CN110564738B - Application of sweet cherry PaPME2 gene in regulating and controlling ripening or softening of sweet cherry fruits - Google Patents

Abstract

The invention discloses the application of sweet cherry PaPME2 gene in regulating the ripening or softening of sweet cherry fruit. The present invention conducts functional research on the pectin methylesterase (PME) genes of 12 sweet cherry fruits and finds that the expression patterns of PaPME2 and PaPME9 genes are closely related to the process of ripening and softening of sweet cherry fruits. The present invention further utilizes VIGS technology to silence the PaPMEs gene of sweet cherry fruit respectively. The results show that the fruit that silences the PaPME2 gene of sweet cherry fruit has significant changes in fruit ripening and softening-related physiological indicators such as the content of firmness, soluble solids and soluble pectin. It is indicated that PaPME2 gene is a key gene affecting the ripening and softening of sweet cherry fruit, and it can be used to regulate the ripening or softening of sweet cherry fruit and cultivate new varieties of sweet cherry with firm flesh and storage and transportation resistance.

Description

Application of sweet cherry PaPME2 gene in regulating sweet cherry fruit ripening or softening

technical field

The invention relates to a new use of the PaPME2 gene of sweet cherry, in particular to the use of the sweet cherry PaPME2 gene in regulating the ripening or softening of sweet cherry fruit, and belongs to the field of new uses of the PaPME2 gene.

Background technique

European sweet cherry (Prunus avium L.), belonging to the Rosaceae Prunus, is the earliest fresh fruit to be marketed in spring in northern China and has high economic value. In addition, the fruit of sweet cherries has high nutritional value and health care effects, and is rich in organic acids, vitamins, amino acids and melatonin, etc., and is deeply loved by consumers. However, sweet cherries soften rapidly after ripening, which makes sweet cherries easy to soften and rot and deteriorate in quality after harvesting, which will cause a lot of losses during transportation and storage, which limits the development of the sweet cherry industry (Xuan Jiping, Wang Gang, Jia Zhanhui, Guo Zhongren. 2015. Research progress on fruit ripening and softening of Prunus genus. China Agronomic Bulletin, 31: 104-118; Brummell D A, Dal Cin V, Crisosto C H, LabavitchJ M. 2004. Cell wall metabolism during maturation,ripening and senescence of peach fruit. Journal of Experimental Botany, 55(405):2029-2039.). Therefore, studying the physiological and molecular mechanism of sweet cherry fruit ripening and softening, improving the storage and transportation resistance of sweet cherry and prolonging the shelf life of the fruit has always been one of the hot issues of research and discussion.

The softening of sweet cherry fruit is mainly caused by the change of cell wall structure and the degradation of cell wall components. The main components of the cell wall are pectin and cellulose (Zhao Yunfeng, Lin Yu, Lin Hetong. Research progress on the relationship between changes in cell wall components and fruit ripening and softening. Food Technology, 2012(12):29-33.). Mainly involved in the degradation of cell wall substances are some hydrolases, such as pectin methylesterase (PME), polygalacturonase (PG), cellulase (Cx) and β-galactoside. Enzyme (β-Galactosidase), etc. (Brummell D A, HarpsterM H. 2001. Cell wall in fruit softening and quality and itsmanipulation in transgenic plants. Plant Molecular Biology, 47: 311-340.; Xuan Jiping, Wang Gang, Jia Zhanhui, Guo Zhongren. 2015. Research progress on fruit ripening and softening of Prunus genus. China Agricultural Science Bulletin, 31: 104-118.; Brummell D A, Dal Cin V, Crisosto C H, Labavitch J M. 2004. Cell wallmetabolism during maturation,ripening and senescence of peach fruit. Journal of Experimental Botany, 55(405):2029-2039.).

Pectin methylesterase genes (PMEs) are a large family of genes widely present in plants, which are divided into TypeⅠand TypeⅡ. PME mainly catalyzes the conversion of pectin ester acid into pectic acid, provides hydrolysis substrate for PG, and cooperates with PG enzyme to soften the fruit (Cheng Jieshan, Shen Huolin, Sun Xiubo, Yang Xueyan, Zhang Mei, Lian Xuhai, Li Rose. The process of fruit ripening and softening Research progress on the role of major related enzymes in China. Northern Horticulture, 2008(01): 49-52.). The study found that in the process of fruit ripening and softening of horticultural crops such as banana, papaya, mango, strawberry, tomato, grape and peach, the enzyme activity of PME increased significantly, indicating that pectin methylesterase plays an important role in fruit ripening and softening (Zhong Manqian, Cong Xinli, Zhang Shiqing, Huang Mianjia. Room temperature preservation effect of papaya treated with exogenous chitosan coating. Chinese Journal of Tropical Biology, 2016, 7(02): 220-223; Ali Z M, Chin L H, Lazan H. 2004. A comparative study on wall degrading enzymes, pectin modifications and softening during ripening of selected tropical fruits. Plant Science, 167(2): 317-327; Muengkaew R, Whangchai K, Chaiprasart P. 2018. Application of calcium–boron improve fruit quality, cell characteristics , and effective softeningenzyme activity after harvest in mango fruit (Mangifera indica L.). Horticulture, Environment, and Biotechnology, 59(4):537-546. Harriman R W, Tieman D M, Handa A K. 1991. Molecular cloning of tomato pectin methylesterase gene and its expression in Rutgers, ripening inhibitor, nonripening, and never ripetomato fruits. Plant Physiology, 97(1): 80-87; Nunan K J, Davies C, Robinson S P, Fincher G B.2001. Expression patterns of cell wall- Modifying enzymes during grape berry development. Planta, 214(2): 257-264; Zhou Houcheng. Study on the genes related to strawberry fruit ripening and softening. Yangling: Northwest; Manganaris G.A, Vasilakakis M, Diam antidis G, Mignani I. 2006. Diverse metabolism of cell wall components of melting and non-meltingpeach genotypes during ripening after harvest or cold storage. Journal of the Science of Food and Agriculture, 86(2):243-250). Since pectin methylesterase is a multi-gene family, and different pectin methylesterase isozymes play different roles in fruit ripening and softening, it is necessary to systematically study the functions of the pectin methylesterase gene family to clarify the Its role in fruit ripening and softening. The ripening and softening of sweet cherry fruit is one of the main characteristics of fruit senescence, accompanied by the decline of fruit quality, such as reduced hardness and soft texture, which seriously affects the taste and its commercial value. Studies have shown that changes in the composition of the cell wall of sweet cherry fruit, especially the changes in pectin components and the interaction of related enzymes are the direct causes of fruit softening (Brummell, 2006; Pelloux et al., 2007; Matas et al., 2009). However, in drupe fruit trees such as sweet cherries, the relationship between PME gene and fruit ripening and softening at the transcriptional level has not been reported yet.

SUMMARY OF THE INVENTION

The main purpose of the present invention is to provide a new application of the sweet cherry PaPME2 gene in regulating the ripening or softening of sweet cherry fruit.

Above-mentioned purpose of the present invention is achieved through the following technical solutions:

The present invention firstly identifies 46 PaPMEs family genes from the sweet cherry genome database and conducts gene expression analysis, and finally confirms that the expression of 12 PaPMEs genes (namely: PaPME1-12 genes) has a certain relationship with fruit development.

The nucleotide sequence of PaPME1 gene is shown in SEQ ID No.1; the nucleotide sequence of PaPME2 gene is shown in SEQ ID No.2; the nucleotide sequence of PaPME3 gene is shown in SEQ ID No.3; The nucleotide sequence of the PaPME4 gene is shown in SEQ ID No.4; the nucleotide sequence of the PaPME5 gene is shown in SEQ ID No.5; the nucleotide sequence of the PaPME6 gene is shown in SEQ ID No.6; the PaPME7 gene The nucleotide sequence of the PaPME8 gene is shown in SEQ ID No.7; the nucleotide sequence of the PaPME8 gene is shown in SEQ ID No.8; the nucleotide sequence of the PaPME9 gene is shown in SEQ ID No.9; the nucleotide sequence of the PaPME10 gene is shown in The nucleotide sequence is shown in SEQ ID No. 10; the nucleotide sequence of the PaPME11 gene is shown in SEQ ID No. 11; the nucleotide sequence of the PaPME12 gene is shown in SEQ ID No. 12.

To further clarify the functions of these 12 PaPMEs genes in the ripening and softening of sweet cherry fruit. The present invention further analyzes the expression patterns of these 12 PaPMEs genes in the ripening and softening process of sweet cherry fruit by using real-time fluorescence quantitative PCR technology, and then uses VIGS technology of sweet cherry fruit to silence the expression of PpPMEs gene in sweet cherry fruit and analyze its Effect on the ripening and softening of sweet cherry fruit; the results showed that the expression of PaPME2 and PaPME9 genes was low in the early stage of fruit development, and the expression was significantly up-regulated in the later stage of fruit development, which continued until the fruit matured, and PaPME2 gene reached the maximum value at the end of fruit ripening and softening; The expression of PaPME9 gene was the highest in the early stage of fruit ripening and softening (42 days after flowering), and then the gene was down-regulated; while the other 10 PaPMEs genes (PaPME1, PaPME3, PaPME4, PaPME5, PaPME6, PaPME7, PaPME8, PaPME10, PaPME11, The expression level of PaPME12) in each stage of fruit development was low and not regular. Silencing the PaPME2 gene in sweet cherry fruit delayed the ripening and softening of sweet cherry fruit, while the fruit firmness, soluble solid content and soluble pectin content of PaPME2 gene-silencing sweet cherry fruit were significantly lower than those in the empty control; while the other PaPMEs in silencing sweet cherry fruit There were no significant changes in genes compared with the empty control. According to these experimental results, PaPME2 is involved in regulating the ripening and softening process of sweet cherry fruit, especially playing an important role in the process of fruit softening, and is a key protein factor leading to the softening of sweet cherry fruit.

Therefore, the present invention provides a method for promoting the ripening of sweet cherry fruit, comprising: constructing a plant expression vector containing the PaPME2 gene; expressing the constructed plant into the sweet cherry, allowing the PaPME2 gene to be overexpressed in the sweet cherry or overexpression.

The present invention also provides a method for cultivating a new variety of early-maturing sweet cherry, which is characterized by comprising: (1) constructing a recombinant plant expression vector containing the PaPME2 gene; (2) transforming the constructed recombinant plant expression vector into a In sweet cherry plant tissue or plant cells; (3) cultivating and screening to obtain a new transgenic sweet cherry variety with early fruit ripening.

Wherein, the promoting fruit ripening includes softening the sweet cherry fruit, increasing the content of soluble solids or soluble pectin in the fruit, and the like.

Wherein, the PaPME2 gene is operably connected to an expression control element to obtain a recombinant plant expression vector that can express the gene in plants; the recombinant plant expression vector Acid sequence and 3' non-coding region, wherein, the 5'-end non-coding region may include promoter sequence, enhancer sequence or/and translation enhancer sequence; the promoter may be a constitutive promoter, an inducible promoter type promoter, tissue- or organ-specific promoter; the 3' non-coding region may contain a terminator sequence, an mRNA cleavage sequence, and the like. Suitable terminator sequences can be taken from the Ti-plasmid of A. tumefaciens, such as the octopine synthase and nopaline synthase termination regions.

In addition, those skilled in the art can optimize the polynucleotide sequence of the PaPME2 gene to enhance or improve the expression efficiency in sweet cherries.

The recombinant plant expression vector may also contain a selectable marker gene for selection of transformed cells. Selectable marker genes are used to select for transformed cells or tissues. Marker genes include: genes encoding antibiotic resistance and genes conferring resistance to herbicidal compounds, among others. In addition, the marker genes also include phenotypic markers, such as β-galactosidase and fluorescent proteins.

The "introduction" refers to genetically transforming the polynucleotide or polypeptide into sweet cherry plants by introducing the PaPME2 gene into the plant cell. Methods for introducing the polynucleotides or polypeptides into plants are known in the art, including, but not limited to, stable transformation, transient transformation, or virus-mediated methods, and the like. "Stable transformation" means that the introduced polynucleotide construct is integrated into the genome of the plant cell and can be inherited by its progeny; "transient transformation" means that the polynucleotide is introduced into the plant but can only be temporarily expressed in the plant or exist.

Suitable methods for introducing the PaPME2 gene into sweet cherry plant cells include: microinjection, electroporation, Agrobacterium-mediated transformation, direct gene transfer, and high-speed ballistic bombardment, among others. The transformed cells can be regenerated into stably transformed plants using conventional methods (McCormick et al. Plant Cell Reports. 1986. 5:81-84).

In the present invention, the PaPME2 gene in sweet cherries can also be knocked out or mutated by gene editing technology (CRISPR/Cas9 transgenic technology) or gene knockout method, such as constructing a gene editing vector or knockout vector of PaPME2 gene, so as to make transgenic plants The ripening process of the sweet cherry fruit is slowed or delayed.

The present invention further provides a method for cultivating a new variety of hard-fleshed, storage- and transportation-resistant sweet cherries, which is characterized by comprising: (1) constructing a gene editing vector or knockout vector containing the PaPME2 gene; (2) converting the The constructed gene editing vector or knockout vector is transformed into sweet cherry plant tissue or plant cells, so that the PaPME2 gene in sweet cherry is knocked out or mutated; (3) Cultivated and screened to obtain a transgenic sweet cherry with firm flesh and storage and transportation resistance New varieties.

The present invention uses VIGS technology to determine that PaPME2 plays an important role in the ripening and softening of sweet cherry fruit. In view of the above research, the present invention determines that PaPME2 may be a key gene related to sweet cherry fruit softening. In order to further analyze the role of PaPME gene family in sweet cherry fruit ripening Physiological functions and action mechanisms in the softening process provide some ideas and experimental foundations, and provide a theoretical basis for clarifying the function of the pectin methylesterase gene in sweet cherry and its role in the process of fruit ripening and softening. The new varieties of sweet cherries provide genetic resources.

Definitions of terms involved in the present invention

Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs.

The term "recombinant host cell" or "host cell" means a cell that includes an exogenous polynucleotide, regardless of the method used for insertion to produce a recombinant host cell, such as direct uptake, transduction, f-pairing, or in the art other known methods. The exogenous polynucleotide can be maintained as a non-integrating vector such as a plasmid or can be integrated into the host genome.

The term "polynucleotide" or "nucleotide" means deoxyribonucleotides, deoxyribonucleosides, ribonucleosides or ribonucleotides and polymers thereof in single- or double-stranded form. Unless specifically limited, the terms encompass nucleic acids containing known analogs of natural nucleotides that have binding properties similar to the reference nucleic acid and are metabolized in a manner similar to naturally occurring nucleotides. Unless otherwise specifically limited, the term also means oligonucleotide analogs, which include PNAs (peptide nucleic acids), DNA analogs used in antisense technology (phosphorothioates, phosphoramidates, etc.) . Unless otherwise specified, conservatively modified variants thereof (including, but not limited to, degenerate codon substitutions) and complementary sequences as well as explicitly specified sequences are also implicitly encompassed by a particular nucleic acid sequence. In particular, degenerate codon substitutions can be achieved by generating sequences in which one or more selected (or all) codons are substituted at position 3 with mixed bases and/or deoxyinosine residues [Batzer et al. , Nucleic Acid Res. 19:5081 (1991); Ohtsuka et al, J. Biol. Chem. 260:2605-2608 (1985); and Cassol et al, (1992); Rossolini et al, Mol Cell. Probes 8:91 -98 (1994)].

The term "operably linked" refers to a functional link between two or more elements, the operably linked elements may be contiguous or non-contiguous.

Term "transformation": A method of introducing a heterologous DNA sequence into a host cell or organism.

Term "expression": Transcription and/or translation of an endogenous gene or transgene in a plant cell.

Term "coding sequence": A nucleic acid sequence that is transcribed into RNA.

Term "recombinant plant expression vector": One or more DNA vectors used to effect plant transformation; these vectors are often referred to in the art as binary vectors. Binary vectors together with vectors with helper plasmids are mostly commonly used for Agrobacterium-mediated transformation. Binary vectors typically include: cis-acting sequences required for T-DNA transfer, selectable markers engineered for expression in plant cells, heterologous DNA sequences to be transcribed, and the like.

Description of drawings

Figure 1. Expression patterns of 12 PaPMEs genes in fruit development.

Figure 2. TRV-mediated detection of relative expression levels of PaPMEs in sweet cherry fruit.

Figure 3. Changes in fruit firmness and soluble solids content of sweet cherry fruits silenced by PaPMEs.

Figure 4. Changes of soluble pectin and cellulose content in sweet cherry fruits silenced by PaPMEs.

Detailed ways

The present invention will be further described below with reference to specific embodiments, and the advantages and characteristics of the present invention will become more apparent with the description. However, these examples are only exemplary and do not constitute any limitation to the scope of the present invention. It should be understood by those skilled in the art that the details and forms of the present invention can be modified or replaced without departing from the spirit and scope of the present invention, but these modifications and replacements all fall within the protection scope of the present invention.

Test Example 1. Silencing PaPMEs gene in sweet cherry fruit by VIGS method and its effect on fruit softening

1. Materials and methods

1.1 Materials

Plant material: The European sweet cherry cultivar 'Zaohongzhu' came from the Cherry Germplasm Resource Garden of Zhengzhou Fruit Tree Research Institute, Chinese Academy of Agricultural Sciences. The rootstock is 'ZY-1', the tree is 6 years old, and the tree grows normally.

Strain and TRV virus vector: VIGS vector tobacco rattle virus (tobacco rattle virus, TRV) pTRV1 and pTRV2 were donated by Professor Liu Yule of Tsinghua University, and Agrobacterium tumefaciens strain GV3101 was preserved in the laboratory.

1.2 Test method

1.2.1 Construction of VIGS recombinant vector and transformation of Agrobacterium

The pTRV2-PaPME1-12 vector was constructed using In-Fusion Cloning technology. The PaPME1-12 gene-specific primer pair PaPME1-2-F/R (Table 1) with 16 overlapping regions (reverse complementary linkers to the pTRV2 fragment linearized by EcoRI and KpnI) were designed respectively, and the target fragment The choice was to silence only the sequences of a single PaPME gene and not other PaPMEs family sequences.

Table 1 Primer sequences

The 12 PaPMEs fragments were amplified with the sweet cherry cDNA as the template, and the fragments of 12 PaPMEs were constructed and connected to EcoRI and KpnI using In-Fusion ^™ HDCloning kit (Clontech, Mount-ain View, CA, United States). On the construction of the linearized pTRV2 by double-enzyme digestion, they were named pTRV2-PaPME1-12 respectively, and were transferred into E. coli DH5α competent. The positive strains were picked and identified by PCR, double-enzyme digestion and sequencing. The pTRV2-PaPME1-12 vectors were transferred into Agrobacterium strain GV3101 for future use.

1.2.3 Transient transformation of sweet cherry fruit by VIGS technology

The VIGS method of sweet cherry fruit was carried out according to the method of Qi Xiliang et al. (Qi Xiliang, Li Ming, Liu Congli, Song Lulu. 2018. TRV mediates the establishment of the VIGS system of European sweet cherry fruit. Journal of Fruit Tree, 35(11):1309-1315), and Three biological replicates were performed.

1.2.4 Semi-quantitative RT-PCR detection and real-time quantitative PCR (qPCR) analysis

The total RNA of sweet cherry fruit samples was extracted and reverse transcribed into cDNA. The Histone2 (Pav_sc0000671.1) gene of sweet cherry was used as an internal reference to adjust the cDNA content of different samples, and then the gene-specific primers of PaPMEs were used to pair PaPME1 -12-J-F/R (Table 1) detected the expression levels of related PaPMEs genes after silencing.

The qPCR reaction was performed on an ABI7500 PCR thermal cycler (Applied Biosystems, Foster City, CA, United States), using the TransStart Top Green qPCR SuperMix (Beijing Quanshijin Biotechnology Co., Ltd., Beijing, China) kit to carry out the reaction with sweet The Histone2 (Pav_sc0000671.1) gene of cherry was used as an internal control for analysis. And three biological replicates were carried out, and the average value was taken.

1.2.5 Determination of fruit firmness and soluble solids

Fruit firmness was measured by GY-4 type hardness tester, and the unit was kg·cm ^-2 . 5 fruits were randomly selected each time, 2 points were selected from different parts of each fruit and repeated three times, and the average value was taken. Determination of soluble solid content The soluble solid content (%) was measured by a hand-held Brix meter (PAL-1, Aituo, Japan), and repeated three times, and the average value was taken.

1.2.6 Determination of soluble pectin and cellulose

The determination of soluble pectin refers to the method of Cao Jiankang, etc., using carbazole-sulfuric acid colorimetric method (Cao Jiankang, Jiang Microwave, Zhao Yumei. 2007. Physiological and Biochemical Experiment Guidance for Postharvest Fruits and Vegetables. China Light Industry Press), the absorbance was measured at 530nm; the cellulose content Refer to Wang Xuekui's method for the determination of α, using anthrone colorimetric method (Wang Xuekui. 2006. Principles and Techniques of Plant Physiological and Biochemical Tests. Education Press, pp: 122-126.), and measure the absorbance at 620 nm.

1.2.7 Data processing

The obtained data were processed and plotted using Microsoft Excel 2010 software; correlation analysis and significant difference (P<0.05) analysis were performed using SPSS17.0 software.

2 Results and Analysis

2.1 Expression pattern analysis of pectin methylesterase gene family in fruit ripening and softening

The results of qPCR analysis of PaPMEs gene family showed that the gene expression levels of PaPME2 and PaPME9 genes were low in the early stage of fruit development (the first expansion stage and hard-core stage), and with the development of fruit, the gene expression levels showed a significant upward-regulated trend. PaPME2 The gene reached the maximum value at the end of fruit ripening and softening; while the expression of PaPME9 gene was the highest at the early stage of fruit ripening and softening (42 days after flowering), and then down-regulated (Fig. 1). The expression levels of other 10 PaPMEs genes (PaPME1, PaPME3, PaPME4, PaPME5, PaPME6, PaPME7, PaPME8, PaPME10, PaPME11, PaPME12) were lower in the fruit development process, and the gene expression did not show a certain regularity. For example, the gene expression levels of PaPME6, PaPME8, PaPME10, PaPME11 and PaPME12 genes are very low in the whole period of fruit development; PaPME1, PaPME3, PaPME4, PaPME5 and PaPME7 genes are relatively low in the early stage of fruit development, and with the development of fruit, the gene expression levels are very low. The expression showed an up-regulated trend, and the gene expression showed a peak in the middle or late fruit development, and then down-regulated (Fig. 1).

2.2. RT-PCR detection of the PaPMEs gene family in sweet cherry fruit silenced by VIGS

In this experiment, the semi-quantitative RT-PCR method was used to detect the silencing efficiency of 12 PaPMEs genes in sweet cherry fruit, because the method in this experiment only amplified the specific target fragments of a single PaPME gene (avoiding the conserved sequences of the PaPMEs gene family). information), so this test only detects whether a single PaPME gene is silenced. Total RNA was extracted from sweet cherry fruits 10 days after inoculation, and RT-PCR analysis was carried out with sweet cherry Histone2 as the internal reference gene.

The detection results showed that the mRNA expressions of 12 PaPMEs genes were significantly decreased in the respectively infected sweet cherry fruits (Figure 2). And through qualitative analysis of Quantity One software, it was found that the expression level of PaPMEs gene silenced sweet cherry fruit decreased by 60% to 85% compared with the negative control (Fig. 2). The above experimental results indicated that the PaPMEs genes in sweet cherry fruit were effectively silenced, respectively.

2.3 Effects of PaPMEs family silencing on fruit softening of sweet cherry

2.3.1 Effects on fruit firmness and soluble solids

Fruit firmness and soluble solids content are important indicators of fruit ripening and softening. In order to analyze the role of PaPMEs genes in fruit ripening and softening, the changes in fruit firmness and soluble solid content of 12 PaPME gene-silenced sweet cherry fruits were analyzed. Compared with the negative control, the fruit firmness was significantly higher than that of the negative control; similarly, the soluble solid content of the PaPME2 gene-silenced sweet cherry fruit was significantly lower than that of the negative control (Figure 3). While other PaPMEs gene-silenced sweet cherry fruits showed no significant fruit firmness and soluble solid content compared with controls (Figure 3). In conclusion, the experimental results showed that the PaPME2 gene was closely related to the fruit softening of sweet cherries.

2.3.2 Effect on soluble pectin content

The content of soluble pectin in fruit is closely related to fruit ripening and softening. In order to further analyze the effect of PaPMEs genes on fruit ripening and softening, the content of soluble pectin in 12 PaPME gene-silenced sweet cherries was measured and analyzed.

The measurement and analysis results are shown in Figure 4: Compared with the control, the content of soluble pectin in the sweet cherry fruit with PaPME2 gene silence was significantly lower than that in the control group; while the content of soluble pectin in the fruit of other PaPMEs gene-silenced sweet cherry was significantly different from that of the control group. The control was not significant (Figure 4). In conclusion, the experimental results show that the expression of PaPME2 gene in sweet cherry fruit is closely related to the content of soluble pectin in the fruit, which further affects the ripening and softening of the fruit.

2.3.3 Effect on cellulose content

In the process of fruit ripening and softening, the content of cellulose is also an important evaluation index reflecting fruit ripening and softening. In order to further clarify the function of PaPMEs genes in the process of fruit ripening and softening. In this experiment, the cellulose content of 12 PaPMEs gene-silenced sweet cherry fruits was determined.

The analysis results showed that the cellulose content of the 12 PaPMEs gene-silenced sweet cherry fruits was basically the same as that of the control (Fig. 4), indicating that the expression of the sweet cherry PaPMEs gene had nothing to do with the change of the cellulose content in the fruit.

sequence listing

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<400> 4

atgtttatgc aaacttgcac tgaaattgaa gaccaaaatt catgcctcac caatgtgcaa 60

gctgagctca aaactatggg ccctgacaat caaaattctg cttcaatcct aactgctgcg 120

attaggcaca cacttaatga agcaagagct gcaatccaaa agatcacaaa gttcagttct 180

ttatccatca gttacagaga acaactagca attgaggatt gcaaagagct cctagacttc 240

tctgtctctg agctggcttg gtctttgggt gagatgaaca aaatccgagg tggtgacaac 300

aatgaacact atgagggaaa cttaaaagct tggctaagtg ctgccctcag taaccaagat 360

acctgccttg aaggttttga gggaactgat agacgtctcg aagatttcgt caggggaagt 420

ttgaagcaag tcacacagct cattggtaat gtcttggcct tgtacactca attacatagc 480

ttacccttta agcctcctag agatcacggc aacccggtga ataaaagttc atcatcagat 540

gatcatttgc cggcatggat cagtgagggt gatcaagagc tgcttagatc caatccacaa 600

tcgggtatgc atgcagatgc tgttgtggca gcagatggga gtggcaagta ccgtacaatc 660

acagaagctg ttaatgcagc tccaaactac agcagcaaga ggtacataat atatgtgaag 720

aagggagtttt atagagaaaa cattgacatg aagaagaaga aaaccaatat tatgtttgta 780

ggggatggga ttggacaaac tgtggtaacg ggtagacgga atttcatgca aggatggact 840

acatttagaa ctgcaactgt tgccgtgtct ggcaagggat ttatagcaag agacatgaca 900

tttagaaaca cggccgggcc ggaaaaccat caaggcgtgg cgcttagagt cgactctgac 960

caatcggcct tcttccggtg cagcatggag ggttaccaag acaccctcta tgctcactcc 1020

ctccgtcaat tttaccgtga atgtagcatc tatggcacca tagacttcat atttggcaac 1080

ggtgcagctg tgctccaaaa ttgcaaaatc tacacaagag ttcccctacc attacaaaag 1140

gtcacaatca cagcccaagg cagaaaaaac ccacatcaaa gcactggatt tgcaatccaa 1200

gacagctata ttcttgccac tcagccaaca tatttgggca ggccatggaa gcaatattcc 1260

aggactgttt ttctgaacac ttatatgagt gggcttgtgc agcccagagg gtggcttgag 1320

tggtatggca actttgcttt gggcaccttg tggtatggtg agtacaagaa ttatgggccg 1380

ggtgctttgc tatccgggcg ggttaaatgg cccggttacc atattatcaa agatgctgca 1440

gcggctagct tctttactgt tggaaggttc attgatggaa gggcttggtt gccatcaaca 1500

ggtgtcaagt ttacagcggg tttgagaaat taa 1533

<210> 5

<211> 2814

<212> DNA

<213> Prunus avium L

<400> 5

atggagagta tgaaaattat gggcctggtt cagggacaga aaatagggtt aagtggactg 60

gatatcatat tatggattat tatgatgcgg caaaattcac agtatcagag tttattattg 120

gtgatgaatg gctgcaggcc acttcatttc cttatgatga tggcatctga tctttgggat 180

ttctgtatga acgaatgttc tatgtacacc aaaggtggaa gttgtatgga tgaggatttc 240

gaccagatat ccgaacgccg aaaggctgag agggcacgta aaatgaggaa gagaattatc 300

atcgcagttg tcgtcgtcgt cctgcttatt ctgattgctg tcggagccta tttacttttg 360

aacaaactta atagtaagaa gggcaatgct aagcaagaca aaacagccaa ttctaagcct 420

gctccagcaa aagctcaacc caagaacgat caagccaaga agaaagtgcc agcaaagggt 480

gagaagatca tgaaggagat gtgtggcgca acagactaca aggacaagtg cgagagcatc 540

atcgaaaagg caaagggcgc atctaaaccg aaggaattca tcaagaccgc tatctcagca 600

gcctcggatg aggccaggat tgcctacagc aaatccagcg agctcacttt taacagccca 660

gaagagaagg gagcatttga ggattgcaaa gtgctgtttg aagatgccat ggacgaatta 720

ggggatgcca tttctcaaat tggcaacaca actgcttcgg ggaagattcg aactggtgtc 780

ttgaacactt ggctgagtgc agtcatatct taccagcaga cgtgtgttga tgggtttcct 840

gatggaaaat tgaagtctga cttggagaag atgttgcagg ccaccaagga attcaccagc 900

aattccttgg ccatgctttc actcctttcc caattccagc taccggttac agcagcagtt 960

tcgggagcaa aacgtcgtct tctagcacag gacaaggacg ggtttcctac ctggatgagc 1020

catgaggagc gaagggtgtt gaagaaaaat gatgagaagc ccacacctaa tgtgactgtg 1080

gcaaaagatg gcagtggaaa cttcaaaacc attagtgaag ccttggcagc catgcctgca 1140

aaatatgaag gacgatatat catctacgtt aaaggaggag tctatgatga gactgtgatt 1200

gtgacaaaaa agatgccaaa tgttaccata tatggtgatg gatcacagaa gagcatcatc 1260

actgggaata agaactatgc agacggagtt aggacattcc aaactgcatc ttttgtaaag 1320

tctcagttca agacctacct tgggaggcca tggaaggaat tctcaagaac catagtgatg 1380

gactcaacaa ttgaggatct gattcaccca gatggatgga caccatggga aggagacttt 1440

gcactgaaaa cgctgtatta tgcagagtat aacaacaagg gaccaggtgc caagactgat 1500

aacagggtca agtggtctgg atacaaagtc attgacaagc aggaggctat gaagtatact 1560

gtagggcctt tcttgaaagg gtatgcttgg ctcagggcca agggagttcc ttttgtggta 1620

gtgttttggg cactgggagg ggcctcagtg ttaggggcag ggcaagccaa tgatagctac 1680

caaatttatg ttaaaaaaga atgcagcttt acaagatatc ccagcatatg tgttcaaacc 1740

atgacggggt ctggttcagg gcatgatcaa caacatgttg atataatgtt ggctcttgca 1800

aacaagacca tatctgagac catgttggcc acctccgagt ttgtcaactt cagctcccaa 1860

ttcaatttgg aaggtgaact tggagcccgt gaagctcaac gtgttcattc cgtcaaagac 1920

tattattgtg gagagctcat gaacatgtcc ttgaagcggc tcgaccaatc cctattggca 1980

ctcaaacaat ctccaaggaa aaacaagcgt gacatccaag catggctcag tgctgcgttg 2040

actttccaag acacttgcaa agactatgct tctggtcaaa tttccaagcg cgtagacaac 2100

gcttctcagt tggtaagtaa cctattagct cttgtcaacc gtatcgcaag taaccataca 2160

acaacggtca caaatcaccg tagtgttgac caagggcttt ttgccaaatg ggtatcgcca 2220

agggaccgga aactacttca ggcaaccgcc ataaaagctg atgccgtggt tgccaaagat 2280

ggatccggca actacaaaac tgtatcggaa gccatcaatg cagcttccgg ggcccggttt 2340

gtgatttatg taaaggcagg ggtttatgac gagaagattc acactaaaaa agatggtatt 2400

acgttgatag gagatggaaa atattccact attattactg gagatgatag cgttgccaaa 2460

ggtgcctcca tgcctggctc agccactttc attaagcact cctacagctc gtatttgggg 2520

aggccatgga agcaatactc tagagccgtc gtcatggaat caaccataga cgacgtcatt 2580

gcaccccaag gctgggtgga gtggcctggg gctggaggat ccagcctcaa gacactgtac 2640

ttcgcggagt atgcgaatgt ggggccgggg gcgggagtgg gcaaaagggt gcaatggcct 2700

gggtttcatg tgattggagc tgacgtggct gttgagttta ctgttgctaa ttttattgct 2760

gggacttcat ggctgccttc tactggagtc actttcgttt ctggcctcca ttga 2814

<210> 6

<211> 2385

<212> DNA

<213> Prunus avium L

<400> 6

atggccacga aaccaagcct acttctctcc atactcattt tctctttcat cttccaaaca 60

gctctctcaa gaagccatca ccactctagc cgtatacgta cttggtgcca gaaaaccccc 120

caccccgagc catgcaacta ctgcatgtca catagccgtc accgtctcat tccgaaacac 180

acctccgaat ttcgaaaaat gttggtgcaa gtggccttgg agagggctct caatgcaaaa 240

tcatacgcct cccaatttgg ccaaaactgt caaaacaatc aacaaaaagc cgcatgggcc 300

gattgcttga aacttttcga ggacaccgtc caccagctca acattaccct tgaaggctta 360

ggcaccaaac gcaattgctc caactttgat gcacagactt ggctcagcgc tgccctcacc 420

aacattcaca cgtgccaagt tgggtctatg gagctgaacg tttcggattt catagcccct 480

atatacacct ccaataacaa tgtgtccgag ctaattagca acggtttggc aattggttca 540

caacttttag gcacgggaga aaattacgca gatgaagagt atccgaattg ggtttcaaag 600

cacgatcgga ggctgttgca agcttcaaaa atcaaggcaa atcttgtggt tgccaaagat 660

ggatctgggc attttcggac ggttcaagcg gccatagatg cagcggctaa gaggaaaata 720

actagtaggt ttattatcta tgtgaagaaa ggtgtttata gagagaacat tgaagttagc 780

aacactaata ataacatcat gctgattggt gctagcatga gatatacaat aatcacagct 840

agccgaagtg tcaatggagg ttccacaacc tataattccg caactgctga tctgaagccc 900

gtggttagtg ctttcaaaac ctacttgggc cggccctgga tgaaatactc tagggttgtt 960

ttcttgaaat gttatctgga cagtttggtc aacccagtgg gctggttgga gtggcaaaga 1020

agtaattttg cgcttagcac tttgtattat ggggagtaca agaattttgg tcctgcttcg 1080

tctaccaggt acagagtgaa gtggcctggt ttccatatca tcaccagtgc aaatgtggcg 1140

tcacagttca ccgtcagcag ccttatcgcc ggtcggtcct ggttgccggg caccggggtt 1200

ccatacacag cgggtcccaa aaccacaagc caatcaacaa gaagcctatc catggcgaca 1260

aaaatgagtt cattccttct atgcttctcc ttctccttac tattctcgcc agccctatct 1320

aacatactca acactggcat caactcttgg tgcagcaaaa ccccctaccc tgaaacctgc 1380

aaatacacct tgacccatgc ccaaaaatac tctctcccga cacgcatgtc cgacttcaag 1440

aaacttgcgg tgcaagtcac aatgcagcag gcactcaaag cccaaagcca caacaagtgg 1500

ctaggtccca agtgcagaaa caagatcgaa aaggctgcat gggccgattg cttgagcctc 1560

taccaagaca ccatcatgct cctcaaccag accatagacc ccgccaccaa atgcaccgac 1620

tatgacgcac agacttggct cagcactgct ctcaccaacc tcgacacgtg ccgggccggg 1680

tttgtggagc tcggggtctc ggactttgtg cttcccctta tgtccaacaa tgtgtccaag 1740

ctcataagta acaccttgtc cattggtaat ggctcaaatg ttccggctgt gacaaatagg 1800

tacaaggagg gttttcctac ttgggtttct cccggtgaca ggaaactgct ccagtcgtcg 1860

ccagccgcgg acgtagttgt tgcccaagac ggatcaggga actacaagac aatcaaagag 1920

gcgttggctg ccgcggcgaa gaggagtggg agcaacaggt ttgtgataca tgtgaagcgt 1980

ggggtttata aggagaacct tgagattaaa ttgaagaaca ttatgctgct tggtgatggt 2040

ctgaggtata ctattatcac cggtagccgt agtgttgtag gtggttctac aactttcaac 2100

tctgcaactg agtattcacg cacgattttt ttgcaaagtt acctggacac tttggttgac 2160

ccagctgggt ggcttgagtg ggatggcaat tttgccctca agactttgta ctatggagag 2220

tacaagaaca ctggaccggg ttcatctacg agcggccggg ttaattgggg tggctatcat 2280

gtgataacta gttcgtcaga ggcatcgaaa tttactgtgg gtaatttcat agctggaagc 2340

tcatggttgc ctgctacaaa cgtgccattt actgctgggc tctga 2385

<210> 7

<211> 879

<212> DNA

<213> Prunus avium L

<400> 7

atgttgggat ccattgctgc agttggtgct tggataagaa agttcaagga gtattatgtt 60

aaattacaat ccctgaggga gaagaaggtt catcaatacc tgatggagat ggaggttcgt 120

ttagaacaca ctaatttaaa ggttatgaaa gatggaagtg gagaattcaa gaccatcact 180

gatgccgtta atagcattcc ggctgacaac acgagatgtg tcattgtgta cattggaggg 240

ggagagtaca atgagaaaat cacaattcca aggaataaac catttgttac atttaatggc 300

tctccaacaa atatgccaac tttgaccttt gctggcacgg cccaaaagca tggaactgtg 360

gacagtgcca cagtgattgc cgaatctgac tacttcgtgg cagctaaccc tattattaag 420

gttgattggt ttcaagacac actttgtgat gacaggggca atcatttttt caaggactgc 480

tttattgaag gcaccgtgga tttcatcttt ggaagtggaa agtctatcta tttggcaatg 540

ccacccaaac tctccctggc ttggaggacc agtcccaggg tggtctttgc ctacactagc 600

atgtccgagg tcatcaccct agccggctgg aacaacaaga accgccccga aagtggccag 660

catgtccgag gtcatcaccc cagtcggctg gaacaacaag aaccgtcccg aaccaccgtg 720

ttctatggag aatacaagtg ttcgggtcca ggttcaagta tggttgggag agtaaaatac 780

accaaacagc tgactgggga acaaatcaaa cctttcctca gccttggcta tattcaaggt 840

tccaaatggc tgcttcctcc tccaaatcca gaagtgtag 879

<210> 8

<211> 694

<212> DNA

<213> Prunus avium L

<400> 8

atgagcggag gagaagcaca gaataagaaa aagaagatcg ccatcatcgg cgtctccgct 60

ttgattctgg tggctatggt agttgctgta actgttggga tcactgtgtc acggcacaaa 120

ggcaaatccg gcggcgagca aacatccacg tcgacaaagg cgatccagtc gatctgccag 180

cccacggact acaaaaagac ctgcgaggac aacctgtcca aggtggctag caacgtcacc 240

gacccaaaag agctggtcaa ggcagggttc caggtcgcca tcgaccagct ccgcgaggtc 300

atcaagaact cgacgacctt gaaggagctt gccaaggacc ccagcacaaa ccaggccttg 360

cagaactgca aggagctctt ggagtatgcc atcgacgatt tgggtgactc gttcgaaaag 420

ctgggtcctt ttgatttcac caagctcgac gcctacgtgg aggatctcaa ggtctggctc 480

agcgccgcca tgacgtacga gcagacttgc ctggatgggt ttgagaacac caccggtgat 540

gctggtgaga agatgaggca gttcttgaag acatctcagg agctcaccag caacggcctt 600

gccatggtaa gcgaagtgtc cacgttgttc aaggctctga acatcaagac cgggcgccgc 660

ctcctccaag ccgctgccac ggccactgat gtga 694

<210> 9

<211> 1695

<212> DNA

<213> Prunus avium L

<400> 9

atggcttcta agctcttttc tcttgtaaca ttttcatcat ttctcataat cttccatttc 60

cttagctccc catctttagc agatgtccct ctagacactc ctctcccacc agaaacaatt 120

tgcaagtcca ctccacaccc ttcctattgc atatctgtcc ttccccacaa aaatgccaat 180

gtctatgact ttggcaggtt ttctgtccaa catgcacttt cccaatccaa taagctcttt 240

gattcaattg aaaaacatct tcaacttggc tcagtcttac cacagcctgc aatccaagcc 300

cttgaggatt gcaagttgct tgcattgttg aacattgatt tcttatcaag ctgccttgaa 360

actgtgaaca agacaagtgg tgttcttgct agcttggatg ctgatgatgt ccaaaccttg 420

ctcagtgcca ttttgaccaa ccaacagact tgttccgatg gccttgaatc tctgccttct 480

gcagctggga gtgtcataaa tgatctctca gcctcaatct ctaacaactc acaattgtgc 540

agtgtctctt tggctctgtt taccaagggt tgggtgccta aggacaaaaa tggagtgcca 600

aagcaaccca agacgcacct ccgattcggg aagggacgct tgaacctgaa gatgtcaagc 660

caagctcgtg caatttatga cgccgctatt aatcatcgga gaagaagact tcttcaggta 720

ggagatgaag aggttttggt gaagggcatt gtggttgtga gtccagatgg aagtggaaac 780

tttaccacca tcaatgctgc cattgctgct gcaccaaaca actccgttgc aagtggtggc 840

tacttcttga tatatgtcac tgctggtgtt tatcaagagt atgtgtcaat tgcatcaaac 900

aagaagtact tgttgatgat cggagacggt atcaaccaga caatcatcac tggaaacaac 960

agcgtaggag atggctcgac aactttcaac tctgccactt tagctgtcac aggactaggg 1020

ttcgtagcag tgaacattac tgttcgtaac acggctggac caagcaaagg gcaggcagtt 1080

gcagtccgaa acggggcaga tttgtcagtt ttctacagct gcagttttga aggctaccaa 1140

gacaccttat acacgcattc tctcagacaa ttctacagag aatgtgatat ctatggaaca 1200

gttgacttca tattcggcaa tgctgcagtt gttttccaaa attgcaacct atatcctcgc 1260

cagcctaacc aagggcagtc caatgccatc acagctcaag gtcgaaccga cccgaatcag 1320

aacacgggga cttctatcca aaactgcact atcaaaccga cgccggattt ggcttcgagt 1380

aatttcactg tcaagaccta tctagggagg ccatggaagg aatattcaag gacagtttac 1440

atgcaaactt tcatggctag tttgattgat cctgctggct ggcttgcatg gagtggagat 1500

tttgctctga gcacactgta ttatgctgag tacaataaca caggccctgg atccaacact 1560

acaaagaggg ttacatggcc tggctaccat gtgatcaatt caaccgttgc tgctaatttt 1620

acagtgacca acttcttgat gggggataat tggttgcctg agactggtgt gccttacact 1680

ggtggattaa tttga 1695

<210> 10

<211> 1143

<212> DNA

<213> Prunus avium L

<400> 10

atggcttctt ctcttttatc atcatcatct tcttcttctt cttacttctt cttggtgtgc 60

accacactca ctgtgttttc cttctggtta tttcatgcag cgccagcggt gacagcggtg 120

agaagctctc gcgtactgct agtccacact attaacaaca atgattcaac atcaaactct 180

accaagaacc accacaagtg ggtggggccc actggccacc gccagatcgc tgtcgacata 240

aatggctccg gggatttcct gtcagtccaa gctgccgtca atgcagtacc ggcgaacaac 300

acggtcgatg ttctcatcct aatcagcccc ggatactaca gagaaaaagt ggttgtgccg 360

gcgacaaagc cgtacatcac gtttcaagga gctgggaaag atgtgaccgt gatcgaatgg 420

catgaccgag ccagtgaccc tgggcccaac ggccagcagc tgaggactta taggacagct 480

tctgttactg ttttcgccaa ctatttctcc gcaagaaata ttagcttcaa gaatacggcg 540

ccggctccga tgccggggat gcaagggtgg caagcggtag cgtttaggat atcaggtgac 600

aaggcatact tctcagggtg tggcttctat ggtgcccagg acacactttg tgacgatgtg 660

gggcgccatt acttcaagga ttgttacatt gagggttcca tagacttcat ttttgggaat 720

ggtcggtcca tgtacaaaga ctgtgagctg cactcgatag caaccaagtt cgggtcaatt 780

gcggcccact acagaaactc agcggatgat aaatcaggct tcgctttcgt gaactgccga 840

gttaccggta cgggtccttt gtatgtgggt cgggccatgg gcaagtactc ccggatcgta 900

tactcctaca cctactttga tgacgtggtc gctcatggcg cctgggatga ctggggcaac 960

acaaccgcca gcactacaac aaagaggagt gtgtttctgg gagtctacaa atgctggggg 1020

ccaggggaag aagccctgcg tggggtgtca tgtgccccag agcttgatta tgaattggcc 1080

catccctttc tggtcaagag tttcgtcaat ggaaggcact ggattgcacc ctctgatgct 1140

tag 1143

<210> 11

<211> 1125

<212> DNA

<213> Prunus avium L

<400> 11

atgaagtcat cctcccattt catttttttc atcatttgtt tctcttctct ttccattgac 60

ccccgcgctg ataatgttga cccccacttg ataaatgttc ctttgcctgc attcaaaaac 120

tcattggaga agaccaagga acttgtgcaa aatgtggcct ctaccatgtc acatatgcat 180

gctggtgaag atttgcttga tggaaattct actgttttgg tcatttcgta ctgtgaggat 240

ttgcttgatg aaactgctgg cgtcttggat tggtcccttt ccacaattga tgatcttaaa 300

gctgatgtcg tttcccatat gagaacatgg ctgaatactt cgcaatccag agaaacgaca 360

tgcgttgacg cttttcaaaa tagcatcgac tcaaatgttg cagagagcct tagacaagtg 420

acaaaatcaa tcgatgaggt ccttggtatg atccaagtgg aggagcaaca tcatcttcat 480

cgtcataatg ctactgcaga tccaggtcca gatcctgatc cttatgatcc tgcactttcc 540

cggacggcag acgtcaccgt ttcacaggat gggagtggga aattcaagag gataatggat 600

gccattgctg ctgcaccgag ccatagccag aagcagttcg tgatatttgt gaagaaagga 660

gtttacaaag aatatgtgaa gattgataaa acaaagacta acttggtgtt gattggagag 720

ggcatgagtg ttaccaccat atctggagat agaagcaatg ctagtgggtt ctcgttccag 780

ttctgcagaa ttgaagcaga ttctgatttg gtgggtaatg tttactctac gaagacgtac 840

ttgggtcgac cttggggcaa atattcacgt accgtgttca tcaagtcttt tatgagtaat 900

attataaggc cagagggttg gctagaatgg agtgggagag ctaatcttga tactttgtat 960

tatgctgagt acaagaacta tggtccgggt gcctcggttg caggccgggt caagtggccg 1020

ggttatcatt tgatttccaa ggtcgattcg tttgcagtgg acaagtttat tggtgggaag 1080

tcatggctgc catccacggg tgtcccattt aaagcagatc tatag 1125

<210> 12

<211> 1014

<212> DNA

<213> Prunus avium L

<400> 12

atgtcatcat gtcttcgtct tgatgctcat tattattgtt catgtgtagc tctaatctta 60

ctaggccttg gattattagg gcaagccaat gcccaaatgt atagagttga tagcaataat 120

aagaagctgg cttattacac catcactgtt gatcaatctg gccatggcaa tttcacctcc 180

attcaatcag ccattgatgc tgttcctata aacaacagga actgggtttc tatcaagatc 240

aaggcaggca cctataagga aaaggtgatc atccccgttg acaagccata cataattctc 300

aaaggagaaa ataggcataa aacactgatt gtttgggatg atcacgactc agttgcacaa 360

agccctactt ttgcctctta tgccgacagt atcattaatt catacaacaa ccctgtgaat 420

aacaaaaatc caagagtgcc agcagtggcg gcaatgattt acggggacaa atcttcattc 480

tatcgatgtg gtttctttgg tttgcaagac actttgtggg atggtcaagg acggcactac 540

tatcaccttt gcaccatcca gggtgccgtt gattttatct tcggcagtgc ccagtccatt 600

tttcagaaat gttcgattca agttcttggg ggagctctgg atcctgggtc ggctggttac 660

atcacagcac agggaagaga caatccaaat gaagcaagtg ggtttgtgtt caaggattgc 720

aaggtgtctg gaacaggttc cacctacttg ggcagggcat ggagaggtta ctctagagtc 780

attttctaca actctaattt ttcgaaaatt gttgtgcctc aaggctggga tgcttggcat 840

ttccaaggca atgagcacca attaacgtat gcagagcatg gttgctatgg gccaggagct 900

gacactttaa agcgtgtgga atgggaaaag aagcttaatg ctgatacggt gcgggagttg 960

actagcttga attttattga tactgatgga tggctcaatg accagccttt ctaa 1014

Claims (1)

1. Sweet cherryPaPME2The application of the gene in regulating and controlling the ripening or softening of the sweet cherry fruit: the regulation and control of the ripening or softening of the sweet cherry fruits comprises the steps of delaying the softening of the fruits, and reducing the content of soluble solids or soluble pectin in the fruits; the method comprises the following steps: construction ofPaPME2Gene editing vector or gene knockout vector of gene, and gene expression vector of sweet cherryPaPME2Knocking out genes; the sweet cherryPaPME2The nucleotide sequence of the gene is shown as SEQ ID No. 2.

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