CN109880830B - Peach polypeptide hormone synthetic gene PpRGF1 and application thereof - Google Patents

Abstract

The invention belongs to the technical field of plant genetic engineering, and relates to a peach polypeptide hormone synthetic genePpRGF1And applications thereof. The invention clones peach polypeptide hormoneGLVMembers of the gene family are obtainedPpRGF1And the expression mode of the gene in the solute type peach fruit ripening process is analyzed, and finally the gene is transferred into Micro-Tom tomatoes by an agrobacterium-mediated method to verify the function of the target gene, so that the function played by polypeptide hormone in the peach ripening process is favorably clarified from a molecular mechanism, the purposeful and targeted quality and character improvement of peaches is further realized, and the method has an active guiding function for cultivating new peach varieties.

Description

Peach Polypeptide Hormone Synthesis Gene PpRGF1 and Its Application

technical field

The invention belongs to the technical field of plant genetic engineering, and relates to a peach polypeptide hormone synthesis gene PpRGF1 and its application.

Background technique

Melting flesh (MF) fruit is a climacteric fruit. Under the action of ethylene, the fruit softens rapidly and reaches commercial maturity. The hard peach (stony hard, SH) fruit does not release ethylene during ripening, and does not soften whether it is left or harvested under the condition of complete color change and accumulation of high concentrations of sugar. The study found that the ethylene synthesis of SH peach was blocked because the expression of the key ethylene synthesis gene ACS1 was always at a low level, while the expression of ACS1 gene in MF peach fruit increased rapidly with the ripening of the fruit. Further research found that in MF peach, auxin induces the expression of ACS1 gene, which in turn induces ethylene synthesis, resulting in fruit softening. Auxin and ethylene jointly regulate the ripening and softening of peach fruit. However, the interaction mechanism between auxin and ethylene is still unclear. The study found that a peach gene CTG134 , which encodes a polypeptide hormone similar to GOLVEN, plays a regulatory role in fruit ripening. The gene was obtained in the mesocarp of peach fruit ripening, expressed during the climacteric stage, and up-regulated during peach fruit ripening. At the same time, the gene responded to NAA and 1-MCP treatments, suggesting that GLV is between auxin and ethylene. may play an intermediate regulatory role.

SUMMARY OF THE INVENTION

One of the objects of the present invention is to provide the peach polypeptide hormone synthesis gene PpRGF1 and the protein it encodes.

The second purpose of the present invention is to provide a recombinant vector of the peach polypeptide hormone synthesis gene PpRGF1 .

The third purpose of the present invention is to provide the application of the peach polypeptide hormone synthesis gene PpRGF1 , the protein encoded by it, and the recombinant vector in tomato breeding, particularly the application in promoting tomato fruit ripening.

To achieve the above object, the present invention adopts the following technical solutions:

The present invention provides a peach polypeptide hormone synthesis gene PpRGF1 , the sequence of which is shown in SEQ ID NO:1.

The present invention also provides the protein encoded by the peach polypeptide hormone synthesis gene PpRGF1 , the amino acid sequence of which is shown in SEQ ID NO:2.

The present invention also provides a recombinant vector comprising the peach polypeptide hormone synthesis gene PpRGF1 .

Preferably, the recombinant vector is an Agrobacterium expression vector.

The invention also provides the application of the peach polypeptide hormone synthesis gene PpRGF1 , the protein encoded by the peach polypeptide hormone synthesis gene PpRGF1 and the recombinant vector of the peach polypeptide hormone synthesis gene PpRGF1 in tomato breeding.

Preferably, the tomato breeding promotes tomato fruit ripening.

Preferably, the tomato breeding comprises the following steps:

A: construct a recombinant vector containing the peach polypeptide hormone synthesis gene PpRGF1 ;

B: transform the constructed recombinant vector into tomato tissue or cells;

C: Breeding and screening to obtain transgenic tomato.

Compared with the prior art, the beneficial effects of the present invention are:

1. The present invention obtains PpRGF1 by cloning members of the peach polypeptide hormone GLV gene family, and analyzes the expression pattern of this gene during the ripening process of solute peach fruit, and finally uses the method mediated by Agrobacterium to transfer it into Micro-Tom tomato to verify the purpose The function of the gene is conducive to clarifying the response mechanism of hormone signals in the process of peach ripening from the molecular mechanism, and has a positive guiding role in further realizing the purposeful and targeted improvement of peach quality and traits and cultivating new peach varieties.

2. Compared with other transgenes that cause local changes in plant phenotype, such as unilateral changes in leaf shape or flower structure, or changes in root length and number of lateral roots, and changes in fruit size or pericarp thickness, PpRGF1 gene promotes fruit development. The expression of ethylene and ripening-related genes stimulates ethylene release, thereby promoting fruit ripening.

Description of drawings

Figure 1 The electrophoresis of PCR amplification of the peach polypeptide hormone synthesis gene PpRGF1 , in the figure - represents the negative control, + represents the positive control, the M in the left band in the figure is the DL 2000 marker, and the size of the gene PpRGF1 gene fragment is 489bp.

Fig. 2 Amino acid multiple sequence alignment of peach PpRGF1 and Arabidopsis AtGLV .

Fig. 3 Evolutionary tree of peach PpRGF1 and Arabidopsis AtGLV .

Fig. 4 The relative expression of peach polypeptide hormone synthesis gene PpRGF1 in CN13 and CN16 fruit ripening stages.

Figure 5 The relative expression of peach polypeptide hormone synthesis gene PpRGF1 in different tissues of CN13 developmental stages.

Fig. 6 Observations of wild-type tomato and transgenic tomato fruits at different developmental stages.

Fig. 7 Comparison of changes in ethylene release from wild-type tomato and transgenic tomato fruits at different ripening stages.

Fig. 8 Comparison of changes in firmness of wild-type tomato and transgenic tomato fruits at different developmental stages.

Fig. 9 Quantitative analysis of ethylene synthesis and signal transduction-related genes and ripening-related genes of wild-type tomato and transgenic tomato fruits at different stages of wild-type tomato and transgenic tomato fruits.

Figure 10 Comparison of postharvest morphology of wild-type tomato and transgenic tomato fruits.

In the attached drawings, GR means green ripening stage, B means color changing period, B+3 means 2 days after color changing, B+6 means 6 days after color changing, R means fruit ripening period, S1 means first exponential growth of fruit period, S2 represents the initial stage of fruit stone hardening, S3 represents the second exponential growth period of fruit, S4I represents the early stage of fruit climax, S4II represents the fruit climax stage, and S4III represents the late stage of fruit climax.

Detailed ways

The following examples are used to illustrate the present invention, but are not intended to limit the protection scope of the present invention. Unless otherwise specified, the technical means used in the examples are conventional means well known to those skilled in the art. The test methods in the following examples are conventional methods unless otherwise specified.

The medium-solute variety 'Zhongyoutao No. 13' (CN13) and the hard-type variety 'Zhongyoutao No. 16' (CN16) of the present invention are provided by the peach breeding garden of Zhengzhou Fruit Tree Research Institute, Chinese Academy of Agricultural Sciences.

Example 1 Isolation and identification of peach polypeptide hormone synthesis gene PpRGF1

experiment method

1. Isolation of the gene PpRGF1

The peach pulp RNA was extracted using the plant polysaccharide polyphenol kit (DP441, Tiangen Biochemical Technology (Beijing) Co., Ltd.), and the single-stranded cDNA was obtained by reverse transcription kit (Tiangen Biochemical Technology (Beijing) Co., Ltd.). cDNA was used as the template, and the following sequences were used as primers to obtain the full-length sequence of the gene PpRGF1 by PCR (kit FastStart SYBR Green Master purchased from Roche Biotechnology Co., Ltd.). The electrophoresis of PCR amplification is shown in Figure 1. The full-length sequence of the gene PpRGF1 is shown in SEQ ID NO: 1 in the sequence listing, with a total of 489 bp, and the amino acid sequence of the co-encoded protein is shown in SEQ ID NO: 2 in the sequence listing, with a total of 162. The amino acid sequences of peach PpRGF1 and the Arabidopsis AtGLV family were aligned and the phylogenetic tree was drawn. The results are shown in Figures 2 and 3.

It can be seen from Figure 2 that both peach PpRGF1 and Arabidopsis AtGLV family proteins contain a conserved C-terminal motif consisting of 13 amino acids.

As can be seen from Figure 3, peach PpRGF1 is most closely related to AtGLV1 in evolution.

The primer sequences are:

Forward PpRGF1 -F: 5'-ATGTCTTCCATTGTTCTTCT-3';

Reverse PpRGF1 -R: 5'-CTAGGACTTTCTATTGTGAA-3'. Annealing temperature for PCR was 58°C.

2. Expression analysis of gene PpRGF1

Fluorescence quantitative PCR operation steps: Polysaccharide and polyphenol RNA extraction kit SK8662 (Sangong, Shanghai) was used to extract total RNA from roots, stems, leaves, flowers and peach fruits at different developmental stages, and reverse transcription kit KR106 (Tiangen, Beijing) to synthesize cDNA, and use this as a template for qRT-PCR detection using Lint-Cycler 480Ⅱ fluorescence quantitative analyzer, and SYBR Green I Master (Roche, Switzerland) for amplification reaction.

The total reaction volume is 15ul, including 100 ng cDNA (1 µL), 2× Lightcycler 480 SYBR Green IMaster (7.5 µL), 0.5 µmol L-1 upstream and downstream primers (0.75 µL each) and RNase-free water (5 µL) .

Reaction program: pre-denaturation at 95 °C for 5 min; denaturation at 95 °C for 30 s, annealing at 60 °C for 30 s, extension at 72 °C for 30 s, a total of 45 cycles. 3 replicates for each sample. Primer specific primers were designed for each transcript sequence using Primer Express 3.0.

The quantitative primer sequences are:

Forward Q PpRGF1 -F: 5'- TCTTCCAATAGGAGCAGCACT -3';

Reverse Q PpRGF1 -R: 5'-CATTTGTGCTTTAGTAAGACGG-3'.

qRT-PCR was used to detect the expression of PpRGF1 gene in different tissues of solute type 'Zhongnectao No. 13' (CN13) and different developmental stages of solute type 'Zhongnectao No. 13' (CN13) and hard type 'Zhongnectao No. 16' (CN16). express the situation. The four sampling time points of the two varieties (CN13 and CN16) were S1, S2, S3, S4I, S4II and S4III. Actin ( ppa007242m ) was selected as the internal reference gene (Brandi et al., 2011), and the relative gene expression was calculated using the 2 ^-ΔΔCT formula (Livak and Schmittgen, 2001). The results are shown in Figures 4 and 5.

As can be seen from Figure 4, by comparing the expression of PpRGF1 gene in different developmental stages of solute-type (CN13) and hard-type (CN16) peach fruits, it was found that the expression of PpRGF1 in the solute-type (CN13) during fruit ripening was significantly higher than that in the hard-type (CN16) peach fruit. type (CN16), while the expression was up-regulated with fruit ripening, while the expression of the hard type (CN16) was almost undetectable.

It can be seen from Figure 5 that the gene PpRGF1 is expressed in different tissues of CN13 roots, stems, leaves, flowers and fruits, and the highest expression is in stems and fruits.

3. Functional identification test of PpRGF1 gene

In order to study whether PpRGF1 gene is involved in the auxin and ethylene signaling pathways during peach ripening, the function of transgenic tomato was analyzed and identified.

3.1 Construction of recombinant vector

The target fragment obtained by PCR was cloned through the pTOPO-blunt vector, and then ligated to the Com-pH2GW7.0 vector using a one-step cloning kit (Vazyme, Nanjing Novizan Biotechnology Co., Ltd.). The primers are as follows:

G- PpRGF1 -F:5'-AAAAAGCAGGCTTCATGTCTTCCATTGTTCTTCT-3';

G- PpRGF1 -R: 5'-AGAAAGCTGGGTcCTAGGACTTTCTATTGTGAA-3'.

Positive clones were detected. The positive clones were then sent to Shanghai Sangon Biotechnology Co., Ltd. for sequencing.

3.2 Screening of transgenic tomato positive strains

Since peach has not yet established an efficient and mature genetic transformation system, the model plant Micro-Tom tomato was used for functional verification of the PpRGF1 gene, and the tomato transformation method was optimized according to Sun et al (2006), Bee Lynn Chew and Yu Pan (University of Nottingham) . Micro-Tom tomato seeds were surface-sterilized with 75% alcohol, rinsed three times with sterile water, and then disinfected with 10% sodium hypochlorite for 1 hour. After taking out, wash 6 times in sterile water and dry on filter paper. Seeds were sown in 1/2 MS medium containing 0.8% agar at pH=5.9. Plants were cultured in a tissue culture chamber with 14 h/10 h light-dark, 25°C, 80% relative humidity, and 250 μmol m ^-2 s ^-1 light intensity.

The plasmid of the sequenced PpRGF1 - Com-pH2GW7.0 vector was extracted and transferred into Agrobacterium GV3101 by liquid nitrogen freeze-thaw method. Adjust the OD of Agrobacterium solution to 0.5, transform Micro-Tom tomato by leaf disc method, and differentiate leaves and roots on rooting medium. After growing into seedlings, DNA was extracted and positive plants were identified by conventional PCR. After harvesting the seeds of the T ₀ generation, positive plants were selected on 1/2 MS medium containing kanamycin. Transgenic positive plants contain antibiotic genes, grow true leaves and taproots on antibiotic-containing medium, and then transplant them into soil for cultivation and harvest T ₁ generation seeds. After the seeds of the T ₁ generation were sown, the difference from the wild type was observed using the T ₂ generation transgenic tomatoes. The following experiments all used the T ₂ generation and progeny homozygous lines.

PCR molecular identification of positive plant primers:

Com-pH2GW7.0F: 5'-TTGGAGAGGACTCCGGTATT-3';

Com-Adapter attB2: 5'-GGGGACCACTTTGTACAAGAAAGCTTGGT-3'.

Used to confirm fragment insertion.

Three transgenic PpRGF1 gene lines ( PpRGF1-6, PpRGF1-8, PpRGF1-15 ) with high expression were selected as representative lines for comparison with wild type.

Example 2 The role of gene PpRGF1 in transgenic tomato

2.1 The effect of gene PpRGF1 on tomato fruit development

Figure 6 is the observation diagram of wild-type tomato and transgenic tomato fruits at different developmental stages. It can be seen from Figure 6 that the transgenic tomato began to turn color at 41d after flowering, and the wild-type tomato began to change color at 45d after flowering, that is, the transgenic tomato matured about 4d earlier than the wild-type tomato. The color of the period is the same.

2.3 Effects of gene PpRGF1 on tomato fruit firmness and ethylene release

The ethylene levels of transgenic and wild-type tomato fruits at different days after flowering (37dpa, 41dpa, 45dpa, 50dpa, 53dpa, 59dpa) were measured. Figure 7 is a comparison of changes in ethylene release from wild-type tomato and transgenic tomato fruits at different ripening stages. It can be seen from Figure 7 that the ethylene release of wild-type and transgenic tomato showed a trend of first increase and then decrease; compared with wild-type tomato, transgenic tomato released higher amount, and the ethylene release peak appeared earlier.

Figure 8 is a comparison of the changes in firmness of wild-type tomato and transgenic tomato fruits at different developmental stages. It can be seen from Figure 8 that the firmness of the tomato fruit gradually decreased during the ripening process, and the firmness of the transgenic tomato was lower than that of the wild-type tomato.

2.4 The regulatory mechanism of gene PpRGF1 on tomato fruit ripening

In order to further study the regulation mechanism of PpRGF1 gene on fruit ripening, the present invention uses qRT-PCR technology to identify ethylene synthesis and signal transduction related genes (such as ACO1 and ACS4 ), ripening related genes (such as E4, E8, RIN and CNR , etc.) Expression in different developmental stages of fruit.

Figure 9 is a quantitative analysis of ethylene synthesis and signal transduction-related genes and ripening-related genes of wild-type tomato and transgenic tomato fruits at different stages. It can be seen from Fig. 9 that the expression trends of ethylene synthesis and signal transduction related genes ACO1 and ACS4 and maturation-related genes E4 and E8 during the development of wild-type and transgenic tomato are the same as the ethylene release trend, and the ethylene release peak of transgenic tomato is higher than that of ethylene release. Wild-type tomato appeared early; and at the fruit ripening stage, the expression levels of ripening-related genes E4 , E8 , RIN and CNR showed that their expression levels in transgenic fruits were significantly higher than those in wild-type tomato fruits. It can be explained that the transgenic tomato promotes the expression of ethylene and ripening-related genes, stimulates the release of ethylene during fruit development, and promotes fruit ripening.

2.5 The effect of gene PpRGF1 on tomato fruit shrinkage and senescence

Figure 10 is a comparison of postharvest morphology of wild-type tomato and transgenic tomato fruits. It can be seen from Figure 10 that the transgenic tomato fruits begin to shrink after being placed at room temperature for 14 days, while the wild-type tomato fruits do not shrink; therefore, compared with the wild-type tomato fruits, the transgenic tomato accelerates the water loss and shrinkage, which is not conducive to harvesting. After storage.

The above-mentioned embodiments are only preferred embodiments of the present invention, and are only used to explain the present invention, but not to limit the scope of implementation of the present invention. It is easy to make other embodiments by replacing or changing the technical content, so all changes and improvements made on the principle of the present invention should be included in the scope of the patent application of the present invention.

SEQUENCE LISTING

<110> Zhengzhou Fruit Tree Research Institute, Chinese Academy of Agricultural Sciences

<120> Peach polypeptide hormone synthesis gene PpRGF1 and its application

<130> 2019

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<170> PatentIn version 3.3

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gcgaaagtct taagtgagac tttaaagtca tcgatgatgc ctaccatctc agttgaatac 180

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aggaaacaag ggcatgccaa tggaccagcc tcaggatatg atattattgc attatcttcc 300

caggttgagc tgaagaaatt gattgaaatg cagggattga agaggcaggc acggacatta 360

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Cys Asn Ser Arg Leu Leu Gly Leu Val Asp Lys Gln Ser Gly Ser Lys

20 25 30

Ser Tyr His Ser Val Glu Asp Val Ala Lys Val Leu Ser Glu Thr Leu

35 40 45

Lys Ser Ser Met Met Pro Thr Ile Ser Val Glu Tyr Gln Ala Gln Gln

50 55 60

Ile Asn Ala Glu Thr Phe Thr His Glu Ser Ile Pro Thr Ala Leu Leu

65 70 75 80

Arg Lys Gln Gly His Ala Asn Gly Pro Ala Ser Gly Tyr Asp Ile Ile

85 90 95

Ala Leu Ser Ser Gln Val Glu Leu Lys Lys Leu Ile Glu Met Gln Gly

100 105 110

Leu Lys Arg Gln Ala Arg Thr Leu Leu Gly Ser Ala Thr His Asn Met

115 120 125

Glu Glu Asp Lys Asp Ser Lys Glu Asp Glu Ala Ile Glu Val Val Gly

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Claims (2)

1. Peach polypeptide hormone synthetic genePpRGF1The application in tomato breeding is characterized in that the tomato breeding is used for promoting the fruit ripening of tomatoes and accelerating the water loss and shrinkage of the tomatoes; the peach polypeptide hormone synthetic genePpRGF1In (1) orderShown in SEQ ID NO 1.

2. Use according to claim 1, characterized in that said tomato breeding comprises the following steps:

a: construction of hormone synthetic gene containing peach polypeptidePpRGF1The recombinant vector of (1);

b: transforming the constructed recombinant vector into tomato tissue or cell;

c: and breeding and screening to obtain the transgenic tomato.

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