Application of transcription factors PbrNAC34a and PbrMYB3 in regulation and control of citric acid accumulation in pear fruits

Abstract

The invention discloses application of transcription factors PbrNAC34a and PbrMYB3 in regulation and control of accumulation of citric acid in pear fruits. The invention discloses that PbrNAC34a in pear fruits regulates expression of transcription factors PbrMYB3 and PbrMYB65, further regulates expression of citric acid metabolism key genes PbrACO2, promotes citric acid isomerization and reduces accumulation of citric acid in the fruits for the first time, and theoretical and practical basis is provided for directional improvement of pear fruit quality characters.

Description

Technical Field

[0001] The present invention belongs to the technical field of molecular biology and genetic engineering, and specifically relates to the application of transcription factor PbrNAC34a or / and PbrMYB3 in regulating the accumulation of citric acid in pear fruit. The present invention has discovered a metabolic pathway PbrNAC34a-PbrMYB3 / 65-PbrACO2 that can regulate the accumulation of citric acid in pear fruit. In the metabolic pathway, the transcription factor PbrNAC34a promotes the expression of the key gene PbrACO2 for citric acid metabolism by regulating the expression of transcription factors PbrMYB3 and PbrMYB65, thereby promoting the isomerization of citric acid and reducing its accumulation in the fruit. Background Art

[0002] As an important economic temperate fruit native to southwest China, pears are widely cultivated around the world and are loved by consumers for their unique sensory properties. In addition to sugar, malic acid and citric acid are the main organic acids in pears, which contribute significantly to the flavor formation of pears.

[0003] The biosynthesis of citrate in horticultural fruits begins with the β-carboxylation of phosphoenolpyruvate (PEPC), which is converted to oxaloacetate (OAA) by cytoplasmic phosphoenolpyruvate carboxylase (PEPC). Subsequently, mitochondrial citrate synthase (CS), the first enzyme in the tricarboxylic acid (TCA) cycle, catalyzes the addition of an acetyl group from acetyl-CoA to OAA to produce citrate. However, analysis of CS and PEPC enzyme activities and related gene expression levels in low / high citrate cultivars and during fruit development revealed no significant correlation with citrate content. Therefore, citrate content in horticultural fruits may be regulated by downstream steps of citrate biosynthesis.

[0004] Since the transcriptional activation or repression of structural genes is regulated by transcription factors (TFs) by binding to the corresponding cis-acting elements in their promoters, the quality formation of horticultural fruits is also controlled by some TFs. Recently, some TFs involved in regulating citric acid metabolism have been identified from horticultural fruits such as apple and kiwifruit, but the molecular mechanism of citric acid metabolism during pear fruit development has not been fully elucidated.

[0005] In the last authorized patent (patent number: 202311391230.0), we revealed that PbrMYB65 can interact with the MYB binding sites in the promoter of the PbrACO2 gene (S1: (-800)-(-795); S2: (-203)-(-198)) to regulate the citric acid isomerization of pear fruit, but its upstream regulatory mechanism has not been reported. At the same time, the expression level of the homologous gene PbrMYB3 of the PbrMYB65 gene found in previous studies is also highly correlated with the expression level of the ACO2 gene ( Figure 1 ), but its regulatory mechanism still needs further study. Summary of the invention

[0006] The purpose of the present invention is to provide an application of NAC family transcription factor PbrNAC34a or a biological material related to the transcription factor PbrNAC34a in regulating citric acid isomerization or citric acid accumulation in the fruit of Rosaceae fruit trees. Studies have shown that PbrNAC34a can bind to the cis-acting element in the promoter of PbrMYB3 or PbrMYB65 genes to induce their expression.

[0007] Another object of the present invention is to provide a MYB family transcription factor PbrMYB3 or a biological material related to the MYB family transcription factor PbrMYB3 for use in regulating citric acid isomerization or citric acid accumulation in the fruit of a Rosaceae fruit tree. Studies have shown that PbrMYB3 can bind to the MYB binding site in the promoter of the PbrACO2 gene, activate its expression, and thus inhibit the accumulation of citric acid in pears.

[0008] Another object of the present invention is to provide a method for inhibiting the accumulation of citric acid in the flesh of Rosaceae fruit trees. By promoting the binding of PbrNAC34a in the nucleus to the promoter of PbrMYB3 gene (binding element: CTTCGTTT) or PbrMYB65 gene (binding element: AGAAAGAA), their transcription is triggered. After translation in the ribosome, the MYB transcription factor PbrMYB3 is transported to the nucleus, interacts with the corresponding MYB binding site (MYBCORE box motif, CAACCG) in the promoter of the PbrACO2 gene, activates its expression, and after translation and transfer to the mitochondria, PbrACO2 isomerizes citric acid to isocitric acid, inhibiting the accumulation of citric acid in pear fruit.

[0009] The objective of the present invention is achieved through the following technical solutions:

[0010] In the first aspect, the present invention claims the use of NAC family transcription factor PbrNAC34a or biological materials related to transcription factor PbrNAC34a in regulating citric acid isomerization or regulating citric acid accumulation in the fruits of Rosaceae fruit trees, wherein the amino acid sequence of the transcription factor PbrNAC34a is shown in SEQ ID NO.3.

[0011] Furthermore, the biological material related to the transcription factor PbrNAC34a is at least one of (a1) to (a7):

[0012] (a1) gene PbrNAC34a encoding the transcription factor PbrNAC34a;

[0013] (a2) an expression cassette containing the gene PbrNAC34a described in (a1);

[0014] (a3) a recombinant vector containing the gene PbrNAC34a described in (a1), or a recombinant vector containing the expression cassette described in (a2);

[0015] (a4) a recombinant microorganism containing the gene PbrNAC34a described in (a1), or a recombinant microorganism containing the expression cassette described in (a2), or a recombinant microorganism containing the recombinant vector described in (a3);

[0016] (a5) a transgenic plant cell line containing the gene PbrNAC34a described in (a1), or a transgenic plant cell line containing the expression cassette described in (a2), or a transgenic plant cell line containing the recombinant vector described in (a3);

[0017] (a6) transgenic plant tissue containing the gene PbrNAC34a described in (a1), or transgenic plant tissue containing the expression cassette described in (a2), or transgenic plant tissue containing the recombinant vector described in (a3);

[0018] (a7) A transgenic plant organ containing the gene PbrNAC34a described in (a1), or a transgenic plant organ containing the expression cassette described in (a2), or a transgenic plant organ containing the recombinant vector described in (a3).

[0019] Furthermore, the nucleotide sequence of the gene PbrNAC34a encoding the transcription factor PbrNAC34a in (a1) is shown as SEQ ID No.4.

[0020] Furthermore, the above application is to overexpress the gene PbrNAC34a encoding the transcription factor PbrNAC34a or increase the protein level or activity of the transcription factor PbrNAC34a, promote the isomerization of citric acid in the fruit, or inhibit the accumulation of citric acid in the fruit. Furthermore, the transcription factor PbrNAC34a activates the expression of the gene PbrMYB3 or / and the gene PbrMYB65 and its downstream gene PbrACO2 by binding to the promoter of the gene PbrMYB3 or / and the gene PbrMYB65, thereby inhibiting the accumulation of citric acid in the fruit; the promoter nucleotide sequence of the gene PbrMYB3 is shown in SEQ ID NO.5, and the promoter nucleotide sequence of the gene PbrMYB65 is shown in SEQ ID NO.6.

[0021] In a specific embodiment of the present invention, it was found that the nuclear transcription factor PbrNAC34a binds to the promoter of the PbrMYB3 gene (binding element: CTTCGTTT) or the PbrMYB65 gene (binding element: AGAAAGAA) to promote their transcription. After translation in the ribosome, the transcription factors PbrMYB3 and PbrMYB65 are transported to the nucleus and further interact with the MYB binding site (MYBCORE box motif, CAACCG) in the promoter of the PbrACO2 gene to activate its expression. Subsequently, PbrACO2 is transported to the mitochondria, isomerizes citric acid to isocitric acid, and inhibits the accumulation of citric acid in pear fruit.

[0022] In the second aspect, the present invention claims protection for the use of MYB family transcription factor PbrMYB3 or biological materials related to MYB family transcription factor PbrMYB3 in regulating citric acid isomerization in the fruits of Rosaceae fruit trees or regulating citric acid accumulation in the fruits of Rosaceae fruit trees, and the amino acid sequence of the transcription factor PbrMYB3 is shown in SEQ ID NO.1.

[0023] Furthermore, the biological material related to the transcription factor PbrMYB3 is at least one of (b1) to (b7):

[0024] (b1) gene PbrMYB3 encoding the transcription factor PbrMYB3;

[0025] (b2) an expression cassette containing the gene PbrMYB3 described in (b1);

[0026] (b3) a recombinant vector containing the gene PbrMYB3 described in (b1), or a recombinant vector containing the expression cassette described in (b2);

[0027] (b4) a recombinant microorganism containing the gene PbrMYB3 described in (b1), or a recombinant microorganism containing the expression cassette described in (b2), or a recombinant microorganism containing the recombinant vector described in (b3);

[0028] (b5) a transgenic plant cell line containing the gene PbrMYB3 described in (b1), or a transgenic plant cell line containing the expression cassette described in (b2), or a transgenic plant cell line containing the recombinant vector described in (b3);

[0029] (b6) transgenic plant tissue containing the gene PbrMYB3 described in (b1), or transgenic plant tissue containing the expression cassette described in (b2), or transgenic plant tissue containing the recombinant vector described in (b3);

[0030] (b7) A transgenic plant organ containing the gene PbrMYB3 described in (b1), or a transgenic plant organ containing the expression cassette described in (b2), or a transgenic plant organ containing the recombinant vector described in (b3).

[0031] Furthermore, the nucleotide sequence of the gene PbrMYB3 encoding the transcription factor PbrMYB3 in (b1) is shown as SEQ ID No.2.

[0032] Furthermore, the above application is that overexpressing the gene PbrMYB3 encoding the transcription factor PbrMYB3 or increasing the protein level or activity of the transcription factor PbrMYB3 can increase the expression of the gene PbrACO2 and increase the activity of the mitochondrial ACO enzyme, thereby inhibiting the accumulation of citric acid in the fruit. Studies have shown that the transcription factor PbrMYB3 can bind to the promoter of PbrACO2, activate the expression of PbrACO2, increase the activity of the mitochondrial ACO (mitACO) enzyme, and thus inhibit the accumulation of citric acid in the fruit.

[0033] In a third aspect, the present invention claims a method for inhibiting the accumulation of citric acid in the fruit of a Rosaceae fruit tree, the method being at least one of the following (c1) and (c2):

[0034] (c1) overexpressing the gene PbrNAC34a encoding the transcription factor PbrNAC34a of claim 1 or increasing the protein level or activity of the transcription factor PbrNAC34a to inhibit the accumulation of citric acid in the fruit;

[0035] (c2) Overexpressing the gene PbrMYB3 encoding the transcription factor PbrMYB3 or increasing the protein level or activity of the transcription factor PbrMYB3 to inhibit the accumulation of citric acid in the fruit.

[0036] Studies have shown that the transcription factor PbrNAC34a can bind to the promoter of gene PbrMYB3 and / or gene PbrMYB65, activate the expression of gene PbrMYB3 and / or gene PbrMYB65 and its downstream gene PbrACO2, thereby inhibiting the accumulation of citric acid in the fruit.

[0037] The Rosaceae fruit tree described in the present invention is pear.

[0038] Research process of the technical solution of the present invention:

[0039] By analyzing the differential transcription factor expression profiles in the peel and flesh of 'Yali' fruit at different growth and development stages and their correlation with the expression level of the PbrACO2 gene, it was found that PbrMYB3 may be involved in regulating the accumulation of citric acid during the growth and development of pear fruit ( Figure 1 ).

[0040] The recombinant plasmid pSAK277-PbrMYB3 was constructed using the fruit DNA of 'Yali' as a template, and the reporter vectors (pGreenII0800-PbrACO2pro-LUC (full-length promoter), p ... frag1 -LUC, pGreenII0800-PbrACO2pro frag2 -LUC, pGreenII0800-PbrACO2pro mut -LUC), and were transformed into Agrobacterium GV3101 and GV3101(psoup) according to the instructions. The LUC / REN ratio in tobacco leaves co-transformed with PbrMYB3 and a reporter gene containing the PbrACO2 gene promoter increased by about 2.2 times ( Figure 2 a).

[0041] In the Y1H experiment, the positive control (pGADT7-p53&p53-pAbAi) and the negative control (pGADT7&PbrACO2pro S1 -pAbAi,pGADT7&PbrACO2pro S2 -pAbAi), bait-prey co-transformant (pGADT7-PbrMYB3&PbrACO2pro S1 -pAbAi,pGADT7-PbrMYB3&PbrACO2pro S2 -pAbAi) and co-transformants containing mutant elements (pGADT7 & PbrACO2pro S1mut-pAbAi,pGADT7&PbrACO2pro S2mut -pAbAi,pGADT7-PbrMYB3&PbrACO2pro S1mut -pAbAi,pGADT7-PbrMYB3&PbrACO2pro S2mut -pAbAi) grew normally on SD / -Leu medium; when AbA was added, the growth of the negative control and transformants containing the mutant binding element was inhibited, while there was no effect on the positive control and the bait-prey co-transformants ( Figure 2 b). ChIP-qPCR studies showed that PbrMYB3 was enriched in the promoter fragment of the PbrACO2 gene containing the MYB binding site ( Figure 2 c).

[0042] The pCAMBIA1300-PbrMYB3 overexpression vector was constructed and transferred into Agrobacterium GV3101 to transform pear callus. After obtaining positive tissues through PCR identification, the positive transgenic tissues and their controls (pCAMBIA1300 empty-transformed callus) were transferred to MS medium with sorbitol and sucrose (1:1, w:w) as carbon sources for growth. The results of qPT-PCR detection technology and mitACO enzyme activity detection kit showed that the mRNA abundance of the PbrMYB3 gene in pear transgenic tissues was positively correlated with the expression level of the PbrACO2 gene and / or the mitACO activity, but negatively correlated with the citric acid content ( Figure 3 ).

[0043] To identify the upstream regulators of PbrMYB3 and PbrMYB65 transcription factors, we performed correlation analysis with 48 other differentially expressed transcription factors. The gene expression levels of 18 members during fruit growth and development showed strong positive correlations (absolute correlation coefficient > 0.6) with PbrMYB3, PbrMYB65, and PbrACO2 mRNA abundance ( Figure 4 Among them, Pbr026635.1 (i.e., PbrNAC34a) showed a relatively high correlation ( Figure 4 ). Further, with the help of the PlantRegMap database and previous reports on common motifs of NAC binding sites, several potential PbrNAC34a binding sites were identified from the promoters of PbrMYB3 and PbrMYB65 ( Figure 5 ).

[0044] Using the fruit cDNA of 'Yali' as a template, the coding sequence of the PbrNAC34a gene was cloned and inserted into the pSAK277 vector to construct the pSAK277-PbrNAC34a recombinant vector. Furthermore, the promoter sequences of the PbrMYB3 and PbrMYB65 genes containing different numbers of PbrNAC34a binding sites were cloned (wherein the PbrMYB3 promoter fragment included PbrMYB3pro (full length), PbrMYB3pro frag1 , PbrMYB3pro frag2 and PbrMYB3pro frag3 ; PbrMYB65 promoter fragments include PbrMYB65pro (full length), PbrMYB65pro frag1 , PbrMYB65pro frag2 , PbrMYB65pro frag3 and PbrMYB65pro frag4 ) (primer sequences are shown in Table 1 in the attached table), inserted into the pGreenII 0800-LUC vector, and constructed a reporter vector (pGreenII0800-PbrMYB3pro-LUC, pGreenII0800-PbrMYB3pro frag1 -LUC, pGreenII0800-PbrMYB3pro frag2 -LUC, pGreenII0800-PbrMYB3pro frag3 -LUC, pGreenII0800-PbrMYB65pro-LUC, pGreenII0800-PbrMYB65pro frag1 -LUC, pGreenII0800-PbrMYB65pro frag2 -LUC, pGreenII0800-PbrMYB65pro frag3 -LUC, pGreenII0800-PbrMYB65pro frag4 -LUC). On this basis, mutation primers were designed (primer sequences are shown in Table 1 in the attached table) to mutate the PbrNAC34a binding site in the promoters of PbrMYB3 and PbrMYB65 genes (CTTCGTTT in the PbrMYB3 promoter was mutated to TTTTTTTT, and PbrMYB3pro mut ; AGAAAGAA in the PbrMYB65 promoter was mutated to CCCCCCCC, PbrMYB65pro mut ), and obtained the mutant promoter sequence PbrMYB3pro mut and PbrMYB65pro mut, inserted into pGreenII0800-LUC vector, and constructed reporter vector pGreenII0800-PbrMYB3pro mut -LUC and pGreenII0800-PbrMYB65pro mut -LUC. The test results showed that the fragments containing the binding element 'CTTCGTTT' (S1 site in the PbrMYB3 promoter: (-1694)-(-1687)) or the binding element 'AGAAAGAA' (S4 site in the PbrMYB65 promoter: (-110)-(-103)) can interact with PbrNAC34a to activate the expression of downstream genes ( Figure 6 a).

[0045] According to the results of the dual luciferase assay, in the Y1H assay, the positive control (pGADT7-p53&p53-pAbAi) and the negative control (pGADT7&PbrMYB3pro S1 -pAbAi,pGADT7&PbrMYB65pro S4 -pAbAi), bait-prey co-transformant (pGADT7-PbrNAC34a&PbrMYB3pro S1 -pAbAi,pGADT7-PbrNAC34a&PbrMYB65pro S4 -pAbAi) and co-transformants containing mutant elements (pGADT7&PbrMYB3pro S1mut -pAbAi,pGADT7&PbrMYB65pro S4mut -pAbAi,pGADT7-PbrNAC34a&PbrMYB3pro S1mut -pAbAi,pGADT7-PbrNAC34a&PbrMYB65pro S4mut -pAbAi) grew normally on SD / -Leu medium; when AbA was added, the growth of the negative control and transformants containing the mutant binding element was inhibited, while there was no effect on the positive control and the bait-prey co-transformants ( Figure 6 b). ChIP-qPCR studies showed that PbrNAC34a was enriched in promoter fragments containing the binding element 'CTTCGTTT' (S1 site in the PbrMYB3 promoter: (-1694)-(-1687)) or the binding element 'AGAAAGAA' (S4 site in the PbrMYB65 promoter: (-110)-(-103)). Figure 6 c).

[0046] The pCAMBIA1300-PbrNAC34a overexpression vector was constructed and transferred into Agrobacterium GV3101 to transform pear callus. After obtaining positive tissues through PCR identification, the positive transgenic tissues and their controls (pCAMBIA1300 empty-loaded callus) were transferred to MS medium with sorbitol and sucrose (1:1, w:w) as carbon sources for growth. Using qPT-PCR detection technology and ultra-performance liquid chromatography (UPLC) determination, it was found that the expression levels of PbrNAC34a, PbrMYB3, PbrMYB65 and PbrACO2 genes in transgenic callus tissues were significantly increased, but the citric acid content was significantly decreased ( Figure 7 ).

[0047] Compared with the prior art, the present invention has the following beneficial effects:

[0048] (1) Revealed the transcription factor PbrMYB3 that regulates the expression of pear PbrACO2 gene.

[0049] (2) For the first time, we revealed that PbrNAC34a is an upstream regulator of PbrMYB3 and PbrMYB65.

[0050] (3) The present invention clarifies the mechanism of action of the PbrNAC34a-PbrMYB3 / 65-PbrACO2 metabolic pathway 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

[0051] Figure 1 Characterization of PbrMYB3 and PbrMYB65 as possible upstream regulators of PbrACO2 gene involved in citric acid metabolism.

[0052] Among them, (a) the number of differentially expressed transcription factors during the development of ‘Yali’ fruit. Differentially expressed transcription factors whose expression levels in the pericarp tissue were consistently higher or lower than those in the cortical tissue of developing ‘Yali’ fruit were identified by DESeq2_EBSeq software according to the following criteria: fold change ≥ 2.0 and FRD < 0.01. (b) Expression profiles of differentially expressed transcription factors in developing P. bretschneideri Rehd. fruits and their correlation with PbrACO2 mRNA abundance. The color scale represents the normalized log2-transformation (FPKM+1), where red, blue, and white represent high, low, and medium expression levels, respectively. The Spearman correlations between different attributes are visualized in the heat map, where red (or light red) lines represent extremely strong (or strong) positive correlations, while green (or light green) lines represent extremely strong (or strong) negative correlations. PbrMYB3 (Pbr003370.1) is highlighted in the red box, and PbrMYB65 (Pbr000749.2) is highlighted in the blue box. (c) Details of two PbrMYB3 / 65 binding sites (MYBCORE box motif, CAACCG) in the PbrACO2 promoter. The binding sites (pink ovals) were predicted by the Plant Regulatory Atlas Database (Tian et al., 2020). ‘Yali’ pear fruits were bagged with three layers of paper bags 34 days after anthesis, while unbagged ‘Yali’ and ‘Dangshansuli’ fruits were also labeled. Pericarp and cortical tissue samples were collected at six developmental stages, including 15, 34, 81, 110, 145, and 160 days after anthesis. Data are from transcriptome analysis and represent the average of three biological replicates, except for the gene expression profile during ‘Dangshansuli’ fruit development (one replicate).

[0053] Figure 2 Analysis of the role of PbrMYB3 in regulating the expression of PbrACO2 gene in citric acid metabolism in pear.

[0054] Among them, (a) dual luciferase assay. PbrMYB3 ORF was introduced into pSAK277 vector, while PbrACO2 promoter fragments of different lengths, containing different numbers of possible PbrMYB3 binding sites (MYBCORE box motif, CAACCG; PbrACO2pro, PbrACO2pro frag1 and PbrACO2pro frag2 ) or mutated binding sites (CAACCG→CTTCCG, PbrACO2pro mut) and introduced into pGreen 0800-LUC vector. Transformants containing empty pSAK277 and each reporter gene were used as controls. (b) Y1H analysis of the interaction between PbrMYB3 and PbrACO2 promoter fragments. The open reading frame (ORF) of PbrMYB3 was amplified into the prey vector pGADT7, while the wild-type (MYBCORE box motif, CAACCG; PbrACO2pro S1 and PbrACO2pro S2 ) or mutated possible PbrMYB3 binding sites (CAACCG→CTTCCG, PbrACO2pro S1mut and PbrACO2pro S2mut ) was inserted into the bait vector pAbAi. Yeast cells co-transformed with pGADT7-p53 and p53-AbAi were used as positive controls, and yeast cells co-transformed with the empty pGADT7 vector and each bait were used as negative controls. The PbrMYB3 binding site in the PbrACO2 promoter was predicted by the Plant Regulatory Atlas Database (Tian et al., 2020). (c) PbrMYB3 activates PbrACO2 expression by binding to its promoter. Figure 3 ChIP-qPCR analysis. Callus overexpressing empty vector served as negative control.

[0055] Among them, (a) Subcellular localization of PbrMYB3. (b) Functional verification of PbrMYB3 in pear callus. (bi) Growth status of pear callus. (b-ii) PbrACO2 expression level and mitACO activity. (b-iii) Chromatogram of samples. (b-iv) PbrMYB3 expression level and citric acid content. Callus transformed with an empty vector was used as a control; the expression levels of PbrACO2 and PbrMYB3 in the control callus were set to 1.0 for RT-qPCR assays. The data represent the mean of three biological replicates, and vertical bars marked with the same letter indicate that there are no significant differences between samples (p<0.05).

[0056] Figure 4 Characterization of PbrNAC34a as an upstream regulator of PbrMYB3 and PbrMYB65.

[0057] Correlation between PbrMYB3 (or PbrMYB65) and 48 other differentially expressed transcription factors during fruit development in P. bretschneideri Rehd. PbrNAC34a (Pbr026635.1) is marked with a red box, and PbrWRKY72 (Pbr009294.1) is marked with a blue box.

[0058] Figure 5 Detailed information of possible PbrNAC34a-binding sites in the promoters of PbrMYB3 and PbrMYB65.

[0059] Among them, (a) PbrMYB3 promoter. (b) PbrMYB65 promoter. The possible PbrNAC34a binding sites in the promoters of PbrMYB3 (pink ellipse) and PbrMYB65 (blue ellipse) were characterized by the Plant Regulatory Atlas Database (Tian et al., 2020) and previous literature reports (Bi et al., 2023; Li et al., 2023).

[0060] Figure 6 Characterization of cis-acting elements involved in PbrNAC34a-induced expression of PbrMYB3 and PbrMYB65.

[0061] Among them, (a) using promoter fragments of different lengths, the activation effect of PbrNAC34a on the transcription of PbrMYB3 (ai) or PbrMYB65 (a-ii) was detected by dual luciferase reporter gene assay. The open reading frame (ORF) of PbrNAC34a was introduced into the pSAK277 vector, and PbrMYB3 and PbrMYB65 promoter fragments of different lengths, containing different numbers of possible PbrNAC34a binding sites (PbrMYB3 promoter fragments including PbrMYB3pro, PbrMYB3pro frag1 ,PbrMYB3pro frag2 , and PbrMYB3pro frag3 ; PbrMYB65 promoter fragments include PbrMYB65pro, PbrMYB65pro frag1 ,PbrMYB65pro frag2 ,PbrMYB65pro frag3 , and PbrMYB65pro frag4 ) or mutated binding sites (CTTCGTTT→TTTTTTTT in PbrMYB3 promoter, PbrMYB3pro mut; AGAAAGAA→CCCCCCCC in the PbrMYB65 promoter, PbrMYB65promut), was introduced into the pGreen 0800-LUC vector. An empty pSAK277 vector and transformants of each reporter gene were used as controls. (b) Y1H experiment of the interaction of PbrNAC34a with the PbrMYB3 (bi) or PbrMYB65 (b-ii) promoter fragments. The open reading frame of PbrNAC34a was amplified into the prey vector pGADT7, while the approximately 200-bp PbrMYB3 and PbrMYB65 promoter fragments, containing the wild type (CTTCGTTT in the PbrMYB3 promoter, PbrMYB3promut) S1 ;AGAAAGAA in PbrMYB65 promoter, PbrMYB65pro S4 ) or mutated PbrNAC34a binding site (CTTCGTTT→TTTTTTTT in the PbrMYB3 promoter fragment, PbrMYB3pro S1mut ;AGAAAGAA→CCCCCCCC in the PbrMYB65 promoter fragment, PbrMYB65pro S4mut ), which were inserted into the bait vector pAbAi. Yeast cells co-transformed with pGADT7-p53 and p53-AbAi served as positive controls, while yeast cells co-transformed with the empty pGADT7 vector and each bait served as negative controls. (c) ChIP-qPCR analysis of the interaction between PbrNAC34a and the promoter fragments of PbrMYB3 (ci) or PbrMYB65 (c-ii). Callus overexpressing the empty vector served as a negative control. Data represent the mean of three biological replicates, and vertical bars labeled with the same letter are not significantly different between samples (p < 0.05).

[0062] Figure 7 Functional validation of PbrNAC34a as an upstream regulator of PbrMYB3 and PbrMYB65.

[0063] Among them, (a) Subcellular localization of PbrNAC34a. AtH2B-mcherry was used as a nuclear marker (Liu et al., 2007). Bars, 10 μm. (b) Functional validation of PbrNAC34a in pear callus. (bi) Growth status of pear callus. (b-ii) Gene expression level. (b-iii) Citric acid content. Callus transformed with empty vector was used as control; the expression level of each gene in control callus was set as 1.0 for RT-qPCR assay. Data represent the mean of three biological replicates, and there is no significant difference between samples marked with the same letter on the vertical bars (p < 0.05). Figure 8 The mechanism of action of PbrNAC34a-PbrMYB3 / 65-PbrACO2 metabolic pathway in regulating citric acid isomerization in pear fruit. DETAILED DESCRIPTION

[0064] 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.

[0065] Example 1 Screening of transcription factor PbrMYB3 and regulation of PbrACO2 gene

[0066] 1. Collection of Tissue Samples

[0067] The experimental materials of this experiment were 'Yali' and 'Dangshan Pear', which were planted in an orchard in Gaoyou, Yangzhou, and were basically the same in age and vigor. 'Yali' was bagged 34 days after the full flowering period, and the unbagged fruits in the same position were used as the control; each treatment was repeated 3 times, and each repeat had 200 fruits. The peel and flesh tissues of the bagged and unbagged 'Yali' and the flesh of the unbagged 'Dangshan Pear' were collected and tested 15 days (S1), 34 days (S2), 81 days (S3), 110 days (S4), 145 days (S5) and 160 days (S6) after the full flowering period.

[0068] 2. Transcriptome Sequencing

[0069] 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 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 align with the 'Dangshan Pear' genome database (http: / / peargenome.njau.edu.cn / ) and calculate gene expression levels (FPKM). Transcriptome sequencing and analysis were completed by Biomarker Biotechnology Co., Ltd. (Beijing, China).

[0070] 3. Screening of transcription factor PbrMYB3

[0071] Previous studies have found (patent number: 202311391230.0) that the citric acid content in the pulp is significantly higher than that in the peel. Therefore, the number of differentially expressed transcription factors between the (un) bagged peel and pulp tissues of 'Yali' during the growth and development of 'Yali' was first analyzed. On this basis, the correlation between the expression level of the PbrACO2 gene and the expression level of differentially expressed genes during the growth and development of 'Yali' and 'Dangshan Pear' fruits was further analyzed to screen out the key transcription factors that regulate the expression of the PbrACO2 gene. The study found that during the growth and development of 'Yali', a total of 50 transcription factors were differentially expressed in the peel and pulp tissues; among them, there was a significant positive correlation between the expression levels of PbrMYB3 / 65 and PbrACO2 (correlation coefficient>0.8)( Figure 1 ). Since we have already studied PbrMYB65 in the previous patent, this patent focuses on PbrMYB3.

[0072] With the help of PlantRegMap database, two potential PbrMYB3 binding sites ((S1: (-800)-(-795); S2: (-203)-(-198))) were identified from the promoter of PbrACO2 gene (2000-bp sequence upstream of transcription start site (ATG)), indicating that PbrMYB3 may be an upstream regulatory factor of PbrACO2 gene ( Figure 1 c).

[0073] Example 2 Regulation of the PbrACO2 gene by the transcription factor PbrMYB3

[0074] 1. Dual luciferase reporter assay

[0075] Using the fruit cDNA of 'Yali' as a template, the coding sequence of the PbrMYB3 gene was cloned (primer sequences are shown in Table 1 in the attached table), inserted into the pSAK277 vector, and the pSAK277-PbrMYB3 recombinant vector was constructed. The protein sequence encoded by the PbrMYB3 gene is shown in SEQ ID NO.1; the nucleotide sequence of the PbrMYB3 gene is shown in SEQ ID NO.2.

[0076] Using the fruit DNA of 'Yali' as template, the promoter sequences of PbrACO2 gene (PbrACO2pro (full length), PbrACO2pro (full length), PbrACO2pro (full length) and PbrACO2pro (full length)) containing different numbers of MYB binding sites (P1: (-800)-(-795); P2: (-203)-(-198)) were cloned. frag1 and PbrACO2pro frag2) (primer sequences are shown in Table 1 in the attached table), inserted into the pGreenII 0800-LUC vector, and constructed a reporter vector (pGreenII0800-PbrACO2pro-LUC, pGreenII0800-PbrACO2pro frag1 -LUC, pGreenII0800-PbrACO2pro frag2 -LUC). On this basis, mutant primers were designed (primer sequences are shown in Table 3 in the attached table) to mutate the MYB binding site in the PbrACO2 gene promoter (CAACCG / CGGTTG→CTTCCG / CGGAAG) to obtain the mutant promoter sequence PbrACO2pro mut , insert into pGreenII0800-LUC vector, construct reporter vector pGreenII0800-PbrACO2pro mut -LUC.

[0077] The transcription factor recombinant plasmid (pSAK277-PbrMYB3) and the reporter vector (pGreenII0800-PbrACO2pro-LUC or pGreenII0800-PbrACO2pro frag1 -LUC or pGreenII0800-PbrACO2pro frag2 -LUC or pGreenII0800-PbrACO2pro mut -LUC) were used to transform Agrobacterium GV3101 and GV3101(psoup) respectively. The positive strains were selected and expanded to OD600 of 0.6-0.8, and then the bacteria were collected, and then the bacteria were resuspended in infection solution (10mM MgCl2, 10mM MES, 200μM acetosyringone, pH=5.7). After induction for 3-5h under dark conditions, tobacco leaves were infected. Before injection, the Agrobacterium bacteria containing pSAK277-PbrMYB3 and the reporter vector were mixed at a ratio of 9:1 (v / v), and the tobacco leaves co-transfected with the Agrobacterium bacteria carrying the pSAK277 empty vector and the corresponding reporter vector were used as controls. 60-72h 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 results showed that pSAK277-PbrMYB3&pGreenII0800-PbrACO2pro-LUC (or pSAK277-PbrMYB3&pGreenII0800-PbrACO2pro frag1-LUC) co-transformed tobacco leaves had a significantly higher Luc / Ren ratio than the control group, while pSAK277-PbrMYB3&pGreenII0800-PbrACO2pro frag2 -LUC(or pSAK277-PbrMYB3&pGreenII0800-PbrACO2pro mut There was no significant difference in the Luc / Ren ratio of tobacco leaves co-transformed with -LUC and its control group ( Figure 2 a).

[0078] 2. Yeastone-hybridassay (Y1H)

[0079] Using the fruit cDNA of 'Yali' as a template, the coding sequence of the PbrMYB3 gene was cloned (primer sequences are shown in Table 1 in the attached table) and inserted into pGADT7 to form the prey vector PbrMYB3-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 1 in the attached table) to form the bait vector (PbrACO2pro S1 -pAbAi, PbrACO2pro S2 -pAbAi, PbrACO2pro S1mut -pAbAi and PbrACO2pro S2mut -pAbAi).

[0080] The bait vector and prey vector were co-transfected into yeast strains, and the Y1H experiment was performed using the Matchmaker Gold Yeast One-Hybrid Library Screening System (Weidi, Shanghai, China) (see the instruction manual for specific steps). S1 / S2 / PbrACO2pro S1mut / S2mut -pAbAi self-activation detection, and select the appropriate AbA concentration for subsequent experiments. Then, the yeast strain co-transformed with the bait vector and the prey vector was cultured on the SD / -Leu deficiency medium supplemented with an appropriate concentration to detect the binding ability of PbrMYB3 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 PbrACO2proS1 / S2 (or PbrACO2pro S1mut / S2mut ) was used as a negative control. The Matchmaker Gold Yeast One-Hybrid Library Screening System was used to detect that PbrMYB3 could bind to a promoter fragment containing a MYB binding site (PbrACO2pro S1 -pAbAi and PbrACO2pro S2 -pAbAi) binding, but after mutation site (PbrACO2pro S1mut -pAbAi and PbrACO2pro S2mut -pAbAi) loses binding ability ( Figure 2 b).

[0081] 3. ChIP-qPCR Experiment

[0082] 10 g of callus containing pCAMBIA1300-PbrMYB3-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 promoter region of PbrACO2 containing the MYB binding site (S1: (-800)-(-795); S2: (-203)-(-198)) (primers are shown in Table 1 in the attached table). The enrichment multiple was detected by qPCR, and it was found that PbrMYB3 could bind to the promoter fragment containing the MYB binding site ( Figure 2 c).

[0083] Example 3 Functional verification of PbrMYB3 gene

[0084] 1. Subcellular localization of the protein encoded by the PbrMYB3 gene

[0085] The coding sequence of PbrMYB3 gene was amplified using the fruit cDNA of 'Yali' as a template (primer sequence is shown in Table 1 in the attached table), inserted into the pBI221-GTP vector to construct the recombinant plasmid pBI221-PbrMYB3-GFP, and co-transformed with the nuclear marker AtH2B-mcherry into Arabidopsis protoplasts. The fluorescence signal was observed using a confocal microscope (Leica Microsystems, Germany).

[0086] 2. Obtaining Pear Callus Overexpressing PbrMYB3

[0087] Using the fruit cDNA of 'Yali' as a template, the coding sequence of the PbrMYB3 gene was amplified and inserted into the pCAMBIA1300 vector to construct the pCAMBIA1300-PbrbMYB3 overexpression vector (primers are shown in the primer pairs in Table 1 Gene Function Validation invivo in the Appendix), and the pear callus (induced by P. communis cv. 'Clapp's Favorite fruitlets) was infected. The infected callus was transferred to MS medium containing hygromycin (Hyg) (carbon source: 30 g.L -1 The positive tissues and the control (transformed empty pCAMBIA1300) were transferred to a sorbitol / sucrose (15 g.L) medium. -1 , 1:1) as the carbon source on MS medium (containing Hyg).

[0088] 3. Analysis of PbrMYB3 and PbrACO2 gene expression in callus

[0089] 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 PbrMYB3 and PbrACO2 genes. The primer sequences of PbrACO2 and PbrMYB3 genes are shown in Table 1 in the attached table. Pear Tublin gene (PbrTub) was used as the housekeeping gene.

[0090] 4. Determination of mitACO enzyme activity in callus

[0091] Mitochondria were purified from pear callus, and then the mitochondrial ACO (mitACO) enzyme activity was determined using an ACO detection 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).

[0092] 5. Determination of citric acid content in callus

[0093] 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.

[0094] Experimental results: PbrMYB3 is located in the nucleus ( Figure 3 a). Overexpression of the PbrbMYB3 gene in pear callus increased the expression of the PbrACO2 gene and the activity of the mitACO enzyme, inhibiting the accumulation of citric acid ( Figure 3 b).

[0095] In summary, the present invention is based on a transcription factor PbrMYB3, which can combine with two MYB binding sites (S1: (-800)-(-795); S2: (-203)-(-198)) in the promoter of the PbrACO2 gene to regulate its transcription level and inhibit the accumulation of pear citric acid.

[0096] Example 4 Screening and regulation of PbrMYB3 upstream regulatory factors

[0097] 1. Screening of transcription factor PbrNAC34a

[0098] To identify upstream regulators of these two MYB transcription factors, we performed correlation analysis between PbrMYB3 / 65 and 48 other differentially expressed transcription factors. The study found that the gene expression levels of 18 members during fruit growth and development showed strong positive correlations (absolute correlation coefficient > 0.6) with PbrMYB3, PbrMYB65, and PbrACO2 mRNA abundance ( Figure 4 Among them, Pbr026635.1 (i.e., PbrNAC34a) showed a relatively high correlation ( Figure 4 ). Further, with the help of the PlantRegMap database and previous reports on common motifs of NAC binding sites, several potential PbrNAC34a binding sites were identified from the promoters of PbrMYB3 and PbrMYB65 ( Figure 5 ), which suggests that PbrNAC34a may be their upstream regulator.

[0099] 2. Dual luciferase reporter assay

[0100] Using the fruit cDNA of 'Yali' as a template, the coding sequence of the PbrNAC34a gene was cloned (primer sequences are shown in Table 1 in the attached table), inserted into the pSAK277 vector, and the pSAK277-PbrNAC34a recombinant vector was constructed. The protein sequence encoded by the PbrNAC34a gene is shown in SEQ ID NO.3; the CDS sequence of the PbrNAC34a gene is shown in SEQ ID NO.4.

[0101] Using 'Yali' fruit DNA as template, clones containing different numbers of PbrNAC34a binding sites ( Figure 5 ) of PbrMYB3 and PbrMYB65 gene promoter sequences (wherein the PbrMYB3 promoter fragment includes PbrMYB3pro (full length), PbrMYB3pro frag1 , PbrMYB3pro frag2 and PbrMYB3pro frag3 ; PbrMYB65 promoter fragments include PbrMYB65pro (full length); PbrMYB65pro frag1 , PbrMYB65pro frag2 , PbrMYB65pro frag3 and PbrMYB65pro frag4 ) (primer sequences are shown in Table 1 in the attached table), inserted into the pGreenII 0800-LUC vector, and constructed a reporter vector (pGreenII0800-PbrMYB3pro-LUC, pGreenII0800-PbrMYB3pro frag1 -LUC, pGreenII0800-PbrMYB3pro frag2 -LUC, pGreenII0800-PbrMYB3pro frag3 -LUC, pGreenII0800-PbrMYB65pro frag1 -LUC, pGreenII0800-PbrMYB65pro frag2 -LUC, pGreenII0800-PbrMYB65pro frag3 -LUC, pGreenII0800-PbrMYB65pro frag4 -LUC). On this basis, mutation primers were designed (primer sequences are shown in Table 1 in the attached table) to mutate the PbrNAC34a binding site in the promoters of PbrMYB3 and PbrMYB65 genes (CTTCGTTT in the PbrMYB3 promoter was mutated to TTTTTTTT, and PbrMYB3pro mut; AGAAAGAA in the PbrMYB65 promoter was mutated to CCCCCCCC, PbrMYB65pro mut ), and obtained the mutant promoter sequence PbrMYB3pro mut and PbrMYB65pro mut , inserted into pGreenII0800-LUC vector, and constructed reporter vector pGreenII0800-PbrMYB3pro mut -LUC and pGreenII0800-PbrMYB65pro mut -LUC.

[0102] The transcription factor recombinant plasmid (pSAK277-PbrNAC34a) and reporter vector (pGreenII0800-PbrMYB3pro-LUC, pGreenII0800-PbrMYB3pro frag1 -LUC, pGreenII0800-PbrMYB3pro frag2 -LUC, pGreenII0800-PbrMYB3pro frag3 -LUC, pGreenII0800-PbrMYB65pro frag1 -LUC, pGreenII0800-PbrMYB65pro frag2 -LUC, pGreenII0800-PbrMYB65pro frag3 -LUC, pGreenII0800-PbrMYB65pro frag4 -LUC, pGreenII0800-PbrMYB3pro mut -LUC and pGreenII0800-PbrMYB65pro mut -LUC) were transformed into Agrobacterium GV3101 and GV3101 (psoup) respectively. The positive strains were selected and expanded to OD 600When the pH value was 0.6-0.8, the bacteria were collected, and then the bacterial solution was resuspended with infection solution (10mMMgCl2, 10mM MES, 200μM acetosyringone, pH=5.7). After induction for 3-5h under dark conditions, tobacco leaves were infected. Before injection, the Agrobacterium bacterial solution containing pSAK277-PbrNAC34a and the reporter vector were mixed at a ratio of 9:1 (v / v), and the tobacco leaves co-transfected with the Agrobacterium bacterial solution carrying the pSAK277 empty vector and the corresponding reporter vector were used as controls. 60-72h 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 fragments containing the binding element 'CTTCGTTT' (S1 site in the PbrMYB3 promoter: (-1694)-(-1687)) or the binding element 'AGAAAGAA' (S4 site in the PbrMYB65 promoter: (-110)-(-103)) can interact with PbrNAC34a to activate the expression of downstream genes ( Figure 6 a).

[0103] 3. Yeastone-hybridassay (Y1H)

[0104] Using the fruit cDNA of 'Yali' as a template, the coding sequence of the PbrNAC34a gene was cloned (primer sequences are shown in Table 1 in the attached table) and inserted into pGADT7 to form the prey vector PbrNAC34a-AD. Using the fruit DNA of 'Yali' as a template, according to the results of the dual luciferase reporter experiment (PbrMYB3 binding site (S1: ((-1694)-(-1687)) or PbrMYB65 binding site (S4: (-110)-(-103))), a 200-bp promoter containing the corresponding binding site or its mutant (PbrMYB3 promoter: CTTCGTTT→TTTTTTTT, PbrMYB3pro S1mut ; PbrMYB65 promoter AGAAAGAA→CCCCCCCC, PbrMYB65pro S4mut ) was inserted into the pAbAi vector (primer sequences are shown in Table 1 in the attached table) to form a bait vector (PbrMYB3pro S1 -pAbAi, PbrMYB65pro S4 -pAbAi, PbrMYB3pro S1mut -pAbAi and PbrMYB65pro S4mut -pAbAi).

[0105] The bait vector and prey vector were co-transfected into yeast strains, and the Y1H experiment was performed using the Matchmaker Gold Yeast One-Hybrid Library Screening System (Weidi, Shanghai, China) (see the instruction manual for specific steps). S1 -pAbAi, PbrMYB65pro S4 -pAbAi, PbrMYB3pro S1mut -pAbAi and PbrMYB65pro S4mut -pAbAi self-activation detection, and select the appropriate AbA concentration for subsequent experiments. Then, the yeast strain co-transformed with the bait vector and the prey vector was cultured on the SD / -Leu deficiency medium supplemented with an appropriate concentration to detect the binding ability of PbrMYB3 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 PbrMYB3pro S1 -pAbAi (or PbrMYB65pro S4 、PbrMYB3pro S1mut or PbrMYB65pro S4mut ) was used as a negative control. Matchmaker Gold Yeast One-Hybrid Library Screening System detected that PbrNAC34a could bind to fragments containing the binding element 'CTTCGTTT' (S1 site in PbrMYB3 promoter: (-1694)-(-1687)) or the binding element 'AGAAAGAA' (S4 site in PbrMYB65 promoter: (-110)-(-103)), but the mutant site (PbrMYB3pro S1mut -pAbAi and PbrMYB65pro S4mut -pAbAi) loses binding ability ( Figure 6 b).

[0106] 4. ChIP-qPCR Experiment

[0107] 10 g of callus containing pCAMBIA1300-PbrNAC34a-GFP or was weighed and crushed, and then 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 promoter region containing the binding element 'CTTCGTTT' (S1 site in the PbrMYB3 promoter: (-1694)-(-1687)) or the binding element 'AGAAAGAA' (S4 site in the PbrMYB65 promoter: (-110)-(-103)) (primers are shown in Table 3 in the attached table). The enrichment multiple was detected by qPCR, and it was found that PbrNAC34a could bind to the promoter fragment containing the PbrMYB3 or PbrMYB65 binding site ( Figure 6 c).

[0108] In summary, the present invention is based on a transcription factor PbrNAC34a, which can bind to the cis-acting element in the PbrMYB3 promoter (S1: (-1694)-(-1687), 'CTTCGTTT') or the cis-acting element in the PbrMYB65 promoter (S4: (-110)-(-103), 'AGAAAGAA') to initiate the expression of downstream genes.

[0109] Example 5 Gene Function Verification of PbrNAC34a

[0110] 1. Subcellular localization of the protein encoded by the PbrNAC34a gene

[0111] The coding sequence of PbrNAC34a gene was amplified using the fruit cDNA of 'Yali' as a template (primer sequences are shown in Table 1 in the attached table), inserted into the pBI221-GTP vector to construct the recombinant plasmid pBI221-PbrNAC34a-GFP, and co-transformed with the nuclear marker AtH2B-mcherry into Arabidopsis protoplasts. The fluorescence signal was observed using a confocal microscope (Leica Microsystems, Germany).

[0112] 2. Obtaining Pear Callus Overexpressing PbrNAC34a Gene

[0113] Using the fruit cDNA of 'Yali' as a template, the coding sequence of the PbrNAC34a gene was amplified and inserted into the pCAMBIA1300 vector to construct the pCAMBIA1300-PbrNAC34a overexpression vector (primers are shown in the primer pairs in Table 1 Gene Function Validation in vivo in the Appendix), and the pear callus (induced from the small fruits of P. communis cv. 'Clapp's Favorite') was infected. The infected callus was transferred to MS medium containing hygromycin (Hyg) (carbon source: 30 g.L -1 The positive tissues and the control (transformed empty pCAMBIA1300) were transferred to a sorbitol / sucrose (15 g.L) medium. -1 , 1:1) as the carbon source on MS medium (containing Hyg).

[0114] 3. Analysis of the expression levels of PbrNAC34a, PbrMYB3, PbrMYB65 and PbrACO2 genes in callus tissue

[0115] 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 PbrNAC34a, PbrMYB3, PbrMYB65, and PbrACO2 genes. The primer sequences of PbrNAC34a, PbrMYB3, PbrMYB65, and PbrACO2 genes are shown in Table 1 in the attached table. Pear Tublin gene (PbrTub) was used as the housekeeping gene.

[0116] 4. Determination of citric acid content in callus

[0117] 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.

[0118] Experimental results: PbrNAC34a is located in the nucleus. Overexpression of the PbrNAC34a gene in pear callus can promote the expression of PbrMYB3 and PbrMYB65 and their downstream gene PbrACO2, thereby inhibiting the accumulation of citric acid in callus ( Figure 7 ).

[0119] In summary, the present invention regulates the accumulation of citric acid during the development of pear fruit based on a PbrNAC34a-PbrMYB3 / 65-PbrACO2 metabolic pathway, that is, PbrNAC34a promotes the isomerization of citric acid by promoting the expression of PbrMYB3 and PbrMYB65 and their downstream gene PbrACO2, thereby affecting the citric acid accumulation and flavor quality of pear fruit, and has great market application potential for cultivating high / low citric acid pear varieties.

[0120] Schedule

[0121] Table 1 Primers involved in the present invention

[0122]

[0123]

[0124]

[0125]

[0126] SEQ ID NO.1

[0127] PbrMYB3

[0128] Protein

[0129] Pyrusbretschneideri

[0130] MRNPPSSKAAAAAASAKMQTTITASSSSSKAAGVAGGTKTPCCAKVGLKRGPWTPEE

[0131] DELLANYIKKEGEGRWRTLPKRAGLLRCGKSCRLRWMNYLRPSVKRGQIAPDEEDLILR

[0132] LHRLLGNRWSLIAGRIPGRTDNEIKNYWNTHLSKKLINQGIDPRTHKPLNPDHHSAADD

[0133] ADLDNTNKSTAVASSSKANDRFSNPNPSPPSDRLVHKEGDPNNSRNGGNIAIDDHDQGT

[0134] IVHGYANMITSINNPDASSSATATGTSLRSNNSHGGVLLGGGGNEEDDDINCCADDVF

[0135] SSFLNSLINEDPHGQHQLQQVLQNGNVSAHAAAAGSENLPLTMTGASTTAPSTFGWE

[0136] SAVLMSSAFIHNDHQRVNDPTE

[0137] SEQ ID NO.2

[0138] PbrMYB3

[0139] CDS

[0140] Pyrusbretschneideri

[0141] ATGAGGAACCCATCGCCTTCGTCGAAAGCAGCAGCAGCAGCAGCAAGTGCTAAGAT

[0142] GCAAACGACGATAACAGCGTCGTCCTCGTCGAGCAAGGCGGCTGGGGTTGCTGGAG

[0143] GGACCAAGACGCCGTGTTGCGCAAAGGTGGGTTTGAAGAGAGGGCCGTGGACTCCCC

[0144] GAAGAGGACGAGCTGCTGGCAAATTACATCAAGAAAGAAGGGGAGGGACGGTGGC

[0145] GGACCCTTCCCAAGCGGGCTGGGTTGCTCCGATGCGGTAAGAGCTGCCGCCTCCGCT

[0146] GGATGAACTATCTCCGCCCTTCCGTCAAGCGCGGCCAGATCGCCCCCGATGAAGAA

[0147] GATCTCATCCTTCGCCTCCATCGCCTTCTGGGCAATCGGTGGTCTTTGATAGCTGGGA

[0148] GGATTCCAGGCCGTACGGACAATGAGATAAAGAACTACTGGAACACACCTGAGC

[0149] AAGAAGCTGATAAACCAAGGCATAGATCCCAGAACCCAAGCCTCTCAATCCAGA

[0150] TCATCACTCTGCTGCTGATGATGCTGACCTGGACAACACAAACAAATCAACTGCTGT

[0151] TGCTTCTTCTTCCAAAGCCAATGATCGGTTCTCAAACCCTAACCCTAGTCCCCCTTCT

[0152] GATCGTCTTGTCCATAAAGAAGGGGATCCAAATAACAGCCGTAATGGTGGAAACAT

[0153] CGCAATTGATGATCATGATCAGGGCACTATAGTCCATGGCTATGCAAATATGATCAC

[0154] GTCCATCAACAATCCCGATGCTTCTTCTTCGGCCACGGCAACGGGTACTTTGAGTTT

[0155] GAGGAGCAACAACAGCCACGGTGGAGTACTACTTGGGGGAGGAGGAAATGAAGAG

[0156] GACGACGACATCAACTGTTGTGCGGACGACGTCTTCTCTTCGTTTCTGAATTCGTTG

[0157] ATCAATGAGGATCCATTTCATGGACAACACCAATTGCAACAAGTACTGCAGAATGG

[0158] GAATGTGAGTGCACACGCAGCTGCTGCTGGTTCCGAGAACCTTCCTTTGATTACTAT

[0159] GACTGGTGCTAGTACTACGGCGCCATCAACATTTGGCTGGGAGTCTGCTGTGCTCAT

[0160] GTCTTCTGCTTTCATCCATAATGATCACCAAAGGGTTAATGATCCAACGGAGTAGSEQ ID NO.3

[0161] PbrNAC34a

[0162] Protein

[0163] Pyrusbretschneideri

[0164] MSHDDSNKADHDHEHEHDEYVDEHEHDMVMPGFRFHPTEEELVEFYLRRKVEGKRFN

[0165] VELITFLDLYRYDPWELPAMAAIGEKEWFFYVPRDRKYRNGDRPNRVTTSGYWKATGA

[0166] DRMIRSENFRSIGLKKTLVFYSGKAPKGIRTSWIMNEYRLPHHETERYQKGEISLCRVYK

[0167] RAGVEDHPSLPRCLPASRASTSRSSVLVAQSTTRSDNKKQHTNNINTVDSVIEKLQAFNE

[0168] GQSQQIHHDHEHQMDQKMNIETEGSSRNSDVTTVLRLSKHKAYPQGHHQHHNIESSTA

[0169] AALGQPGAPTHPVDQEEGMAAFSHHSANSKQSAANCSSVISSTGNSCAALFTSGSSSVLS

[0170] SNMNAIDDLHRLLNYQPVNVQQQAASNHHQHQQYVNHHLVQYYDHHHHHPQPSNLF

[0171] STNFPSAAAALSTIQPQQQQLPSNTLPTAFSDRLWDWNPISEPTRPDYNNLFKSEQ ID NO.4

[0172] PbrNAC34a

[0173] CDS

[0174] Pyrusbretschneideri

[0175] ATGAGCCACGACGACTCCAACAAGGCCGACCATGATCATGAGCATGAGCATGATGA

[0176] GTATGTTGATGAGCATGAGCACGACATGGTGATGCCGGGCTTTCGCTTCCACCCTAC

[0177] TGAGGAGGAACTTGTAGAGTTCTACCTTCGCCGTAAGGTTGAGGGCAAGCGCTTCA

[0178] ATGTTGAACTCATTACTTTTCTCGATCTTTATCGCTATGACCCTTGGGAACTTCCAGC

[0179] TATGGCAGCAATTGGGGAGAAGGAATGGTTCTTTTATGTGCCAAGAGACCGCAAGT

[0180] ACCGCAACGGCGATAGGCCAAACCGGGTAACTACTTCTGGGTATTGGAAGGCAACT

[0181] GGAGCCGACCGCATGATTCGAAGCGAAAACTTCCGCTCAATCGGCCTCAAGAAAAC

[0182] CCTAGTCTTTTACTCCGGGAAAGCTCCCAAAGGCATTCGAACTAGTTGGATCATGAA

[0183] CGAATATCGCTTGCCGCATCATGAAACTGAACGATATCAAAAGGGAGAAATATCAC

[0184] TTTGCCGAGTATACAAGAGAGCCGGAGTTGAAGACCACCCATCACTCCCTCGTTGTC

[0185] TCCCAGCATCCAGGGCATCCACATCTAGATCATCAGTACTAGTGGCTCAATCGACGA

[0186] CCAGGTCAGACAATAAAAAGCAGCACACCAATAATATCAACACAGTGGATAGTGTG

[0187] ATTGAAAAACTCCAAGCTTTTAATGAAGGCCAATCGCAGCAGATTCATCATGATCAT

[0188] GAACATCAGATGGATCAAAAGATGAACATCGAAACTGAAGGTAGCAGCAGAAATTC

[0189] TGATGTGACAACAGTTTTGCGGCTTTCCAAGCATAAAGCGTACCCTCAGGGCCACCA

[0190] CCAGCACCACAACATTGAGAGTAGTACCGCTGCCGCACTTGGACAACCGGGTGCCC

[0191] CAACTCATCCTGTGGATCAGGAAGAAGGGATGGCTGCTTTCTCGCACCACTCAGCAA

[0192] ACTCAAAGCAATCTGCTGCTAATTGCTCTTCGGTGATCTCGAGTACTGGCAATTCAT

[0193] GTGCAGCACTCTTCACGAGTGGTTCTTCTTCTGTTTTGTCTTCCAATATGAATGCGAT

[0194] TGATGATCTTCATAGACTTTTGAATTACCAACCTGTAAACGTACAGCAGCAGGCTGC

[0195] TTCCAATCATCACCAACATCAACAATATGTAAATCATCATCTAGTGCAATACTATGA

[0196] TCATCATCATCATCATCCCCAACCCAGCAACCTGTTCTCAACAAATTTCCCTTCAGCA

[0197] GCGGCAGCACTATCAACTATACAGCCACAGCAGCAGCAGCTCCCCTCCAACACACT

[0198] TCCGACTGCTTTCTCTGACCGCCTATGGGACTGGAATCCAATATCAGAGCCAACCCG

[0199] GCCGGACTACAACAATCTGTTCAAGTAA

[0200] SEQ ID NO.5

[0201] PbrMYB3

[0202] Promoter

[0203] Pyrusbretschneideri

[0204] TGTTATTTACCGATTCAATTGTGGATAAGACGTTAATTAAGGAGGTGGTACTAGTATTG

[0205] ATGGTGTGCCTCTTTGACTAGATTGATTCTGCTCTGAGCAAAGCAGAAATACAAGCAC

[0206] CTAAGATTAGAAACCTTGCCAACATGAATTTATACAGTAACGTTTGTAAATGAGTAAAT

[0207] GGTTTTCTTTCGGGATCACTCTTGGGTTGTGATTGCCAGAATATTTATATGGAAATTCA

[0208] ACAGTCGGTGGATTTATAAACAAGAGGGCGCATGCGTGTTAATCCGTGTGATCGATTA

[0209] GCAGCAATATAGTGTTCTAACAACTTACAATCCAAGTATTATCCTTCGTTTTGAGATGA

[0210] TTTTTTGTACTATAAATGGAACCCATCAGCTTTAAATTTCATCCGTGAAGGTAGAGAA

[0211] ACTTGCAATTGGATCCTCTCCTGAGCAGGAGATCAGGATCCTCTTGATCTACTAACAT

[0212] GGATCGTTAGATCCAACAGTTACAATTATTATAATTTTTAGAAGAATTTCCTGT

[0213] TTGTAATCGTTGAATCAAAATCCAACAACCCGTGTTAGTGAGTAAGGTGAATCCTAAT

[0214] CTCCTGCTCAGGAATTCCGAGAAACTTGATCTCAGACCCATCAATTTCTCAAAGTTTT

[0215] GCCGGGGAGTGGGAAGTAGCAAGTCTCAGTGTCTCTCGTCTCATTACTGGGGGTTGG

[0216] GATCAGGTGTGTATGCGGTGCATGATTTAATCGGTGAACAGAGAAAAATCCAGATCGTT

[0217] GATTATGCGAAGACTTGGGAGTGGCACTGGCTGGTAATCATTTTTTTAAAATTTTCT

[0218] TCTGAAAATAATATAGAAAATAAAAGAAAGGTGGCTGAGTGCTCTGCGGGTCCT

[0219] ACCTTTAACCATCCTCGCTGTCCTTTGCTGCTTCACTTCATGATTCAAACTCCCACACT

[0220] CCCACGCGCATTCAACTAAGGTTAGTCTAAAACTCTCCCACACGCGGTGTAATTGAAA

[0221] GAAATAGAGAACTGAAAGTATAAACAAAAGCAAAAGGTTGTATGTTTGTAAGT

[0222] AATTAAGATGATTAACTCATGCATTCAAGGTCTTTTGTCCAATAAGTGAATAATGAT

[0223] TAAAATCAATGACTGAAAGTCGTCATATAAGTGCGCAAGTCACTATTATTTTTTCGTAA

[0224] TTCAACTAATAAGAAAATTATACTATAGAAATTAGTAATTTTGAGATACATTAA

[0225] TGTAAGAACTCCAGTACCATGAAACAATAAAAGTAAAAACATTGTGAGTGCACTAAA

[0226] TTGAGATCAAATTTAAACCAAATTTTGTAAATAGTTGTTAATGATTTAATTATTACTC

[0227] AACTGTTGTTGATTAACATTCTTTTTTTCAAACCTATGCCGTCATGCAAGAGTGCAAC

[0228] ACATTTCCACTACTGTAGTAAAGGATCACTTGTTTGTTGATTAATATTCTTATTTTCTAT

[0229] TGGTGAAACATCATTTTATTTACAAATCTGTTTAAAAATTTGGTCCGGCTTAATATTATCA

[0230] TTTTTTAGTAAGGTCATCAAGCCGAGTCATTCATTCGATTCAACAAGAAATCACTCAG

[0231] CTACGCCCTCGACACGTGAGGCTGAATTAACCAAGCAAGCAGACAACGATATTTTT

[0232] GTTAGTGAGATAGTGGAGCCTAAGAATATCACTCAGCTACCCCCTCAATTAATATAGCA

[0233] CTAATAATGCGAGTGGTGTTAAAGCAATAGAGTGGTGCGTGAGAGTGTATATTCAGCT

[0234] GCCTATTAGAATATTATTACAACAAAACCCAGCACTCTGTGTCTCGGCTATCCATCTCT

[0235] CATCTCTCTCTCTCTATCTCTCATCCCCCATCCTCAATACCAAACAAATTAAAGATGCA

[0236] AAACTAAATCTACCTACACACACAGACACAGCATCTTAAAACCTCACAGAAGTAAGA

[0237] AAAACAAGATCAGGTTCTCAAACTATCAAAATCTCCATTTGTATTCGCATTCGATCCAT

[0238] TCCTACTCAG

[0239] SEQ ID NO.6

[0240] PbrMYB65

[0241] Promoter

[0242] Pyrusbretschneideri

[0243] TAAGACGTTAATTAAGAGGTAGACCTAGCTGTTTAGAAAACTATATGAAATCTGATC

[0244] GTTGTAAGATCCGTTCATTCCATATGGATTGCATTTGTTTTAAATTTGGCGAAGTATTAT

[0245] CGAATCCTGCATTTTATGTAATTGGACTCATTTTCTGCTTAACAATTTGATGTCTAATCT

[0246] TAGTTTGGTTTATGTTATCATTCCTGTATCCTAATGTAAGTCTAGTAATTACTTGGAGG

[0247] GATAATGATGATGTGCCCGTTTGATCTAGATTGATGCTGCTTTGCAAGAGGGAGTTAAT

[0248] ATATTGAAGGTCGTGCTTTTTAAAGGCTTCGCAGAAAGTGCGAGACGCTTCAATTCAG

[0249] AGCAGAAATACAAGCATCTAAGAGTAGAAACCTTGCCAACATGAATTTCACACTAG

[0250] CATCTGCAAATGAGTAAATGGGTTTTTGTTTTTTGGGATAACACTTGGGTCGTGATTGT

[0251] CAGAATATATGGAAACTCGACCGGTTGTTGAATCTAAAATCAAGAGGACCTACGGGC

[0252] GTTGCATTGTTGACAACCCATGTCATCGAGTAGGGTTCTAACAATCACGGCTCACGGA

[0253] TTATCTTTGTTTTGAGATCGTCTTTTGTGCTATAAATGTGACCCATCACCCATCAGCTTT

[0254] AAATTTCATCAATGAAGGTAAAGCGATAAACGACCTCAGACTCATCATTTCCCAAAGT

[0255] TTTGCCGGGGAGTAGGCAGTCGGAAGTCTCAATGTCTCATGACTGGGGGTTGGGATC

[0256] CGGTGGGCACCCAGTGCATGATTTTATTCATAAATGCCCATTGTATGCAGTTTTATTTGC

[0257] ATTGAAGTTGTTTTCACGATTCGAACGATCTTTCGACCATAAAAAAAATAATATTTTCGT

[0258] TTCATGAAGACTCGTCTTTCGGTCAACTCGTTGAATGAAGCGGAGACTGGGGAGTGG

[0259] CTGCCGCTGGTTGTAGTTGTATTTGTAAGGTTGTCTGAAAAATAATATAGAAAAGAAA

[0260] AGAAAATAATGTCTGAGTGCTCTGCGGGTCCCGTTCTGCGGCCACCCCGTGCTGTCATT

[0261] TCTGCTGCACTTTATGATTCAAACTCCGCCAGTCCCACGCGCATTCGAGTAAAGGTTA

[0262] GACTGATACTCTCCCATACGCAAAGCGTAACTGAAAGGAATATGAAAAAATAAAGTTA

[0263] AAACAGGGTAGAGGTTTGTAGGTTTCTAACTCTGAGTAGTTTAGACGATTAATTCATGC

[0264] ACTCGAGTTATATTGGAGGTTAAAAAATATAGAATTATATACATACCGTCTAATATTTTC

[0265] GTGGATATAGTGCTTCAAAGCGTCTTGGGTTTGAAACTTTATTTAAAAAAAAATTATTG

[0266] TAATATTTTCAAACCTTTCCACTTCTCTTAATAATAATAAATTAAAAAAAATACATTATTA

[0267] TAAAATAAATCAATAAAAATCAATGACTAAAAATAGTCATATAAATGCTTAAAAGCTAC

[0268] TAATAACTAAGAGATATACATCAAGTGTATCTAATTAATATAGATGCACATAGCCACAAT

[0269] GGGAGAACTCCATACTGAAATTTTAAGTAAGGAAAAACATTGTGCAATAAATTGATGC

[0270] AATTTTTTTTATAAGGCATCACTTAGTCGGATACATAAGGTCATTTAGTAAGAAATTATC

[0271] ACGTTGACACTAGTAAGACGGAATTAACCAGCTGTCCCTAAATTCCAGTAGAGAAAAT

[0272] ATAGCTCTGGTAAAGCAAGTGGTGTTAAAGCAATAGATAGGCGCGTGAGAATATATTC

[0273] AGCTGCCTATCACAATATTATTATAACAAATATCCAGCACTGTGTCTCGGCTCCCCATCT

[0274] TTTATCTCCCGATCTCATCTCCCATCCTCCTCAATACCAAAACTATATCCGCTTAAACAC

[0275] AAACAAAAAAAGAAAGAAAAACAAGATCAGGTTGTCAAGATATCAAAATCTCCATTT

[0276] GTCTTCGCATTCGATCCACTCCTACTCAGAAGAAGAAGAAAAACAACACTGAAAAA

[0277] GCTTTACA。

Claims

1. Application of NAC family transcription factor PbrNAC34a or biological materials related to transcription factor PbrNAC34a in regulating citric acid isomerization or citric acid accumulation in the fruits of Rosaceae fruit trees, wherein the amino acid sequence of the transcription factor PbrNAC34a is shown in SEQ ID NO.

3.

2. The use according to claim 1, characterized in that: The biological material related to the transcription factor PbrNAC34a is at least one of (a1) to (a7): (a1) gene PbrNAC34a encoding the transcription factor PbrNAC34a; (a2) an expression cassette containing the gene PbrNAC34a described in (a1); (a3) a recombinant vector containing the gene PbrNAC34a described in (a1), or a recombinant vector containing the expression cassette described in (a2); (a4) a recombinant microorganism containing the gene PbrNAC34a described in (a1), or a recombinant microorganism containing the expression cassette described in (a2), or a recombinant microorganism containing the recombinant vector described in (a3); (a5) a transgenic plant cell line containing the gene PbrNAC34a described in (a1), or a transgenic plant cell line containing the expression cassette described in (a2), or a transgenic plant cell line containing the recombinant vector described in (a3); (a6) transgenic plant tissue containing the gene PbrNAC34a described in (a1), or transgenic plant tissue containing the expression cassette described in (a2), or transgenic plant tissue containing the recombinant vector described in (a3); (a7) A transgenic plant organ containing the gene PbrNAC34a described in (a1), or a transgenic plant organ containing the expression cassette described in (a2), or a transgenic plant organ containing the recombinant vector described in (a3).

3. The use according to claim 2, characterized in that: The nucleotide sequence of the gene PbrNAC34a encoding the transcription factor PbrNAC34a in (a1) is shown in SEQ ID No.

4.

4. The use according to claim 1 or 2, characterized in that: Overexpression of the gene PbrNAC34a encoding the transcription factor PbrNAC34a or increasing the protein level or activity of the transcription factor PbrNAC34a promotes the isomerization of citric acid in the fruit or inhibits the accumulation of citric acid in the fruit.

5. The use according to claim 5, characterized in that: The transcription factor PbrNAC34a activates the expression of gene PbrMYB3 and / or gene PbrMYB65 and its downstream gene PbrACO2 by binding to the promoter of gene PbrMYB3 and / or gene PbrMYB65, thereby inhibiting the accumulation of citric acid in the fruit; the promoter nucleotide sequence of gene PbrMYB3 is shown in SEQ ID NO.5, and the promoter nucleotide sequence of gene PbrMYB65 is shown in SEQ ID NO.

6.

6. Application of MYB family transcription factor PbrMYB3 or biological materials related to MYB family transcription factor PbrMYB3 in regulating citric acid isomerization or citric acid accumulation in the fruits of Rosaceae fruit trees, wherein the amino acid sequence of the transcription factor PbrMYB3 is shown in SEQ ID NO.

1.

7. The use according to claim 6, characterized in that: The biological material related to the transcription factor PbrMYB3 is at least one of (b1) to (b7): (b1) gene PbrMYB3 encoding the transcription factor PbrMYB3; (b2) an expression cassette containing the gene PbrMYB3 described in (b1); (b3) a recombinant vector containing the gene PbrMYB3 described in (b1), or a recombinant vector containing the expression cassette described in (b2); (b4) a recombinant microorganism containing the gene PbrMYB3 described in (b1), or a recombinant microorganism containing the expression cassette described in (b2), or a recombinant microorganism containing the recombinant vector described in (b3); (b5) a transgenic plant cell line containing the gene PbrMYB3 described in (b1), or a transgenic plant cell line containing the expression cassette described in (b2), or a transgenic plant cell line containing the recombinant vector described in (b3); (b6) transgenic plant tissue containing the gene PbrMYB3 described in (b1), or transgenic plant tissue containing the expression cassette described in (b2), or transgenic plant tissue containing the recombinant vector described in (b3); (b7) A transgenic plant organ containing the gene PbrMYB3 described in (b1), or a transgenic plant organ containing the expression cassette described in (b2), or a transgenic plant organ containing the recombinant vector described in (b3).

8. The use according to claim 7, characterized in that: The nucleotide sequence of the gene PbrMYB3 encoding the transcription factor PbrMYB3 in (b1) is shown in SEQ ID No.

2.

9. The use according to claim 7, characterized in that: Overexpressing the gene PbrMYB3 encoding the transcription factor PbrMYB3 or increasing the protein level or activity of the transcription factor PbrMYB3 can increase the expression amount of the gene PbrACO2 and increase the activity of the mitochondrial ACO enzyme, thereby inhibiting the accumulation of citric acid in the fruit.

10. A method for inhibiting the accumulation of citric acid in the fruit of a Rosaceae fruit tree, characterized in that: The method is at least one of the following (c1) and (c2): (c1) overexpressing the gene PbrNAC34a encoding the transcription factor PbrNAC34a of claim 1 or increasing the protein level or activity of the transcription factor PbrNAC34a to inhibit the accumulation of citric acid in the fruit; (c2) Overexpressing the gene PbrMYB3 encoding the transcription factor PbrMYB3 of claim 6 or increasing the protein level or activity of the transcription factor PbrMYB3 to inhibit the accumulation of citric acid in the fruit.

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