CN117660474B - Application of interaction between pear transcription factor PbrMYB65 and PbrACO2 gene promoter in regulating citric acid isomerization in fruit - Google Patents

Abstract

The invention discloses an application of pear transcription factor PbrMYB and PbrACO gene promoter interaction in regulating and controlling fruit citric acid isomerization. The nucleotide sequence of the pear MYB family transcription factor PbrMYB is shown as SEQ ID NO.1 in a sequence table, and the amino acid sequence of the gene coding protein is shown as SEQ ID NO. 2. The invention discloses a transcription factor PbrMYB65 for regulating and controlling the gene expression of pear PbrACO for the first time. The action mechanism of interaction of PbrMYB and MYB binding sites (P1: (-800) - (-795) and P2: (-203) - (-198)) in PbrACO gene promoters in regulating and controlling the isomerization of citric acid of pear fruits is elucidated, and theoretical and practical basis is provided for realizing directional improvement of quality traits of pear fruits.

Description

Application of the interaction between the pear transcription factor PbrMYB65 and the promoter of the PbrACO2 gene in regulating fruit citric acid isomerization

Technical Field

The invention belongs to the technical field of molecular biology and genetic engineering, and specifically relates to a pear transcription factor PbrMYB65 which promotes citric acid isomerization by regulating the expression of a key citric acid metabolism gene PbrACO2.

Background Art

Pear is the king of fruits; rich in nutrients, it is favored by consumers all over the world. Malic acid and citric acid are the main organic acids in fruits and play an important role in the formation of fruit flavor quality.

The content of organic acids in fruits is closely related to their own synthesis, degradation and compartmental distribution. For horticultural crops, phosphoenolpyruvate carboxylase (PEPC) in the cytoplasm and citrate synthase (CS) in the mitochondria are the key enzymes for citric acid biosynthesis: phosphoenolpyruvate is carboxylated to produce oxaloacetate under the action of PEPC; after entering the mitochondria, the latter combines with acetyl-CoA (Ac-CoA) to produce citric acid under the catalysis of CS. However, analysis of the enzyme activities and related gene expression levels of CS and PEPC in low/high citric acid varieties and during fruit development found that there was no significant correlation between them and citric acid content. Therefore, the accumulation of citric acid in fruits may be mainly regulated by downstream degradation pathways.

Aconitases (ACOs) are iron-sulfur enzymes containing a 4Fe-4S cluster that catalyze the reversible isomerization of citrate. Plant ACOs are located in the cytoplasm (cytACOs) and mitochondria (mytACOs) and are mainly involved in the isomerization of citrate to aconitic acid. Overexpression of the rice OsACO1 gene or the citrus CitACO3 gene inhibited the accumulation of citrate in plant tissues; on the other hand, knocking out the rice OsACO1 gene or silencing the tomato SlACO1 gene promoted the accumulation of citrate in tissues.

Transcription factors (TFs) can bind to cis-acting elements in the promoters of downstream structural genes and regulate their expression. They play an important role in plant growth and development and stress. They mainly include WRKYs, bZIPs and MYBs family genes. PuWRKY31 can bind to the W-box element in the promoter of PuSWEET15, activate the transcription of PuSWEET15, and promote the accumulation of soluble sugars in ‘Nanguo’ pears. CitWRKY1 can interact with CitNAC62 to synergistically regulate the expression of CitACO3 gene in citrus and inhibit the accumulation of citric acid in the fruit. So far, there are few reports on the ACO gene family members involved in citric acid metabolism in pear fruit and their regulatory mechanisms.

Summary of the invention

The object of the present invention is to provide an application of a PbrACO2 gene or a biological material related to the PbrACO2 gene in regulating the isomerization of citric acid in the fruit of a Rosaceae fruit tree or regulating the accumulation of citric acid in the fruit of a Rosaceae fruit tree.

Another object of the present invention is to provide the use of MYB family transcription factor PbrMYB65 or biological materials related to MYB family transcription factor PbrMYB65 in regulating citric acid isomerization in the fruit of Rosaceae fruit trees or regulating citric acid accumulation in the fruit of Rosaceae fruit trees. Studies have shown that the interaction between MYB family transcription factor PbrMYB65 and the PbrACO2 gene promoter can regulate citric acid accumulation in pear fruit. Transcription factor PbrMYB65 can specifically bind to the MYB binding site (P1: (-800)-(-795); P2: (-203)-(-198)) in the upstream promoter of the PbrACO2 gene, promoting the transcription of the PbrACO2 gene.

Another object of the present invention is to provide a method for inhibiting the accumulation of citric acid in the fruit of Rosaceae fruit trees.

The objective of the present invention is achieved through the following technical solutions:

In a first aspect, the present invention claims protection for the use of the PbrACO2 gene or biological materials related to the PbrACO2 gene in regulating citric acid isomerization or regulating citric acid accumulation in the fruits of Rosaceae fruit trees, wherein the nucleotide sequence of the PbrACO2 gene is shown in SEQ ID NO.3.

Furthermore, the biological material related to the PbrACO2 gene is at least one of (1) to (8):

(1) PbrACO2 protein with an amino acid sequence as shown in SEQ ID NO.4;

(2) an expression cassette containing the PbrACO2 gene;

(3) a recombinant vector containing the PbrACO2 gene;

(4) a recombinant vector containing the expression cassette described in (2);

(5) a recombinant microorganism containing the PbrACO2 gene;

(6) A recombinant microorganism containing the expression cassette described in (2);

(7) A recombinant microorganism containing the recombinant vector described in (3);

(8) A recombinant microorganism containing the recombinant vector described in (4).

Furthermore, the PbrACO2 protein is a protein encoded by the PbrACO2 gene.

Furthermore, overexpression of the PbrACO2 gene inhibits citric acid accumulation in fruit, or promotes citric acid isomerization.

In the second aspect, the present invention claims protection for the use of MYB family transcription factor PbrMYB65 or biological materials related to MYB family transcription factor PbrMYB65 in regulating citric acid isomerization in the fruits of Rosaceae fruit trees or regulating citric acid accumulation in the fruits of Rosaceae fruit trees. The amino acid sequence of the transcription factor PbrMYB65 is shown in SEQ ID NO.2.

Furthermore, the biological material related to the transcription factor PbrMYB65 is at least one of the following (1) to (8):

(1) a gene PbrMYB65 encoding the transcription factor PbrMYB65;

(2) an expression cassette containing the gene PbrMYB65 described in (1);

(3) a recombinant vector containing the gene PbrMYB65 described in (1);

(4) a recombinant vector containing the expression cassette described in (2);

(5) a recombinant microorganism containing the gene PbrMYB65 described in (1);

(6) A recombinant microorganism containing the expression cassette described in (2);

(7) A recombinant microorganism containing the recombinant vector described in (3);

(8) A recombinant microorganism containing the recombinant vector described in (4).

Furthermore, the nucleotide sequence of the gene PbrMYB65 encoding the transcription factor PbrMYB65 in (1) is shown as SEQ ID No.1.

Furthermore, the interaction between the MYB family transcription factor PbrMYB65 and the PbrACO2 gene promoter can regulate the citric acid isomerization or citric acid accumulation in pear fruit. Furthermore, the MYB family transcription factor PbrMYB65 promotes the expression of the PbrACO2 gene by binding to the MYB binding sites (P1: (-800)-(-795); P2: (-203)-(-198)) in the PbrACO2 gene promoter, thereby increasing the activity of the mitochondrial ACO (mitACO) enzyme and inhibiting the accumulation of citric acid in the fruit. The P1 binding site sequence is (+/-)CAACCG/CGGTTG; the P2 binding site sequence is (+/-)CGGTTG/CAACCG.

In a fourth aspect, the present invention claims a method for inhibiting the accumulation of citric acid in the fruit of a Rosaceae fruit tree, overexpressing the gene PbrMYB65 encoding the transcription factor PbrMYB65 or/and the PbrACO2 gene to inhibit the accumulation of citric acid in the fruit; the nucleotide sequence of the gene PbrMYB65 encoding the transcription factor PbrMYB65 is shown in SEQ ID No.1; the nucleotide sequence of the PbrACO2 gene is shown in SEQ ID No.3.

In the above application and method, the Rosaceae fruit tree is pear.

Research process of the technical solution of the present invention:

All PbrACOs genes (6 gene family members) were identified based on the pear genome database; further, by analyzing the expression profile of PbrACOs genes in different growth and development stages of ‘Yali’ fruit and its correlation with citric acid content, the family member PbrACO2 that may be involved in regulating citric acid isomerization was screened out.

The recombinant plasmid pBI221-PbrACO2-GFP was constructed and co-transformed with the mitochondrial marker protein MSTP-mcherry into Arabidopsis protoplasts. GFP fluorescence was observed using a laser confocal microscope, and it was found that PbrACO2 was located in the mitochondria.

The pCAMBIA1300-PbrACO2 overexpression vector was constructed and transferred into Agrobacterium GV3101 to transform pear callus. After positive tissue was obtained by PCR identification, the positive transgenic tissue and its control (callus transformed with pCAMBIA1300 empty vector) were transferred to MS medium with sorbitol and sucrose (1:1, w:w) as carbon sources for growth.

Using qPT-PCR detection technology and mitACO enzyme activity detection kit, it was found that the PbrACO2 gene expression level and mitochondrial enzyme activity in pear PbrACO2 positive transgenic tissues were significantly increased.

Ultra-high performance liquid chromatography (UPLC) detection revealed that the citric acid content was significantly reduced in pear PbrACO2-positive transgenic tissues.

The promoter of PbrACO2 gene was cloned and submitted to PlantCARE database (http://bioinformatics.psb.ugent.be/webtools/plantcare/html/), and the cis-regulatory elements were predicted. W-box, G-box and MYB binding elements were found, indicating that it may be regulated by PbrWRKYs, PbrbZIPs and PbrMYBs transcription factors. Combined with the expression trend of related transcription factors during the growth and development of pear fruit (the correlation coefficient with PbrACO2 expression was set to >0.8 or <-0.8), the transcription factors PbrMYB3, PbrMYB65 and PbrMYB81 that may regulate the expression of PbrACO2 gene were screened. Because PbrMYB65 has the highest correlation with the expression level of PbrACO2, we chose it for further study.

The recombinant plasmid pSAK277-PbrMYB65 was constructed, and the reporter vectors (pGreenII0800-PbrACO2pro-LUC (full-length promoter), pGreenII 0800-PbrACO2pro ^frag1 -LUC, pGreenII0800-PbrACO2pro ^frag2 -LUC, pGreenII 0800-PbrACO2pro ^mut -LUC) were constructed with the PbrACO2 gene promoter sequence containing different numbers of MYB binding sites or its mutants (CAACCG/CGGTTG→CTTCCG/CGGAAG) and transformed into Agrobacterium GV3101 and GV3101(psoup), respectively. Tobacco leaves were co-infected with Agrobacterium strains carrying pSAK277-PbrMYB65 and a reporter vector, and the activities of firefly luciferase and Renilla luciferase were detected. It was found that the Luc/Ren ratio of tobacco leaves co-transformed with pSAK277-PbrMYB65&pGreenII 0800-PbrACO2pro-LUC (or pSAK277-PbrMYB65&pGreenII 0800-PbrACO2pro ^frag1 -LUC) was significantly higher than that of the control group, while there was no significant difference in the Luc/Ren ratio of tobacco leaves co-transformed with pSAK277-PbrMYB65&pGreenII 0800-PbrACO2pro ^frag2 -LUC (or pSAK277-PbrMYB65&pGreenII 0800-PbrACO2pro ^mut -LUC) and its control group.

The prey vector PbrMYB65-AD was constructed, and the bait vectors (PbrACO2pro S1-pAbAi, PbrACO2pro S2-pAbAi, PbrACO2pro ^S1mut- pAbAi and PbrACO2pro ^S2mut -pAbAi) were constructed with the PbrACO2 promoter fragment containing the MYB binding site (P1: (-800)-(-795); P2: (-203)-(-198)) or its mutants (CAACCG/CGGTTG→CTTCCG/ ^CGGAAG ). The Matchmaker Gold Yeast One-Hybrid Library Screening System was used to detect that PbrMYB65 could bind to the promoter fragments containing the MYB binding site (PbrACO2pro ^S1 -pAbAi and PbrACO2pro ^S2 -pAbAi), but the mutant site (PbrACO2pro ^S1mut - ^pAbAi ) could not bind to the promoter fragments containing the MYB binding site. -pAbAi and PbrACO2pro ^S2mut -pAbAi), to which PbrMYB65 could not bind.

Pear callus carrying pCAMBIA1300-GFP was used as a control. Chromatin immunoprecipitation quantitative PCR (ChIP-qPCR) analysis was performed on the PbrACO2 promoter fragment containing the MYB binding site (P1: (-800)-(-795); P2: (-203)-(-198)), and it was found that PbrMYB65 could bind to the promoter fragment containing the MYB binding site.

Combined with further electrophoretic mobility shift assay (EMSA) results, it was concluded that PbrMYB65 activated the expression of PbrACO2 gene by binding to the MYB binding sites (P1: (-800)-(-795); P2: (-203)-(-198)) in the upstream promoter of the gene.

The recombinant plasmid pBI221-PbrMYB65-GFP was constructed and co-transformed with the nuclear marker protein AtH2B-mcherry into Arabidopsis protoplasts. GFP fluorescence was observed using a laser confocal microscope, and it was found that PbrMYB65 was located in the nucleus.

The pCAMBIA1300-PbrbMYB65 overexpression vector was constructed and transformed into Agrobacterium GV3101 to infect pear callus. After positive tissue was obtained by PCR identification, the positive transgenic tissue and its control (callus transformed with pCAMBIA1300 empty vector) were transferred to MS medium with sorbitol and sucrose (1:1) as carbon sources for growth.

The results of qPT-PCR and mitochondrial ACO (mitACO) enzyme activity detection kit showed that the expression levels of PbrMYB65 and PbrACO2 genes and the activity of mitACO enzyme in PbrbMYB65 positive transgenic callus of pear significantly increased.

Ultra-performance liquid chromatography (UPLC) detection revealed that the citric acid content was significantly reduced in pear PbrbMYB65-positive transgenic callus.

Compared with the prior art, the present invention has the following beneficial effects:

1. The transcription factor PbrMYB65 that regulates the expression of the PbrACO2 gene in pear fruit was revealed for the first time.

2. The present invention clarifies the mechanism of interaction between PbrMYB65 and the MYB binding sites in the promoter of the PbrACO2 gene (P1: (-800)-(-795); P2: (-203)-(-198)) in regulating citric acid isomerization in pear fruit, providing a theoretical and practical basis for the targeted improvement of pear fruit quality traits.

BRIEF DESCRIPTION OF THE DRAWINGS

The present invention will be further described in detail below in conjunction with the accompanying drawings and specific embodiments.

Figure 1 Dynamic changes in the content of organic acids (citric acid, oxalic acid, tartaric acid, malic acid, shikimic acid and total acid) in the peel and pulp of bagged and unbagged ‘Yali’ fruits during their growth and development. (a) Unbagged ‘Yali’; (b) Bagged ‘Yali’. Different lowercase letters represent significant differences between samples (p<0.05).

Fig. 2 The ratio of citric acid/malic acid (or oxalic acid) during the growth and development of bagged and unbagged ‘Yali’ fruits. (a) Citric acid/malic acid ratio; (b) Citric acid/oxalic acid ratio. Different lowercase letters represent significant differences between samples (p<0.05).

Figure 3 Dynamic changes in PbrACOs gene expression in peel and flesh of bagged and unbagged ‘Yali’ fruits during growth and development. (a) Transcriptome analysis of PbrACOs gene expression. Red indicates high level, green indicates low level, and black indicates medium level. (b) qRT-PCR verification of PbrACO2 gene expression pattern.

Fig. 4 Correlation between PbrACOs gene expression, organic acid content, and citric acid/malic acid (or oxalic acid) ratio. Negative correlation is indicated in green, and positive correlation is indicated in red.

Fig. 5 PbrACO2 localizes to mitochondria in Arabidopsis protoplasts.

Figure 6 PbrACO2 gene function verification. (a) Callus growth diagram; (b) PbrACO2 gene expression and mitochondrial ACO (mitACO) enzyme activity; (c) Organic acid content. Different lowercase letters represent significant differences between samples (p<0.05).

Figure 7 Screening of upstream transcription factors involved in regulating the expression of PbrACO2 gene. (a) Distribution of cis-acting elements in the promoter of PbrACO2 gene; (b) Information of cis-acting elements in the promoter of PbrACO2 gene; (c) Changes in the expression levels of PbrWRKYs, PbrbZIPs and PbrMYBs transcription factors during the growth and development of ‘Yali’ fruit and their correlation with the expression level of PbrACO2 gene.

Figure 8 PbrMYB65 can bind to the MYB binding site in the PbrACO2 gene and activate its expression. (a) Dual luciferase assay; (b) Yeast one-hybrid assay; (c) Chromatin immunoprecipitation qPCR assay (ChIP-PCR); (d) Electrophoretic mobility shift assay (EMSA). Different lowercase letters represent significant differences between samples (p<0.05).

Fig. 9 PbrMYB65 is localized in the nucleus.

Figure 10 PbrMYB65 gene function verification. (a) Callus growth diagram; (b) PbrACO2 gene expression and mitACO enzyme activity; (c) PbrMYB65 gene expression and citric acid content. Different lowercase letters represent significant differences between samples (p<0.05).

Figure 11 The mechanism by which the MYB family transcription factor PbrMYB65 interacts with the PbrACO2 gene promoter to regulate citric acid accumulation in pear fruit.

DETAILED DESCRIPTION

The present invention is described in detail below in conjunction with specific embodiments. The examples are provided for a better understanding of the present invention, but are not intended to limit the present invention. The experimental methods in the following embodiments are all conventional methods, and the experimental reagents involved are all conventional biochemical reagents.

Example 1 PbrACOs gene expression profile and its correlation with citric acid content:

1. Collection of tissue samples

The ‘Yali’ fruit with similar tree age and vigor planted in the orchard in Gaoyou, Yangzhou was used as the experimental material of this experiment. The fruit was bagged 34 days after the full flowering period, and the fruit without bagging in the same position was used as the control. Each treatment was repeated 3 times, and each replicate had 200 fruits. The peel and flesh tissues of the bagged and unbagged ‘Yali’ fruit were collected 15 days (S1), 34 days (S2), 81 days (S3), 110 days (S4), 145 days (S5) and 160 days (S6) after the full flowering period to determine the content of citric acid and the amount of PbrACOs genes.

2 Determination of citric acid content

Ultra-high performance liquid chromatography (UPLC) was used to determine the content of organic acids in ‘Yali’ tissue. Weigh 0.5 g of tissue, grind it thoroughly in liquid nitrogen, transfer it to a 10 mL glass graduated tube, add 5 mL of ultrapure water, and extract it by ultrasonic for 15 minutes after 80°C water bath for 30 minutes. Then transfer the extract to a 2 mL centrifuge tube, centrifuge it at 4°C and 12000 rpm for 20 minutes, and collect the supernatant. Take 1.5 mL of the supernatant and filter it with a 0.45 μm Sep-Pak water system microporous filter membrane to obtain the organic acid extract. The organic acid content was determined by liquid chromatography equipped with a Waters 1525 system: Zorbar SB-Aq column (4.6 mm × 250 mm × 5.0 μm), 2% methanol and 98% 20 mM potassium dihydrogen phosphate buffer (pH 2.4) as the mobile phase (flow rate 0.7 mL/min), column temperature 5°C, aters 2487 UV detector, detection wavelength 210 nm, injection volume 5 μL. The content of shikimic acid, oxalic acid, tartaric acid, malic acid and citric acid in the tissue was calculated based on the sample peak area and the standard curve.

3 Transcriptome sequencing

Total RNA was extracted from the peel and pulp tissues of pears using the EASYspin Plus Plant RNA Rapid Extraction Kit (RN3802, Aidelai, Beijing). The purity, concentration, and integrity of the sample RNA were identified using a NanoDrop 2000 spectrophotometer (Thermo Scientific, USA) and an Agilent 2100 Bioanalyzer (Agilent Technologies, Santa Clara, CA, USA). The transcriptome library was constructed using the NEBNext UltraTM RNA Library Prep Kit for Illumina (E7770, NEB, USA). The constructed library was then sequenced using the HiSeq X-ten sequencing platform. After removing low-quality reads, clean reads were obtained for subsequent data analysis. HISAT2 software was used to compare with the ‘Dangshan Pear’ genome database (http://peargenome.njau.edu.cn/), and gene expression levels (FPKM) were calculated. Transcriptome sequencing and analysis were completed by Biomarker Biotechnology Co., Ltd. (Beijing, China). Based on the transcriptome annotation, we discovered six ACO encoding genes in the pear genome (see Table 1 in the Appendix).

4qRT-PCR detection

The total RNA from the 'Yapear' tissue was extracted using a plant total RNA extraction kit (RE-05011, Chengdu Fuji Biotechnology Co., Ltd.), and the extraction method was referred to the kit instructions. The centrifuge tubes and various types of gun tips used in the extraction process were all RNase-free. The extracted total RNA was reversed into cDNA using the Quanshijin AE301-02EasyScript cDNA First Chain Synthesis Kit (AE301-02, Beijing Quanshijin Biological). Finally, the PrimeScript ^TM RT-PCR Kit (RR014A, Baoriyi Biotechnology (Beijing) Co., Ltd.) was used to detect the relative expression of PbrACO2 at different developmental stages. The pear Tublin gene (PbrTub) was used as the housekeeping gene. The primer sequence of the PbrACO2 gene is shown in Table 2 in the attached table.

5 Screening of genes involved in citric acid metabolism in pear

The correlation between PbrACOs gene expression and citric acid content during the growth and development of ‘Yali’ fruit was analyzed (correlation coefficient <-0.6), and the key genes involved in citric acid isomerization during the growth and development of the fruit were screened out.

Experimental results: During the growth and development of ‘Yali’ fruit, the changing trends of the contents of five organic acids in the peel and pulp were different (Figure 1); among them, shikimic acid, oxalic acid, tartaric acid and malic acid showed a downward trend (Figure 1), while the ratio of citric acid/malic acid (or oxalic acid) showed an upward trend (Figure 2). Bagging did not change the changing trend of organic acids in the fruit. Further studies found that the citric acid content and citric acid/malic acid ratio in the pulp were significantly higher than those in the peel (Figures 1 and 2). According to transcriptome annotation, we discovered 6 ACO encoding genes (PbrACO1-6) in the pear genome (Table 1); the expression trends of different genes in the growth and development of bagged and unbagged ‘Yali’ fruits were different (PbrACO6 gene was not expressed) (Figure 3a). Correlation analysis found that during the growth and development of ‘Yali’ fruit, only PbrACO2 had significantly higher gene expression in bagged and unbagged peels than in the flesh, and it was significantly correlated with citric acid content (correlation coefficient <-0.6) (Figure 4). qRT-PCR verified the expression trend of PbrACO2 during fruit growth and development (Figure 3b). Therefore, we selected the PbrACO2 gene for further study.

Example 2 Functional verification of PbrACO2 gene

1Determination of subcellular localization of protein encoded by PbrACO2 gene

Using the fruit cDNA of ‘Yali’ as a template, the coding sequence of the PbrACO2 gene was amplified (primer sequences are shown in Table 3 in the attached table), inserted into the pBI221-GTP vector to construct the recombinant plasmid pBI221-PbrACO2-GFP, and co-transformed with the mitochondrial marker MSTP-mcherry into Arabidopsis protoplasts. The fluorescence signal was observed using a laser confocal microscope (Leica Microsystems, Germany), and it was found that PbrACO2 was located in the mitochondria. The CDS sequence of the PbrACO2 gene is shown in SEQ ID No.3; the protein sequence encoded by the PbrACO2 gene is shown in SEQ ID No.4.

Obtaining callus tissue of pear overexpressing PbrACO2 gene

Using the fruit cDNA of 'Yali' as a template, the coding sequence of the PbrACO2 gene was amplified and inserted into the pCAMBIA1300 vector to construct the pCAMBIA1300-PbrACO2 overexpression vector, which was then transformed into Agrobacterium GV3101 and infected with pear callus (induced by P. communis cv. 'Clapp's Favorite' small fruit). The infected callus was transferred to MS medium (carbon source: 30 g·L ^-1 sucrose) containing hygromycin (Hyg) and cultured for 1.5-2 months. After PCR identification, the callus was expanded. The positive tissue and the control (transformed empty pCAMBIA1300) were co-transferred to MS medium (containing Hyg) with sorbitol/sucrose (15 g·L ^-1 , 1:1) as the carbon source for growth.

3 Analysis of PbrACO2 gene expression in callus tissue

Total RNA from callus was extracted using a plant total RNA extraction kit (RE-05011, Chengdu Fuji Biotechnology Co., Ltd.) and reverse transcribed to synthesize cDNA. Real-time qRT-PCR was used to detect the expression of PbrACO2 gene. The primer sequences of PbrACO2 gene are shown in Table 2. Pear Tublin gene (PbrTub) was used as the housekeeping gene.

4 Determination of mitACO enzyme activity in callus

Mitochondria were purified from pear callus, and then the mitochondrial ACO (mitACO) enzyme activity was determined using an ACO test kit (ACO-1-Z, Suzhou Keming Biotechnology Co., Ltd., Suzhou, China). The protein concentration in the crude enzyme solution was determined using a BCA protein concentration assay kit (A045-4, Nanjing Jiancheng Bioengineering Institute, Nanjing, China).

5 Determination of citric acid content in callus tissue

Weigh 0.5g of tissue, grind it thoroughly in liquid nitrogen, transfer it to a 10mL glass graduated tube, add 5mL of ultrapure water solution, place it in a water bath at 80℃ for 30min, and then extract it by ultrasonic for 15min. Transfer the extract to a 2mL centrifuge tube, centrifuge it at 4℃, 12000rpm for 20min, and collect the supernatant. Take 1.5mL of the supernatant and filter it with a 0.45μm Sep-Pak water system microporous filter membrane to obtain an organic acid extract. Determine the citric acid content using a liquid chromatograph.

Experimental results: PbrACO2 is located in the mitochondria of Arabidopsis protoplasts (as shown in Figure 5). Using qPT-PCR detection technology and mitACO enzyme activity detection kit, it was found that the expression of PbrACO2 gene and mitochondrial enzyme activity in pear PbrACO2 positive transgenic callus tissue increased significantly. Using ultra-high performance liquid chromatography (UPLC) detection, it was found that the citric acid content was significantly reduced in pear PbrACO2 positive transgenic tissue (as shown in Figure 6). Overexpression of PbrACO2 gene in pear callus tissue can increase mitACO enzyme activity and inhibit citric acid accumulation.

Example 3 Screening of PbrACO2 upstream regulatory genes and their regulatory mechanism

Screening of upstream regulatory genes of 1PbrACO2

Using the fruit DNA of ‘Yali’ as a template, the 2000bp sequence upstream of the promoter of the PbrACO2 gene (SEQIDNo.5) was cloned and submitted to the PlantCARE database (http://bioinformatics.psb.ugent.be/webtools/plantcare/html/), and the cis-regulatory elements were predicted. W-box, G-box and MYB binding elements were found (Figure 7a-b), indicating that it may be regulated by PbrWRKYs, PbrbZIPs and PbrMYBs transcription factors. Based on the gene expression levels of PbrWRKYs, PbrbZIPs and PbrMYBs during the growth and development of ‘Yali’ fruit and their correlation with the expression level of PbrACO2 gene (the screening threshold was set to >0.8 or <-0.8), the transcription factors PbrMYB3, PbrMYB65 and PbrMYB81 that may regulate the expression of the PbrACO2 gene were screened. Because PbrMYB65 had the highest correlation with PbrACO2 expression levels ( Figure 7 c), we selected it for further study.

2 Dual luciferase reporter assay

Using the fruit cDNA of 'Yali' as a template, the coding sequence of the PbrMYB65 gene was cloned (primer sequences are shown in Table 3 in the attached table), inserted into the pSAK277 vector, and the pSAK277-PbrMYB65 recombinant vector was constructed. The CDS sequence of the PbrMYB65 gene is shown in SEQ ID No. 1; the protein sequence encoded by the PbrMYB65 gene is shown in SEQ ID No. 2.

Using 'Yali' fruit DNA as a template, the promoter sequences of PbrACO2 gene (PbrACO2pro (full length), PbrACO2pro frag1 and PbrACO2pro ^frag2 ) containing different numbers of MYB binding sites (P1: (-800)-(-795); P2: (-203)-(- ¹⁹⁸ )) were cloned (primer sequences are shown in Table 3 in the Appendix), inserted into the pGreenII0800-LUC vector, and reporter vectors (pGreenII 0800-PbrACO2pro-LUC, pGreenII 0800-PbrACO2pro ^frag1 -LUC, pGreenII 0800-PbrACO2pro ^frag2 -LUC) were constructed. On this basis, mutation primers were designed (primer sequences are shown in Table 3 in the appendix), and the MYB binding site in the PbrACO2 gene promoter was mutated (CAACCG/CGGTTG→CTTCCG/CGGAAG) to obtain the mutant promoter sequence PbrACO2pro ^mut , which was inserted into the pGreenII 0800-LUC vector to construct the reporter vector pGreenII 0800-PbrACO2pro ^mut -LUC.

The transcription factor recombinant plasmid (pSAK277-PbrMYB65) and the reporter vector (pGreenII0800-PbrACO2pro-LUC or pGreenII 0800-PbrACO2pro ^frag1 -LUC or pGreenII 0800-PbrACO2pro ^frag2 -LUC or pGreenII 0800-PbrACO2pro ^mut -LUC) were transformed into Agrobacterium GV3101 and GV3101 (psoup) respectively. The positive strains were selected for expansion culture and collected when OD ₆₀₀ was 0.6-0.8, and then the bacterial liquid was resuspended in infection solution (10mMMgCl ₂ , 10mM MES, 200μM acetosyringone, pH=5.7), and inducing for 3-5h under dark conditions and then infecting tobacco leaves. Before injection, the Agrobacterium containing pSAK277-PbrMYB65 and the reporter vector were mixed at a ratio of 9:1 (v/v), and the tobacco leaves co-transfected with the Agrobacterium carrying the pSAK277 empty vector and the corresponding reporter vector were used as controls. 60-72 hours after infection, the leaves were collected and the activities of firefly luciferase and Renilla luciferase were detected using the Dual-Luciferase Reporter Assay System kit (Promega, USA) (see the instructions for specific steps). The test results showed that the Luc/Ren ratio of tobacco leaves co-transformed with pSAK277-PbrMYB65&pGreenII 0800-PbrACO2pro-LUC (or pSAK277-PbrMYB65&pGreenII 0800-PbrACO2pro ^frag1 -LUC) was significantly higher than that of the control group, while there was no significant difference in the Luc/Ren ratio of tobacco leaves co-transformed with pSAK277-PbrMYB65&pGreenII 0800-PbrACO2pro ^frag2 -LUC (or pSAK277-PbrMYB65&pGreenII 0800-PbrACO2pro ^mut -LUC) and its control group (see Figure 8a).

3. Yeast one-hybrid assay (Y1H)

Using the fruit cDNA of 'Yali' as a template, the coding sequence of the PbrMYB65 gene was cloned (primer sequences are shown in Table 3 in the attached table) and inserted into pGADT7 to form the prey vector PbrMYB65-AD. Using the fruit DNA of 'Yali' as a template, according to the distribution of MYB binding sites (P1: (-800)-(-795); P2: (-203)-(-198)), a 200-bp promoter fragment of the PbrACO2 gene containing the MYB binding site or its mutant (CAACCG/CGGTTG→CTTCCG/CGGAAG) was inserted into the pAbAi vector (primer sequences are shown in Table 3 in the attached table) to form bait vectors (PbrACO2pro ^S1 -pAbAi, PbrACO2pro ^S2 -pAbAi, PbrACO2pro ^S1mut -pAbAi, and PbrACO2pro ^S2mut -pAbAi).

The bait vector and prey vector were co-transformed into yeast strains, and the Y1H experiment was performed using the Matchmaker Gold Yeast One-Hybrid Library Screening System (Weidi, Shanghai, China) (see the instructions for specific steps). The self-activation test of PbrACO2pro ^S1/S2 /PbrACO2pro ^S1mut/S2mut -pAbAi was performed on SD/-Ura deficiency medium supplemented with aureobasidin A (AbA), and the appropriate AbA concentration was selected for subsequent experiments. Then, the yeast strains co-transformed with the bait vector and the prey vector were cultured on SD/-Leu deficiency medium supplemented with an appropriate concentration to detect the binding ability of PbrMYB65 to the PbrACO2 promoter. The yeast strain co-transformed with pGAD7-p53 and p53-AbAi was used as a positive control, and the yeast strain co-transformed with pGADT7-AD and PbrACO2pro ^S1/S2 (or PbrACO2pro ^S1mut/S2mut ) was used as a negative control. The Matchmaker Gold Yeast One-Hybrid Library Screening System yeast single hybrid library screening system detected that PbrMYB65 could bind to the promoter fragment containing the MYB binding site (PbrACO2pro ^S1 -pAbAi and PbrACO2pro ^S2 -pAbAi), but after the mutation site (PbrACO2pro ^S1mut -pAbAi and PbrACO2pro ^S2mut -pAbAi), PbrMYB65 could not bind to it (see Figure 8b).

4. ChIP-qPCR Experiment

10 g of callus containing pCAMBIA1300-PbrMYB65-GFP was weighed, crushed, and cross-linked for 15-30 min in 1% (v/v) formaldehyde cross-linking buffer. Glycine was added to terminate the cross-linking, and the cross-linked product was ground with liquid nitrogen and cell lysis to obtain chromatin, which was further interrupted by ultrasound to obtain soluble sheared chromatin (average DNA length 200-500 bp). A portion of the soluble sheared chromatin was used as input, and the remaining portion was used for immunoprecipitation (IP) with GFP antibody after washing and pre-cleaning. Pear callus carrying pCAMBIA1300-GFP was used as a control. Specific fluorescent quantitative primers were designed in the PbrACO2 promoter region containing the MYB binding site (P1: (-800)-(-795); P2: (-203)-(-198)) (primers are shown in Table 3 in the Appendix), and the enrichment multiple was detected by qPCR. It was found that PbrMYB65 can bind to the promoter fragment containing the MYB binding site (see Figure 8c).

5. EMSA Experiment

Using the fruit cDNA of ‘Yali’ as a template, the full length of PbrMYB65 CDS was amplified (primer sequences are shown in Table 3 in the attached table) and inserted into the pCold-TF expression vector. The correctly sequenced pCold-PbrMYB65 plasmid was transformed into Escherichia coli BL21 (DE3) to obtain the recombinant protein His-PbrMYB65 with a His tag, and the pCold-TF empty protein was expressed as a control. The crude protein was purified and used for subsequent experiments. According to the sequence information of the MYB binding site in the promoter of the PbrACO2 gene (P1: (-800)-(-795); P2: (-203)-(-198)), a DNA probe of about 30-40 bp was synthesized, including a biotin-labeled probe carrying an unmutated (or mutated) MYB binding site and an unlabeled competitive probe carrying a MYB binding site. The DNA probe was synthesized by Suzhou Jinweizhi Biotechnology Co., Ltd. (probe sequences are shown in Table 3 in the attached table). EMSA experiments were performed according to the manual provided by Chemiluminescent EMSA Kit (Biyuntian, China). The experimental results showed that PbrMYB65 activated the expression of PbrACO2 gene by binding to the MYB binding sites (P1: (-800)-(-795); P2: (-203)-(-198)) in the upstream promoter of the gene (see Figure 8d).

Experimental results: PbrMYB65 can bind to the MYB binding sites in the promoter of PbrACO2 gene (P1: (-800)-(-795); P2: (-203)-(-198)) to promote its transcription. The sequence of the P1 binding site is (+/-)CAACCG/CGGTTG; the sequence of the P2 binding site is (+/-)CGGTTG/CAACCG.

Example 4 Functional verification of PbrMYB65

1Determination of subcellular localization of protein encoded by PbrMYB65 gene

The coding sequence of the PbrMYB65 gene was amplified using the ‘Yali’ fruit cDNA as a template (primer sequences are shown in Table 3 in the Appendix), inserted into the pBI221-GTP vector to construct the recombinant plasmid pBI221-PbrMYB65-GFP, and co-transformed with the nuclear marker AtH2B-mcherry into Arabidopsis protoplasts. Fluorescence signals were observed using a confocal microscope (Leica Microsystems, Germany).

Obtaining callus from pear overexpressing PbrMYB65 gene

Using the fruit cDNA of 'Yali' as a template, the coding sequence of the PbrMYB65 gene was amplified and inserted into the pCAMBIA1300 vector to construct the pCAMBIA1300-PbrbMYB65 overexpression vector, which was used to infect pear callus (induced by small fruits of P. communis cv.'Clapp'sFavorite'). The infected callus was transferred to MS medium (carbon source: 30 g·L ^-1 sucrose) containing hygromycin (Hyg) and cultured for 1.5-2 months. After PCR identification, the callus was expanded. The positive tissue and the control (transformed empty pCAMBIA1300) were co-transferred to MS medium (containing Hyg) with sorbitol/sucrose (15 g·L ^-1 , 1:1) as the carbon source for growth.

3 Analysis of PbrMYB65 and PbrACO2 gene expression in callus

Total RNA from callus was extracted using a plant total RNA extraction kit (RE-05011, Chengdu Fuji Biotechnology Co., Ltd.), and reverse transcribed to synthesize cDNA. Real-time qRT-PCR was used to detect the expression of PbrMYB65 and PbrACO2 genes. The primer sequence of PbrACO2 gene is shown in Table 2, and the primer sequence of PbrMYB65 gene is shown in Table 3 in the attached table. Pear Tublin gene (PbrTub) was used as the housekeeping gene.

4 Determination of mitACO enzyme activity in callus

Mitochondria were purified from pear callus, and then the mitochondrial ACO (mitACO) enzyme activity was determined using an ACO test kit (ACO-1-Z, Suzhou Keming Biotechnology Co., Ltd., Suzhou, China). The protein concentration in the crude enzyme solution was determined using a BCA protein concentration assay kit (A045-4, Nanjing Jiancheng Bioengineering Institute, Nanjing, China).

5 Determination of citric acid content in callus tissue

Weigh 0.5g tissue, grind it thoroughly in liquid nitrogen, transfer it to a 10mL glass graduated tube, add 5mL ultrapure water solution, place it in a water bath at 80℃ for 30min, and then extract it by ultrasonic for 15min; transfer the extract to a 2mL centrifuge tube, centrifuge it at 4℃, 12000rpm for 20min, collect the supernatant; take 1.5mL of the supernatant, filter it with a 0.45μm Sep-Pak water system microporous filter membrane, and obtain the organic acid extract. The citric acid content was determined by liquid chromatography.

Experimental results: PbrMYB65 is located in the nucleus (Figure 9). Overexpression of the PbrbMYB65 gene in pear callus can increase the expression of the PbrACO2 gene and the activity of the mitACO enzyme, and inhibit the accumulation of citric acid (Figure 10).

In summary, the present invention is based on a transcription factor PbrMYB65, which can combine with the MYB binding sites (P1: (-800)-(-795); P2: (-203)-(-198)) in the promoter of the PbrACO2 gene to regulate its transcription level (as shown in Figure 11), affecting the citric acid accumulation and flavor quality of pear fruit, and has great market application potential for breeding high/low citric acid pear varieties.

The above-mentioned specific implementation methods of the present invention do not constitute a limitation on the protection scope of the present invention. It should be pointed out that without departing from the technical principle of the present invention, any improvement, equivalent replacement, etc. made by those skilled in the art should be included in the protection scope of the claims of the present invention.

Claims (3)

1. Use of PbrACO gene or biological material related to PbrACO gene in promoting isomerization of citric acid in pear fruit or inhibiting accumulation of citric acid in pear fruit, wherein the nucleotide sequence of PbrACO gene is shown in SEQ ID NO. 3;

The biological material related to PbrACO genes is at least one of (1) - (8):

(1) PbrACO2 protein with an amino acid sequence shown as SEQ ID NO. 4;

(2) An expression cassette comprising the PbrACO gene;

(3) A recombinant vector containing the PbrACO gene;

(4) A recombinant vector comprising the expression cassette of (2);

(5) A recombinant microorganism containing the PbrACO gene;

(6) A recombinant microorganism comprising the expression cassette of (2);

(7) A recombinant microorganism comprising the recombinant vector of (3);

(8) A recombinant microorganism comprising the recombinant vector of (4);

overexpression of the PbrACO gene inhibits citric acid accumulation in fruits or promotes citric acid isomerization.

Use of MYB family transcription factor PbrMYB or a biological material related to MYB family transcription factor PbrMYB in promoting citric acid isomerization in pear fruit or inhibiting citric acid accumulation in pear fruit, wherein the amino acid sequence of transcription factor PbrMYB65 is shown in SEQ ID No. 2;

the biological material related to the transcription factor PbrMYB is at least one of the following (1) - (8):

(1) A gene PbrMYB with a nucleotide sequence shown as SEQ ID No.1 and encoding the transcription factor PbrMYB;

(2) An expression cassette comprising the gene PbrMYB of (1);

(3) A recombinant vector comprising the gene PbrMYB of (1);

(4) A recombinant vector comprising the expression cassette of (2);

(5) A recombinant microorganism comprising the gene PbrMYB of (1);

(6) A recombinant microorganism comprising the expression cassette of (2);

(7) A recombinant microorganism comprising the recombinant vector of (3);

(8) A recombinant microorganism comprising the recombinant vector of (4);

interaction of MYB family transcription factor PbrMYB with PbrACO gene as defined in claim 1 regulates citric acid isomerization or accumulation of citric acid in pear fruit; the MYB family transcription factor PbrMYB promotes the expression of PbrACO genes by combining with MYB binding sites in a PbrACO gene promoter, improves the activity of mitochondrial ACO enzymes, and further inhibits the accumulation of citric acid in fruits.

3. A method for inhibiting the accumulation of citric acid in pear fruits, characterized in that the overexpression of the gene PbrMYB or/and PbrACO2 encoding the transcription factor PbrMYB65 inhibits the accumulation of citric acid in fruits; the nucleotide sequence of the gene PbrMYB65 for encoding the transcription factor PbrMYB is shown as SEQ ID No. 1; the nucleotide sequence of PbrACO gene is shown in SEQ ID No. 3.