Apple MdERF105 gene and application of protein coded by apple MdERF105 gene in regulation and control of apple powdering

Abstract

The invention discloses an application of an apple MdERF105 gene and a protein coded by the apple MdERF105 gene in regulation and control of apple powdering. The invention belongs to the field of gene engineering, and particularly relates to an application of an apple MdERF105 gene and a protein coded by the apple MdERF105 gene in regulation and control of apple powdering. The MdERF105 protein or the substance for regulating and controlling the activity or content of the protein or regulating and controlling the expression of the coding gene of the protein can be applied in the following aspects: 1) regulating and controlling the pulverization degree of plant pulp; 2) application in preparation of a product for regulating and controlling the pulverization degree of plant pulp; 3) application in cultivation of plants with changed pulverization degree of pulp; 4) application in preparation of products for cultivating plants with changed pulverization degree of pulp; and 5) application in plant breeding. By regulating and controlling the MdERF105 gene, the pulverization degree of the picked apple fruits can be controlled, the pulverization process of the picked fruits can be accurately regulated and controlled, and apple variety improvement can be carried out.

Description

Technical Field

[0001] The present invention belongs to the field of genetic engineering, and particularly relates to the application of the apple MdERF105 gene and the protein encoded thereby in regulating the flouriness of apples. Background Art

[0002] The apple industry is one of the important pillar industries of China's agricultural economy. The flouriness of apples is a texture change phenomenon that occurs during post - ripening and storage, presenting a soft, spongy, juice - lacking, and gritty taste when chewed. Floury apples, due to their soft and easily swallowable pulp, are especially suitable for people with limited chewing ability such as the elderly and infants, and have characteristics such as being suitable for specific consumption scenarios and having stable processing. For example, Huaniu and Huangyuanmai apples are used in baby food processing due to their floury texture, while Qinguan apples are commonly used in baking (apple pies, purees) and canned food production.

[0003] However, there are obvious differences in the time of flouriness occurrence among different apple varieties, which results in consumers and processing enterprises being unable to control the apples to reach the ideal floury state in a timely manner, affecting the consumption experience and production cycle. In the prior art, the regulation of post - harvest flouriness of apples relies on natural selection, bud mutation selection, or optimization of storage conditions, which have problems such as long cycle and uncontrollability, and there is an urgent need for precise regulation means based on molecular mechanisms.

[0004] The occurrence of fruit flouriness is accompanied by the dissolution of the middle lamella and the rupture of the primary cell wall, which is closely related to pectin metabolism. As one of the largest transcription factor families in plants, AP2 / ERF can interact with pectin metabolism - related genes such as PG, GAL, and PME to regulate the texture of fruits, but the regulation technology of ERF transcription factors on the flouriness of apple fruits is still unknown. Summary of the Invention

[0005] The technical problem to be solved by the present invention is how to improve the degree of fruit pulp flouriness and soften the fruit pulp texture.

[0006] In order to solve the problems existing in the prior art, the present invention provides the application of a protein or a substance that regulates the activity or content of the protein or regulates the expression of the encoding gene of the protein in regulating the degree of fruit pulp flouriness of plants.

[0007] The application of the protein or the substance for regulating gene expression or the substance for regulating the activity or content of the protein provided by the present invention in any one of the following:

[0008] 1) Application in regulating the degree of fruit pulp flouriness of plants;

[0009] 2) Application in preparing a product for regulating the degree of fruit pulp flouriness of plants;

[0010] 3) Application in cultivating plants with changed degrees of fruit pulp flouriness;

[0011] 4) Use in the product for preparing plants with altered pulp powderiness degree;

[0012] 5) Use in plant breeding;

[0013] The protein is any of the following proteins:

[0014] a1) The protein with the amino acid sequence of SEQ ID No: 2;

[0015] a2) The protein with the amino acid sequence shown in SEQ ID No: 2 through substitution and / or deletion and / or addition of one or several amino acid residues and having the same function;

[0016] a3) The protein with more than 75% identity to the amino acid sequence defined in any of a1) or (a2) and having the same function;

[0017] a4) The fusion protein obtained by connecting a tag to the end of the protein defined in any of a1)-(a3).

[0018] The name of the protein described in a1) above is MdERF105.

[0019] In order to facilitate the purification or detection of the protein in a1), a tag protein can be connected to the amino terminus or carboxyl terminus of the protein composed of the amino acid sequence shown in SEQ ID No: 2 in the sequence listing.

[0020] The tag protein includes but is not limited to: GST (glutathione S-transferase) tag protein, His6 tag protein (His-tag), MBP (maltose-binding protein) tag protein, Flag tag protein, SUMO tag protein, HA tag protein, Myc tag protein, eGFP (enhanced green fluorescent protein), eCFP (enhanced cyan fluorescent protein), eYFP (enhanced yellow-green fluorescent protein), mCherry (monomeric red fluorescent protein) or AviTag tag protein.

[0021] The above-mentioned protein can be artificially synthesized, or its coding gene can be synthesized first and then obtained through biological expression.

[0022] Those skilled in the art can easily mutate the nucleotide sequence encoding the protein MdERF105 of the present invention by using known methods, such as directed evolution or point mutation. Those nucleotides that have been artificially modified and have 75% or more identity to the nucleotide sequence of the protein MdERF105 isolated from the present invention, as long as they encode the protein MdERF105 and have the function of the protein MdERF105, are all derived from the nucleotide sequence of the present invention and are equivalent to the sequence of the present invention.

[0023] The above-mentioned identity of 75% or more may be an identity of 80%, 85%, 90% or 95% or more.

[0024] In this article, identity refers to the identity of amino acid sequences or nucleotide sequences. The identity of amino acid sequences or nucleotide sequences can be determined using homology search sites on the Internet, such as the BLAST web page of the NCBI homepage website. For example, in Advanced BLAST 2.1, by using blastp as the program, setting the Expect value to 10, setting all Filters to OFF, using BLOSUM62 as the Matrix, setting the Gap existence cost, Per residue gap cost, and Lambda ratio to 11, 1, and 0.85 (default values) respectively, and performing a search to calculate the identity of a pair of amino acid sequences or nucleotide sequences, and then the identity value (%) can be obtained.

[0025] In this article, the above-mentioned identity of 80% or more may be an identity of at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99%.

[0026] In this article, the above-mentioned identity of 90% or more may be an identity of at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99%.

[0027] In the above application, the protein is derived from Malus pumila Mill.

[0028] In this article, the substance that regulates the activity and / or content of the protein may be a substance that regulates gene expression, and the gene encodes the protein MdERF105.

[0029] In the above text, the substance that regulates gene expression may be a substance that performs at least one of the following 6 regulations:

[0030] 1) Regulation carried out at the transcriptional level of the gene;

[0031] 2) Regulation carried out after transcription of the gene (that is, regulation of the splicing or processing of the primary transcript of the gene);

[0032] 3) Regulation of the RNA transport of the gene (that is, regulation of the transport of the mRNA of the gene from the nucleus to the cytoplasm);

[0033] 4) Regulation of the translation of said gene;

[0034] 5) Regulation of the mRNA degradation of said gene;

[0035] 6) Post-translational regulation of said gene (i.e., regulation of the activity of the protein translated from said gene).

[0036] In the present invention, the regulation may be up-regulation or enhancement or increase. The regulation may also be inhibition or decrease or down-regulation.

[0037] In this article, the up-regulation or enhancement or increase of the expression level of the coding gene of the aforementioned protein in the recipient plant, or / and the enhancement, increase or up-regulation of the activity and / or content of the coding gene of the above-mentioned protein are achieved by introducing the coding gene of the above-mentioned protein into the recipient plant.

[0038] In this article, the regulation of the expression of the coding gene of the protein may also be inhibition or decrease or down-regulation of the expression of the coding gene. The inhibition or decrease or down-regulation of the expression of the coding gene can be achieved by gene knockout or gene silencing.

[0039] The so-called gene knock-out refers to the phenomenon that a specific target gene is inactivated by gene editing technology. Gene knock-out inactivates a specific target gene through changes in DNA sequences, including but not limited to those based on Zinc-finger nucleases (ZFN), transcription activator-like effector nucleases (TALEN), and the CRISPR / Cas system. CRISPR (clustered regulatory interspaced short palindromic repeat) is a locus in the genome containing multiple short repeat sequences. The Cas9 protein can cleave the target sequence recognized by crRNA–tracrRNA under the mediation of RNA.

[0040] The gene silencing refers to the phenomenon that a gene is not expressed or is expressed at a low level without damaging the original DNA. Gene silencing enables a gene not to be expressed or to be expressed at a low level on the premise of not changing the DNA sequence. Gene silencing can occur at two levels. One is transcriptional gene silencing caused by DNA methylation, heterochromatinization, and position effect, etc. The other is post-transcriptional gene silencing, that is, at the post-transcriptional level of a gene, the gene is inactivated by specifically inhibiting the target RNA, including antisense RNA, co-suppression, quelling, RNA interference (RNAi), and translation inhibition mediated by microRNA (miRNA), etc.

[0041] In the above applications, the substance for regulating the expression of a gene or regulating the activity or content of the protein may be a biological material related to the protein described above, and the biological material may be any one of the following:

[0042] c1) A nucleic acid molecule encoding the protein described above;

[0043] c2) An expression cassette containing the nucleic acid molecule described in c1);

[0044] c3) A recombinant vector containing the nucleic acid molecule described in c1), or a recombinant vector containing the expression cassette described in c2);

[0045] c4) A recombinant microorganism containing the nucleic acid molecule described in c1), or a recombinant microorganism containing the expression cassette described in c2), or a recombinant microorganism containing the recombinant vector described in c3);

[0046] c5) A transgenic plant cell line containing the nucleic acid molecule described in c1), or a transgenic plant cell line containing the expression cassette described in c2);

[0047] c6) A transgenic plant tissue containing the nucleic acid molecule described in c1), or a transgenic plant tissue containing the expression cassette described in c2);

[0048] c7) A transgenic plant organ containing the nucleic acid molecule described in c1), or a transgenic plant organ containing the expression cassette described in c2);

[0049] e1) A nucleic acid molecule that inhibits or reduces or silences the expression of the protein-coding gene described above;

[0050] e2) An expression cassette containing the nucleic acid molecule described in e1);

[0051] e3) A recombinant vector containing the nucleic acid molecule described in e1), or a recombinant vector containing the expression cassette described in e2);

[0052] e4) A recombinant microorganism containing the nucleic acid molecule described in e1), or a recombinant microorganism containing the expression cassette described in e2), or a recombinant microorganism containing the recombinant vector described in e3);

[0053] e5) A transgenic plant cell line containing the nucleic acid molecule described in e1), or a transgenic plant cell line containing the expression cassette described in e2);

[0054] e6) A transgenic plant tissue containing the nucleic acid molecule described in e1), or a transgenic plant tissue containing the expression cassette described in e2);

[0055] e7) A transgenic plant organ containing the nucleic acid molecule described in e1), or a transgenic plant organ containing the expression cassette described in e2).

[0056] In the above applications, the nucleic acid molecule described in c1) can be a DNA molecule shown as any of the following:

[0057] d1) A DNA molecule with a nucleotide sequence shown as SEQ ID No: 3;

[0058] d2) A DNA molecule with a coding region sequence shown as SEQ ID No: 1;

[0059] d3) A DNA molecule having 90% or more identity with the nucleotide sequence defined by d1) or d2) and encoding the protein described above;

[0060] d4) A DNA molecule that hybridizes with the nucleotide sequence defined by d1) or d2) under stringent conditions and encodes the protein described above.

[0061] In the above applications, the nucleic acid molecule described in e1) can be a DNA molecule with a nucleotide sequence shown as SEQ ID No: 4.

[0062] The nucleic acid molecules described herein can be DNA, such as cDNA, genomic DNA, or recombinant DNA; the nucleic acid molecules can also be RNA, such as gRNA, mRNA, siRNA, shRNA, sgRNA, miRNA, or antisense RNA.

[0063] The vectors described herein are well-known to those skilled in the art and include, but are not limited to: plasmids, phages (such as λ phage or M13 filamentous phage, etc.), cosmids (i.e., cosmid plasmids), Ti plasmids, or viral vectors. Specifically, they can be vectors pSAK277 and TRV2.

[0064] An existing plant expression vector can be used to construct a recombinant expression vector containing the MdERF105 gene. The plant expression vector includes, but is not limited to, binary Agrobacterium vectors and vectors that can be used for plant microprojectile bombardment, etc. The plant expression vector may also contain the 3'-untranslated region of the foreign gene, that is, it contains a polyadenylation signal and any other DNA fragments involved in mRNA processing or gene expression. The polyadenylation signal can direct the addition of polyadenylic acid to the 3' end of the mRNA precursor. For example, it includes, but is not limited to, the genes of the Agrobacterium crown gall tumor-inducing (Ti) plasmid (such as the nopaline synthase Nos gene), and the 3'-transcribed untranslated regions of plant genes (such as soybean storage protein genes) all have similar functions.

[0065] When using the MdERF105 gene to construct a recombinant plant expression vector, any enhanced promoter or constitutive promoter can be added before its transcription start nucleotide, including but not limited to the cauliflower mosaic virus (CAMV) 35S promoter, the ubiquitin promoter of maize (ubiquitin), etc. They can be used alone or in combination with other plant promoters. In addition, when using the gene of the present invention to construct a plant expression vector, enhancers can also be used, including translation enhancers or transcription enhancers. These enhancer regions can be the ATG start codon or the adjacent region start codon, etc., but must be in the same reading frame as the coding sequence to ensure the correct translation of the entire sequence. The sources of the translation control signal and the start codon are extensive and can be natural or synthetic. The translation initiation region can come from the transcription initiation region or the structural gene.

[0066] For the convenience of identifying and screening transgenic plant cells or plants, the used plant expression vector can be processed, such as adding genes that can be expressed in plants and encode enzymes or luminescent compounds that can produce color changes (GUS gene, luciferase gene, etc.), antibiotic markers with resistance (gentamicin marker, kanamycin marker, etc.) or anti-chemical reagent marker genes (such as herbicide-resistant genes), etc. Considering the safety of transgenic plants, no selective marker gene can be added, and the transformed plants can be directly screened under stress.

[0067] Using any vector that can direct the expression of foreign genes in plants, the MdERF105 gene or gene fragment provided by the present invention can be introduced into plant cells or recipient plants to obtain transgenic cell lines and transgenic plants with altered flesh powderiness. The expression vector carrying the MdERF105 gene can transform plant cells or tissues by conventional biological methods such as using Ti plasmids, Ri plasmids, plant virus vectors, direct DNA transformation, microinjection, electroporation, Agrobacterium-mediated transformation, etc., and the transformed plant tissues can be cultivated into plants.

[0068] The present invention also provides a method for promoting the pulverization of plant pulp. The method includes step M, and step M is to enhance, increase or up-regulate the activity and / or content of the aforementioned protein in the target plant, and / or to enhance, increase or up-regulate the expression level of the coding gene of the aforementioned protein, so as to promote the pulverization of plant pulp.

[0069] In a specific embodiment, the method for up-regulating, enhancing or increasing the pulverization of plant pulp may include the following steps: introducing an expression vector containing the MdERF105 gene into a recipient plant to over-express the MdERF105 gene, so that the pulverization process of the pulp of the obtained transgenic plant is advanced.

[0070] The expression vector containing the MdERF105 gene is pSAK277-MdERF105, and the structure of the vector pSAK277-MdERF105 is described as follows: a DNA fragment with the sequence of SEQ ID No:1 is inserted between the two restriction enzyme sites of EcoRI and HindⅢ of the starting vector pSAK277, and other sequences of the vector pSAK277 are kept unchanged to obtain a recombinant vector. The pSAK277-MdERF105 vector can express MdERF105, and its amino acid sequence is SEQ ID No:2.

[0071] The present invention also provides a method for delaying the pulverization of plant pulp. The method includes step P, and step P is to inhibit, reduce or silence the activity and / or content of the aforementioned protein in the target plant, and / or to inhibit, reduce or silence the expression level of the coding gene of the aforementioned protein, so as to delay the pulverization of plant pulp.

[0072] In the above method, reducing the expression level and / or activity of the coding gene of the protein MdERF105 in the target plant may be: using gene mutation, gene knockout, gene editing or gene knockdown technology to reduce the activity or inactivate the coding gene of the protein MdERF105 in the genome of the target plant.

[0073] In a specific embodiment, the method for delaying the pulverization of plant pulp may include the following steps: introducing an expression vector that inhibits, reduces or silences the MdERF105 gene into a recipient plant to inhibit, reduce or silence the expression of the MdERF105 gene, so that the pulverization process of the pulp of the obtained transgenic plant is delayed.

[0074] The expression vector for suppressing, reducing or silencing the MdERF105 gene is TRV2-MdERF105. The structure of the vector TRV2-MdERF105 is described as follows: It is a recombinant vector obtained by inserting a DNA fragment with the sequence of SEQ ID No: 4 between the EcoRI and XhoI restriction enzyme sites of the starting vector TRV2, while keeping other sequences of the vector TRV2 unchanged.

[0075] The present invention provides a method for cultivating a plant with an advanced pulp powdering process, including enhancing, increasing or upregulating the expression of the coding gene of the above-mentioned protein and / or the content and / or activity of the above-mentioned protein in the target plant, or / and enhancing, increasing or upregulating the activity and / or content of the coding gene of the above-mentioned protein, to obtain a plant with an advanced pulp powdering process.

[0076] In one embodiment of the present invention, the breeding method for cultivating a plant with an advanced pulp powdering process includes the following steps:

[0077] (1) Construct a recombinant expression vector for enhancing, increasing or upregulating the coding gene of the protein described above;

[0078] (2) Transfer the recombinant expression vector constructed in step (1) into a receptor plant to obtain a plant with an earlier pulp powdering process than the receptor plant.

[0079] In the present invention, the purpose of plant breeding may include cultivating a plant with an advanced pulp powdering process or cultivating a plant with a delayed pulp powdering degree.

[0080] In the present invention, the improvement of the pulp powdering degree is specifically manifested as: the cross-section of the pulp is complete and dispersed, severely wrinkled, the texture of the pulp is softened, and the hardness is decreased.

[0081] In the present invention, the plant may be as follows:

[0082] N1) Dicotyledonous plants;

[0083] N2) Plants of the Rosales order;

[0084] N3) Plants of the Rosaceae family;

[0085] N4) Plants of the genus Malus;

[0086] N5) Apples.

[0087] The MdERF105 gene identified in the present invention that participates in the regulation of apple fruit powdering can control the degree of postharvest apple fruit powdering through variety improvement, precisely regulate the postharvest powdering process of the fruit, so as to meet the diverse needs of different consumers, shorten the market cycle of apple processed products, and has important application value. Description of the Drawings

[0088] Figure 1 It is a phenotypic observation diagram after transient infection of apple MdERF105. Among them, A is the phenotypic diagram after injecting the fruit; B is the scanning electron microscope diagram of the injected pulp tissue part. Among them, the MdERF105-OE group is the overexpression group of the MdERF105 gene; the MdERF105-VIGS group is the gene silencing group of MdERF105; pSAK277 is the control group of the overexpression empty plasmid; pTRV is the control group of the silencing empty plasmid.

[0089] Figure 2 It is the experimental result diagram of transient infection of apple MdERF105. Among them, A is the result of the degree of flouriness after overexpression and silencing of apple MdERF105; B is the result of the hardness change after overexpression and silencing of apple MdERF105; C is the relative expression level diagram of cell wall metabolism genes after overexpression and silencing of apple MdERF105. Among them, the MdERF105-OE group is the overexpression group of the MdERF105 gene; the MdERF105-VIGS group is the gene silencing group of MdERF105. Specific implementation manners

[0090] The present invention will be further described in detail below in conjunction with specific implementation manners. The provided embodiments are only for clarifying the present invention, rather than limiting the scope of the present invention. The following provided embodiments can be used as a guide for those of ordinary skill in the art to make further improvements, and do not constitute any limitation to the present invention in any way.

[0091] The experimental methods in the following embodiments are all conventional methods unless otherwise specified, and are carried out according to the techniques or conditions described in the literature in this field or according to the product instructions. The materials, reagents, etc. used in the following embodiments can be obtained from commercial channels unless otherwise specified.

[0092] Unless otherwise specified, the quantitative tests in the following embodiments are all set with three repeated experiments, and the results are averaged.

[0093] The "Red Star" apples in the following embodiments are purchased from Beijing Lizhen Mingyu Planting Center.

[0094] pSAK277 in the following examples has been described in: Xu R.R. et al. PsERF1B-PsMYB10.1-PsbHLH3 module enhances anthocyanin biosynthesis in the flesh-reddening of amber-fleshed plum (cv. Friar) fruit in response to cold storage. Horticulture Research, 2023, 10, doi: https: / / doi.org / 10.1093 / hr / uhad091. The biological material can be obtained from the applicant. This biological material is only used for repeating the experiments of the present invention and cannot be used for other purposes.

[0095] pTRV2 in the following examples has been described in: Fu D Q et al. Virus-induced gene silencing in tomato fruit. Plant Journal, 2005, 43: 299-308. The biological material can be obtained from the applicant. This biological material is only used for repeating the experiments of the present invention and cannot be used for other purposes.

[0096] Example 1: Cloning of Apple MdERF105 Gene

[0097] The coding sequence (CDS) of the MdERF105 gene in the apple cultivar 'Red Star' is SEQ ID No: 1, and the MdERF105 protein encoded by the amino acid sequence is SEQ ID No: 2. In the genomic DNA of the apple cultivar 'Red Star', the genomic gene encoding the MdERF105 protein is shown as SEQ ID No: 3 in the sequence listing.

[0098] RNA extraction and cDNA synthesis were performed using the flesh of 'Red Star' apples with powdery flesh after postharvest storage as the material. Using the specific primer pair MdERF105-F: 5'-ATGGCATCAGAAGACTCTTCAG-3' and MdERF105-R: 5'-TCAAGTAACCATGAGCTGAGAA-3', the coding region sequence of MdERF105 (nucleotide sequence is SEQ ID No: 1) was obtained by PCR amplification. The PCR reaction system was 50 μL, and the components included: PrimeSTAR Max Premix (2X) 25 μL, upstream and downstream primers (10 μmmol / L) 1 μL each, cDNA 2 μL, and H2O 21 μL. The PCR program was: pre-denaturation at 98 °C for 5 min, 35 cycles of 98 °C for 10 s, 58 °C for 15 s, and 72 °C for 30 s, and 72 °C for 5 min.

[0099] Example 2. Preparation of Overexpression Recombinant Plasmid and Overexpression Recombinant Genetically Engineered Bacteria Containing the MdERF105 Gene

[0100] Using the specific primer pair: 5’-ACTAGTGGATCCAAAGAATTC ATGGAGATGGAATCTGCT-3’ (the underlined part is the EcoRⅠ restriction site) and 5’-AGAAGTACTCTCGAG AAGCTT CTAATAGCTTTGTTCC-3’ (the underlined part is the HindⅢ restriction site), using the PCR product obtained in Example 1 as a template for PCR reaction. The PCR reaction system is 50 μL, and the components include: PrimeSTAR Max Premix (2X) 25 μL, upstream and downstream primers (10 μmmol / L) 1 μL each, cDNA 2 μL, H2O 21 μL. The PCR program is: pre-denaturation at 98℃ for 5 min, 35 cycles of 98℃ for 10 s, 58℃ for 15 s, and 72℃ for 30 s, and extension at 72℃ for 5 min.

[0101] After recovering the obtained PCR product, the target fragment was ligated into the pSAK277 vector digested with EcoRⅠ and HindⅢ using the Vazyme ClonExpress II One Step Cloning Kit, and verified by sequencing to obtain the recombinant plasmid pSAK277-MdERF105 containing the MdERF105 gene.

[0102] The structure of the pSAK277-MdERF105 vector is described as follows: a DNA fragment with the sequence of SEQ ID No:1 was inserted between the two restriction sites of EcoRⅠ and HindⅢ of the starting vector pSAK277, and the other sequences of the vector pSAK277 were kept unchanged to obtain the recombinant vector. The pSAK277-MdERF105 vector can express MdERF105, and its amino acid sequence is SEQ ID No:2.

[0103] Then the positive plasmid pSAK277-MdERF105 was introduced into the Agrobacterium strain GV3101 to obtain the recombinant genetically engineered bacteria containing the MdERF105 gene, that is, the Agrobacterium strain GV3101-OE-MdERF105 containing the overexpression vector.

[0104] At the same time, the pSAK277 plasmid was introduced into the Agrobacterium strain GV3101 to obtain the Agrobacterium strain GV3101-pSAK277.

[0105] Example 3. Preparation of Specific Fragment Silencing Recombinant Plasmid and Silencing Recombinant Genetically Engineered Bacteria Containing the MdERF105 Gene

[0106] Using specific primer pairs: 5’-ATTCTGTGAGTAAGGTTACC GAATTC AGAACGGATCACCGAAAG-3’ (the underlined part is the EcoRⅠ restriction site) and 5’-TCTTCGGGACATGCCCGGGC CTCGAG AACGGAGACAGAGGAGGC-3’ (the underlined part is the XhoI restriction site), using the product obtained in Example 1 as a template for PCR reaction. The PCR reaction system is 50 μL, and the components include: PrimeSTAR Max Premix (2X) 25 μL, upstream and downstream primers (10 μmmol / L) 1 μL each, cDNA 2 μL, H2O 21 μL. The PCR program is: pre-denaturation at 98 °C for 5 min, 35 cycles of 98 °C for 10 s, 58 °C for 15 s, and 72 °C for 30 s, and 72 °C for 5 min.

[0107] After recovering the obtained PCR product, the target fragment was ligated into the pTRV2 vector digested with EcoRⅠ and XhoI using the Vazyme ClonExpress II One Step Cloning Kit, and verified by sequencing to obtain a recombinant plasmid containing the specific fragment of the MdERF105 gene.

[0108] The structure of the pTRV2-MdERF105 vector is described as follows: It is a recombinant vector obtained by inserting a DNA fragment with the sequence of SEQ ID No:4 between the two restriction sites of EcoRⅠ and XhoI of the starting vector pTRV2, while keeping other sequences of the vector pTRV2 unchanged.

[0109] Then the positive plasmid pTRV2-MdERF105 was introduced into the Agrobacterium strain GV3101 to obtain a recombinant genetically engineered bacterium containing the specific fragment of the MdERF105 gene, that is, the Agrobacterium strain GV3101-TRV2-MdERF105 containing the silencing expression vector.

[0110] At the same time, the pTRV1 plasmid was introduced into the Agrobacterium strain GV3101 to obtain the Agrobacterium strain GV3101-TRV1. The pTRV2 plasmid was introduced into the Agrobacterium strain GV3101 to obtain the Agrobacterium strain GV3101-TRV2.

[0111] Example 4. Verification of the function of the MdERF105 gene

[0112] Apple pulp was selected to verify the function of apple MdERF105 in regulating flouriness.

[0113] The experimental steps are as follows: Resuspend GV3101-pSAK277, GV3101-pTRV1, GV3101-pTRV2, GV3101-OE-MdERF105, and GV3101-TRV2-MdERF105 in infection buffer (10 mmol / L MES, 10 mmol / L MgCl2, 100 μmol / L acetosyringone) to an OD 600 of 0.8. Mix equal volumes of the bacterial suspensions containing GV3101-pTRV1 and GV3101-pTRV2, GV3101-TRV2-MdERF105, add 2% red ink, and let it stand in the dark at room temperature for 3 hours.

[0114] Select Redstar apples at commercial maturity, create a pinhole with a depth of 2 - 3 cm using a 1 mL disposable sterile syringe, and aspirate 200 μL from the infection solutions containing GV3101-pSAK277, GV3101-pTRV1 + pTRV2, GV3101-OE-MdERF105, and GV3101-VIGS-MdERF105 (GV3101-pTRV1 + TRV2-MdERF105). Make 3 injection holes along the equatorial plane of each fruit, and use the empty vector as a negative control. Slowly push the needle into the fruit part to inject the infection solution, and use a round sticker to mark the position of the pinhole. Incubate in the dark at room temperature for 24 h, then place it in an environment with a temperature of 23 °C and an air humidity of 70% for cultivation. Regularly collect the pulp tissue in the area near the infection during storage, and use a scanning electron microscope to observe the microscopic morphology of the pulp tissue to screen and identify positive fruits ( Figure 1 in A).

[0115] Take 3 apples, longitudinally cut the pulp tissue 5 mm under the peel along the equatorial part, with a volume of 4×3×2 mm, and immediately fix it in 3 mL of phosphate buffer (0.1 mol / L, pH 7.2) containing 2% paraformaldehyde (w / v) and 0.1% glutaraldehyde (v / v), and store it at 4 °C for later use. Wash the sections with phosphate buffer and dehydrate them in ethanol with a concentration gradient of 10% - 100%. Dry them with a critical point dryer using liquid CO2 as the replacement liquid. Paste the dried sections on the surface of a positive aluminum oxide metal stage with double-sided tape, coat them with a gold film using a sputter coater, and store them in a desiccator. Observe the surface morphology of the samples under a scanning electron microscope (S-3400N, Hitachi), with an accelerating voltage of 15 kV, and take the most representative photos at a magnification of 200×.

[0116] The phenotypic results are as Figure 1As shown in B: The SEM results showed that the apple pulp tissue at 0 d was arranged neatly, tightly and firmly, with very small cell gaps. At 9 d, the cross-section of the pulpy flesh in the powdered state was complete and dispersed, and it shrank severely. The Red Star apples injected with Agrobacterium tumefaciens GV3101-OE-MdERF105 showed obvious powdering at 5 d, and the degree of powdering further increased at 9 d; the cross-section of the Red Star apples injected with Agrobacterium tumefaciens GV3101-VIGS-MdERF105 remained relatively neat at 9 d after harvest, without obvious powdering phenomenon.

[0117] Example 5: Determination of the degree of apple pulp powdering

[0118] The Fruit Disc Shaking Method was used to evaluate the degree of apple powdering. Three discs (10 mm in diameter and 5 mm in thickness) were taken from the equatorial plane of the apple fruit with a puncher. The discs were soaked in 12% sucrose solution for 45 min under vacuum conditions. After soaking, the excess water was wiped off with a gauze, and the discs were weighed and recorded as Wi. Then the discs were transferred to a test tube containing 10 mL of 12% sucrose solution and oscillated in a continuous oscillator for 7 h, and reweighed and recorded as Ws. The degree of powdering was calculated using the following formula. Ten fruits were taken at each sampling point, and the results were averaged and expressed as %.

[0119] Degree of powdering (%) = (Wi - Ws) / Wi × 100.

[0120] The results were as Figure 2 shown in A: The degree of powdering of the control group pulp showed an upward trend during postharvest storage, reaching 35% at 9 d, and it was considered to have reached the powdered state. The degree of powdering of the Red Star apple pulp tissue injected with Agrobacterium tumefaciens GV3101-OE-MdERF105 exceeded 35% at 5 d and exceeded 40% at 9 d, indicating that the powdering phenomenon had occurred at 5 d; compared with the control, injecting Agrobacterium tumefaciens GV3101-VIGS-MdERF105 significantly reduced the degree of powdering of Red Star apples at 9 d, only about 20%, and did not reach the powdered state.

[0121] The results showed that MdERF105 had a significant positive promoting effect on the occurrence of apple pulp powdering.

[0122] Example 6: Determination of the hardness of apple pulp

[0123] Six apples were taken at each sampling point. Three points were symmetrically taken along the equatorial part and peeled. The fruits were fixed on the plate of a Brookfield CT3 physical property analyzer, and the texture of the fruits was analyzed using the TPA mode. A cylindrical probe TA42 with a diameter of 3 mm was selected, the initial force was 0.4 N, the test rate was 60 mm / min, and the compression deformation degree was 10%. The hardness (Hardness, N) was recorded and analyzed using TexturePro CT software: the peak pressure reached when the sample was extruded for the first time.

[0124] The results are as Figure 2 shown in B: The hardness of the pulp of the control group apples showed a continuous downward trend during postharvest storage and decreased to 5 N at 9 d. The hardness of the pulp tissue of Red Star apples injected with Agrobacterium tumefaciens GV3101 - OE - MdERF105 was significantly lower than that of the control group at 5 d. The hardness of Red Star apples injected with Agrobacterium tumefaciens GV3101 - VIGS - MdERF105 decreased relatively slowly during postharvest storage. Especially after 5 d, the hardness was still significantly higher than that of the control group at 9 d and 13 d.

[0125] The results indicate that MdERF105 plays an important role in the softening of the pulp texture of Red Star apples.

[0126] Example 7, Real - time Fluorescent Quantitative PCR Detection of the Relative Expression Level of Genes

[0127] Approximately 0.3 g of the apple pulp sample treated in Example 4 was taken and ground using liquid nitrogen for ribonucleic acid RNA extraction. RNA extraction was performed using the Tiangen RNAprep Pure Polysaccharide and Polyphenol Plant Total RNA Extraction Kit. The quality of RNA was detected using a 1.2% agarose gel. The RNA concentration was detected using a NanoDrop instrument. The synthesis of the first - strand cDNA was performed using the One - Step Transcript gDNA Removal and cDNA Synthesis SuperMix Kit (TransGen Biotech Co., Ltd., Beijing). The reverse transcription conditions were as follows: incubation at 42 °C for 15 min; heating at 85 °C for 5 s for heat inactivation of RT / RI and gDNA Remover. The cDNA was taken out and stored at - 20 °C for later use. The real - time fluorescent quantitative PCR system was 20 μL, containing approximately 100 ng of cDNA, 1 μL of each primer, and 10 μL of 2×TS Reaction Mix (TransGen Biotech, Beijing). The amplification program was as follows: 95 °C for 3 min; 95 °C for 10 s, 60 °C for 30 s for 40 cycles. EF - 1α and Mdactin were selected as internal reference genes. The expression level was calculated using the 2 - ΔΔCT method.

[0128] Table 1. Primer Information for Real - time Fluorescent Quantitative PCR Detection

[0129]

[0130] The results are as Figure 2 shown in C below: After separately injecting Agrobacterium tumefaciens GV3101-OE-MdERF105, the relative expression level of MdERF105 in apple pulp was significantly higher than that of the control. After injecting Agrobacterium tumefaciens GV3101-VIGS-MdERF105, the relative expression level of MdERF105 in apple pulp was significantly lower than that of the control, indicating that MdERF105 was successfully transiently infected into the pulp and expressed. In addition, after overexpressing MdERF105, the relative expression levels of MdPG, MdPL5, and MdAF in the pulp tissue increased significantly. After silencing MdERF105, the relative expression levels of MdPG, MdPL5, and MdAF in the pulp tissue decreased significantly, indicating that MdERF105 may interact with the above cell wall metabolism genes.

[0131] The present invention has been described in detail above. For those skilled in the art, without departing from the spirit and scope of the present invention and without unnecessary experiments, the present invention can be implemented within a relatively wide range under equivalent parameters, concentrations, and conditions. Although specific embodiments of the present invention are given, it should be understood that the present invention can be further improved. In short, according to the principle of the present invention, this application intends to cover any modifications, uses, or improvements to the present invention, including changes made using conventional techniques known in the art that depart from the scope disclosed in this application.

Claims

1. Use of a protein or a substance that regulates the activity or content of the protein or regulates the expression of the encoding gene of the protein in any of the following: 1) Use in regulating the degree of powdery texture of plant pulp; 2) Use in preparing a product for regulating the degree of powdery texture of plant pulp; 3) Use in cultivating plants with altered degree of powdery texture of pulp; 4) Use in preparing a product for cultivating plants with altered degree of powdery texture of pulp; 5) Use in plant breeding; The protein is any of the following proteins: a1) A protein with an amino acid sequence of SEQ ID No: 2; a2) A protein obtained by substituting and / or deleting and / or adding one or several amino acid residues in the amino acid sequence shown in SEQ ID No: 2 and having the same function; a3) A protein having more than 80% identity with the amino acid sequence defined in a1) or a2) and having the same function; a4) A fusion protein obtained by connecting a tag to the end of the protein defined in any of a1)-a3).

2. The application according to claim 1, characterized in that: The protein is derived from apple.

3. The application according to claim 1 or 2, characterized in that: The substance that regulates the activity or content of the protein or regulates the expression of the encoding gene of the protein is a biological material related to the protein in the application described in claim 1 or 2, and the biological material is any of the following: c1) A nucleic acid molecule encoding the protein; c2) An expression cassette containing the nucleic acid molecule described in c1); c3) A recombinant vector containing the nucleic acid molecule described in c1), or a recombinant vector containing the expression cassette described in c2); c4) A recombinant microorganism containing the nucleic acid molecule described in c1), or a recombinant microorganism containing the expression cassette described in c2), or a recombinant microorganism containing the recombinant vector described in c3); c5) A transgenic plant cell line containing the nucleic acid molecule described in c1), or a transgenic plant cell line containing the expression cassette described in c2); c6) A transgenic plant tissue containing the nucleic acid molecule described in c1), or a transgenic plant tissue containing the expression cassette described in c2); c7) A transgenic plant organ containing the nucleic acid molecule described in c1), or a transgenic plant organ containing the expression cassette described in c2); e1) A nucleic acid molecule that inhibits or reduces or silences the expression of the protein-encoding gene; e2) An expression cassette containing the nucleic acid molecule described in e1); e3) A recombinant vector containing the nucleic acid molecule described in e1), or a recombinant vector containing the expression cassette described in e2); e4) A recombinant microorganism containing the nucleic acid molecule described in e1), or a recombinant microorganism containing the expression cassette described in e2), or a recombinant microorganism containing the recombinant vector described in e3); e5) A transgenic plant cell line containing the nucleic acid molecule described in e1), or a transgenic plant cell line containing the expression cassette described in e2); e6) A transgenic plant tissue containing the nucleic acid molecule described in e1), or a transgenic plant tissue containing the expression cassette described in e2); e7) A transgenic plant organ containing the nucleic acid molecule described in e1), or a transgenic plant organ containing the expression cassette described in e2).

4. The application according to claim 3, characterized in that: The nucleic acid molecule described in c1) is any of the following DNA molecules, d1) A DNA molecule with a nucleotide sequence of SEQ ID No: 3; d2) The coding region sequence is the DNA molecule shown in SEQ ID No: 1; d3) A DNA molecule having 90% or more identity with the nucleotide sequence defined in d1) or d2) and encoding the protein described in claim 1; d4) A DNA molecule that hybridizes with the nucleotide sequence defined in d1) or d2) under stringent conditions and encodes the protein described in claim 1.

5. A method for promoting the degree of powdery texture of plant pulp, characterized in that: The method includes step M, and step M is to enhance, increase or up-regulate the activity and / or content of the protein described in claim 1 or 2 in the target plant, and / or, enhance, increase or up-regulate the expression level of the coding gene of the protein described in claim 1 or 2 to promote the flesh powderiness of the plant.

6. A method for delaying the degree of flesh powderiness of plants, characterized in that: The method includes step P, and step P is to inhibit, reduce or silence the activity and / or content of the protein described in claim 1 or 2 in the target plant, and / or, inhibit, reduce or silence the expression level of the coding gene of the protein described in claim 1 or 2 to delay the degree of flesh powderiness of the plant.

7. A plant breeding method for advancing the process of pulp powderization, characterized in that: A plant including enhancing, increasing or up-regulating the expression level of the coding gene of the protein described in claim 1 or 2 in the recipient plant, and / or, the activity and / or content of the protein, and the flesh powderiness process of the plant is advanced earlier than that of the recipient plant.

8. The method according to claim 7, wherein: It includes the following steps: 1) Construct a recombinant expression vector that enhances, increases or up-regulates the coding gene of the protein described in claim 1 or 2; 2) Transfer the recombinant expression vector constructed in step 1) into the recipient plant to obtain a plant whose flesh powderiness process is earlier than that of the recipient plant.

9. The protein described in claim 1 or 2 and / or the biological material described in claim 3 or 4.

10. The application according to any one of claims 1 to 4, and / or, the method according to any one of claims 5 to 8, characterized in that: The plant is any one of the following: N1) Dicotyledonous plants; N2) Plants of Rosales; N3) Plants of Rosaceae; N4) Plants of Malus; N5) Apple.

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