CN114107317A

CN114107317A - Peach fruit ethylene response factor PpRAP2.12 gene and cloning method and application thereof

Info

Publication number: CN114107317A
Application number: CN202111232471.1A
Authority: CN
Inventors: 邵兴锋; 曹可枫; 韦莹莹; 陈义; 姜舒
Original assignee: Ningbo University
Current assignee: Ningbo University
Priority date: 2021-10-22
Filing date: 2021-10-22
Publication date: 2022-03-01
Anticipated expiration: 2041-10-22
Also published as: CN114107317B

Abstract

The invention discloses a peach fruit ethylene response factor PpRAP2.12 gene and a cloning method and application thereof, which are characterized in that the peach fruit ethylene response factor PpRAP2.12 has a nucleotide sequence shown as SEQIDNO: 1 is shown in the specification; the amino acid sequence of the protein coded by the peach fruit ethylene response factor PpRAP2.12 is shown as SEQ ID NO: 2, the cloning method comprises the following steps: (1) extracting total RNA of peach fruits and performing reverse transcription to obtain cDNA serving as a template; (2) designing a primer according to the gene sequence of PpRAP2.12; (3) and (3) PCR amplification: the PpRAP2.12 gene amplification product is obtained by PCR amplification, and has the advantages that the PpRAP2.12 and PpVIN2 promoters have DNA-protein interaction, the PpRAP2.12 positively controls the transcription activity of PpVIN2, the expression level of the PpRAP2.12 is effectively increased in peach fruits, the gene expression and the activity of the acid invertase PpVIN2 can be obviously increased, and the decomposition of sucrose is increased.

Description

Peach fruit ethylene response factor PpRAP2.12 gene and cloning method and application thereof

Technical Field

The invention belongs to the field of plant molecular biology, and particularly relates to a peach fruit ethylene response factor PpRAP2.12 gene for regulating and controlling peach fruit acid invertase PpVIN2, and a cloning method and application thereof.

Background

Peach (A)Prunus persica) The rose fruit juice is a rose and peach plant, is easy to cause cold damage during refrigeration, and has the symptoms of internal browning, juice reduction, flocculent spoilage and the like, thereby losing the commodity value. Peach fruits are often accompanied by changes in soluble sugar content and activity of metabolic-related enzymes under low temperature stress. Sucrose is the soluble sugar with the highest content in peach fruits, and the content of the sucrose not only affects the taste and flavor of the peaches, but also affects the cold resistance of the peaches. The sucrose content in the peach fruits rapidly decreases under low-temperature storage, and the occurrence of cold damage is accompanied. The vacuole type acid invertase (VIN) is the most important enzyme participating in sucrose decomposition, wherein the PpVIN2 gene is induced by low temperature, plays an important role in the sucrose decomposition process and is closely related to the cold resistance of peach fruits.

The yeast two-hybrid system (Y2H) is a system in which two proteins to be studied are cloned (fused) into a DNA binding domain (DNA-BD) and a transcription Activation Domain (AD) of a transcription activator (e.g., GAL 4) of a yeast expression plasmid, respectively, to construct a fusion expression vector, and the interaction between the two proteins is analyzed on the basis of the expression product and a color reaction. The interaction protein PpVIN2 can be effectively screened out by a yeast two-hybrid system.

The plant transient overexpression technology (transient overexpression) is a technology for obtaining transient high-level expression of a target gene, can reflect the function of the gene, and has wide application in aspects of promoter analysis, protein interaction, gene function analysis and the like. A regulation element is added at the upstream of a target gene in an artificial construction mode, so that the gene can realize a large amount of transcription and translation under the condition of artificial control, and the overexpression of a gene product is realized, wherein the agrobacterium-mediated gene transient expression is simple and rapid and is widely applied. At present, the successful application of transient overexpression PpRAP2.12 in peach fruits is not available, so that the gene function analysis and the application of the target gene PpRAP2.12 transient overexpression in the peach fruits by an agrobacterium transformation method are particularly important.

Disclosure of Invention

The invention aims to solve the technical problem of providing a peach fruit ethylene response factor PpRAP2.12 gene for positively regulating and controlling acid invertase PpVIN2 of a peach fruit and participating in sucrose metabolism, and a cloning method and application thereof.

The technical scheme adopted by the invention for solving the technical problems is as follows: a peach fruit ethylene response factor PpRAP2.12 gene has a nucleotide sequence shown as SEQ ID NO: 1 is shown.

The amino acid sequence of the protein coded by the peach fruit ethylene response factor PpRAP2.12 gene is shown as SEQ ID NO: 2, respectively.

The method for cloning the peach fruit ethylene response factor PpRAP2.12 gene comprises the following steps:

(1) extracting total RNA of peach fruits and performing reverse transcription to obtain cDNA serving as a template;

(2) designing a primer according to the PpRAP2.12 gene sequence: the sequence of the upstream primer is as follows: 5 '-ATGACGTCGGATGGAGCCA-3', downstream primer sequence: 5 '-TTAACTTCGAGGCTTACCACTTCC-3';

(3) and (3) PCR amplification: the PpRAP2.12 gene amplification product is obtained by PCR amplification.

The application of the peach fruit ethylene response factor PpRAP2.12 gene in preparing the promoter for the activity of peach fruit acid invertase PpVIN 2.

The application of the peach fruit ethylene response factor PpRAP2.12 gene in cultivating a cold-resistant peach variety with PpRAP2.12 gene silencing.

Compared with the prior art, the invention has the advantages that:

1. the ethylene response factor PpRAP2.12 gene of peach fruit is proved for the first time to be combined with the promoter of acid invertase PpVIN2, and the activity of the promoter is positively regulated to influence the catabolism of sucrose.

2. The overexpression system of the PpAPp2.12 gene is successfully constructed in peach fruits for the first time, and for the 'Yulu' peach fruits which effectively increase the expression quantity of the PpAP2.12 gene, the activity of acid invertase VIN can be effectively increased, the decomposition of cane sugar is increased, the content of cane sugar serving as an anti-freezing protective agent is reduced, and thus the cold resistance of the fruits is reduced.

In conclusion, the invention provides a clone of a key gene PpAPp2.12 for regulating and controlling peach fruit acid invertase PpVIN2 and application thereof, proves that the PpAPp2.12 and PpVIN2 promoters have an interaction relationship between DNA and protein, and proves that PpRAP2.12 positively regulates and controls the transcription activity of PpVIN2 through a dual-luciferase experiment. The target gene PpAPp2.12 is over-expressed in peach fruits by an agrobacterium transient transformation method, and the gene expression and the enzyme activity of PpVIN2 can be obviously improved. The PpARP 2.12 gene has an effective function in the PpVIN2 mediated sucrose metabolic process, improves peach fruit varieties by silencing the gene, provides an important theoretical basis for improving the cold resistance of fruits and reducing the cold damage of the fruits, and has wide application prospect.

Drawings

FIG. 1 is a Y1H system demonstrating that PpRAP2.12 can bind the promoter of PpVIN2 gene;

FIG. 2 shows the results of dual luciferase experiments to verify that PpRAP2.12 promotes the transcription activity of PpVIN 2;

FIG. 3 shows that PpVIN2 gene expression and enzyme activity (A, B, C) are promoted by overexpression of PpRAP2.12 in peach fruits by an agrobacterium transient transformation method. Note: (A) analysis of PpRAP2.12 expression quantity after agrobacterium transient transformation, (B) analysis of PpVIN2 expression quantity after agrobacterium transient transformation, and (C) effect on VIN activity after successful agrobacterium transient transformation.

Detailed Description

The invention is described in further detail below with reference to the accompanying examples.

Detailed description of the preferred embodiment

Peach fruit ethylene response factor PpRAP2.12 gene clone and sequence analysis

1. Extracting total RNA from the Yulu peach fruit, and inverting the total RNA into cDNA serving as a PCR template; the method comprises the following specific steps: adopting EasttepTM total RNA extraction kit (Promega, United States) of Promega to extract total RNA of peach fruits, and then adopting HiScript II Q Select RT Super Mix for qPCR (Vazyme, Nanjing, China) kit to perform reverse transcription to obtain cDNA serving as a template of PCR reaction;

2. the online website NCBI-PRIMER (https:// www.ncbi.nlm.nih. gov/tools/PRIMER-blast /) was used to design specific amplification PRIMERs for the CDS region of the peach fruit PpAPp2.12 Gene (Gene ID: LOC 18783672), upstream PRIMER sequence: 5 '-ATGTGTGGAGGTGCTATAATATCCG-3', downstream primer sequence: 5 '-TCAGAAACCTCCCCCAATCAG-3';

3. and (3) PCR amplification: obtaining PpRAP2.12 gene amplification product through PCR amplification, wherein the reaction system of the PCR amplification is as follows: 1 muL cDNA, 12.5 muL 2 x Phanta Max Master Mix (PCR amplification high fidelity enzyme: 2 x Phanta Max Master Mix purchased from Nanjing Nodezam Biotech Co., Ltd.), 1 muL of each of the upstream and downstream primers, 9.5 muL ddH 2O; the PCR amplification procedure was: pre-denaturation at 95 ℃ for 3 min; denaturation at 95 ℃ for 15s, annealing at 60 ℃ for 15s, extension at 72 ℃ for 1min, and 35 cycles; completely extending at 72 ℃ for 5 min;

4. colony PCR and sequencing comparison: recovering and purifying PCR amplification products, connecting the PCR amplification products with a PMD18-T carrier, transferring escherichia coli DH5 alpha (CWBIO, Beijing, China), coating the products on an LB-Amp plate which is uniformly coated with X-Gal and IPTG, and performing inversion culture at 37 ℃ for overnight; and (3) sequencing after further verification of PCR (polymerase chain reaction), and obtaining a PpAPp2.12 sequence matched with peach fruit genome data, wherein the sequence is represented by SEQ ID NO: 1, and the following components:

ATGTGTGGAGGTGCTATAATATCCGATTTCATAGCGCCGACTCGGTCCCGGCTCACCGCTGACTACCTCTGGCCCGATCTCAAAAAACCCAGTTCGGATAAGCGGTTCTCGAAGCCTCTCAGGCCCGAAATCATTGACTTAGACCACGACTTCGAGGCTGATTTCCAGGAATTCAAGGACGAGTCTGATGTGGACGAAGATGATGAAATGGTTGAGGCCAAGCCCTCTGCTTTCTCTGCTGGAAAGCCCTCTTCTGCTCGTGGCTCTACGACTGTGAAATTAGTGGAGTTCAATGGGCAGGCTGAGAAATCTGCAAAGAGAAAGAGGAAGAACCAGTACAGGGGAATTCGCCAGCGCCCATGGGGTAAGTGGGCTGCAGAGATCCGAGACCCAAGGAAAGGGGTCCGGGTTTGGCTTGGAACTTTCAACACTGCAGAAGAAGCTGCAAGGGCTTATGATTCCGAGGCACGTAGAATTCGTGGCAAGAAAGCCAAGGTTAATTTCCCTGATGAAACCCCACGATCTTCTGCAAAGCGTTCTGTCAAGGCAAATCCTCAGAAAATGCTACCCAAGACAAACACGAATGCCGTTCAGCCTAACCTGAACCAGAATATCAATTTTGTGAATGACCCAAATCAGGACTACTACAATGCTATGGGTTTTCTGGATGAAAAGCCAGCAACTAATAACTTTGGGTTTATGTCCACCTTCCCGGCCAATGAGGATGTTGCGCTGAAATCCTCTACCCCATCCGATGCTGTCCCGCTGTATTTCAGCTCTGATCAGGGAAGCAATTCTTTTGATTGTTCTGACTTTGGCTGGGGAGAACAAGGTTCAAAGACTCCAGAAATATCATCTGTTCTTTCATGTGTTATGGAAGAAAGTGATGACTCACTGTTTCTGGAGGATGCTAGCCCAACGAAGAAACTGAAGTCTAACCCAGAGGATCTGGTGCCTGTTCAGGATAATGCAGGAAAGACACTGACTGATGAGCTCTCAGCTTTTGAGATGAAGTACTTTCAGACGCCATATCTTGATGGGAGCTGGGATGCTTCAGTGGACGCCTTCCTCAGCACAGATGCAACTCAGGATGGTGGCAACTCAGTGGACTTTTGGAGCTTCGATGACCTGATTGGGGGAGGTTTCTGA。

the amino acid sequence of the protein coded by the peach fruit ethylene response factor PpRAP2.12 gene is shown as SEQ ID NO: 2, as shown in the figure:

MCGGAIISDFIAPTRSRLTADYLWPDLKKPSSDKRFSKPLRPEIIDLDHDFEADFQEFKDESDVDEDDEMVEAKPSAFSAGKPSSARGSTTVKLVEFNGQAEKSAKRKRKNQYRGIRQRPWGKWAAEIRDPRKGVRVWLGTFNTAEEAARAYDSEARRIRGKKAKVNFPDETPRSSAKRSVKANPQKMLPKTNTNAVQPNLNQNINFVNDPNQDYYNAMGFLDEKPATNNFGFMSTFPANEDVALKSSTPSDAVPLYFSSDQGSNSFDCSDFGWGEQGSKTPEISSVLSCVMEESDDSLFLEDASPTKKLKSNPEDLVPVQDNAGKTLTDELSAFEMKYFQTPYLDGSWDASVDAFLSTDATQDGGNSVDFWSFDDLIGGGF。

detailed description of the invention

The yeast single-hybrid system (Y1H) was used to confirm that PpRAP2.12 can bind to the promoter of PpVIN2 gene

1. Construction and identification of decoy recombinant vector pAbAi-PpVIN2pro-A and prey recombinant vector pGADT7-PpRAP2.12 containing PpVIN2 promoter truncation (PpVIN 2 pro-A)

The PpVIN2 promoter contains abundant transcription factor binding sites, and because the PpVIN2 promoter sequence obtained by cloning is longer, and the front end of the promoter sequence contains a potential binding site GCC-box (ACCGAC) of PpRAP2.12, the PpVIN2 promoter is planned to be truncated, and the front end part of the promoter is taken and named as PpVIN2pro-A to verify that the PpVIN 2.12 transcription factor can be bound or not.

Specific amplification PRIMERs of peach fruit PpAPp2.12 Gene and PpVIN2 Gene promoter truncations (Gene IDs LOC18783672 and LOC18776102 respectively) are designed by using an online website NCBI-PRIMER (https:// www.ncbi.nlm.nih. gov/tools/PRIMER-blast /), wherein the two ends of the PRIMERs are respectively provided with a proper enzyme cutting site and a proper protection base (Table 1).

TABLE 1 construction of primer sequences for recombinant vectors pGADT 7-PpARAP2.12 and pAbAi-PpVIN2pro-A

Note: underlined indicates a protected base; the bold shows the restriction sites, SmaI, XhoI, NdeI and BamHI in this order

The reaction system of PCR amplification is 25 muL: 1 muL cDNA, 12.5 muL 2 x Phanta Max Master Mix, 1 muL each of the upstream and downstream primers, 9.5 muL LddH 2O. The PCR amplification program of the target fragments PpRAP2.12 and PpVIN2pro-A is pre-denaturation at 95 ℃ for 3 min; denaturation at 95 ℃ for 15s, annealing at 60 ℃ for 15s, and extension at 72 ℃ for 1min, (35 cycles); extension was complete for 5min at 72 ℃.

And recovering and purifying the PCR amplification product, connecting the PCR amplification product with a PMD18-T vector, transferring Escherichia coli DH5 alpha (CWBIO, Beijing, China), coating the Escherichia coli DH5 alpha (CWBIO, Beijing) on an LB-Amp plate uniformly coated with X-Gal and IPTG, carrying out inversion culture at 37 ℃ overnight, then picking out a single colony for PCR verification, and carrying out Plasmid extraction on a subclone positive bacteria sample with correct colony PCR identification and sequencing result by using an E.Z.N.A. Plasmid DNA Mini Kit I purchased from OMEGA according to the instruction.

The extracted plasmid and the vector to be connected are subjected to double enzyme digestion by corresponding restriction enzymes, and then are recovered and purified by an OMEGA Gel recovery and purification Kit Gel Extraction Kit. The promoter subcloned plasmid PpVIN2pro-A after restriction enzyme purification is connected with the yeast single-hybrid bait expression vector pAbAi through T4 DNA Ligase, and the PpRAP2.12 subcloned plasmid is connected with the yeast single-hybrid protein expression vector pGADT7 through T4 DNA Ligase, and the connection method refers to the instruction of T4 DNA Ligase (Takara, Japan). According to the requirements of DH5 alpha complete Cell (CWBIO, Beijing, China), the recombinant plasmid is respectively transferred into Escherichia coli DH5 alpha, then coated on LB-Amp/kana plates which are uniformly coated with X-Gal and IPTG, inverted cultured overnight at 37 ℃, single clone is selected for colony PCR identification, and the positive clone containing the target fragment is selected and sent to Shanghai bioengineering company Limited for sequencing. After the error is ensured, the bacterial liquid is propagated and preserved, and plasmids are extracted to respectively obtain positive recombinant bait plasmid pAbAi-PpVIN2pro-A and recombinant protein expression plasmid pGADT 7-PpRAP2.12.

2. Bait plasmid self-activation detection and interaction identification

The yeast recombinant bait plasmid pAbAi-proVIN2pro-A was linearized with FD BpiI restriction enzyme. The Y1H competence was prepared according to the Matchmaker Gold yeast single hybrid System (Takara, Japan) instructions and the linearized pAbAi-proVIN2pro-A vector was transferred into it. The optimal resistance concentration of the yeast decoy strain AbA was selected according to the instructions. The obtained yeast decoy strain Y1H [ pAbAi-proVIN2A ] was made competent according to the Matchmaker Gold yeast single hybrid system (Takara, Japan) instructions, and the yeast recombinant protein expression plasmid pGADT 7-PpAP2.12 was transformed into the competent cells to obtain a yeast expression strain in which the promoter truncation and the PpAPp2.12 protein were co-transformed. The empty pGADT7 plasmid was transformed into the bait strain Y1H [ pAbAi-proVIN2A ] yeast competence as a negative control. pGADT7-53 was transformed into Y1H [ pAbAi-53] yeast competent as positive control. Each co-transformed strain was plated on SD/-Leu/AbA-deficient medium (background concentration of optimum AbA resistance of each bait strain) and incubated at 30 ℃ for 3-5 days in an incubator.

As shown in FIG. 1, SD/-Leu/AbA¹⁵⁰Yeast strains containing pAbAi-PpVIN2pro-A and PGADT 7-PpAP 2.12 on the deficient medium grew well, while the negative controls were SD/-Leu/AbA¹⁵⁰Sterile colony growth on a defect culture medium proves that the PpAPpAPp2.12 protein serving as a transcription factor can be mutually identified with a peach fruit acidic PpVIN2 promoter truncation PpVIN2pro-A to generate DNA-protein interaction.

Detailed description of the preferred embodiment

Results of using dual-luciferase experiment to verify that PpRApp 2.12 promotes the transcription activity of PpVIN2

1. The PpVIN2 promoter full length was constructed on pGreen II 0800-LUC as a reporter vector, and the CDS sequence of PpRAP2.12 was constructed on pGreen II 62-sk vector as a control and effector vector.

Specific amplification PRIMERs for peach fruit PpAPp2.12 Gene and PpVIN2 Gene promoters (Gene ID LOC18783672 and LOC18776102, respectively) were designed using the online website NCBI-PRIMER (https:// www.ncbi.nlm.nih. gov/tools/PRIMER-blast /), with appropriate cleavage sites and protected bases or vector homologous sequences at both ends of the PRIMERs (Table 2).

TABLE 2 construction of primer sequences for recombinant vectors pGreen II 62-sk-PpRAp2.12 and pGreen II 0800-PpVIN2pro

Note: underlined indicates a protected base; the thick indicates the restriction enzyme site, XhoI, SmaI, BamHI, SmaI in this order

The reaction system of PCR amplification is 25 muL: 1 muL cDNA, 12.5 muL 2 x Phanta Max Master Mix, 1 muL each of the upstream and downstream primers, 9.5 muL ddH 2O. The PCR amplification program of the target fragment PpRAP2.12 is pre-denaturation at 95 ℃ for 3 min; denaturation at 95 ℃ for 15s, annealing at 60 ℃ for 15s, and extension at 72 ℃ for 1min, (35 cycles); extension was complete for 5min at 72 ℃. The PCR amplification program of the target segment PpVIN2pro is pre-denaturation at 95 ℃ for 3 min; denaturation at 95 ℃ for 15s, annealing at 60 ℃ for 15s, and extension at 72 ℃ for 2 min, (35 cycles); extension was complete for 5min at 72 ℃.

According to the Clon Express II recombinant reaction system, the using amount of each component in the recombinant reaction is calculated according to the concentration of the carrier and the recovered products of the target fragments, and the recombinant reaction is completed. The PpVIN2 promoter was ligated to expression vector pGreen II 0800, and the recombinant vector was named pGreen II 0800-PpVIN2 pro.

The PCR recovery product of PpRAP2.12 is firstly connected with a PMD18-T vector, and is connected to a pGreen II 62-sk vector through T4 ligase after double digestion, and the recombinant vector is named as pGreen II 62-sk-PpRAP2.12.

2. Double-luciferase detection is carried out after agrobacterium infects tobacco

The recombinant vector plasmids pGreenII 0800-PpVIN2pro and pGreenII 62-sk-PpAPp 2.12 were transformed into GV3101pSoup (CWBIO, Beijing, China) Agrobacterium and expanded. The Agrobacterium was collected into a prepared osmotic culture medium (containing Kan, Gen, Rif, MES, MgCl)₂AS), OD600=0.75 was adjusted. pGreenII 62-sk-transcription factor and pGreenII 0800-VIN2 invasion liquid are mixed according to the proportion of 10:1Cooperative as an experimental group, pGreenII 62-sk and pGreenII 0800-VIN2 invaded liquid were mixed at a ratio of 10:1 as a control group.

Injecting tobacco with the seedling age of about one month, injecting 3-5 leaves into each tobacco plant, and repeating the biological processes of each group by 4. And carrying out dual-luciferase detection after the infected tobacco is normally cultured for 72 h. Punching a hole at a leaf infection part, sampling, grinding by 100 muL 1 XPBS, taking 50 muL supernatant after sufficient grinding, and detecting by using a dual-luciferase reporter gene detection kit (Promega, United States), wherein the detection steps refer to the kit instruction. In the case of renilla luciferase as an internal reference, the RLU value obtained by the firefly luciferase assay was divided by the RLU value obtained by the renilla luciferase assay. Based on the obtained ratio, we can determine that pprap2.12 promotes the transcription activity of PpVIN2, as shown in fig. 2.

Detailed description of the invention

The PpRAP2.12 is transiently overexpressed in peach fruits by an agrobacterium transient transformation method, and the VIN activity is obviously increased.

The tested variety of "Yulu" honey peach (A.B.)Prunus persicaL, Batscch) was collected from the institute of industrialized juicy peaches of Nibo city, Zhejiang province, and peach fruits in green ripe stage with uniform size and without damage by diseases and insects and mechanical damage were selected for agrobacterium infection.

1. Construction and identification of the recombinant vector PBI 121-PpRAp2.12: the on-line website NCBI-PRIMER (https:// www.ncbi.nlm.nih. gov/tools/PRIMER-blast /) was used to design specific amplification PRIMERs for peach fruit PpAPp2.12 (Gene ID: LOC 18783672) with appropriate cleavage sites at both ends and homologous sequences of the expression vector (Table 3).

TABLE 3 primer sequence for constructing recombinant vector PBI121-PpRAp2.12

Note: the homologous sequences of the vector are underlined; bold indicates the cleavage site, BamHI and SmaI in that order.

The reaction system of PCR amplification is 25 muL: 1 muL cDNA, 12.5 muL 2 x Phanta Max Master Mix, 1 muL each of the upstream and downstream primers, 9.5 muL ddH 2O. The PCR amplification program of the target fragment PpRAP2.12 is pre-denaturation at 95 ℃ for 3 min; denaturation at 95 ℃ for 15s, annealing at 60 ℃ for 15s, and extension at 72 ℃ for 1min, (35 cycles); extension was complete for 5min at 72 ℃.

According to the Clon Express II recombinant reaction system, the using amount of each component in the recombinant reaction is calculated according to the concentration of the carrier and the recovered products of the target fragments, and the recombinant reaction is completed. The PpRAP2.12 is connected with an expression vector PBI121, and the recombinant vector is named as PBI 121-PpRAP2.12.

According to the requirement of DH5 alpha complete Cell (CWBIO, Beijing, China), the ligation solution PBI 121-PpARAP2.12 was transformed into E.coli DH5 alpha by heat shock method, then spread on LB/kana plate coated with X-Gal and IPTG uniformly, cultured overnight at 37 ℃ in inverted mode, and single clone was picked up for colony PCR identification. The positive clones containing the target fragment were picked and sent to Shanghai bioengineering GmbH for sequencing. Obtaining PpRAP2.12 full-length sequence matched with peach fruit genome data.

2. The recombinant plasmid transforms agrobacterium and infects peach fruit

The recombinant plasmid carrying the target gene fragment and the empty load are transformed into Agrobacterium tumefaciens GV3101 (CWBIO, Beijing, China) respectively by a freeze-thaw method. Culturing in an LB solid culture medium (kana, 50 mug/mL; Gen, 50 mug/mL; rif, 50 mug/mL) at 28 ℃ for 2-3 days, picking up a single clone, inoculating the single clone into a fresh LB liquid culture medium (kana, 50 mug/mL; Gen, 50 mug/mL; rif, 50 mug/mL), activating at 28 ℃ for 12-16h, and culturing in the fresh LB liquid culture medium (kana, 50 mug/mL; rif, 50 mug/mL; MES, 10 mM; AS, 40 mM) at 28 ℃ for 16-24 h to OD600=0.8-1.0 by shaking at 200 rpm. The cells were collected by centrifugation at 5000g for 10min at room temperature, and the supernatant was discarded. This was resuspended in permeation buffer (10 mM MgCl 2; 10mM MES, pH 5.6; 200. mu.M AS), OD600 was adjusted to 1.0, and then the resuspension buffer was allowed to stand in the dark for 3 hours.

Respectively injecting the heavy suspension buffer solution into the yin and yang sides of the Yulu peach fruits by using a sterile injector, wherein the needle head is approximately 1cm away from the skin of the peach fruits and does not touch the peach pits, and the infected peach fruits are stored at the temperature of 20 ℃ and the humidity of 85-90%. One group of peach fruits was injected with a heavy suspension of GV3101-PBI121 as a negative control, the other group of peach fruits was injected with a heavy suspension of GV3101-PBI 121-PpAPpAP 2.12, all peach fruits were sampled 1 and 3 days after infection, respectively, and the samples were frozen in a container at-80 ℃.

3. Analysis of basic expression quantity of PpRApp 2.12 and PpVIN2 of peach fruit after transient transformation of agrobacterium

qPCR analysis showed that the expression levels of PpRAP2.12 and PpVIN2 in the GV3101-PBI121-PpRAP2.12 group were significantly higher than the control GV3101-PBI121 when the peach fruits were infected and stored at 20 ℃ for one day. The expression levels of the PpAPp2.12 gene and the PpVIN2 gene are respectively increased by 40 percent and 76 percent compared with the control group, which indicates that the PpAP2.12 gene in the peach fruit is effectively overexpressed, and a system for overexpressing the PpAP2.12 gene in the peach fruit by an agrobacterium transient transformation method is successfully constructed. And the change of the PpVIN2 gene expression level in the peach fruit after the PpRAP2.12 gene is over-expressed can further prove that the PpRAP2.12 promotes the transcription activity of PpVIN 2.

4. Activity analysis of VIN in peach fruit with agrobacterium tumefaciens transiently overexpressing PpRApp 2.12

As shown in fig. 3, compared to the control group, the VIN enzyme activity in the experimental group overexpressing pprap2.12 increased by 49% on day 1, indicating that effective increase of pprap2.12 in the peach fruit can significantly increase the VIN enzyme activity. The over-expression of the PpRAP2.12 gene can cause the massive decomposition of sucrose as an anti-freezing protective agent, and aggravate the cold damage of peach fruits. The discovery of the PpAPp2.12 gene provides a theoretical basis for obtaining a peach variety with higher cold resistance by a molecular breeding technology. Through molecular breeding technology, PpRAP2.12 genes in the fruits can be silenced, so that the PpVIN2 activity in the peach fruits is reduced, and the peach variety with higher cold resistance is bred.

The above description is not intended to limit the present invention, and the present invention is not limited to the above examples. Those skilled in the art should also realize that changes, modifications, additions and substitutions can be made without departing from the true spirit and scope of the invention.

Sequence listing

<110> Ningbo university

<120> peach fruit ethylene response factor PpRAP2.12 gene and cloning method and application thereof

<160> 9

<170> PatentIn version 3.3

<210> 1

<211> 1149

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<213> full length of ethylene response factor PpRAP2.12 gene of peach fruit (ATGTGTGGAGGTGCTATAATATCCGATTTCATAGCGCCGACTCGGTCCCGGCTCACCGCTGACTACCTCTGGCCCGATCTCAAAAAACCCAGTTCGGATAAGCGGTTCTCGAAGCCTCTCAGGCCCGAAATCATTGACTTAGACCACGACTTCGAGGCTGATTTCCAGGAATTCAAGGACGAGTCTGATGTGGACGAAGATGATGAAATGGTTGAGGCCAAGCCCTCTGCTTTCTCTGCTGGAAAGCCCTCTTCTGCTCGTGGCTCTACGACTGTGAAATTAGTGGAGTTCAATGGGCAGGCTGAGAAATCTGCAAAGAGAAAGAGGAAGAACCAGTACAGGGGAATTCGCCAGCGCCCATGGGGTAAGTGGGCTGCAGAGATCCGAGACCCAAGGAAAGGGGTCCGGGTTTGGCTTGGAACTTTCAACACTGCAGAAGAAGCTGCAAGGGCTTATGATTCCGAGGCACGTAGAATTCGTGGCAAGAAAGCCAAGGTTAATTTCCCTGATGAAACCCCACGATCTTCTGCAAAGCGTTCTGTCAAGGCAAATCCTCAGAAAATGCTACCCAAGACAAACACGAATGCCGTTCAGCCTAACCTGAACCAGAATATCAATTTTGTGAATGACCCAAATCAGGACTACTACAATGCTATGGGTTTTCTGGATGAAAAGCCAGCAACTAATAACTTTGGGTTTATGTCCACCTTCCCGGCCAATGAGGATGTTGCGCTGAAATCCTCTACCCCATCCGATGCTGTCCCGCTGTATTTCAGCTCTGATCAGGGAAGCAATTCTTTTGATTGTTCTGACTTTGGCTGGGGAGAACAAGGTTCAAAGACTCCAGAAATATCATCTGTTCTTTCATGTGTTATGGAAGAAAGTGATGACTCACTGTTTCTGGAGGATGCTAGCCCAACGAAGAAACTGAAGTCTAACCCAGAGGATCTGGTGCCTGTTCAGGATAATGCAGGAAAGACACTGACTGATGAGCTCTCAGCTTTTGAGATGAAGTACTTTCAGACGCCATATCTTGATGGGAGCTGGGATGCTTCAGTGGACGCCTTCCTCAGCACAGATGCAACTCAGGATGGTGGCAACTCAGTGGACTTTTGGAGCTTCGATGACCTGATTGGGGGAGGTTTCTGA)

<400> 1

<210> 2

<211> 382

<212> PRT

<213> peach fruit ethylene response factor PpRAP2.12 gene coded protein (MCGGAIISDFIAPTRSRLTADYLWPDLKKPSSDKRFSKPLRPEIIDLDHDFEADFQEFKDESDVDEDDEMVEAKPSAFSAGKPSSARGSTTVKLVEFNGQAEKSAKRKRKNQYRGIRQRPWGKWAAEIRDPRKGVRVWLGTFNTAEEAARAYDSEARRIRGKKAKVNFPDETPRSSAKRSVKANPQKMLPKTNTNAVQPNLNQNINFVNDPNQDYYNAMGFLDEKPATNNFGFMSTFPANEDVALKSSTPSDAVPLYFSSDQGSNSFDCSDFGWGEQGSKTPEISSVLSCVMEESDDSLFLEDASPTKKLKSNPEDLVPVQDNAGKTLTDELSAFEMKYFQTPYLDGSWDASVDAFLSTDATQDGGNSVDFWSFDDLIGGGF)

<400> 2

<210> 3

<211> 25

<212> DNA

<213> specific amplification upstream primer of CDS region of peach fruit PpRAP2.12 gene (5'-ATGTGTGGAGGTGCTATAATATCCG-3')

<400> 3

<210> 4

<211> 21

<212> DNA

<213> specific amplification downstream primer of CDS region of peach fruit PpRAP2.12 gene (5'-TCAGAAACCTCCCCCAATCAG-3')

<400> 4

<210> 5

<211> 29

<212> DNA

<213> pAbAi-PpVIN2pro-A-F（TCCCCCGGG TCACCCGTACTGTCCATTTA）

<400> 5

<210> 6

<211> 29

<212> DNA

<213> pAbAi-PpVIN2pro-A-R（CCGCTCGAG GCTATCTTGCAGCCTCCACC）

<400> 6

<210> 7

<211> 34

<212> DNA

<213> pGADT7-PpRAP2.12-F（TCCCATATG ATGTGTGGAGGTGCTATAATATCCG）

<400> 7

<210> 8

<211> 30

<212> DNA

<213> pGADT7-PpRAP2.12-R（CCGGGATCC TCAGAAACCTCCCCCAATCAG）

<400> 8

<210> 9

<211> 45

<212> DNA

<213> pGreen Ⅱ 0800-PpVIN2pro-F（GGTACCGGGCCCCCCCTCGAGTTATTCATGTGGTCACCCGTACTG）

<400> 9

<210> 10

<211> 46

<212> DNA

<213> pGreen Ⅱ 0800-PpVIN2pro-R（AGAACTAGTGGATCCCCCGGGCTTCCGGGTCTCCGGGGCTCCTCCA）

<400> 10

<210> 11

<211> 34

<212> DNA

<213> pGreen Ⅱ 62-sk-PpRAP2.12-F（TCCGGATCC ATGTGTGGAGGTGCTATAATATCCG）

<400> 11

<210> 12

<211> 30

<212> DNA

<213> pGreen Ⅱ 62-sk-PpRAP2.12-R（CCGCCCGGGTCAGAAACCTCCCCCAATCAG）

<400> 12

<210> 13

<211> 46

<212> DNA

<213> PBI121-PpRAP2.12-F（ACGGGGGACTCTAGAGGATCC ATGTGTGGAGGTGCTATAATATCCG）

<400> 13

<210> 14

<211> 42

<212> DNA

<213> PBI121-PpRAP2.12-R（ATAAGGGACTGACCACCCGGG TCAGAAACCTCCCCCAATCAG）

<400> 14

Claims

1. A peach fruit ethylene response factor PpRAP2.12 gene is characterized in that: the nucleotide sequence is shown as SEQID NO: 1 is shown.

2. A protein encoded by the peach fruit ethylene response factor pprap2.12 of claim 1, wherein: the amino acid sequence is shown as SEQIDNO: 2, respectively.

3. A method for cloning a peach fruit ethylene response factor PpRAP2.12 gene as claimed in claim 1, which comprises the steps of: the method comprises the following steps:

(2) designing a primer according to the PpRAP2.12 gene sequence: the sequence of the upstream primer is as follows: 5 '-ATGTGTGGAGGTGCTATAATATCCG-3', downstream primer sequence: 5 '-TCAGAAACCTCCCCCAATCAG-3';

4. An application of the peach fruit ethylene response factor PpRAP2.12 gene of claim 1 in preparing an activity promoter of peach fruit acid invertase PpVIN 2.

5. An application of the peach fruit ethylene response factor PpRAP2.12 gene of claim 1 in cultivating a cold-resistant peach variety with PpRAP2.12 gene silent.