mechanism, and to provide technical support for improving the quality of sweet cherries.

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

[0006] Application of the PavMYB1 gene in regulating the synthesis of sweet cherry anthocyanins, wherein 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 in SEQ ID NO.2.

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

[0009] Preferably, the construction method comprises 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 by the PavMYB1 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 PavMYB1 gene using the forward primer PavMYB1-FV and the reverse primer PavMYB1-RV, and connecting the amplified fragment to a 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 anthocyanin.

[0014] The present invention also provides a method for regulating the synthesis of anthocyanins in sweet cherries, 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 cells 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 anthocyanin synthesis. The present invention verifies the regulatory function of PavMYB1 in the anthocyanin metabolic pathway by genetic and molecular means. The pigment accumulation of fruits overexpressing PavMYB1 is significantly increased, and the expression levels of the key anthocyanin biosynthesis genes CHS, ANS, DFR, and UFGT are significantly increased, thereby promoting the synthesis of sweet cherry anthocyanins and helping to improve the quality of the fruit.

[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 the understanding of the mechanism. The present 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 bacterial 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 of PavMYB1 gene 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 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 PavMYB1 gene from sweet cherry

[0028] Input the PavMYB1 gene CDS sequence into the website (https: / / crm.vazyme.com / cetool / ) to design the PavMYB1 gene cloning primers. The selected sequence needs to have an appropriate length (15-25bp), Tm value (50-60℃) and GC content (about 50%). The Tm values ​​of the two-chain primers 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 the 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 30s; 98℃ denaturation 15s; 58℃ annealing 15s; 72℃ extension 10s; 35 cycles; 72℃ final extension 5min; 4℃ storage. 50μL 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 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 ⅡOne Step Cloning Kit was used to connect the purified PCR products to vector pBI121 and vector pTRV2 respectively to obtain the overexpression vector pBI121-PavMYB1 and the VIGS gene silencing vector pTRV2-PavMYB1.

[0034] Specific:

[0035] Construction of 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 amplified to obtain the PavMYB1 gene coding frame, and the coding frame of the PavMYB1 gene was connected to the vector pBI121 by homologous recombination to obtain the overexpression vector pBI121-PavMYB1.

[0036] Construction of VIGS gene silencing vector pTRV2-PavMYB1: Using sweet cherry DNA as a template, the 3' non-conserved region fragment 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), and the amplified fragment was connected to the vector by homologous recombination to obtain the VIGS gene silencing vector pTRV2-PavMYB1.

[0037] Then the constructed vectors pBI121-PavMYB1 and pTRV2-PavMYB1 were transferred 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 PavMYB1 gene overexpression test group included pBI121-PavMYB1 solution, and the control group was pBI121 empty solution. The gene silencing test group included pTRV2-PavMYB1 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 an ultra-low temperature refrigerator at -80℃ 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 PavMYB1 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] 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 increased significantly, while the pigment accumulation of fruits silencing PavMYB1 decreased significantly ( 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] 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, ANS, DFR, and UFGT 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.

[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 anthocyanin key biosynthesis genes CHS, ANS, DFR, and UFGT in sweet cherry fruits with overexpression of PavMYB1 gene were significantly increased, while the expression levels of anthocyanin key biosynthesis genes CHS, ANS, DFR, and UFGT in sweet cherry fruits with silenced PavMYB1 gene were all decreasing, especially CHS and ANS, which were significantly reduced ( Figure 4 ). This indicates that PavMYB1 can positively regulate the expression of key genes CHS and ANS in anthocyanin biosynthesis, 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 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 PavMYB1 gene in regulating the synthesis of sweet cherry anthocyanins, the nucleotide sequence of the PavDREB1 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 PavMYB1 gene is shown in SEQ ID NO.

2.

3. An overexpression vector constructed by the PavMYB1 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 PavMYB1 gene coding frame was amplified using forward primer PavMYB1-FO and reverse primer PavMYB1-RO, and the PavMYB1 gene coding frame was connected to the 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.

5. A VIGS gene silencing vector constructed with the PavMYB1 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-conserved region fragment of the PavMYB1 gene was amplified using the forward primer PavMYB1-FV and the reverse primer PavMYB1-RV, and the amplified fragment was connected 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.

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 synthesis of sweet cherry anthocyanins.

9. A method for regulating the synthesis of sweet cherry anthocyanins, 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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