Application of PavDREB1 gene in regulating aging direction of sweet cherry fruits

Abstract

The invention provides application of a PavDREB1 gene in regulating and controlling the aging direction of sweet cherry fruits, and belongs to the technical field of molecular breeding. The nucleotide sequence of the PavDREB1 gene is as shown in SEQ ID NO.1, and the amino acid sequence of the protein coded by the PavDREB1 gene is as shown in SEQ ID NO.2. The invention also provides an overexpression vector constructed by the gene, a VIGS gene silencing gene vector, a construction method of the overexpression vector and the VIGS gene silencing gene vector, and a recombinant microorganism containing the vectors. Engineering bacteria containing related carriers are injected and infected at the equator of fruits 25 days after sweet cherries blossom, it is found that overexpression of the PavDREB1 gene can accelerate fruit senescence and increase the expression level of senescence-related genes, and silencing of the gene can slow down fruit senescence and related gene expression. The invention provides a novel molecular assisted breeding strategy and theoretical basis for the preservation of the picked sweet cherry fruits.

Description

Technical Field

[0001] The invention belongs to the technical field of molecular breeding, and particularly relates to application of a PavDREB1 gene in regulating the senescence direction of sweet cherry fruits. Background Art

[0002] Sweet cherry fruits are rich in nutrients such as anthocyanins, vitamin C and carotenoids, and are deeply loved by consumers. In recent years, the sweet cherry industry has developed rapidly, with the planting area and output increasing year by year. However, the high rate of bad fruit and short shelf life faced by sweet cherry fruits in the post-harvest aging stage are becoming increasingly serious, which not only limits its circulation in the market, but also seriously restricts the further development of the sweet cherry industry. In order to overcome these challenges, researchers have explored a variety of preservation technologies in recent years, including physical methods (such as low temperature, cold shock, gas conditioning and irradiation), chemical methods (such as the use of chlorine dioxide, ethanol, acetic acid, calcium chloride and 1-methylcyclopropene, etc.) and biological methods (such as the use of plant extracts, chitosan, essential oils, phenolic substances and yeast, etc.). These technologies effectively maintain fruit quality and extend shelf life by controlling microbial growth, reducing mechanical damage, increasing fruit hardness or delaying fruit aging and softening. Despite this, there are relatively few means of delaying fruit aging based on molecular breeding technology. Therefore, screening and studying the key transcription factor encoding genes in the senescence process of sweet cherry fruit and in-depth analysis of their transcriptional regulatory mechanisms are of great scientific significance and practical application value for deepening our understanding of the molecular basis of fruit senescence, developing new molecular-assisted breeding strategies, and improving the post-harvest preservation of sweet cherry fruit.

[0003] DREB transcription factors (Dehydration responsive element binding protein), also known as drought response element binding proteins, usually bind to DRE / CRT elements (core sequence is A / GCCGAC). DRE / CRT elements are often present in stress response genes such as drought and cold response-related genes. Therefore, DREB transcription factors are believed to be mainly involved in the abiotic stress response of plants through ABA-dependent or -independent signal transduction pathways. At present, different numbers of DREB transcription factors have been excavated and identified in various plants such as Arabidopsis, tomato, corn, wheat, and rice, and their functional diversity in physiological processes such as plant growth and development, stress resistance, hormone signal transduction, and aging has also been gradually reported. However, in sweet cherry, the function of DREB transcription factors and their effects on fruit aging are relatively rare, which limits our in-depth understanding of the transcriptional regulatory mechanism of sweet cherry fruit aging. Therefore, studying the role of DREB transcription factors in sweet cherry fruit aging is of great scientific significance for revealing its transcriptional regulatory mechanism. Summary of the invention

[0004] In view of this, the purpose of the present invention is to provide an application of the PavDREB1 gene in regulating the senescence of sweet cherry fruit, to reveal its mechanism of action in regulating the senescence 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 PavDREB1 gene in regulating the senescence direction of sweet cherry fruit, 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 PavDREB1 gene is shown in SEQ ID NO.2.

[0008] The present invention also provides an overexpression vector constructed by the PavDREB1 gene.

[0009] Preferably, the construction method comprises the following steps: using sweet cherry cDNA as a template, amplifying the PavDREB1 gene coding frame using the forward primer PavDREB1-FO and the reverse primer PavDREB1-RO, and connecting the PavDREB1 gene coding frame to a vector by homologous recombination; the nucleotide sequence of the forward primer PavDREB1-FO is shown in SEQ ID NO.3, and the nucleotide sequence of the reverse primer PavDREB1-RO is shown in SEQ ID NO.4.

[0010] The present invention also provides a VIGS gene silencing vector constructed by the PavDREB1 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 PavDREB1 gene using the forward primer PavDREB1-FV and the reverse primer PavDREB1-RV, and connecting the amplified fragment to a vector by homologous recombination; the nucleotide sequence of the forward primer PavDREB1-FV is shown in SEQ ID NO.5, and the nucleotide sequence of the reverse primer PavDREB1-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 senescence direction of sweet cherry fruit.

[0014] The present invention also provides a method for regulating the aging 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 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) Improving the storability of sweet cherry fruit. The present invention can affect the senescence process of sweet cherry fruit by regulating the expression of the sweet cherry transcription factor PavDREB1 gene, thereby improving the storability of the fruit and reducing post-harvest losses.

[0018] (2) Providing a new strategy for molecular-assisted breeding. The present invention clarifies the role of the sweet cherry transcription factor PavDREB1 gene in regulating the senescence of sweet cherry fruit, and provides a new target and direction for molecular-assisted breeding of sweet cherry. Breeders can use this gene to carry out targeted variety improvement and cultivate sweet cherry varieties with better preservation performance.

[0019] (3) Deepen the understanding of the molecular mechanism of fruit aging. The present invention studies the role of the sweet cherry transcription factor PavDREB1 gene in sweet cherry fruit aging, which helps to further reveal the transcriptional regulatory mechanism of sweet cherry fruit aging. This is not only of great significance to the sweet cherry industry, but also provides a reference and reference for the aging research of other fruits, and promotes a deeper understanding of the molecular basis of fruit aging. BRIEF DESCRIPTION OF THE DRAWINGS

[0020] Figure 1 The gene expression level of PavDREB1 in sweet cherry fruit injected with PavDREB1 gene overexpression bacterial solution;

[0021] Figure 2 is the gene expression level of PavDREB1 in sweet cherry fruit injected with PavDREB1 gene VIGS gene silencing vector;

[0022] Figure 3 The color change of sweet cherry fruit with overexpression of PavDREB1 gene and silencing of PavDREB1 gene;

[0023] Figure 4 The expression levels of NCED, PL1, and PL2 in sweet cherry fruits with overexpression of PavDREB1 gene and silencing of PavDREB1 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 PavDREB1 gene from sweet cherry

[0028] Input the CDS sequence of the PavDREB1 gene into the website (https: / / crm.vazyme.com / cetool / ) to design the PavDREB1 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 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 PavDREB1 gene sequence had 1167 bases (SEQ ID NO.1) and the encoded protein contained 388 amino acids (SEQ ID NO.2).

[0031] Example 2

[0032] Construction and infection of PavDREB1 gene overexpression and VIGS gene silencing vectors

[0033] This example 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-PavDREB1 and the VIGS gene silencing vector pTRV2-PavDREB1.

[0034] Specific:

[0035] Construction of overexpression vector pBI121-PavDREB1: Using sweet cherry cDNA as a template, the PavDREB1 gene coding frame was amplified using forward primer PavDREB1-FO (SEQ ID NO.3) and reverse primer PavDREB1-RO (SEQ ID NO.4), and the coding frame of the PavDREB1 gene was connected to the vector pBI121 by homologous recombination to obtain the overexpression vector pBI121-PavDREB1.

[0036] Construction of VIGS gene silencing vector pTRV2-PavDREB1: Using sweet cherry DNA as a template, the 3' non-conserved region fragment of the PavDREB1 gene was amplified using the forward primer PavDREB1-FV (SEQ ID NO.5) and the reverse primer PavDREB1-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-PavDREB1.

[0037] Then the constructed vectors pBI121-PavDREB1 and pTRV2-PavDREB1 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 PavDREB1 gene overexpression test group included pBI121-PavDREB1 solution, and the control group was pBI121 empty solution. The gene silencing test group included pTRV2-PavDREB1 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 with liquid nitrogen, and stored in an ultra-low temperature refrigerator at -80°C 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 PavDREB1 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 Novazon Biotech Co., Ltd.

[0039] Among them, the primers used for detecting the expression level of the sweet cherry PavDREB1 gene based on the qRT-PCR method include the forward primer PavDREB1-FQ (SEQ ID NO.7) and the reverse primer PavDREB1-RQ (SEQ ID NO.8).

[0040] The results showed that the gene expression level of PavDREB1 in sweet cherry fruits injected with PavDREB1 gene overexpression solution was significantly increased compared with the control ( Figure 1 ), while the gene expression level of PavDREB1 was significantly reduced in sweet cherry fruits injected with VIGS gene silencing vector ( Figure 2 ). At the same time, the color change of fruits overexpressing PavDREB1 was accelerated, while the color change of fruits with silenced PavDREB1 was slowed down ( Figure 3 ). These results indicate that the transcription factor PavDREB1 can regulate the senescence process of sweet cherry fruit.

[0041] Example 3

[0042] Detection of expression levels of genes related to senescence in sweet cherry

[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 senescence-related genes NCED, PL1, and PL2 in 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 aging-related gene NCED based on the qRT-PCR method include forward primer NCED-FQ (SEQ ID NO.9) and reverse primer NCED-RQ (SEQ ID NO.10); the primers for detecting the sweet cherry aging-related gene PL1 include forward primer PL1-FQ (SEQ ID NO.11) and reverse primer PL1-RQ (SEQ ID NO.12); the primers for detecting the sweet cherry aging-related gene PL2 include forward primer PL2-FQ (SEQ ID NO.13) and reverse primer PL2-RQ (SEQ IDNO.14).

[0045] The results showed that compared with the control, the expression levels of senescence-related genes NCED, PL1, and PL2 in sweet cherry fruits overexpressing the PavDREB1 gene were significantly increased, while the expression levels of senescence-related genes NCED, PL1, and PL2 in sweet cherry fruits silencing the PavDREB1 gene showed a downward trend ( Figure 4 ). This indicates that PavDREB1 can regulate the expression of genes related to fruit senescence, and silencing the PavDREB1 gene can slow down the senescence 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 PavDREB1 gene in regulating the senescence direction of sweet cherry fruit, 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 PavDREB1 gene is shown in SEQ ID NO.

2.

3. An overexpression vector constructed by the PavDREB1 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 a template, the PavDREB1 gene coding frame was amplified using forward primer PavDREB1-FO and reverse primer PavDREB1-RO, and the PavDREB1 gene coding frame was connected to the vector by homologous recombination. The nucleotide sequence of the forward primer PavDREB1-FO is shown in SEQ ID NO.3, and the nucleotide sequence of the reverse primer PavDREB1-RO is shown in SEQ ID NO.

4.

5. A VIGS gene silencing vector constructed with the PavDREB1 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 PavDREB1 gene was amplified using the forward primer PavDREB1-FV and the reverse primer PavDREB1-RV, and the amplified fragment was connected to the vector by homologous recombination. The nucleotide sequence of the forward primer PavDREB1-FV is shown in SEQ ID NO.5, and the nucleotide sequence of the reverse primer PavDREB1-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 senescence direction of sweet cherry fruit.

9. A method for regulating the aging 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 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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