Application of VvMYB APL gene in regulation and control of plant fruit maturation

Abstract

The invention discloses application of a VvMYB APL gene in regulation and control of plant fruit ripening, and belongs to the technical field of plant genetic engineering. The invention finds that the VvMYB APL gene in a grape fruit treated by hydrogen peroxide has a down-regulated expression phenomenon in different stages, which indicates that the VvMYB APL gene may negatively regulate the development of the grape fruit. A VvMYB APL gene is further cloned from 'Kyoho' grapes, subcellular localization shows that the VvMYB APL gene is localized in a cell nucleus, a GUS staining test verifies the promoter activity of MYB APL, and a result shows that the promoter of the gene can start GUS reporter protein, and H2O2 treatment can enhance the activity of the gene. Furthermore, a VvMYB APL gene overexpression and silent vector transformation experiment shows that the silence of the VvMYB APL gene obviously promotes the ripening of grape fruits, and the overexpression inhibits the ripening of the fruits.

Description

Technical Field

[0001] The present invention relates to VvMYB APL The application of genes in regulating the ripening of plant fruits belongs to the technical field of plant genetic engineering. Background Art

[0002] Grape is one of the most widely cultivated fruit trees in the world. my country is an important producer of table grapes. The maturity of its fruit is closely related to the economic benefits of agriculture. In the field of fruit research, the regulation of fruit maturity has always been a hot topic of concern and research. Fruit ripening is not only a complex biological process involving physiological and biochemical changes, but also an oxidative process regulated by reactive oxygen species. The exploration of early-maturing varieties can not only effectively respond to the diverse needs of consumers, but also further enrich the grape germplasm resource bank. Therefore, exploring the factors that affect grape fruit ripening is of great significance to the grape-related industry for the use of molecular methods to conduct early molecular breeding of grapes to develop early-maturing grape varieties.

[0003] As one of the largest transcription factor families in plants, MYB transcription factors have multiple biological functions, participate in controlling the construction of cell morphology and the metabolism of plant secondary products, and play an important role in various abiotic stresses and plant growth and development. Studies have reported that in bananas, MaMYB8 Participates in regulating banana fruit ripening by regulating starch degradation and cell wall metabolism; in tomatoes, RR-type MYB transcription factors SlMYBR2 Participates in the energy conversion and metabolism of fruits, affecting the ripening process of tomatoes, and the expression level of this gene increases significantly with the ripening of fruits; knockout SlMYBR2 Significantly delaying the ripening process of tomato fruit. In short, MYB transcription factors are widely present in higher plants and are involved in regulating the growth and development of flowers, fruits, leaves and roots.

[0004] Recent studies have shown that MYB APL may be closely related to plant development. APL (ALTEREDPHLOEMDEVELOPMENT) protein is a member of the MYB transcription factor family and is required for the normal formation of sieve elements and companion cells. MYB APL is an important transcription factor complex in plants, involved in regulating plant growth and development and response to the environment. However, the regulatory role of MYB APL in the ripening process of grapes is still unclear. Summary of the invention

[0005] The object of the present invention is to provide VvMYB APL The application of genes in regulating the ripening of plant fruits provides a new regulatory site involved in the ripening process of plant fruits.

[0006] In order to achieve the above object, the present invention VvMYB APLThe technical solution adopted for the application of genes in regulating the ripening of plant fruits is: VvMYB APL Application of genes in regulating plant fruit ripening, the VvMYB APL The nucleotide sequence of the coding region of the gene is shown in SEQ ID NO.1.

[0007] The beneficial effects of the above technical solution are: VvMYB APL The application of genes in regulating the ripening of plant fruits is a pioneering invention. The previous research of this invention found that hydrogen peroxide (H2O2) treatment can effectively promote the early ripening of grape fruits. Then, the normal grape fruits and the grape fruits treated with H2O2 were analyzed and it was found that the grape fruits treated with H2O2 had VvMYB APL Genes were differentially expressed.

[0008] To further clarify MYB APL The subcellular localization test showed that it was localized in the cell nucleus. MYB APL The results showed that the promoter of this gene can activate the GUS reporter protein, and H2O2 treatment can enhance its activity. MYB APL The gene may work synergistically with other genes to regulate fruit development.

[0009] To make it clear MYB APL Effects on fruit development, construct MYB APL Gene overexpression and silencing vectors were transiently transformed into grapes. The results showed that MYB APL Gene silencing significantly promoted the color change of grape berries, while MYB APL Overexpression of the gene did not have this effect. An important indicator of grape ripening is the change in the color of the grape skin. Therefore, the above results fully demonstrate that MYB APL The gene is involved in regulating the ripening of grape fruit. The present invention provides a new mechanism for revealing the regulation of fruit development and ripening, provides a theoretical basis for early molecular breeding and ripening regulation of grapes, and is also a new mechanism for regulating the ripening of grape fruit. MYB APL On the basis that the gene can regulate the development of plant phloem, it provides new functions for the gene and enriches its functional system.

[0010] As a further improvement, the regulating the ripening of plant fruits is regulating the color change of plant fruits or the ripening of fruit pods.

[0011] As a further improvement, overexpression VvMYB APL Genes that inhibit the ripening of plant fruits; VvMYB APL The expression of genes promotes the ripening of plant fruits.

[0012] As a further improvement, the overexpression VvMYB APL Gene for construction VvMYB APL Gene overexpression vectors are transformed into plants to VvMYB APL Gene overexpression.

[0013] As a further improvement, the inhibition VvMYB APL Gene expression constructs VvMYB APL The RNAi vector of the gene is transformed into plants. VvMYB APL Inhibit gene expression.

[0014] As a further improvement, the plant is grape or Arabidopsis thaliana.

[0015] As a further improvement, the grapes are 'Kyoho' grapes. BRIEF DESCRIPTION OF THE DRAWINGS

[0016] Figure 1 The results are shown in Table 1. VvMYB APL Differential expression analysis of genes; Figure 2 In Example 2 of the present invention VvMYB APL Gene cloning and sequence analysis (where A is VvMYB APL A is the electrophoresis detection of the gene, B is the phylogenetic tree of VvMYB APL, and C is the amino acid homologous sequence analysis of VvMYB APL); Figure 3 In Example 3 of the present invention VvMYB APL Subcellular localization of (scale bar in the figure = 50 μL); Figure 4 In Example 4 of the present invention VvMYB APL Analysis of gene promoter activity; Figure 5 For the overexpression in Example 5 of the present invention VvMYB APL Phenotypic observation of transgenic Arabidopsis; Figure 6 Phenotypic observation and analysis of transgenic 'Kyoho' grapes in Example 6 of the present invention MYB APL Analysis of gene expression (in the figure, A is the phenotype observation of transgenic 'Kyoho' grapes, B is the phenotype observation of transgenic 'Kyoho' grapes MYB APL Gene expression analysis, * represents P < 0.05, *** represents P < 0.001). DETAILED DESCRIPTION

[0017] The present invention firstly analyzes the transcriptome data of grape berries after H2O2 treatment and finds that MYB APL Gene differential expression, mainly after H2O2 treatment MYB APL Down-regulation of gene expression indicates MYB APL This gene may negatively regulate fruit development. MYB APL The present invention also uses Agrobacterium-mediated transient transformation, Agrobacterium-mediated stable inheritance, subcellular localization and GUS staining experiments to investigate the role of genes in fruit development. MYB APL The function of the gene was analyzed. MYB APL The gene can effectively regulate the ripening process of grape fruit. Specifically, when the gene is overexpressed, the ripening of the grape fruit is inhibited to a certain extent, while inhibiting the expression of the gene can promote the early ripening of the grape fruit.

[0018] The present invention is further described in detail below in conjunction with specific examples. Unless otherwise specified, the equipment and reagents used in each embodiment, experimental example and comparative example can be obtained from commercial sources.

[0019] In the following examples, unless otherwise specified, the examples are all based on conventional experimental conditions, such as Sambrook et al. Molecular cloning: alaboratory manual (Sambrook J & Russell DW, Molecular cloning: alaboratory manual, 2001), or the conditions recommended by the manufacturer's instructions.

[0020] Plant material: In the embodiment of the present invention, the 'Kyoho' grapes from the grape garden of the experimental base of Henan University of Science and Technology were used as materials, the grape fruits treated with H2O2 and the control were subjected to transcriptome sequencing, and the grape RNA and DNA were extracted from the samples. Grape fruits under natural development were used as transgenic test materials. Nicotiana benthamiana was used as the material for subcellular localization and promoter activity analysis.

[0021] The present invention VvMYB APL Specific examples of the application of genes in regulating plant fruit ripening: Example 1 Transcriptome data of grape berries after H2O2 treatment In the previous research of the inventors of the present invention, it was found that the use of hydrogen peroxide (H2O2) to treat Kyoho grape berries can promote the early ripening of Kyoho grape berries (for details, see: Guo DL, Wang ZG, Li Q, et al. Hydrogen peroxide treatment promotes early ripening of Kyoho grape[J]. Australian journal of grape and wine research, 2019, 25(3): 357-362.). Further analysis of the transcriptome data after H2O2 treatment revealed that MYB APLGenes were differentially expressed ( Figure 1 ), specifically, after H2O2 treatment, VvMYB APL Down-regulation of gene expression indicates VvMYB APL The gene may negatively regulate fruit ripening.

[0022] Example 2 VvMYB APL Gene cloning and sequence analysis In this example, RNAprep Pure Polysaccharide and Polyphenol Plant Total RNA Extraction Kit (DP441) (Tiangen Biochemical Technology (Beijing) Co., Ltd.) was used to extract total RNA from Kyoho grape fruit. Hi Fi Script cDNA Synthesis Kit (Beijing Kangwei Biotechnology Co., Ltd.) was used to reverse transcribe the extracted total RNA to obtain cDNA. FastPurePlant DNA Isolation Mini Kit-BOX2 (DC104-01) (Nanjing Novozyme Biotechnology Co., Ltd.) DNA extraction kit was used to extract DNA from Kyoho grape fruit. For specific operations, refer to the kit instructions.

[0023] Obtained from NCBI website (https: / / www.ncbi.nlm.nih.gov / ) VvMYB APL The CDS sequence of the VIT_19s0085g00890 gene was obtained. The required primers were designed using Premier 5 software (primer information is shown in Table 1). Using cDNA as a template, high-fidelity enzymes were used for cloning. VvMYB APL For the full-length gene coding region, the reaction system was 10μL 2× M5HiPer plus Taq HiFi PCR mix (with blua dye), 0.5μL forward primer, 0.5μL reverse primer, 100ngcDNA, and 10μL of H2O to make up to 10μL. The PCR amplification program was 95℃ for 3min, then 34 cycles of 94℃ for 25s, 58℃ for 25s, and 72℃ for 20s, and finally 72℃ for 5min, 4℃ forever.

[0024] Table 1 Gene cloning primer information The PCR amplification product was subjected to 1% agarose gel electrophoresis to detect the band size, and the target band was recovered using a gel recovery kit.

[0025] Use the Expasy-Translate tool (https: / / web.expasy.org / translate) to translate VvMYB APLThe CDS sequences were translated into protein sequences, and homology comparison and analysis were performed through the NCBI website (https: / / www.ncbi.nlm.nih.gov / ). MEGA 7.0 software was used to perform homology comparison, amino acid sequence analysis, and phylogenetic tree analysis on the sequences.

[0026] In this example, the cDNA of mature 'Kyoho' grapes was used as a template for gene cloning, and a specific band of 1071 bp was obtained ( Figure 2 (shown in A) VvMYB APL The nucleotide sequence of the coding region of the gene is shown in SEQ ID NO. 1. The homologous sequence of grape VvMYB APL was downloaded from the NCBI database (https: / / www.ncbi.nlm.nih.gov / ), and the amino acid sequence of VvMYB APL was compared with that of MYB APL proteins of other species using software ( Figure 2 As shown in C), MYB APL was found to contain two relatively conserved domains. The MYB APL protein phylogenetic tree was constructed using MEGA 7.0 software. In the clustering of MYB APL ( Figure 2 As shown in B), grapes ( Vitis vinifera L.) and apple ( Malus pumila Mill. ) are clustered into one category, and their homology is relatively close.

[0027] Example 3 Subcellular localization of VvMYB APL In this example, primers were designed using primier5.0 (primer information is shown in Table 2), and homologous recombination was used to construct the target gene recombinant plasmid. VvMYB APL The CDS sequence of the gene was connected to the 101LYFP vector. The specific operation is as follows: Table 2 Information of primers for subcellular localization gene cloning Refer to the instructions of the 2× KeyPo Master Mix (Dye Plus) kit to clone the target gene, refer to the instructions of the Seamless Cloning Kit produced by Biyuntian, and use DNA ligase to connect the cloned target gene fragment with the 101LYFP vector double-digested with EcoR I and BamHI, and then transfer it to DH5α Escherichia coli. The positive clones screened by PCR and sequencing were stored at -80℃. The recombinant plasmid was further transformed into the Agrobacterium tumefaciens GV3101 strain. The resuspended solution of the recombinant plasmid and the YFP empty Agrobacterium were respectively prepared with the resuspended solution of the nuclear maker Agrobacterium and the resuspended solution of the auxiliary plasmid p19 Agrobacterium in a ratio of 4:3:3, and the mixed solution was aspirated with a 1mL syringe and injected into tobacco leaves (Nicotiana benthamiana). After the injection, it was cultured in the dark for 24 hours, and then cultured in weak light for 2-3 days for transient expression of the bacterial solution, and then the fluorescence signal was detected using a confocal laser scanning microscope.

[0028] Construct VvMYB APL-YFP vector and observe VvMYB APL Subcellular localization in tobacco leaves. Through subcellular localization analysis, it was found that the transcription factor VvMYB APL All transfected cells showed green fluorescence signals in the nucleus ( Figure 3 ), while the green fluorescence of the control YFP protein is distributed in the nucleus and cytoplasm of the cell ( Figure 3 ), the results show VvMYB APL Localized in the cell nucleus.

[0029] Example 4 VvMYB APL Gene promoter activity analysis The primers were designed by homologous recombination method (primer information is shown in Table 3). VvMYB APL and 35S The promoters of the two genes were connected to the 0390-GUS vector digested with HindⅢ and EcoRⅠ, respectively. Then, the vectors were transformed into DH5α Escherichia coli. The positive clones were screened by PCR and sequencing, and the bacterial solution was stored at -80℃. Then, the recombinant plasmids of the positive strains were extracted, and the recombinant plasmids and 35S plasmids were introduced into the Agrobacterium tumefaciens GV3101 strain respectively.

[0030] Table 3 Primer information for promoter activity analysis gene cloning After resuspending the recombinant plasmid and 35S empty bacterial solution with resuspension solution, detached tobacco leaves were immersed in the mixed bacterial solution with the distal side facing downward, covered with a 0.22μM microporous filter membrane, and then vacuum-permeated (3 to 5 leaves were used for one gene, resuspension solution was used for negative control, and 35S empty vector was used for positive control).

[0031] Build VvMYB APL::GUS Carrier, Observation VvMYB APL The promoter activity of VvMYB APL The promoter can normally regulate VvMYB APL Gene expression ( Figure 4 After the tobacco leaves were treated with H2O2 and then stained with GUS, the color of the tobacco leaves became darker ( Figure 4 ), the results showed that the promoter of VvMYB APL could respond to H2O2 signals.

[0032] Example 5 VvMYB APL Gene negatively regulates fruit ripening in Arabidopsis This embodiment first constructs VvMYB APL The overexpression vector of the gene was then transformed into Arabidopsis thaliana, and the phenotype of Arabidopsis thaliana was observed. The specific implementation operations are as follows: 1. Construction of overexpression vector and stable transformation The primers were designed by homologous recombination method (primer information is shown in Table 4). VvMYB APL Overexpression vector VvMYB APL The gene was ligated into the pCAMBIA2300-GFP vector digested with BamHI, and the resulting construct was named 2300GFP-MYB APL . Then the cells were transferred into DH5α Escherichia coli, and the positive clones screened by PCR and sequencing were stored at -80℃. Then the recombinant plasmids of the positive strains were extracted and introduced into Agrobacterium tumefaciens GV3101 strain.

[0033] Table 4 Transgenic gene cloning primer information By using the floral dip method, place the flower buds of Arabidopsis in a centrifuge tube containing Agrobacterium infiltration solution for about 10 minutes. After infection, place them on absorbent paper to gently wipe off the excess bacterial solution, turn them upside down, and culture them in the dark for 24 hours before normal culture. To improve the transformation efficiency of Arabidopsis, perform Arabidopsis floral dip infection once every 3-4 days (when Arabidopsis grows well), and then perform Arabidopsis floral dip infection again after another three days (when Arabidopsis grows well).

[0034] 2. Results After the Arabidopsis seeds are mature and harvested (Arabidopsis T0 generation seeds), transgenic Arabidopsis seeds will be verified.

[0035] The received Arabidopsis seeds (T0) were planted on a culture medium containing kanamycin and positive seedlings were selected. The phenotype of Arabidopsis plants can be observed after the T3 generation is selected. The observation results show (such as Figure 5 Compared with the wild type, MYB APL Transgenic Arabidopsis thaliana is resistant to Kan; compared with the wild type, MYB APL Transgenic delayed the growth and development of Arabidopsis, and the maturity of Arabidopsis pods was significantly delayed by about 5 days. MYB APL It will cause Arabidopsis seeds to mature late. MYB APL Negatively regulates Arabidopsis pod ripening.

[0036] Example 6 VvMYB APL Gene negatively regulates grape berry ripening This embodiment first constructs VvMYB APL The overexpression and RNAi vectors of the genes were then transformed into grape berries, and real-time fluorescence quantitative PCR was used to detect the expression of genes in the transformed grape berries. VvMYB APL The expression level of the gene was detected and the role of the gene in grape fruit ripening was analyzed. The specific operations are as follows: 1. Construction of overexpression and silencing vectors and transient transformation The primers were designed by homologous recombination method (primer information is shown in Table 5). VvMYB APL Overexpression vector VvMYB APL The gene was ligated into the pIR-GUS vector digested with BamHI and XbaI to replace the GUS coding gene, and the resulting construct was named pIR-VvMYB APL-OE To build VvMYB APL Silencing the vector VvMYB APL The gene was reversely subcloned into the pIR-GUS vector digested with BamHI and XbaI to replace the GUS coding gene, and the resulting construct was named pIR- VvMYB APL-RNAi The plasmid was then transferred to DH5α E. coli, and the positive clones screened by PCR and sequencing were stored at -80°C. The VvMYB APL E. coli solution was purified using the Vigras plasmid large-scale extraction and purification kit.

[0037] Table 5 Transgenic gene cloning primer information According to the method of Jia et al. (see: Jia H, Zuo Q, Sadeghnezhad E, et al. HDAC19recruits ERF4 to the MYB5a promoter anddiminishes anthocyanin accumulationduring grape ripening. Plant J, 2023, 113(1): 127-144.), pierce the grape stalk with the needle of a 5mL syringe, insert the capillary into the hole, and suck about 200 ng of DNA (50μL) into the capillary until it is completely absorbed by the grape bunch. Inject the second time about one week later, using the IL-60-BS vector as an auxiliary plasmid (the construction of the recombinant plasmid requires the auxiliary plasmid for replication, movement and expression). Inject twice in total, and measure the gene expression level 3-5 days after the second injection.

[0038] 2. Real-time fluorescence quantitative PCR detection Designed using Primer Blast (https: / / www.ncbi.nlm.nih.gov / tools / primer-blast / ) in the online website NCBI VvMYB APL The fluorescent quantitative primers of the genes (as shown in Table 6) were detected using the ChamQTM Universal SYBR q PCR Master Mix fluorescent dye of Novozymes and the Quantageneq225 fluorescent quantitative PCR instrument of Beijing Coolbo Technology Co., Ltd. qPCR program: In a 10 μL reaction mixture consisting of 5 μL 2× SYBR Green PCR Master Mix, 0.3 μL primers and 200 ng cDNA, the amplification reaction was carried out at 95°C for 5 minutes, followed by 40 cycles of 95°C for 10 seconds, 58°C for 30 seconds and 95°C for 15 seconds. Three replicates were performed for each sample. Used in all experiments Actin As an internal reference gene, according to 2 （-ΔΔCT） The algorithm normalizes the expression levels of the genes being tested.

[0039] Table 6 Primer information for fluorescence quantitative PCR 3. Results To verify MYB APL The regulatory role in the ripening process of grape fruit has constructed pIR:MYB APL Overexpression vector ( pIR-VvMYB APL-OE )and pIR:MYB APL Silencing vector ( pIR-VvMYB APL-RNAi ), in the grape body MYB APLGene overexpression and silencing, pIR Empty vector was used as control. Different bunches of grapes on the same grapevine were injected separately. pIR-VvMYB APL-OE , pIR-VvMYB APL-RNAi and pIR Empty vector plasmid, the size of the injected fruit is about 40 days after flowering, the injection is divided into two times, and the growth status of the fruit is observed about one week after each injection. The specific observation time is at the time of injection (2024.6.18), about one week after the first injection (2024.6.26), and about one week after the second injection (2024.7.02). The observation results show ( Figure 6 As shown in A), the injection was carried out about 40 days after the flowering of the 'Jufeng' grape. pIR-VvMYB APL-RNAi The grapes injected with the plasmid showed obvious color change. pIR-VvMYB APL-OE and pIR The growth of grapes with empty vector plasmid did not change significantly, and the fruit color did not turn red. pIR- VvMYB APL-RNAi The grapes with the plasmid had a deeper color change and a larger number of fruits. pIR-VvMYB APL-OE There was no obvious change in the color of the grape fruit. pIR Some grapes in the empty vector plasmid changed color. On the 4th day after the second injection, we took transgenic grape samples, extracted RNA and reverse transcribed it, and performed fluorescence quantitative experiments. The results of fluorescence quantitative data analysis showed that ( Figure 6 Compared with the empty vector control, the silencing MYB APL After the gene, the grape fruit MYB APL The gene expression level decreased to about 1 / 2 of the control, and the corresponding grape phenotype was that the fruit color turned red; overexpression MYB APL After the gene, the grape fruit MYB APL The gene expression level increased by about 15 times that of the control, and the corresponding grape phenotype was that the fruit color did not change significantly. The above results show that MYB APL The gene acts as a negative regulator to regulate grape fruit ripening.

[0040] Finally, it is noted that the above embodiments are only used to illustrate the technical solutions of the present invention, rather than to limit it. Although the present invention has been described in detail with reference to the aforementioned embodiments, those skilled in the art should understand that they can still modify the technical solutions described in the aforementioned embodiments, or replace some or all of the technical features therein with equivalents. However, these modifications or replacements do not cause the essence of the corresponding technical solutions to deviate from the scope of the technical solutions of the embodiments of the present invention.

Claims

1. VvMYB APL The application of the gene in regulating the ripening of plant fruits is characterized by: Said VvMYB APL The nucleotide sequence of the coding region of the gene is shown in SEQ ID NO.

1.

2. according to claim 1 VvMYB APL The application of the gene in regulating the ripening of plant fruits is characterized by: The regulating plant fruit ripening is regulating the color change of plant fruits or the ripening of fruit pods.

3. The method according to claim 1 or 2 VvMYB APL The application of the gene in regulating the ripening of plant fruits is characterized by: Overexpression VvMYB APL Genes that inhibit the ripening of plant fruits; VvMYB APL The expression of genes promotes the ripening of plant fruits.

4. according to claim 3 VvMYB APL The application of the gene in regulating the ripening of plant fruits is characterized by: The overexpression VvMYB APL Gene for construction VvMYB APL Gene overexpression vectors are transformed into plants to VvB APL Gene overexpression.

5. According to claim 3 VvMYB APL The application of the gene in regulating the ripening of plant fruits is characterized by: The inhibition VvMYB APL Gene expression constructs VvMYB APL The RNAi vector of the gene is transformed into plants. VvB APL Inhibit gene expression.

6. The method according to claim 1 or 2 VvMYB APL The application of the gene in regulating the ripening of plant fruits is characterized by: The plants are grape and Arabidopsis thaliana.

7. According to claim 6 VvMYB APL The application of the gene in regulating the ripening of plant fruits is characterized by: The grapes are 'Kyoho' grapes.

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