Application of PavDREB1 gene in regulating fruit senescence of sweet cherry

Abstract

The application provides application of PavDREB1 gene in regulation of aging direction of sweet cherry fruits, and belongs to the technical field of molecular breeding.The nucleotide sequence of the PavDREB1 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 gene vector constructed from the gene and a construction method of the vector, and recombinant microorganisms containing the vector.The application finds that overexpression of the PavDREB1 gene can accelerate fruit aging and increase the expression level of aging-related genes by injecting the engineering bacteria containing the related vector into the equator of the fruits 25 days after flowering of the sweet cherry, and silencing the gene can slow down fruit aging and the expression of related genes.The application provides a new molecular assisted breeding strategy and theoretical basis for postharvest preservation of sweet cherry fruits.

Description

Technical Field

[0001] This invention belongs to the field of molecular breeding technology, and in particular relates to the application of the PavDREB1 gene in regulating the senescence of sweet cherry fruits. Background Technology

[0002] Sweet cherries are rich in nutrients such as anthocyanins, vitamin C, and carotenoids, making them popular with consumers. In recent years, the sweet cherry industry has developed rapidly, with planting area and yield increasing year after year. However, the high rate of spoilage and short shelf life of sweet cherries during the post-harvest senescence stage are becoming increasingly serious problems. This not only limits their circulation in the market but also severely restricts the further development of the sweet cherry industry. To overcome these challenges, researchers have explored various preservation technologies in recent years, including physical methods (such as low temperature, cold shock, modified atmosphere packaging, and irradiation), chemical methods (such as using chlorine dioxide, ethanol, acetic acid, calcium chloride, and 1-methylcyclopropene), and biological methods (such as utilizing plant extracts, chitosan, essential oils, phenolic substances, and yeast). These technologies effectively maintain fruit quality and extend shelf life by controlling microbial growth, reducing mechanical damage, increasing fruit firmness, or delaying fruit senescence and softening. Nevertheless, methods for delaying fruit senescence based on molecular breeding technology are relatively limited. Therefore, screening and studying the genes encoding key transcription factors in the senescence process of sweet cherry fruit, and deeply analyzing 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 postharvest preservation effect of sweet cherry fruit.

[0003] DREB transcription factors (Dehydration-responsive element-binding proteins), also known as drought-responsive element-binding proteins, typically bind to DRE / CRT elements (with the core sequence A / GCCGAC). DRE / CRT elements are commonly found in stress-responsive genes, such as those related to drought and cold responses. Therefore, DREB transcription factors are believed to participate in plant abiotic stress responses primarily through ABA-dependent or non-dependent signal transduction pathways. Currently, varying numbers of DREB-like transcription factors have been identified and discovered in various plants, including Arabidopsis, tomato, maize, wheat, and rice, and their diverse functions in physiological processes such as plant growth and development, stress resistance, hormone signal transduction, and senescence are being reported. However, research on the function of DREB transcription factors and their effects on fruit senescence in sweet cherry is relatively limited, restricting our in-depth understanding of the transcriptional regulatory mechanisms of sweet cherry fruit senescence. Therefore, studying the role of DREB transcription factors in sweet cherry fruit senescence is of significant scientific importance for revealing its transcriptional regulatory mechanisms. Summary of the Invention

[0004] In view of this, the purpose of this invention is to provide the 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] To achieve the above-mentioned objectives, the present invention provides the following technical solution:

[0006] The application of the PavDREB1 gene in regulating the senescence of sweet cherry fruit, the nucleotide sequence of which 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 from the above-mentioned PavDREB1 gene.

[0009] Preferably, the construction method includes the following steps: using sweet cherry cDNA as a template, the PavDREB1 gene coding frame is amplified using the forward primer PavDREB1-FO and the reverse primer PavDREB1-RO, and the PavDREB1 gene coding frame is ligated 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.

[0010] The present invention also provides a VIGS gene silencing vector constructed from the above-mentioned PavDREB1 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 PavDREB1 gene is amplified using the forward primer PavDREB1-FV and the reverse primer PavDREB1-RV, and the amplified fragment is ligated 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 application of the above-mentioned overexpression vector, or VIGS gene silencing vector, or recombinant microorganism in regulating the senescence of sweet cherry fruit.

[0014] The present invention also provides a method for regulating the senescence direction of sweet cherry fruit, wherein the above-mentioned overexpression vector is transformed into DH5α Escherichia coli to prepare engineered bacteria, or the above-mentioned VIGS vector is transformed 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 the fruit.

[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) Improve the storage resistance of sweet cherry fruit. This 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 storage resistance of the fruit and reducing post-harvest losses.

[0018] (2) Providing a new strategy for molecular-assisted breeding. This invention clarifies the role of the sweet cherry transcription factor PavDREB1 gene in the regulation of sweet cherry fruit senescence, providing a new target and direction for molecular-assisted breeding of sweet cherries. Breeders can utilize this gene for targeted variety improvement to cultivate sweet cherry varieties with better preservation performance.

[0019] (3) Deepening the understanding of the molecular mechanisms of fruit senescence. This invention studies the role of the sweet cherry transcription factor PavDREB1 gene in sweet cherry fruit senescence, which helps to further reveal the transcriptional regulatory mechanism of sweet cherry fruit senescence. This is not only of great significance to the sweet cherry industry, but also provides a reference for the study of senescence in other fruits, promoting a deeper understanding of the molecular basis of fruit senescence. Attached Figure Description

[0020] Figure 1 The gene expression level of PavDREB1 in sweet cherry fruits injected with bacteria that overexpress the PavDREB1 gene;

[0021] Figure 2 The gene expression level of PavDREB1 in sweet cherry fruits injected with the PavDREB1 gene VIGS gene silencing vector.

[0022] Figure 3 The color change of sweet cherry fruits with overexpression of the PavDREB1 gene and silence of the PavDREB1 gene;

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

[0028] Input the PavDREB1 gene CDS sequence into the website (https: / / crm.vazyme.com / cetool / ) to design PavDREB1 gene cloning primers. The selected sequence needs to have an 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 primer. Select the KpnI restriction site on the website (https: / / crm.vazyme.com / cetool / singlefragment.html) and input a sequence 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 PavDREB1 gene sequence has a total of 1167 bases (SEQ ID NO.1) and encodes a protein containing 388 amino acids (SEQ ID NO.2).

[0031] Example 2

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

[0033] This embodiment uses the homologous recombination method, employing materials purchased from Nanjing Novizan. II. The One Step Cloning Kit was used to ligate the purified PCR products into vectors pBI121 and pTRV2, respectively, to obtain the overexpression vector pBI121-PavDREB1 and the VIGS gene silencing vector pTRV2-PavDREB1.

[0034] Specifically:

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

[0036] Construction of the 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). The amplified fragment was then ligated into the vector using homologous recombination to obtain the VIGS gene silencing vector pTRV2-PavDREB1.

[0037] The constructed vectors pBI121-PavDREB1 and pTRV2-PavDREB1 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 PavDREB1 gene overexpression group included pBI121-PavDREB1 bacterial suspension, while the control group consisted of pBI121 empty vector bacterial suspension. The gene silencing group included pTRV2-PavDREB1 and pTRV1 bacterial suspensions, while the control group consisted of pTRV2 and pTRV1 empty vector bacterial suspensions. Each treatment had three 30 biological replicates. Sweet cherry fruits were collected 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 PavDREB1 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 to detect the expression level of the PavDREB1 gene in sweet cherry using 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, compared with the control, the gene expression level of PavDREB1 in sweet cherry fruits injected with PavDREB1 gene overexpression bacterial solution was significantly increased. Figure 1 In contrast, the expression level of PavDREB1 was significantly reduced in sweet cherry fruits injected with the VIGS gene silencing vector. Figure 2 Meanwhile, overexpression of PavDREB1 accelerated fruit color change, while silencing PavDREB1 slowed down fruit pigment color change. Figure 3 These results all indicate that the transcription factor PavDREB1 can regulate the senescence process of sweet cherry fruits.

[0041] Example 3

[0042] Detection of aging-related gene expression levels 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 senescence-related genes 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] Among them, the primers for detecting the senescence-related gene NCED in sweet cherry based on qRT-PCR include the forward primer NCED-FQ (SEQ ID NO. 9) and the reverse primer NCED-RQ (SEQ ID NO. 10); the primers for detecting the senescence-related gene PL1 in sweet cherry include the forward primer PL1-FQ (SEQ ID NO. 11) and the reverse primer PL1-RQ (SEQ ID NO. 12); and the primers for detecting the senescence-related gene PL2 in sweet cherry include the forward primer PL2-FQ (SEQ ID NO. 13) and the reverse primer PL2-RQ (SEQ ID NO. 14).

[0045] The results showed that, compared with the control, the expression levels of aging-related genes NCED, PL1, and PL2 were significantly increased in sweet cherry fruits overexpressing the PavDREB1 gene, while the expression levels of aging-related genes NCED, PL1, and PL2 were decreased in sweet cherry fruits with silenced PavDREB1 gene. Figure 4 This indicates that PavDREB1 can regulate the expression of genes related to fruit senescence, and that silencing the PavDREB1 gene can slow down the senescence 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. The application of a VIGS gene silencing vector constructed targeting the PavDREB1 gene or a recombinant Agrobacterium containing a VIGS gene silencing vector constructed targeting the PavDREB1 gene in slowing down the senescence of sweet cherry fruit, wherein the nucleotide sequence of the PavDREB1 gene is shown in SEQ ID NO.

1.

2. The application according to claim 1, characterized in that, The construction of the VIGS gene silencing vector targeting the PavDREB1 gene includes the following steps: Using sweet cherry DNA as a template, the 3' non-conserved region fragment of the PavDREB1 gene was amplified using forward primer PavDREB1-FV and reverse primer PavDREB1-RV. The amplified fragment was then ligated into a vector using 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.

3. A method for slowing down the senescence of sweet cherry fruits, characterized in that, The VIGS gene silencing vector constructed for the PavDREB1 gene as described in claim 1 was transformed into GV3101 Agrobacterium competent cells to prepare engineered bacteria, and the bacterial solution was injected into the equator of the fruit 25 days after flowering to infect it.

4. The application of the VIGS gene silencing vector constructed targeting the PavDREB1 gene as described in claim 1, or the recombinant Agrobacterium containing the VIGS gene silencing vector constructed targeting the PavDREB1 gene, in cherry breeding with better preservation performance.

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