CN110922459B

CN110922459B - Application of SlSNAT1 protein and related biological material thereof in regulation and control of plant seed aging resistance

Info

Publication number: CN110922459B
Application number: CN201911102418.2A
Authority: CN
Inventors: 张娜; 王小云; 刘颖; 郭仰东; 谢倩; 王志荣; 吕红梅; 张姣姣
Original assignee: China Agricultural University
Current assignee: China Agricultural University
Priority date: 2019-11-12
Filing date: 2019-11-12
Publication date: 2021-02-19
Anticipated expiration: 2039-11-12
Also published as: CN110922459A

Abstract

The invention relates to the technical field of biology, in particular to application of a SlSNAT1 protein and a related biological material thereof in regulation and control of plant seed aging resistance. The invention discovers that the tomato SlSNAT1 protein positively regulates the anti-aging performance of plant seeds, improves the expression quantity and/or activity of SlSNAT1 protein in plants, obviously improves the germination activity of naturally aged plant seeds, and further effectively prolongs the service life of the seeds. The discovery of the new function of the SlSNAT1 provides gene resources and a new method for breeding new plant varieties with seed aging resistance, and has wide application prospect and higher application value.

Description

Application of SlSNAT1 protein and related biological material thereof in regulation and control of plant seed aging resistance

Technical Field

The invention relates to the technical field of biology, in particular to application of SlSNAT1 protein and related biomaterials in regulation and control of plant seed aging resistance.

Background

Tomato (tomato lycopersicon L.) is a herbaceous plant in the genus of tomato of the family Solanaceae, originates in south America, is originally used only as ornamental cultivation, and is discovered to have rich nutrient substances and unique flavor, so that the tomato is gradually one of the most popular fruits and vegetables cultivated all over the world and is also one of important facility-cultivated vegetables. The seeds are an important root base for agricultural production, and the vitality of the seeds directly influences the level of agricultural production. The natural aging of seeds is an inevitable phenomenon during seed storage, and can directly affect the germination rate of seeds and finally the yield. Therefore, the method for improving the vitality and the storage period length of the tomato seeds has important application value for increasing the yield and the output value of the facility vegetables.

Studies show that the life span and germination rate of seeds can be obviously improved after the sunflower gene HaHSFA9 is transferred into tobacco (Prieto-Dapena P., Castano R., Almoguera C., et al, improved resistance to controlled determination in transgenic seeds plant Physiology,2006,142(3): 1102-1112.). In 2012, french scientists identified 4 LEA family proteins that could improve the life and vitality of seeds by about 30 times using alfalfa, and this family is considered to have an important role in the loss of seed vitality and in the regulation of seed storability (charelain e., hundretmark m., leprime o., et al. Studies in the model plant arabidopsis thaliana found that important components ABI3 and ABI5 in the ABA signaling pathway play an important role in the seed dormancy and germination process, and that ABI3 and ABI5 mutants exhibited a dormancy-attenuated phenotype (zuyu, 2018, BES1 interacting with ABI5 and participating in the molecular mechanism regulating the germination process of arabidopsis thaliana seeds, university of china, doctrine chessman paper). In addition, the decomposed eupatorium adenophorum leaching liquor and polyamine can be compounded to form a specific germinator to promote the aged seeds to germinate (Chinese patent CN 105961444A).

At present, few reports are provided about the regulation mechanism and related genes of seed aging and vigor in tomatoes, and the discovery of the novel related genes for regulating seed aging and vigor has important significance for improving the germination rate of plant seeds and the yield of plants.

Disclosure of Invention

In order to solve the technical problems in the prior art, the invention aims to provide the application of the SlSNAT1 protein and related biological materials thereof in regulating and controlling the anti-aging performance of plant seeds.

The invention discovers that the tomato SlSNAT1 protein (shown as SEQ ID NO.1 in sequence) is related to the anti-aging performance of plant seeds, improves the expression level of SlSNAT1 protein in plants, can obviously improve the anti-aging performance of the plant seeds, prolongs the service life of the plant seeds, and improves the vitality of the aged plant seeds.

Specifically, the technical scheme of the invention is as follows:

in a first aspect, the invention provides the use of a SlSNAT1 protein or a nucleic acid encoding it or a biological material comprising a nucleic acid encoding a SlSNAT1 protein for modulating the vigor of a plant seed.

Preferably, the seed vigor is the vigor of an aged seed. More preferably, the vigor is germination vigor.

The seed vigor may be a germination vigor of naturally aged seeds. The natural aging refers to a natural aging process of storing the seeds under the room temperature condition (25-28 ℃ and the air humidity of 30-60%).

In a second aspect, the invention provides the use of a SlSNAT1 protein or a nucleic acid encoding it or a biological material comprising a nucleic acid encoding a SlSNAT1 protein for modulating the longevity of plant seeds.

In a third aspect, the invention provides the use of a SlSNAT1 protein or a nucleic acid encoding the same or a biological material comprising a nucleic acid encoding a SlSNAT1 protein in the genetic breeding of plant seed for tolerance to aging.

The plant seed aging-resistant genetic breeding can be realized by introducing a coding nucleic acid of the SlSNAT1 protein or a biological material containing the coding nucleic acid of the SlSNAT1 protein into the plant by using a genetic engineering technical means, or by carrying out cross breeding on the plant constructed by using the genetic engineering technical means and other plants.

Preferably, in the above application, the activity of plant seeds is improved or the life of the plant seeds is prolonged by increasing the expression level and/or activity of the SlSNAT1 protein in the plant.

The increase in the expression level and/or activity of the SlSNAT1 protein in the plant can be achieved by means of conventional techniques in the art. For example: an expression vector carrying a gene encoding the SlSNAT1 protein is introduced into the plant.

In the invention, the SlSNAT1 protein has any one of the following amino acid sequences:

(1) an amino acid sequence shown as SEQ ID NO. 1;

(2) the amino acid sequence of the protein with the same function is obtained by replacing, inserting or deleting one or more amino acids in the amino acid sequence shown as SEQ ID NO. 1;

(3) a fusion protein obtained by connecting a label to the N end and/or the C end of the SlSNAT1 protein;

(4) an amino acid sequence having at least 90% homology with the amino acid sequence shown as SEQ ID No. 1; preferably, the homology is at least 95%; more preferably 99%.

The amino acid sequence shown as SEQ ID No.1 is the amino acid sequence of tomato SlSNAT1 protein, and a person skilled in the art can substitute, delete and/or add one or more amino acids according to the amino acid sequence of tomato SlSNAT1 protein, conservative substitution of amino acids and other conventional technical means in the art on the premise of not influencing the activity of the tomato SlSNAT1 protein, so that the SlSNAT1 protein mutant with the same function as the tomato SlSNAT1 protein is obtained.

In the above-mentioned tag linked to the N-terminal and/or C-terminal, the tag (tag) refers to a polypeptide or protein that is expressed by fusion with a target protein using in vitro DNA recombination technology, so as to facilitate the expression, detection, tracing and/or purification of the target protein. The tag may be one or more of Flag tag, His tag, MBP tag, HA tag, myc tag, GST tag, SUMO tag.

In the invention, the encoding nucleic acid of the SlSNAT1 protein has any one of the following nucleotide sequences:

(1) a nucleotide sequence shown as SEQ ID NO.2 or a complementary sequence thereof;

(2) the coding sequence is a nucleotide sequence of a sequence shown as SEQ ID NO. 2;

(3) a nucleotide sequence that hybridizes with the nucleotide sequence of (1) or (2) under stringent conditions and encodes the SlSNAT1 protein.

The encoding nucleic acid may be DNA, including but not limited to cDNA, genomic DNA, or recombinant DNA, or RNA.

The nucleotide sequence shown as SEQ ID NO.2 is the CDS sequence of SlSNAT1 protein in tomato. All nucleotide sequences encoding the SlSNAT1 protein are within the scope of the invention in view of codon degeneracy.

In the present invention, the biological material includes an expression cassette, a vector, a host cell, an engineering bacterium, a transgenic plant cell line, a transgenic plant tissue, a transgenic plant organ, a transgenic plant, a tissue culture produced by a regenerable cell of the transgenic plant, or a protoplast produced by the tissue culture.

The biological material may be:

(1) an expression cassette comprising a nucleic acid encoding the SlSNAT1 protein;

(2) a vector comprising a nucleic acid encoding the SlSNAT1 protein, or a vector comprising (1) the expression cassette;

(3) a host cell containing a nucleic acid encoding the SlSNAT1 protein, or a host cell containing (1) the expression cassette, or a host cell containing (2) the vector;

(4) an engineering bacterium containing the encoding nucleic acid of the SlSNAT1 protein, or an engineering bacterium containing the expression cassette (1), or an engineering bacterium containing the vector (2);

(5) a transgenic plant cell line comprising a nucleic acid encoding the SlSNAT1 protein, or a transgenic plant cell line comprising (1) the expression cassette, or a transgenic plant cell line comprising (2) the vector;

(6) a transgenic plant tissue comprising a nucleic acid encoding the SlSNAT1 protein, or a transgenic plant tissue comprising (1) the expression cassette, or a transgenic plant tissue comprising (2) the vector;

(7) a transgenic plant organ comprising a nucleic acid encoding the SlSNAT1 protein, or a transgenic plant organ comprising (1) the expression cassette, or a transgenic plant organ comprising (2) the vector;

(8) a transgenic plant containing the encoding nucleic acid of the SlSNAT1 protein, or a transgenic plant containing the expression cassette of (1), or a transgenic plant containing the vector of (2);

(9) a tissue culture produced from regenerable cells of the transgenic plant of (8);

(10) protoplasts produced from the tissue culture of (9).

In a fourth aspect, the present invention provides a method of modulating the vigor or longevity of a plant seed, comprising: regulating the expression level and/or activity of the SlSNAT1 protein in the plant; the SlSNAT1 protein has any one of the following amino acid sequences:

(1) an amino acid sequence shown as SEQ ID NO. 1;

Preferably, the method of modulating the vigor or longevity of plant seeds comprises: increasing seed vigor or prolonging seed longevity of the plant by increasing the expression level and/or activity of the SlSNAT1 protein in the plant.

The above-mentioned increase of the expression level of the SlSNAT1 protein in the plant can be achieved by means of conventional techniques in the art, such as: an expression vector carrying a gene encoding the SlSNAT1 protein is introduced into the plant.

As a preferred embodiment of the present invention, the expression vector carrying the coding gene of the SlSNAT1 protein may be a pBI121 vector carrying the coding gene of the SlSNAT1 protein.

The expression vector carrying the coding gene of the SlSNAT1 protein can be used for transforming plant cells or tissues by using Ti plasmid, Ri plasmid, plant virus vector, direct DNA transformation, microinjection, conductance, agrobacterium mediation and other conventional biological methods, and culturing the transformed plant cells or tissues into plants.

In the present invention, the plant is a monocotyledon or a dicotyledon. Such plants include, but are not limited to, tomato, Arabidopsis, rice, wheat, corn, soybean, cotton, peanut, and the like.

Preferably, the plant is a plant of the genus Lycopersicon.

The invention has the beneficial effects that:

the invention discovers that the SlSNAT1 protein participates in regulation and control of the plant seed aging process, the SlSNAT1 protein positively regulates the plant seed aging resistance, the expression level of the SlSNAT1 protein in plants is improved, and the plant seed aging resistance is obviously improved. Experiments prove that the germination rate of the transgenic tomato seeds over-expressing the SlSNAT1 protein after natural aging for 2-3 years is obviously higher than that of wild tomato seeds, so that the service life of the seeds is effectively prolonged. The discovery of the new function of the SlSNAT1 provides gene resources and a new method for breeding new plant varieties with seed aging resistance, and has wide application prospect and higher application value.

Drawings

FIG. 1 is a gel electrophoresis image of PCR amplification products of the SlSNAT1 gene in example 1 of the invention; wherein M is DL2000 DNA Marker; the remaining lanes are PCR amplification products of the SlSNAT1 gene.

FIG. 2 is a gel electrophoresis image of the SLSNAT1 overexpression plasmid transformed Agrobacterium tumefaciens in example 2 of the present invention; wherein M is DL2000 DNA Marker; p is the positive control OX-SlSNAT1 overexpression vector plasmid, and the remaining lanes are monoclonal Agrobacterium.

FIG. 3 is a gel electrophoresis image of validation of a SlSNAT1 overexpression transgenic line in example 2 of the invention; wherein M is DL2000 DNA Marker; p is positive control recombinant plasmid OX-SlSNAT 1; WT is tomato Micro Tom wild type plant; OX1-OX6 are different overexpression transgenic lines.

FIG. 4 shows the RT-PCR verified expression level of the overexpression transgenic line of SlSNAT1 in example 2 of the present invention; WT is tomato Micro Tom wild type plant; OX1-OX6 are different overexpression transgenic lines.

FIG. 5 shows the germination of naturally aged seeds of a transgenic plant overexpressed by SlSNAT1 in example 2 of the present invention; wherein, WT is tomato Micro Tom wild type plant, OX6 is overexpression transgenic line.

FIG. 6 is the statistics of the germination rate of naturally aged seeds of SlSNAT1 overexpression transgenic plants in example 2 of the present invention; wherein A is the statistics of the germination rate of the naturally aged seeds for two years, and B is the statistics of the germination rate of the naturally aged seeds for three years; WT is tomato Micro Tom wild type plant, OX is overexpression transgenic line OX 6.

Detailed Description

Preferred embodiments of the present invention will be described in detail with reference to the following examples. It is to be understood that the following examples are given for illustrative purposes only and are not intended to limit the scope of the present invention. Various modifications and alterations of this invention will become apparent to those skilled in the art without departing from the spirit and scope of this invention.

The experimental procedures used in the following examples are all conventional procedures unless otherwise specified.

Materials, reagents and the like used in the following examples are commercially available unless otherwise specified. Among them, tomato Micro Tom (wild type plant, WT) was purchased from Nanjing Fengshou horticulture Co., Ltd., cat # 3558.

Example 1 cloning of the SlSNAT1 Gene

1. Obtaining of test materials

Planting the tomato Micro Tom in an illumination incubator of the gardening academy of Chinese agricultural university, wherein the illumination incubator is used for culturing under the condition that the illumination density is 10000 LUX; illuminating for 12h at 26 ℃; dark 12h, temperature 16 ℃. And (3) when the tomato seedlings grow to the 4 th leaf, taking the mature leaves, quickly putting the mature leaves into liquid nitrogen for freezing, and storing the mature leaves in a refrigerator at the temperature of minus 80 ℃ for later use.

2. Extraction of RNA

Total RNA from tomato leaves was extracted using an RNA extraction kit (purchased from Wash-ocean Biotech Co., Ltd., cat # 0416-50).

3. Obtaining of cDNA

Using the total RNA extracted in step 2 as a template, a reverse transcription reaction was carried out using a reverse transcription kit (purchased from Takara, Inc., cat # RR047A) to obtain cDNA, and the cDNA solution was further diluted to 100 ng/. mu.l to serve as a reaction template for the PCR described below.

Wherein, the reaction system and the reaction program of the reverse transcription are as follows: the following were added to ice and reacted at 70 ℃ for 10 minutes: total RNA 1ng, Oligo dT 2. mu.l, RNase Free dH₂O is complemented to 10 mu l; after the reaction was completed, the reaction mixture was quickly placed on ice for 2 minutes,then the following were added in order: reverse transcription Buffer 4. mu.l, dNTP mix 1. mu.l, RNase inhibitor 0.5. mu.l, M-mlv 2. mu.l, RNase Free dH₂O0.5. mu.l, reacted at 42 ℃ for 1 hour for reverse transcription, and reacted at 70 ℃ for 15 minutes to inactivate the enzyme, thereby finally obtaining cDNA.

4. Amplification of target Gene SlSNAT1

And 3, taking the cDNA obtained in the step 3 as a template, carrying out PCR amplification by using primers SlSNAT1S and SlSNAT1A to obtain a PCR product shown as SEQ ID NO.2, storing at 4 ℃ after the reaction is finished, and carrying out detection by using 1% agarose gel electrophoresis, wherein the result is shown in figure 1, the size of a target band is expected to be about 774bp, the band obtained by cloning is located about 750bp DNA Marker, and the result is consistent with the expectation, so that the full-length sequence (CDS) of the open reading frame of the target gene SlSNAT1 shown as SEQ ID NO.2 is obtained.

The primer sequences are as follows:

SlSNAT1S：5′-ATGCAAATGCAAACTCTCCAC-3′；

SlSNAT1A：5′-ATACATGGGGTACCAGAACATT-3′。

the PCR reaction system is shown in Table 1:

TABLE 1 PCR reaction system of target gene SlSNAT1 open reading frame full-length sequence

The PCR reaction procedure is shown in table 2:

TABLE 2 PCR reaction procedure

Example 2 application of SlSNAT1 in tomato aged seed germination regulation

Construction of SlSNAT1 overexpression vector

Connecting the target gene fragment with a vector by using a Clonexpress II recombinant cloning kit (purchased from Nanjing Nodezam Biotechnology Co., Ltd., product number C112-01) to obtain a connection product; wherein, the recombination reaction comprises the following steps:

1. and (3) amplification of the insert: by introducing homologous sequences at the ends of the linearized cloning vector at the 5 ' end of the primers, the 5 ' and 3 ' extreme ends of the amplified product of the insert are provided with sequences corresponding to the two ends of the linearized cloning vector, respectively.

According to the principle and the sequence of the gene SlSNAT1, an amplification primer with a restriction enzyme cutting site SmaI and a homologous sequence introduced into the tail end of the linearized cloning vector at the 5' end is designed, and the sequence of the primer is as follows:

SlSNAT1S′：5′-CGCACAATCCCACTATCCTTCATGCAAATGCAAACTCTCCAC-3′；

SlSNAT1A′：5′-ATCCAGACTGAATGCCCACAGGATACATGGGGTACCAGAACATT-3′。

the cDNA obtained in example 1 is used as a template, the primers SlSNAT1S 'and SlSNAT 1A' are used for PCR amplification to obtain PCR products, the PCR products are subjected to electrophoresis detection by 1% agarose gel, and then gel cutting and recovery are carried out to obtain an insert containing the target gene SlSNAT 1.

The PCR product is cut and recovered by using a glue recovery kit (purchased from Tiangen Biotechnology technology (Beijing) Co., Ltd., product number DP209-02), and the method specifically comprises the following steps:

A. the insert was cut out and placed in a 1.5ml centrifuge tube, 600. mu.l of the sol solution was added, and the gel was heated at 65 ℃ for 10 minutes to completely dissolve the gel mass to obtain a solution.

B. The obtained liquid is cooled to room temperature and then added into an adsorption column, the mixture is kept stand for 2 minutes at room temperature, 12000g of the mixture is centrifuged for 1 minute at room temperature, and the liquid is discarded.

C. 600 microliters of washing solution was added to the adsorption column, centrifuged at 12000g at room temperature for 1 minute, the permeate was discarded, and the procedure was repeated.

D. Centrifuge at 12000g for 5 minutes at room temperature and transfer the column to a 1.5ml centrifuge tube.

E. Adding 40 microliter TE Buffer preheated at 65 ℃, standing for 5 minutes, centrifuging at 12000g at room temperature for 2 minutes, and collecting liquid to obtain the insert containing the target gene SlSNAT 1.

2. And (3) carrying out recombination reaction of the fragment and the vector:

selecting endonuclease SmaI to perform enzyme digestion on the vector pBI121 (purchased from vast Ling plasmid platform, the product number P0274) to obtain a linearized vector pBI 121; and carrying out homologous recombination on the insert containing the target gene SlSNAT1 and the linearized vector pBI121 to obtain a ligation product pBI121-SlSNAT 1.

Wherein, the enzyme digestion system is shown in table 3:

TABLE 3 digestion system

The reaction system for homologous recombination is shown in Table 4:

TABLE 4 homologous recombination reaction System

After the system is prepared in an ice-water bath, the system is placed in a metal bath at 37 ℃ for reaction for 30 minutes, and then the reaction is stopped by rapidly cooling the bath in ice for 3 minutes to obtain a connecting product.

3. Transformation of the ligation products into E.coli competence

A. Taking out Escherichia coli competence DH5 alpha stored in a refrigerator at-80 ℃, and thawing on ice;

B. sucking 50 μ L of competent cells in a clean bench, adding 10 μ L of ligation product pBI121-SlSNAT1, blowing, stirring, and standing on ice for 30 min;

C. after the ice bath is finished, putting the mixture into a metal bath with the temperature of 42 ℃ for heat shock for 80s, and rapidly carrying out ice bath for 3 min;

D. adding 500 μ L LB liquid culture medium into a clean bench, blowing, mixing, and shake culturing at 37 deg.C and 180rpm for 2 h;

E. 100. mu.L of the culture solution was aspirated and uniformly spread on an LB selective solid medium using a spreader. After the bacterial liquid is completely dried, the sealed flat plate is placed in an incubator at 37 ℃ in an inverted mode and cultured overnight;

F. after the bacterial plaque grows out, selecting a single clone, performing shake culture on 700 mu L LB liquid selective medium at 37 ℃ and 180rpm for about 3h, verifying PCR after the bacterial liquid is turbid, performing PCR amplification by using primers SlSNAT1S and SlSNAT1A (the PCR reaction system and the program are shown in tables 1 and 2) by the same method as example 1, and selecting the bacterial liquid containing the target fragment SlSNAT 1.

4. Plasmid extraction

And selecting a bacterial solution containing a target fragment verified by bacterial solution PCR, extracting a plasmid for sequencing, comparing the sequence with a known nucleotide sequence, and verifying to obtain an overexpression vector of the SlSNAT1, namely the recombinant plasmid OX-SlSNAT 1.

The plasmid extraction is carried out by adopting a plasmid small-extraction medium-volume kit (purchased from Tiangen Biotechnology technology (Beijing) Co., Ltd., product number DP106), and the specific steps are as follows:

(1) taking 10ml of the overnight cultured bacterial liquid in the step 3, centrifuging at 12000g for 1 minute, and removing the supernatant;

(2) adding 500 mu L of solution P1, and shaking by a shaker until the bacterial cells are completely suspended;

(3) adding 500. mu.L of solution P2, and gently turning up and down for 6-8 times;

(4) adding 700 μ L of solution P3, immediately turning gently up and down for 6-8 times, and centrifuging at 12000g for 10 min;

(5) adding the supernatant into an adsorption column, centrifuging at 12000g for 1 min, and removing the penetrating liquid;

(6) adding 600 μ L of rinsing liquid into the adsorption column, centrifuging at 12000g for 1 min, removing the penetrating liquid, and repeating once;

(7) placing the adsorption column in a collection tube, and centrifuging at 12000g for 5 minutes;

(8) the adsorption column was placed in a clean centrifuge tube, 200. mu. LEB Buffer was added to the adsorption column, left at room temperature for 5 minutes, and centrifuged at 12000g for 2 minutes, and the plasmid was collected in the centrifuge tube.

(II) recombinant plasmid transformation of Agrobacterium GV3101

1. mu.L of the recombinant plasmid OX-SlSNAT1 was added to 50. mu.L of Agrobacterium GV3101 (purchased from Shanghai Weidi Bio Inc., cat # AC1001), mixed well, and ice-cooled for 10 minutes;

2. quick freezing with liquid nitrogen for 5 min, at 37 deg.C for 5 min, and ice-cooling again for 3 min;

3. adding 500 μ L YEB liquid culture medium without antibiotic, culturing at 28 deg.C and 200rpm for 4 hr;

4. uniformly coating 100 mu L of bacterial liquid on a YEB solid culture medium containing rifampicin, and performing inverted culture for 2 days;

5. selecting a single clone to carry out PCR verification on bacteria liquid, wherein the PCR verification result is shown in figure 2, selecting and respectively storing agrobacterium infection liquid successfully transformed with recombinant plasmid OX-SlSNAT1 for later use, and naming the agrobacterium successfully transformed with recombinant plasmid OX-SlSNAT1 as OX-SlSNAT1-GV 3101;

(III) obtaining of SlSNAT1 overexpression transgenic plant

1. OX-SlSNAT1-GV3101 is used for infecting tomatoes to obtain transgenic T₀Plant generation plant

(1) Seeding

200 tomato Micro Tom seeds are placed in a 10ml disposable centrifuge tube, the shaking table is used for shaking up and disinfecting for 15 minutes by 4 percent sodium hypochlorite, and the shaking table is fully washed for 7 to 8 times by sterile water in a super clean workbench to thoroughly remove the residual disinfectant. Seeds were placed on sterilized filter paper and blotted to dry the residual liquid and then sown on seed germination medium (MS 4.43g/L + sucrose 30g/L + gel 2.5g/L, pH 5.8).

(2) Seed germination T0

Culturing the sown seeds in a dark room for 3-4 days, culturing the seeds in the light for 3-4 days after the seeds germinate, and culturing the seeds in the tissue when cotyledons are flattened and true leaves are exposed.

(3) Preculture stage T1

Taking cotyledon part of tomato plantlet growing for 7-8 days as tissue culture material, cutting off leaf tip with scissors, and cutting the rest part into 5 x 5mm square blocks as explant. Placing the treated explants on a pre-culture medium (MS 4.43g/L + sucrose 30g/L + gel 2.5g/L +1 mg/L6-BA +0.1mg/L IAA, pH 5.8), spreading filter paper before the culture medium is lifted, placing the cotyledon with the back side facing upwards, placing the cotyledon with the interval of 5-10mm as good, and culturing for two days in a dark place.

(4) Co-cultivation stage T1

Soaking the explant after two days of preculture in the staining solution (OD-OD) of OX-SlSNAT1-GV3101₆₀₀0.15-0.17), continuously shaking during soaking, pouring off the infection liquid after infecting for 5 minutes, sucking dry by filter paper, placing on a pre-culture medium, and culturing in a dark room for 2 days.

(5) Bud Induction phase T21

Taking out the explants after 2 days of co-culture from a dark room, putting all the explants in a bud induction culture medium T21(MS 4.43g/L + sucrose 30g/L + gelatin 2.5g/L +1mg/L ZT +0.1mg/L IAA +200mg/L Tim + antibiotics), transferring the explants into a new T21 culture medium after 7 days of light culture for continuous subculture, and after the first subculture, carrying out next subculture generally every 2 weeks until the explants completely germinate.

(6) Bud elongation stage T22

After bud induction, when the length of the sprouting bud of the explant is about 2-3cm, the explant is transferred into a bud elongation culture medium T22(MS 4.43g/L + sucrose 30g/L + gelatin 2.5g/L +0.5mg/L ZT +1mg/L GA +200mg/L Tim + antibiotics) and cultured for 3-4 weeks.

(7) Root stage Tr

When the shoots were elongated to 4-5cm, the shoots were transferred to rooting medium Tr (MS 4.43g/L + sucrose 30g/L + gelatin 2.5g/L +2mg/L IBA + +150mg/L Tim +1/2 antibiotics) after cutting out the callus and cultured for 3-4 weeks.

(8) Period of soil culture

Transferring the plantlets which have vigorous rooting and grow to a certain height into a soil pot in time to obtain transgenic T₀And (5) plant generation.

2. Transgenic T₀PCR identification of generation plants

Extraction of tomato Micro Tom (WT) and transgenic T₀Total DNA of leaf blades of the plants was identified by PCR using the above total DNA and recombinant plasmid OX-SlSNAT1(CK) as templates (the PCR reaction system and the reaction program are shown in tables 1 and 2, respectively) using primers (the overexpression identification S and the overexpression identification A in Table 5), and the results are shown in FIG. 3,no target band was found in lanes WT, 2, 4, and 5, while no target band was found in lanes P (positive control plasmid) and 1, 3, 6, 7, and 8, indicating that the transgenic lines corresponding to

lanes

1, 3, 6, 7, and 8 are positive T₀The generation SlSNAT1 gene overexpression strain is named as OX1-6 in sequence.

TABLE 5 transgenic identification primer List

3. Identification of relative expression quantity of SlSNAT1 in transgenic line

Tomato Micro Tom (WT) and transgenic line (OX1-6) leaves are taken as materials to extract RNA, reverse transcription is carried out to synthesize first strand cDNA, and the cDNA obtained by reverse transcription is diluted to 50 ng/. mu.l to be used as a template of RT-PCR. Tomato Ef α was used as an internal reference gene, and the above cDNA was used as a template for RT-PCR amplification using primers (qSNAT 1S and qSNAT1A and Ef α S and EF α A in Table 5), and the reaction system and reaction procedure were set up in three parallel replicates using ABI PRISM 7500 and 2 as shown in tables 6 and 7, respectively, using an instrument^-△△CTThe method analyzes the expression of the SlSNAT1 gene in different strains. The results are shown in figure 4, the gene expression levels of SNAT1 in OX4, OX5 and OX6 are remarkably increased and are respectively 3-5 times of the gene expression levels of SlSNAT1 in WT, and transgenic strains OX4, OX5 and OX6 are further proved to be positive over-expression strains, namely SlSNAT1 gene over-expression strains, wherein the OX6 is highest in up-regulation expression level, so that the germination rate of aged seeds is identified by adopting OX 6.

TABLE 6 RT-PCR reaction System

TABLE 7 reaction procedure

(IV) phenotypic testing of transgenic plants

1. Natural ageing treatment of seed

The same amount of tomato Micro Tom seeds (WT) and transgenic line seeds ((OX6-SlSNAT1)) are bagged with seeds respectively in 2016 (9 months), and the seeds are stored at room temperature (28 ℃), air humidity of 30% and in dark to reach a natural aging state.

2. Naturally aged seed germination

After tomato seeds are harvested, the initial value of seed germination rate is determined in the current harvest year, namely 2016. 100 seeds of WT and OX6 were taken respectively to test the initial germination rate, each group was repeated 3 times, the measured germination rate was about 98%, and WT and OX6 were not different.

The first germination test was performed in 2018 after tomato seeds were naturally aged for two years. A130 mm disposable square culture dish (Haimen department's instrument laboratory instrument factory, product number 130a) is used as a germination box, 3 layers of filter paper are padded in the germination box, 100 tomato Micro Tom seeds (WT) and transgenic line seeds (OX6-SlSNAT1) are placed on the filter paper respectively, 10ml of deionized water is added to submerge the seeds, the culture dish is placed in a constant-temperature culture room at 25 ℃ to be germinated in a dark place, photographing is carried out in the dark place, the germination condition is counted, and each group is repeated for three times. Wherein, the seed germination is determined as the emergence of white emergence of the radicle.

And after the tomato seeds are naturally aged for three years, carrying out a second germination test in 2019 and 9 months, wherein the test process is the same as 2018, and counting the germination condition.

The calculation formula of the seed germination rate is as follows: germination rate ═ (number of germinated seeds/number of test seeds) × 100%.

The germination conditions of the naturally aged seeds for two and three years are shown in figure 5, the statistical results of the germination rates are shown in figure 6, and the results show that the germination rate of the seeds of the over-expressed SlSNAT1 transgenic plant is obviously improved compared with that of the wild type, wherein the germination rate of the seeds of the over-expressed SlSNAT1 transgenic plant is improved by 21% compared with that of the wild type in 2 days of natural aging, and the germination rate of the seeds of the over-expressed SlSNAT1 transgenic plant is improved by 16% compared with that of the wild type in 5 days of germination; and (3) naturally aging for three years, wherein the germination rate of the seeds of the transgenic plant of the over-expression SlSNAT1 is improved by 29 percent compared with that of the wild type when the seeds germinate for 2 days, and the germination rate of the seeds of the transgenic plant of the over-expression SlSNAT1 is improved by 35.7 percent compared with that of the wild type when the seeds germinate for 5 days.

Although the invention has been described in detail hereinabove by way of general description, specific embodiments and experiments, it will be apparent to those skilled in the art that many modifications and improvements can be made thereto based on the invention. Accordingly, such modifications and improvements are intended to be within the scope of the invention as claimed.

Sequence listing

<110> university of agriculture in China

<120> application of SlSNAT1 protein and related biological materials thereof in regulation and control of plant seed aging resistance

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<213> Artificial Sequence (Artificial Sequence)

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Met Gln Met Gln Thr Leu His Leu Val Ser Thr Ser Thr Val Ala Ser

1 5 10 15

Ser Ser Ser Ser Ser Ser Leu Pro Thr Ile Val Ser Leu Asn Cys Cys

20 25 30

Arg Cys Gln Pro Ser Asn Gln Leu Pro Phe Pro Asn Ser Asn Leu Gly

35 40 45

Phe Leu Lys Val Lys Arg Gln Pro Lys Val Ser Asn Leu Lys Ala Ser

50 55 60

Phe Trp Asp Ser Ile Arg Ser Gly Phe Gly Lys Asn Asn Ile Ile Gln

65 70 75 80

Val Ile Asp Thr Pro Ser Ser Glu Glu Glu Glu Glu Glu Pro Leu Pro

85 90 95

Glu Glu Phe Val Leu Val Glu Lys Thr Gln Pro Asp Gly Thr Val Glu

100 105 110

Gln Ile Ile Phe Ser Ser Gly Gly Asp Val Asp Val Tyr Asp Leu Gln

115 120 125

Asp Leu Cys Asp Lys Val Gly Trp Pro Arg Arg Pro Leu Ser Lys Leu

130 135 140

Ala Ala Ala Leu Lys Asn Ser Tyr Ile Val Ala Thr Leu His Ser Arg

145 150 155 160

Lys Phe Ser Ser Gly Glu Glu Gly Ser Gly Glu Lys Lys Leu Ile Gly

165 170 175

Met Ala Arg Ala Thr Ser Asp His Ala Phe Asn Ala Thr Ile Trp Asp

180 185 190

Val Leu Val Asp Pro Ser Tyr Gln Gly Gln Gly Leu Gly Lys Val Leu

195 200 205

Ile Glu Lys Leu Ile Arg Thr Leu Leu Gln Arg Asp Ile Gly Asn Ile

210 215 220

Ser Leu Phe Ala Asp Ser Lys Val Val Glu Phe Tyr Arg Asn Leu Gly

225 230 235 240

Phe Glu Pro Asp Pro Glu Gly Ile Lys Gly Met Phe Trp Tyr Pro Met

245 250 255

Tyr

<210> 2

<211> 774

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

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atgcaaatgc aaactctcca cttagtatcc acttctactg ttgcttcttc ttcttcttct 60

tcttccctac ccactattgt ttctcttaat tgctgccgtt gtcaaccttc aaatcagttg 120

ccatttccca attctaattt gggttttctg aaagttaaga ggcaaccaaa agtttctaac 180

ttgaaggcta gcttttggga ttccatcaga tccgggtttg gcaagaataa cataatacag 240

gttatagata caccatccag tgaagaagaa gaggaagaac ctttgcctga ggaatttgtt 300

ctagttgaaa agactcaacc tgatggaaca gttgaacaga ttatattctc ttctggagga 360

gatgttgatg tgtatgatct ccaagattta tgtgataagg ttggttggcc tcgaagacca 420

ctgtctaagc tagctgcagc tctgaaaaat agctatatag ttgcaacttt gcattctagg 480

aaattctcat caggagaaga ggggagtgga gaaaagaagc tgataggcat ggcccgtgca 540

acatcagatc atgcattcaa tgcaacaatt tgggatgttc ttgttgatcc ttcctatcag 600

ggacaaggac ttggaaaagt tcttatcgag aaactgatac gaacccttct ccaaagggac 660

atcggaaata tttcactgtt tgcagatagt aaagttgtgg aattttacag gaatcttggt 720

tttgaacctg atccagaggg aattaaggga atgttctggt accccatgta ttag 774

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<212> DNA

<213> Artificial Sequence (Artificial Sequence)

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atgcaaatgc aaactctcca c 21

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<212> DNA

<213> Artificial Sequence (Artificial Sequence)

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atacatgggg taccagaaca tt 22

<210> 5

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<213> Artificial Sequence (Artificial Sequence)

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cgcacaatcc cactatcctt catgcaaatg caaactctcc ac 42

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<213> Artificial Sequence (Artificial Sequence)

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atccagactg aatgcccaca ggatacatgg ggtaccagaa catt 44

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<212> DNA

<213> Artificial Sequence (Artificial Sequence)

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cgcacaatcc cactatcctt c 21

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atccagactg aatgcccaca gg 22

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<212> DNA

<213> Artificial Sequence (Artificial Sequence)

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ggaacttgag aaggagccta ag 22

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<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 10

caacaccaac agcaacagtc t 21

<210> 11

<211> 21

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 11

tgatccagag ggaattaagg g 21

<210> 12

<211> 22

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 12

tctcagaatc caacagccac at 22

Claims

The application of the SlSNAT1 protein or coding nucleic acid thereof or biological material containing the coding nucleic acid of the SlSNAT1 protein in improving the germination rate of naturally aged tomato seeds is disclosed, wherein the amino acid sequence of the SlSNAT1 protein is shown as SEQ ID No.1, the coding nucleic acid of the SlSNAT1 protein is shown as SEQ ID No.2, and the biological material is an expression cassette, a vector, a host cell, an engineering bacterium, a transgenic plant cell line, a transgenic plant tissue, a transgenic plant organ, a transgenic plant, a tissue culture produced by regenerable cells of the transgenic plant or a protoplast produced by the tissue culture.
2. The use according to claim 1, wherein the germination rate of naturally aging tomato seeds is increased by increasing the expression level and/or activity of the SlSNAT1 protein in tomato.
The application of the SlSNAT1 protein or coding nucleic acid thereof or biological material containing the coding nucleic acid of the SlSNAT1 protein in prolonging the life of tomato seeds is disclosed, wherein the amino acid sequence of the SlSNAT1 protein is shown as SEQ ID No.1, the coding nucleic acid of the SlSNAT1 protein is shown as SEQ ID No.2, and the biological material is an expression cassette, a vector, a host cell, an engineering bacterium, a transgenic plant cell line, a transgenic plant tissue, a transgenic plant organ, a transgenic plant, a tissue culture produced by a regenerable cell of the transgenic plant or a protoplast produced by the tissue culture.
4. The use according to claim 3, wherein the longevity of tomato seeds is increased by increasing the expression level and/or activity of the SlSNAT1 protein in tomato.
The application of the SlSNAT1 protein or coding nucleic acid thereof or biological material containing the coding nucleic acid of the SlSNAT1 protein in tomato seed aging-resistant genetic breeding is disclosed, wherein the amino acid sequence of the SlSNAT1 protein is shown in SEQ ID No.1, the coding nucleic acid of the SlSNAT1 protein is shown in SEQ ID No.2, and the biological material is an expression cassette, a vector, a host cell, an engineering bacterium, a transgenic plant cell line, a transgenic plant tissue, a transgenic plant organ, a transgenic plant, a tissue culture produced by a regenerable cell of the transgenic plant or a protoplast produced by the tissue culture.
6. A method for increasing the germination rate of naturally-aged tomato seeds or prolonging the life of tomato seeds, comprising: increasing the germination rate of naturally aged tomato seeds or prolonging the seed life of tomatoes by increasing the expression level and/or activity of SlSNAT1 protein in the tomatoes;

the amino acid sequence of the SlSNAT1 protein is shown in SEQ ID NO. 1.