CN110564738A - application of sweet cherry PaPME2 gene in regulating and controlling ripening or softening of sweet cherry fruits - Google Patents

Abstract

The invention discloses an application of a sweet cherry PaPME2 gene in regulating and controlling the ripening or softening of a sweet cherry fruit. According to the invention, the function research of Pectin Methylesterase (PME) genes of 12 sweet cherry fruits shows that the expression patterns of the PaPME2 and the PaPME9 genes are closely related to the ripening and softening process of the sweet cherry fruits. According to the invention, the VIGS technology is further utilized to silence the PaPMEs genes of the sweet cherry fruits respectively, and the result shows that the hardness, the content of soluble solid matters, the content of soluble pectin and other fruit ripening and softening related physiological indexes of the fruits with the silent PaPME2 genes of the sweet cherry fruits are remarkably changed, so that the PaPME2 gene is a key gene influencing the ripening and softening of the sweet cherry fruits, and can be applied to the regulation and control of the ripening or softening of the sweet cherry fruits, the cultivation of new varieties of hard meat and sweet cherry with storage and transportation resistance.

Description

Application of sweet cherry PaPME2 gene in regulating and controlling ripening or softening of sweet cherry fruits

Technical Field

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

Background

The European sweet cherry (Prunus avium L.) belongs to the Rosaceae, belongs to the Prunus plant, is the earliest fresh fruit on the market in the northern spring of China, and has high economic value. Moreover, the sweet cherry fruits have extremely high nutritional value and health care effect, contain rich organic acids, vitamins, amino acids, melatonin and the like, and are deeply loved by consumers. However, the rapid softening of the sweet cherry fruit after ripening leads to the extreme softening and rotting of the picked sweet cherry, the quality of the sweet cherry is reduced, and the sweet cherry is greatly lost during transportation and storage, which limits the development of the sweet cherry industry (Nuanzai, Wanggang, Jiazhui, Guo loyalty. 2015. plum fruit ripening research progress. Chinese agricultural bulletin, 31: 104-118; Brummell D A, Dal Cin V, Crissoto C H, LabavitchJ M.2004.cell wall metabolism during the formation, ripping and senseice of the peach fruit journal of the national laboratory Botany,55 (202405): 9-2039.). Therefore, the research on the physiological and molecular mechanisms of the ripening and softening of the sweet cherry fruits, the improvement of the storage and transportation resistance of the sweet cherries and the prolongation of the shelf life of the fruits is one of the hot problems in research and discussion.

Sweet cherry fruit softening is mainly caused by the change of cell wall structure and the degradation of cell wall components, and the main components of the cell wall are pectin and cellulose (Zhao Yufeng, forest Yu, Lin river Tong. research on the relation between the change of cell wall components and the ripening and softening of fruits advances. food science and technology 2012(12): 29-33.). The major contributors to cell wall material degradation are some hydrolases, if Pectinesterase (PME), Polygalacturonase (PG), cellulase (Cx) and beta-Galactosidase (beta-Galactosidase) etc. (Brummell D A, Harpser M H.2001.cell wall metabolism in fruit watering and quality and evaluation in genetic plants Biology,47: 311-340.; Duckweed, Wanggang, Jia Zhan, Guo Kenren. 2015. plum plant fruit ripening research progress. Chinese agrimony report, 31: 104-118.; Brummell D A, Dal Cin V, Cristo C H, Labavitch J.2004. cell metabolism and evaluation, 9. journal of 9. 20255. J.9. cell metabolism and 9. supplement).

Pectate methylesterase genes (PMEs) are a large family of genes widely present in plants, and are classified into Type I and Type II. PME mainly catalyzes the conversion of pectate acid to pectate, provides a hydrolysis substrate for PG, and acts synergistically with PG enzyme to soften fruit (progress in the study of major related enzyme actions in Chengjie, Shenhulin, Sunwu, Yanyan, Zhang Mei, Neilai, Lirose, ripening and softening of fruit North horticulture, 2008(01): 49-52.). It was found that the enzymatic activity of PME was significantly increased during the ripening and softening of fruits in horticultural crops such as bananas, papayas, mangoes, strawberries, tomatoes, grapes and peaches, indicating that pectin methylesterase plays an important role in ripening and softening of fruits (Carpion, Shizu, Zhang, Huangmianjia, Anmianjia, Normal temperature fresh-keeping effect of exogenous chitosan coating treatment of papaya, Hippo et al, 2016,7(02): 220-223; Ali Z M, Chin L H, Lazan H.2004.A comparative study on wall grading enzymes, cultivation modifications and growing of selected vertical fruits plant Science,167(2): 317-327; Muengkaew R, artificial chai K, Chapsart P.2018.application of calcium-boron engineering, culture of fruit, culture of rice, culture of, tiemann D M, Handa A K.1991.molecular cloning of bottom mounted methyl ethylene reagents and expressions in Rutgers, ending inhibitor, ending, and near end mounted yield 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 duringvane replacement. plant, 214(2): 257-264; the maturity of the week, the research of the related gene of the ripening and softening of strawberry fruits, the northwest of Yangling; manganaris G.A, Vasilakakis M, Diamantidis G, Mignani I.2006. overture metabolism of cell wall components of long and non-long peptides along with rear axle design or cold storage. journal of the science of Food and Agriculture,86(2): 243-250). Since pectin methylesterase is a multigene family, and different pectin methyl ester isozymes play different roles in fruit ripening and softening, it is necessary to perform systematic functional research on the pectin methylesterase gene family to clarify the role in fruit ripening and softening. Ripening and softening of sweet cherry fruits are one of the main characteristics of fruit aging, and the taste and the commodity value of the sweet cherry fruits are seriously influenced along with the reduction of the fruit quality, such as reduction of hardness, softening of texture and the like. Studies have shown that alterations in the cell wall composition of sweet cherry fruits, especially changes in the pectin composition and the interaction of related enzymes, are a direct cause of fruit softening (Brummell, 2006; Pelloux et al, 2007; Matas et al, 2009). However, in stone fruit trees such as sweet cherries, studies on PME gene and fruit ripening and softening from the transcription level have not been reported yet.

Disclosure of Invention

The invention mainly aims to provide a new application of a sweet cherry PaPME2 gene in regulating and controlling the ripening or softening of a sweet cherry fruit.

The above object of the present invention is achieved by the following technical solutions:

According to the invention, 46 PaPMEs family genes are firstly identified from a sweet cherry genome database and subjected to gene expression quantity analysis, and finally, the expression of 12 PaPMEs genes (namely, PaPME1-12 genes) is determined to have a certain relation with the development of fruits.

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

To further clarify the function of these 12 PaPMEs genes in the ripening and softening of sweet cherry fruits. The expression mode of the 12 PaPMEs genes in the ripening and softening process of the sweet cherry fruits is further refined and analyzed by using a real-time fluorescent quantitative PCR technology, and then the expression of the PpPMEs genes in the sweet cherry fruits is silenced by using the VIGS technology of the sweet cherry fruits and the influence of the PpPMEs genes on the ripening and softening of the sweet cherry fruits is analyzed; the results show that the expression levels of the PaPME2 and the PaPME9 genes are low in the early development stage of fruits, the expression is obviously up-regulated in the late development stage of the fruits and is consistently continued until the fruits are mature, and the PaPME2 gene reaches the maximum value at the terminal stage of mature and softening of the fruits; the expression level of the PaPME9 gene is highest in the early stage (42 days after flowers) of fruit ripening and softening, and then the gene is down-regulated; and the other 10 PaPMEs genes (PaPME1, PaPME3, PaPME4, PaPME5, PaPME6, PaPME7, PaPME8, PaPME10, PaPME11 and PaPME12) have lower expression level and no certain regularity at each stage of fruit development. Silencing the PaPME2 gene of the sweet cherry fruit delays the ripening and softening of the sweet cherry fruit, and meanwhile, silencing the fruit hardness, soluble solid content and soluble pectin content of the sweet cherry fruit by using the PaPME2 gene is obviously lower than that of an unloaded control; while other PaPMEs genes of the silenced sweet cherry fruit were not significantly changed compared to the empty control. According to the test results, the PaPME2 is involved in regulating the ripening and softening process of the sweet cherry fruits, plays an important role in the fruit softening process and is a key protein factor for causing the sweet cherry fruits to soften.

Accordingly, the present invention provides a method for promoting ripening of a sweet cherry fruit, comprising: constructing a plant expression vector containing a PaPME2 gene; the constructed plant is expressed and transformed into the sweet cherry, and the PaPME2 gene is overexpressed or overexpressed in the sweet cherry.

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

Wherein, the promotion of fruit ripening comprises softening the sweet cherry fruit, increasing the soluble solid content or soluble pectin content in the fruit, and the like.

Wherein, the PaPME2 gene is operably connected with an expression regulatory element to obtain a recombinant plant expression vector which can express the gene in plants; the recombinant plant expression vector can consist of a 5 ' non-coding region, a polynucleotide sequence of a PaPME2 gene and a 3 ' non-coding region, wherein the 5 ' non-coding region can comprise a promoter sequence, an enhancer sequence or/and a translation enhancement sequence; the promoter can be a constitutive promoter, an inducible promoter, a tissue or organ specific promoter; the 3' non-coding region may comprise a terminator sequence, an mRNA cleavage sequence, and the like. Suitable terminator sequences can be taken from the Ti-plasmid of Agrobacterium tumefaciens, for example the octopine synthase and nopaline synthase termination regions.

In addition, the polynucleotide sequence of the PaPME2 gene can be optimized by one skilled in the art to enhance or improve 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 transformed cells or tissues. The marker genes include: genes encoding antibiotic resistance, genes conferring resistance to herbicidal compounds, and the like. In addition, the marker gene also comprises phenotypic markers, such as beta-galactosidase, fluorescent protein and the like.

By "introducing" is meant genetically transforming a polynucleotide or polypeptide into a sweet cherry plant in such a manner that the PaPME2 gene is introduced inside the plant cell. Methods for introducing such polynucleotides or polypeptides into plants are well known in the art and include, but are not limited to, stable transformation, transient transformation, or virus-mediated methods. "stably transformed" means that the introduced polynucleotide construct is integrated into the genome of the plant cell and is capable of being inherited by progeny thereof; "transient transformation" refers to a polynucleotide that is introduced into a plant but is only transiently expressed or present in the plant.

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).

The invention can knock out or mutate the PaPME2 gene in the sweet cherry by a gene editing technology (CRISPR/Cas9 transgenic technology) or a gene knock-out method, such as constructing a gene editing vector or a knock-out vector of the PaPME2 gene, so that the ripening process of the sweet cherry fruit of the transgenic plant is slowed down or lagged.

The invention further provides a method for cultivating a new variety of hard-meat sweet cherries which are durable to storage and transportation, which is characterized by comprising the following steps: (1) constructing a gene editing vector or a knockout vector containing the PaPME2 gene; (2) transforming the constructed gene editing vector or knockout vector into sweet cherry plant tissues or plant cells to knock out or mutate the PaPME2 gene in the sweet cherry; (3) and breeding and screening to obtain a new variety of the transgenic sweet cherry with hard meat and storage and transportation resistance.

In view of the research, the PaPME2 is determined to be a key gene related to the softening of the sweet cherry fruits, some ideas and experimental bases are provided for further analyzing the physiological functions and action mechanisms of a PaPME gene family in the process of the softening of the sweet cherry fruits, theoretical bases are provided for determining the functions of the sweet cherry pectin methylesterase gene and the action in the process of the softening of the fruits, and gene resources are provided for cultivating new sweet cherry varieties with hard meat and tolerance in storage and transportation.

Definitions of terms to which the invention relates

Unless defined otherwise, 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 the recombinant host cell, e.g., direct uptake, transduction, f-pairing, or other methods known in the art. The exogenous polynucleotide may remain as a non-integrating vector, such as a plasmid, or may integrate into the host genome.

The term "polynucleotide" or "nucleotide" means deoxyribonucleotides, deoxyribonucleosides, ribonucleosides, or ribonucleotides and polymers thereof in either single-or double-stranded form. Unless specifically limited, the term encompasses 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, and the like). Unless otherwise specified, a particular nucleic acid sequence also implicitly encompasses conservatively modified variants thereof (including but not limited to) degenerate codon substitutions) and complementary sequences as well as the sequence explicitly specified. In particular, degenerate codon substitutions may be achieved by generating sequences in which the 3 rd position of one or more selected (or all) codons is substituted with mixed base 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. Probess 8:91-98(1994) ].

The term "operably linked" refers to a functional linkage between two or more elements that may be operably linked and may or may not be contiguous.

The term "conversion": a method for introducing a heterologous DNA sequence into a host cell or organism.

The term "expression": transcription and/or translation of endogenous genes or transgenes in plant cells.

The term "coding sequence": a nucleic acid sequence transcribed into RNA.

The term "recombinant plant expression vector": one or more DNA vectors for effecting plant transformation; these vectors are often referred to in the art as binary vectors. Binary vectors, together with vectors with helper plasmids, are most commonly used for agrobacterium-mediated transformation. Binary vectors typically include: cis-acting sequences required for T-DNA transfer, selectable markers engineered to be capable of expression in plant cells, heterologous DNA sequences to be transcribed, and the like.

Drawings

FIG. 1.12 expression patterns of PaPMEs genes in fruit development.

FIG. 2 TRV mediated detection of the relative expression of PaPMEs genes in sweet cherry fruits.

FIG. 3 variation of fruit firmness and soluble solids content of sweet cherry fruits with PaPMEs gene silencing.

FIG. 4. changes in soluble pectin, cellulose content of sweet cherry fruits with PaPMEs gene silencing.

Detailed Description

The invention is further described below in conjunction with specific embodiments, the advantages and features of which will become apparent from the description. These examples are illustrative only and do not limit the scope of the present invention in any way. It will be understood by those skilled in the art that various changes in form and details may be made therein without departing from the spirit and scope of the invention, and that such changes and modifications may be within the scope of the invention.

Experimental example 1VIGS method for silencing PaPMEs gene in sweet cherry fruit and testing influence on fruit softening

1. Materials and methods

1.1 materials

Plant material: the European sweet cherry cultivar 'Zao hong zhu' is from a cherry germplasm resource garden of Zhengzhou fruit tree institute of Chinese academy of agricultural sciences, the stock is 'ZY-1', the tree age is 6 years, and the tree body grows normally.

Strain and TRV viral vector: VIGS vector tobacco rattle virus vector (TRV) pTRV1 and pTRV2 were offered by professor Liuyule, university of Qinghua, and Agrobacterium tumefaciens strain GV3101 was stored in the laboratory.

1.2 test methods

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) was designed with 16 overlapping regions (linkers reverse complementary to each other with the EcoRI and KpnI linearized pTRV2 fragments), respectively, and the target fragment was selected to silence only the sequence of a single PaPME gene, but not other PaPMEs family sequences.

TABLE 1 primer sequences

Respectively amplifying 12 PaPMEs fragments by using sweet cherry cDNA as a template and utilizing In-Fusion^TMHD Cloning kit (Clontech, Mount-ain View, CA, United States) constructs fragments of 12 PaPMEs respectively to construct pTRV2 linearized by EcoRI and KpnI double digestion, which are named as pTRV2-PaPME1-12 respectively, and transfers the pTRV 5 alpha competence into escherichia coli DH5 alpha, picks up positive strains, and transfers 12 pTRV2-PaPME1-12 vectors into agrobacterium GV3101 for standby after PCR identification, double digestion identification and correct sequencing.

1.2.3 transient transformation of sweet cherry fruits Using the VIGS technique

The VIGS method of sweet cherry fruits is carried out by referring to the method of Qixilian et al (Qixilian, Li Ming, Liu Smart, Song Lu, 2018.TRV mediated establishment of VIGS system of European sweet cherry fruits, fruit tree academy, 35(11):1309-1315) and carrying out three biological repetitions.

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

Extracting total RNA of a sweet cherry fruit sample, performing reverse transcription to obtain cDNA, regulating the cDNA content of different samples by taking a Histone2(Pav _ sc0000671.1) gene of sweet cherry as an internal reference, and detecting the expression level of related PaPMEs genes after silencing by using gene specific primers of the PaPMEs to PaPME1-12-J-F/R (Table 1).

The qPCR reaction was performed on ABI7500 PCR thermocycler (Applied Biosystems, Foster City, CA, United States), and analyzed using the TransStart Top Green qPCR SuperMix (beijing holo gold biotechnology limited, beijing, china) kit with the Histone2(Pav _ sc0000671.1) gene of sweet cherry as an internal reference. And three biological replicates were performed, and the average was taken.

1.2.5 measurement of fruit hardness and soluble solids

The fruit hardness GY-4 durometer measurement is in kg cm^-2. Randomly selecting 5 fruits each time, selecting 2 points at different parts of each fruit for measurement, repeating the measurement for three times, and taking an average value. The soluble solid content was measured by a handheld brix meter (PAL-1, aituo, japan) by measuring the soluble solid content (%) and repeating three times, and the average value was taken.

1.2.6 measurement of soluble pectin and cellulose

A method for measuring soluble pectin by reference to Caojiakang and the like adopts a carbazole sulfuric acid colorimetric method (Caojiakang, ginger microwave, Zhao Yumei 2007. guidance of physiological and biochemical experiments after fruit and vegetable harvest. China light industry Press) to measure absorbance at 530 nm; the cellulose content is determined by reference to the method of Wangcoquini, and the absorbance is determined at 620nm by adopting anthrone colorimetric method (Wangcoquini 2006 plant physiological and biochemical test principle and technology education publisher, pp: 122-126.).

1.2.7 data processing

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

2 results and analysis

2.1 analysis of expression Pattern of pectin methylesterase Gene family in fruit ripening and softening

The qPCR analysis result of the PaPMEs gene family shows that: the gene expression levels of the PaPME2 and PaPME9 genes are low in the early fruit development stage (the first expansion stage and the sclerous stage), the gene expression levels are in a remarkable up-regulation trend along with the fruit development, and the PaPME2 gene reaches the maximum value at the terminal stage of fruit ripening and softening; the expression level of the PaPME9 gene is highest in the early stage of fruit ripening and softening (42 days after flowers), and then is reduced (figure 1). The expression levels of other 10 PaPMEs genes (PaPME1, PaPME3, PaPME4, PaPME5, PaPME6, PaPME7, PaPME8, PaPME10, PaPME11 and PaPME12) are lower in the fruit development process, and the expression of the genes does not show a certain rule. For example, the gene expression levels of the PaPME6, PaPME8, PaPME10, PaPME11 and PaPME12 genes are very low during the whole period of fruit development; the expression level of the PaPME1, the PaPME3, the PaPME4, the PaPME5 and the PaPME7 genes is low in the early fruit development stage, the expression of the genes is up-regulated along with the development trend of fruits, and the expression level of the genes shows a peak in the middle or later fruit development stage and is then down-regulated (figure 1).

RT-PCR detection of VIGS silencing sweet cherry fruit PaPMEs gene family

The silencing efficiency of 12 PaPMEs of sweet cherry fruits is respectively detected by adopting a semi-quantitative RT-PCR method, and because only a specific target fragment of a single PaPME gene is amplified (conservative sequence information of a PaPMEs gene family is avoided) in the method of the test, the test only needs to respectively detect whether the single PaPME gene is silenced. And respectively extracting the total RNA of the sweet cherry fruits after 10 days of inoculation, and carrying out RT-PCR analysis by taking the sweet cherry Histone2 as an internal reference gene.

And (3) displaying a detection result: the expression level of mRNA of 12 PaPMEs genes in the respectively infected sweet cherry fruits is obviously reduced (figure 2). And qualitative analysis of quality One software shows that the expression level of the sweet cherry fruits with the silenced PaPMEs gene is reduced by 60-85% compared with that of negative control (figure 2). The experimental results show that the PaPMEs genes in the sweet cherry fruits are effectively silenced respectively.

2.3 Effect of PaPMEs family silencing on sweet cherry fruit softening

2.3.1 Effect on fruit firmness and soluble solids

Fruit hardness and soluble solids content are important indicators reflecting fruit ripening and softening. To analyze the role of PaPMEs genes in fruit ripening and softening, the present experiment analyzed the changes in fruit firmness and soluble solids content of 12 PaPME gene silenced sweet cherry fruits, and the results showed: compared with a negative control, the fruit hardness of the sweet cherry fruit with the PaPME2 gene silencing effect is obviously higher than that of the negative control; likewise, the soluble solids content of PaPME2 gene-silenced sweet cherry fruits was significantly lower than that of the negative control (fig. 3). While the fruit firmness and soluble solids content of other PaPMEs gene-silenced sweet cherry fruits were not significant compared to the control (fig. 3). The experimental results show that: the PaPME2 gene is closely related to fruit softening of sweet cherries.

2.3.2 Effect on soluble pectin content

The content of soluble pectin in the fruits is closely related to the mature softening of the fruits, and in order to further analyze the influence of PaPMEs genes on the mature softening of the fruits, the test carries out measurement and analysis on the content of soluble pectin in 12 sweet cherry fruits with PaPME genes silenced.

The results of the measurement and analysis are shown in FIG. 4: compared with the control group, the content of soluble pectin in sweet cherry fruits with the silenced PaPME2 gene is obviously lower than that in the control group; while the content of soluble pectin in other sweet cherry fruits with PaPMEs gene silenced was not significant compared to the control (fig. 4). The experimental results show that: the expression level of the PaPME2 gene in the sweet cherry fruit is closely related to the content of soluble pectin in the fruit, and further influences the ripening and softening of the fruit.

2.3.3 Effect on cellulose content

In the fruit ripening and softening process, the content of cellulose is also an important evaluation index for reflecting the fruit ripening and softening. To further clarify the function of the PaPMEs gene in the ripening and softening process of fruits. The test assay analyzed the cellulose content in 12 sweet cherry fruits with PaPMEs gene silenced.

The analysis results show that: the cellulose content of 12 sweet cherry fruits with PaPMEs gene silenced is basically consistent with that of a control (figure 4), and the expression level of the PaPMEs gene of the sweet cherry fruits is unrelated to the change of the cellulose content of the fruits.

Sequence listing

<110> Zhengzhou fruit tree institute of Chinese academy of agricultural sciences

Application of <120> sweet cherry PaPME2 gene in regulating and controlling ripening or softening of sweet cherry fruits

<130> HN-2002-190826A

<160> 12

<170> SIPOSequenceListing 1.0

<210> 1

<211> 627

<212> DNA

<213> Prunus avium L

<400> 1

atgggagctc aaactttcac aaccccaacc tcaaattcaa acaatgctct cacatttctc 60

ctcccaattc tcctattgat cttcatgcca aacatgcaca caaccttggc aacttcttct 120

tcttctcaga cctacaaaac ctacgtcaaa actgcctgca acacaaccac atacccacta 180

atttgctaca aatctctctc ctcatacgcc tccaaagtca aatccgatcc tcataagctt 240

tgtacctatg ccctctccgt taccctaaaa gcagcaaaaa acgcctcttc cgtagtttca 300

aagctatata aaaatacagg attaacccca tccgagaaag gggtggtcga ggattgtata 360

gacaatatta aggactccat cgacgagctc aaacagtcag tgagttccat gagcaatttg 420

ggggtttcgg gttccgacgt gcaattacaa ttggatgata taaagacttg ggttagcgcc 480

gtcatcacgg atgacgctac ttgcacggat ggatttgatg gtgtgaaggt aagcaccgcg 540

gttaagaccg ccatcaagaa cagcattgtg aatgctgcta ggttggctag caatgctctt 600

tctctcattg acagcctcat ttactag 627

<210> 2

<211> 747

<212> DNA

<213> Prunus avium L

<400> 2

atggcaccat ttcttgcttc caacctcatc accatcatca tgatcacgat catcacaacc 60

tcaccgcaac tcgttctgac tcacccgaac ctcatccctc cttacctctc ccaactcgac 120

acgtggatcg tccacaacat gagggaccac gccaaccgca aggccaccca agccactctc 180

cacttcgact ctaaattgct ctccgccgag gacgccgtca aaataataac agtcaagcaa 240

gacggcactg gagacttcaa aacagtcact gacgccgtca acagcattcc gtcgtggaac 300

acgcgtcgcg tcgtggtgtt catcggcggg ggtgagtaca gagaaaagat tctggtcgac 360

tcgtcgcggc cgttcgtgac cttttacggg gataagaacg acgtgccgtc gatcacgttt 420

gatggcacgg cgttgaagta cgggacgtgg gatagtgcca cggtggccgt cgaggccgac 480

tacttcgtgg ccgtcaacat tgcgttcgtg gtaataataa taatacgtta ttatatgact 540

tgccgttttg ctaatattaa ttctgatgtc gtttttcatc tctgcatgac tgtgtattat 600

ggagagtaca agtgtatggg accgggttcg agctcaacgg gtcgggtcaa gtatgcaaag 660

atgttatctg atgaagaagc aaagcccttc ctcggcatga cttttatcag aggaacaaag 720

tgggttctcc cacctcccaa gctctga 747

<210> 3

<211> 741

<212> DNA

<213> Prunus avium L

<400> 3

atgccaacat tccaacacat tttctatgtt attattgctc ttcttctctt caattcaacc 60

caaacacaat gccacagcaa gggacttcga cctgggaatt cagctgggaa agtacatttg 120

accaagaaca tgacacaagc ccaattctca gaacagcaat tcatgaagtg ggtgaggttt 180

gttggaaggc tgaaacactc tgtgttcaag acagccaaaa ataagctctt cccttcttac 240

actctgcatg tggctaagaa ccctgctgct ggagacttca caaccattca ggacgccatt 300

gactctctcc cattcatcaa tctcttgaga gtggtcatca aggtccatgc aggggtctac 360

gcggagaagg ttaatatacc tccattgaaa tcatatataa ccatagaagg agcaggagca 420

gacaaaacaa ttgttcaatg gggagacact gctcaaacac catctggtcc aaagaaacaa 480

ccgatgggga ctttcaattc tgcaactttt gctgtgaatt ccccttattt cattgccaag 540

aacatcacat tcaagaacac aacatttaac tttggctgtt gtcaaatcat gaggactgtg 600

ttttatgggc aatacaagtg cactggacca ggagctcgct ttgcagggag ggtttcatgg 660

tctagagagc tcactgatga ggaagccaag ccatttattt cccttacctt catagatggt 720

tctgagtgga tcaaattgta a 741

<210> 4

<211> 1533

<212> DNA

<213> Prunus avium L

<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

aagggagttt 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 (10)

1. Use of the sweet cherry PaPME2 gene in regulating the ripening or softening of a sweet cherry fruit.

2. Use according to claim 1, wherein said fruit ripening or softening comprises: the fruit becomes soft, and the soluble solid content or soluble pectin content in the fruit is increased.

3. Use according to claim 1, characterized in that: the regulating and controlling the ripening or softening of the sweet cherry fruits comprises softening the fruits, and increasing the content of soluble solids or soluble pectin in the fruits.

4. Use according to claim 1, characterized in that: the regulating and controlling the ripening or softening of the sweet cherry fruits comprises delaying the softening of the fruits, and reducing the content of soluble solids or soluble pectin in the fruits.

5. Use according to claim 1, characterized in that: the nucleotide sequence of the sweet cherry PaPME2 gene is shown in SEQ ID No. 2.

6. A method of promoting ripening of a sweet cherry fruit comprising: (1) constructing a recombinant plant expression vector containing the PaPME2 gene; (2) the constructed recombinant plant is expressed and transformed into the sweet cherry, so that the PaPME2 gene is over-expressed or over-expressed in the sweet cherry.

7. A method for cultivating a new early-maturing sweet cherry variety is characterized by comprising the following steps: (1) constructing a recombinant plant expression vector containing the PaPME2 gene; (2) transforming the constructed recombinant plant expression vector into sweet cherry plant tissues or plant cells; (3) and breeding and screening to obtain a new variety of the transgenic sweet cherry with early fruit ripening.

8.A method for delaying the ripening of a sweet cherry fruit, comprising: constructing a gene editing vector or gene knockout vector of the PaPME2 gene, and knocking out or mutating the PaPME2 gene in the sweet cherry.

9. A method for cultivating a new variety of hard-meat sweet cherries which are durable to storage and transportation is characterized by comprising the following steps: (1) constructing a gene editing vector or a gene knockout vector containing the PaPME2 gene; (2) transforming the constructed gene editing vector or gene knockout vector into sweet cherry plant tissues or plant cells to knock out or mutate the PaPME2 gene in the sweet cherry; (3) and breeding and screening to obtain a new variety of the transgenic sweet cherry with hard meat and storage and transportation resistance.

10. The method according to any one of claims 6 to 9, wherein the nucleotide sequence of the sweet cherry PaPME2 gene is represented by SEQ ID No. 2.