CN112391404B

CN112391404B - Application of strawberry SnRK2.1 gene in regulation and control of strawberry fruit ripening and quality formation

Info

Publication number: CN112391404B
Application number: CN202010877476.9A
Authority: CN
Inventors: 李冰冰; 毛文文; 陈亚婷; 刘婷; 冯倩倩; 李莉
Original assignee: China Agricultural University
Current assignee: China Agricultural University
Priority date: 2020-08-27
Filing date: 2020-08-27
Publication date: 2022-08-23
Anticipated expiration: 2040-08-27
Also published as: CN112391404A

Abstract

The invention discloses application of a strawberry SnRK2.1 gene in regulation and control of strawberry fruit ripening and quality formation. The invention relates to a technical scheme for controlling the application of a substance for controlling the expression of a plant SnRK2.1 gene in controlling the sugar content of a plant fruit. The amino acid sequence of the protein coded by the SnRK2.1 gene can be the protein of SEQ ID No.1 in a sequence table. Experiments prove that compared with a plant with a pH7WG 2D-transferred and an empty carrier 1, the SnRK2.1-transferred gene can promote the fruit ripening and sugar accumulation of strawberries; when the fruit grows to 23 days after blossom, the fructose content of the strawberry fruit of the SnRK2.1 transgenic plant is improved by 7.87 percent, the glucose content is improved by 9.84 percent, P is less than 0.05, the sucrose content is improved by 18.61 percent, and P is less than 0.01. The SnRK2.1 gene can be applied to the regulation and control of the mature period of strawberry fruits and the quality improvement in the actual production.

Description

Application of strawberry SnRK2.1 gene in regulation and control of strawberry fruit ripening and quality formation

Technical Field

The invention relates to the field of genetic engineering, in particular to application of a strawberry SnRK2.1 gene in regulation and control of strawberry fruit ripening and quality formation.

Background

Sucrose non-glycolytic protein kinase 2(SnRK2) is a serine-threonine protein kinase and plays an important role in stress, and researches show that 10 SnRK2 members in rice can be induced by salt stress, and 11 SnRK2 members in corn play an important role in stress. In the arabidopsis SnRK2 family, all but snrk2.9 are activated by high osmotic and salt stress. The SnRK2s plays a very important role in an ABA signal transduction pathway, and the family is divided into three classes according to the response to ABA, wherein one class is SnRK2 kinase strongly induced by ABA, the second class is SnRK2 kinase slightly induced by ABA, and the third class is kinase not induced by ABA.

With the development of economy, fruits become essential food in daily life of people, and the fruits are rich in various nutrient substances such as sucrose, glucose, fructose, vitamins and the like, so that the fruits have important economic value. The ripening and quality formation of strawberry fruit are a complex biological process, which involves various metabolic systems and is regulated and controlled by various factors such as environment and hormone. The strawberry cultivation mainly in the greenhouse is limited by the cultivation environment, poor fruit coloring and low sugar content are easily caused by the greenhouse content, and the industrial value of the strawberries is reduced. Therefore, the selection of good strawberry varieties is crucial to production.

In strawberry, there are 9 SnRK2 family members, among which snrk2.6 negatively regulate fruit ripening, while no related studies on other members have been reported. Related members of SnRK2 kinase are cloned and separated, and the method has important significance for realizing strawberry gene improvement by utilizing genetic engineering in the future and improving the quality of strawberry fruits on a molecular level.

Disclosure of Invention

The technical problem to be solved by the invention is how to regulate the ripening of the strawberry fruits or how to improve the quality of the strawberry fruits.

In order to solve the above technical problems, the present invention firstly provides any one of the following uses of a substance regulating expression of snrk2.1 gene derived from strawberry:

f1, the application of the substance for regulating gene expression in regulating the content of sucrose, glucose and/or fructose in plant fruits;

f2, the application of the substance for regulating gene expression in regulating the sugar content of plant fruits;

f3, and the application of the substance for regulating gene expression in the preparation of products for improving the sugar content of plant fruits;

f4, and the application of the substance for regulating gene expression in the preparation of products for increasing the contents of sucrose, glucose and/or fructose in plant fruits;

f5, the application of the substance for regulating gene expression in regulating the mature period of plant fruit;

f6, and application of a substance for regulating gene expression in preparing a product for shortening the maturation period of strawberry fruits;

f7, the application of the substance for regulating gene expression in plant breeding;

f8, the application of the substance for regulating gene expression in improving plant quality;

F1-F8, the gene encodes the following protein A1), A2) or A3):

A1) the amino acid sequence is protein of SEQ ID No.1 in a sequence table;

A2) a protein which is obtained by substituting and/or deleting and/or adding one or more amino acid residues of the amino acid sequence shown as SEQ ID No.1 in the sequence table, is derived from A1) and has the same function, or has more than 80 percent of identity with the protein shown as A1) and has the same function;

A3) a fusion protein obtained by connecting protein tags at the N-terminal or/and the C-terminal of A1) or A2).

The protein is derived from strawberry, and the plant is any one of the following:

C1) (ii) a monocotyledonous plant,

C2) a dicotyledonous plant, a plant selected from the group consisting of dicotyledonous plants,

C3) a plant of the order Rosales,

C4) a plant of the family Rosaceae,

C5) a plant of the genus Fragaria,

C6) and (5) strawberry.

In the protein, SEQ ID No.1 in a sequence table consists of 337 amino acid residues.

The protein can be artificially synthesized, or can be obtained by synthesizing the coding gene and then performing biological expression.

In the above protein, the protein tag (protein-tag) refers to a polypeptide or protein that is expressed by fusion with a target protein using in vitro recombinant DNA technology, so as to facilitate expression, detection, tracking and/or purification of the target protein. The protein tag may be a Flag tag, a His tag, an MBP tag, an HA tag, a myc tag, a GST tag, and/or a SUMO tag, among others.

In the above proteins, identity refers to the identity of amino acid sequences. The identity of the amino acid sequences can be determined using homology search sites on the Internet, such as the BLAST web pages of the NCBI home website. For example, in the advanced BLAST2.1, by using blastp as a program, setting the value of Expect to 10, setting all filters to OFF, using BLOSUM62 as a Matrix, setting Gap existence cost, Per residual Gap cost, and Lambda ratio to 11, 1, and 0.85 (default values), respectively, and performing a calculation by searching for the identity of a pair of amino acid sequences, a value (%) of identity can be obtained.

In the above protein, the 80% or more identity may be at least 81%, 82%, 85%, 86%, 88%, 90%, 91%, 92%, 95%, 96%, 98%, 99% or 100% identity.

In the above application, the substance for regulating gene expression may be a substance for regulating at least one of the following 6 kinds of regulation: 1) regulation at the level of transcription of said gene; 2) regulation after transcription of the gene (i.e., regulation of splicing or processing of a primary transcript of the gene); 3) regulation of RNA transport of the gene (i.e., regulation of nuclear to cytoplasmic transport of mRNA of the gene); 4) regulation of translation of the gene; 5) regulation of mRNA degradation of the gene; 6) post-translational regulation of the gene (i.e., regulation of the activity of a protein translated from the gene).

In the above application, the regulation of gene expression may be to enhance or increase the gene expression.

In the above application, the substance for regulating gene expression may be an agent for enhancing or increasing the gene expression.

In order to solve the technical problems, the invention also provides any one of the following applications of the protein in the application:

p1, the application of the protein in regulating the content of sucrose, glucose and/or fructose in plant fruits;

p2, the application of the protein in regulating and controlling the sugar content of plant fruits;

p3, the application of the protein in preparing products for improving the sugar content of plant fruits;

p4, the application of the protein in preparing products for improving the content of sucrose, glucose and/or fructose in plant fruits;

p5, the application of the protein in regulating the mature period of the plant fruit;

p6 and application of the protein in preparation of products for shortening the ripening cycle of strawberry fruits;

p7, use of the above protein in plant breeding;

p8 and the application of the protein in improving the quality of the plant.

In order to solve the technical problem, the invention also provides any one of the following applications of the biological material related to the protein in the application:

q1, use of the biomaterial for regulating the sucrose, glucose and/or fructose content of a plant fruit;

q2, the use of the biomaterial in regulating the sugar content of plant fruits;

q3, and the application of the biological material in preparing products for improving the sugar content of plant fruits;

q4, the application of the biological material in preparing products for improving the content of sucrose, glucose and/or fructose in plant fruits;

q5, the use of the biomaterial in regulating the ripening cycle of a plant fruit;

q6, and the application of the biological material in preparing products for shortening the ripening cycle of strawberry fruits;

q7, use of the biomaterial in plant breeding;

q8, use of the biomaterial in plant quality improvement;

the biomaterial is any one of the following B1) to B9):

B1) a nucleic acid molecule encoding the protein;

B2) an expression cassette comprising the nucleic acid molecule of B1);

B3) a recombinant vector containing the nucleic acid molecule of B1) or a recombinant vector containing the expression cassette of B2);

B4) a recombinant microorganism containing B1) the nucleic acid molecule, or a recombinant microorganism containing B2) the expression cassette, or a recombinant microorganism containing B3) the recombinant vector;

B5) a transgenic plant cell line containing the nucleic acid molecule according to B1) or a transgenic plant cell line containing the expression cassette according to B2);

B6) transgenic plant tissue comprising the nucleic acid molecule of B1) or transgenic plant tissue comprising the expression cassette of B2);

B7) a transgenic plant organ containing the nucleic acid molecule of B1), or a transgenic plant organ containing the expression cassette of B2);

B8) a nucleic acid molecule that inhibits or reduces gene expression of the protein;

B9) an expression cassette, a recombinant vector, a recombinant microorganism or a transgenic plant cell line comprising the nucleic acid molecule according to B8).

In the above biological material, the nucleic acid molecule of B1) may be a gene encoding the protein as shown in B1) B2) or B3):

b1) the coding sequence of the coding chain is a cDNA molecule or a DNA molecule of the nucleotide of SEQ ID No.2 in the sequence table;

b2) the nucleotide is cDNA molecule or DNA molecule of SEQ ID No.2 in the sequence table,

b3) a cDNA or DNA molecule which hybridizes with the cDNA or DNA molecule defined in b2) and encodes a protein having the same function.

In the above biological materials, the expression cassette containing a nucleic acid molecule described in B2) refers to a DNA capable of expressing the protein described in the above application in a host cell, and the DNA may include not only a promoter for initiating transcription of the gene encoding the protein but also a terminator for terminating transcription of the gene encoding the protein. Further, the expression cassette may also include an enhancer sequence. Promoters useful in the present invention include, but are not limited to: constitutive promoters, tissue, organ and development specific promoters, and inducible promoters. Examples of promoters include, but are not limited to: constitutive promoter 35S of cauliflower mosaic virus; the wound-inducible promoter from tomato, leucine aminopeptidase ("LAP", Chao et al (1999) Plant Physiology 120: 979-992); chemically inducible promoter from tobacco, pathogenesis-related 1(PR1) (induced by salicylic acid and BTH (benzothiadiazole-7-carbothioic acid S-methyl ester)); tomato proteinase inhibitor II promoter (PIN2) or LAP promoter (both can be obtained from jasmineKetonic acid methyl ester induction); heat shock promoters (U.S. patent 5,187,267); tetracycline-inducible promoters (U.S. Pat. No. 5,057,422); seed-specific promoters, such as the millet seed-specific promoter pF128(CN101063139B (Chinese patent 200710099169.7)), seed storage protein-specific promoters (e.g., the promoters of phaseolin, napin, oleosin, and soybean beta conglycin (Beachy et al (1985) EMBO J.4: 3047-3053)). They can be used alone or in combination with other plant promoters. All references cited herein are incorporated herein in their entirety. Suitable transcription terminators include, but are not limited to: agrobacterium nopaline synthase terminator (NOS terminator), cauliflower mosaic virus CaMV 35S terminator, tml terminator, pea rbcS E9 terminator and nopaline and octopine synthase terminators (see, e.g., Odell et al (I) ⁹⁸⁵ ) Nature 313: 810; rosenberg et al (1987) Gene,56: 125; guerineau et al (1991) mol.gen.genet,262: 141; proudfoot (1991) Cell,64: 671; sanfacon et al Genes Dev.,5: 141; mogen et al (1990) Plant Cell,2: 1261; munroe et al (1990) Gene,91: 151; ballad et al (1989) Nucleic Acids Res.17: 7891; joshi et al (1987) Nucleic Acid Res, 15: 9627).

The recombinant expression vector containing the protein coding gene expression cassette can be constructed by using the existing plant expression vector. The plant expression vector comprises a binary agrobacterium vector, a vector for plant microprojectile bombardment and the like. Such as pAHC25, pWMB123, pBin438, pCAMBIA1302, pCAMBIA2301, pCAMBIA1301, pCAMBIA1300, pBI121, pCAMBIA1391-Xa or pCAMBIA1391-Xb (CAMBIA Corp.) and the like. The plant expression vector may also comprise the 3' untranslated region of the foreign gene, i.e., a region comprising a polyadenylation signal and any other DNA segments involved in mRNA processing or gene expression. The polyadenylation signal can direct polyadenylic acid to the 3 'end of the mRNA precursor, and similar functions can be found in untranslated regions transcribed from the 3' end of Agrobacterium crown gall inducible (Ti) plasmid genes (e.g., nopaline synthase gene Nos) and plant genes (e.g., soybean storage protein gene). When the gene of the present invention is used to construct a plant expression vector, enhancers, including translational or transcriptional enhancers, may be used, and these enhancer regions may be ATG initiation codon or initiation codon of adjacent regions, etc., but must be in the same reading frame as the coding sequence to ensure correct translation of the entire sequence. The translational control signals and initiation codons are widely derived, either naturally or synthetically. The translation initiation region may be derived from a transcription initiation region or a structural gene. In order to facilitate identification and screening of transgenic plant cells or plants, plant expression vectors to be used may be processed, for example, by adding genes encoding enzymes or luminescent compounds which produce a color change (GUS gene, luciferase gene, etc.), marker genes for antibiotics which are expressible in plants (e.g., nptII gene which confers resistance to kanamycin and related antibiotics, bar gene which confers resistance to phosphinothricin which is a herbicide, hph gene which confers resistance to hygromycin which is an antibiotic, dhS gene which confers resistance to methatrexate, EPSPS gene which confers resistance to glyphosate), or marker genes for chemical resistance (e.g., herbicide resistance), mannose-6-phosphate isomerase gene which provides the ability to metabolize mannose, etc. From the safety of transgenic plants, the transgenic plants can be directly screened and transformed in a stress environment without adding any selective marker gene.

In the above biological material, the recombinant microorganism may be specifically yeast, bacteria, algae and fungi.

In the above application, the sugar content may be sucrose, glucose and/or fructose content; the content of sucrose, glucose and/or fructose in plant fruits is regulated and controlled to be increased; the regulation and control of the fruit ripening cycle of the plant is to accelerate the fruit ripening cycle of the plant.

Any of the following products comprising the substance of the regulatory gene and/or the protein and/or the biological material are also within the scope of the present invention:

d1, products for increasing the sucrose, glucose and/or fructose content of plant fruits;

d2, a product for increasing the sugar content of plant fruits;

d3, shortening the mature period of plant fruit.

In the above application or product, the plant is any one of:

C1) (ii) a monocotyledonous plant,

C3) a plant of the order Rosales,

C4) a plant of the family Rosaceae,

C5) a plant of the genus Fragaria,

C6) and (4) strawberry.

The gene of SnRK2.1 cloned from a diploid variety Dibosco of strawberry is constructed in a pH7WG2D, and after a vector is expressed by 1, a diploid strawberry tissue culture seedling is transformed to obtain the strawberry with the transformed SnRK2.1 gene. The observation result of the fruit ripening period shows that T ₀ Compared with the plants with empty carriers, the positive plants with the transferred SnRK2.1 genes can promote the plants to mature and shorten the maturation period of strawberry fruits; the statistical analysis result of the fruit index shows that T is 23d after flowering ₀ Compared with the empty vector plant, the fruit of the positive plant with the gene of the SnRK2.1 is obviously increased in sugar content, wherein the content of fructose and glucose is obviously increased, and P is<0.05, the sucrose content is extremely obviously increased, P<0.01. The SnRK2.1 gene is shown to have important effect on the development and quality formation of strawberry fruits, and can be applied to the regulation and control of the ripening cycle of the strawberry fruits in actual production and the quality improvement.

Drawings

FIG. 1 is a vector diagram of pH7WG2D, 1.

FIG. 2 is T ₀ And (4) screening a generation SnRK2.1 transgenic line. "ACTIN represents" the detection band of the ACTIN gene, "the bands corresponding to" P5, P6 "represent the detection of the target gene: CK represents the control plant of the empty vector and 1-15 represents the SnRK2.1 transgenic plant to be identified.

FIG. 3 is T ₀ And (4) observing the mature period of the fruit of the generation SnRK2.1 transgenic plant. OE for snrk2.1 transgenic plants; CK represents empty vector plants.

FIG. 4 is T ₀ And analyzing the fruit index of the generation SnRK2.1 transgenic plant. The ordinate is the sugar content, and the unit is mg/g FW; the abscissa DPA21 represents 21 days after the plant blooms, and DPA23 represents 23 days after the plant blooms; CK stands for empty vector plants and OE for snrk2.1 transgenic plants.

Detailed Description

The present invention is described in further detail below with reference to specific embodiments, and the examples are given only for illustrating the present invention and not for limiting the scope of the present invention. The examples provided below serve as a guide for further modifications by a person skilled in the art and do not constitute a limitation of the invention in any way.

The experimental procedures in the following examples, unless otherwise indicated, are conventional and are carried out according to the techniques or conditions described in the literature in the field or according to the instructions of the products. Materials, reagents and the like used in the following examples are commercially available unless otherwise specified.

The quantitative tests in the following examples, all set up three replicates and the results averaged. The data were processed using GraphPad Prism statistical software, the experimental results are expressed as mean ± standard deviation, and differential significance analysis was performed using t-test.

Example 1: application of strawberry SnRK2.1 gene in regulation and control of strawberry fruit ripening and quality formation

Transforming diploid strawberry Dibosco (hereinafter referred to as wild strawberry, WT) tissue culture seedlings by using SnRK2.1 gene expression strain EHA105/pH7WG 2D-SnRK2.1. Wherein, THE cloning of THE SnRK2.1 gene is from diploid strawberry Dibosco (purchased from THE HEIRLOOM SEED STORE, and THE purchasing website is https:// www.theheirloomseedstore.com/product/strawberry-fragola-di-bosc o), THE cloning of THE SnRK2.1 gene, THE construction process of pH7 RKWG 2D-SnRK2.1, SnRK2.1 gene expression bacteria EHA105/pH7WG2D-SnRK2.1 and empty vector transformation bacteria EHA105/pH7WG2D are shown in THE following step three.

1) Respectively activating SnRK2.1 gene expression strain EHA105/pH7WG2D-SnRK2.1 and empty vector transformation strain EHA105/pH7WG2D, and shaking the concentration of the strain liquid to OD ₆₀₀ Respectively pouring the obtained mixture into 50ml centrifuge tubes at 5000rpm for 10min, pouring out the centrifuged supernatant in a super clean bench, adding MS activating solution (solvent is water, solute and content thereof is MS4.4g/L, sucrose 30g/L, 1M NaOH to adjust pH to 5.8, sterilizing at high temperature and high pressure, and adding acetosyringone with final concentration of 100 μ M before use) to suspend thallusActivation at 28 ℃ for 1 h.

2) Preparing plant materials during bacterial liquid activation, taking young and tender leaves of diploid strawberry Dibosco tissue culture seedlings, shearing into proper sizes, placing the leaves into MS activating solution, pouring suspended bacterial liquid into the leaves, infecting for 30min to 1h, transferring explants onto a co-culture medium after infecting (the solvent is water, the solute content is MS4.4g/L, the sucrose content is 30g/L, the agar powder content is 7g/L, the 6-BA content is 0.1mg/L, the 2,4-D content is 0.01mg/L, the TDZ content is 2mg/L, the pH value is adjusted to 5.8 by 1M NaOH, sterilizing at high temperature and high pressure), and culturing in dark for 2D.

3) The dark cultured explants are carefully transferred to a screening medium (the solvent is water, the solute content is MS4.4g/L, the sucrose content is 30g/L, the agar powder content is 7g/L, 6-BA 0.1mg/L, 2, 4-D0.01 mg/L, TDZ 2mg/L, 1M NaOH is used for adjusting the pH value to 5.8, Hyg 2mg/L and Tim (timentin) 400mg/L are added after high-temperature and high-pressure sterilization), and the buds growing for about 40 days are cultured.

4) As the carrier is provided with the GFP reporter gene, the resistant bud bundle can be irradiated under fluorescence, the fluorescent bud bundle is reserved, when the resistant plants grow to 2-3 cm, the plants are transferred to a rooting culture medium (the solvent is water, the solute and the content thereof are MS4.4g/L, 30g/L of cane sugar, 7g/L of agar powder and 1M NaOH are used for regulating the pH value to 5.8, and Hyg 3mg/L and Tim 400mg/L are added after high-temperature and high-pressure sterilization).

5) Transplanting seedlings with roots growing to 4-5cm into soil, carefully removing a root culture medium, covering the seedlings with a preservative film for moisture preservation, and removing the preservative film after 15d to finally obtain a SnRK2.1 transgenic plant to be identified (a plant obtained by impregnating SnRK2.1 gene expression bacteria EHA105/pH7WG2D-SnRK2.1) and a no-load control plant (a plant obtained by impregnating empty vector transformation bacteria EHA105/pH7WG 2D).

Second, obtaining transgenic positive plants and identifying phenotype

2.1 obtaining transgenic Positive plants

Respectively extracting RNA of a plant (numbered 1-15) of the SnRK2.1 transgenic to be identified and an unloaded control plant (CK), inverting the RNA into cDNA, and identifying by PCR, wherein primers are as follows:

P5：5’-ATGGAGCAGAAACTCATCTCTGAAGAGGATCTG-3’

P6：5’-CAACGCACATACAAAATCACCAC-3’

the constitutively expressed actin gene was used as an internal control. Wherein the positive plants of the SnRK2.1 transgenic plants can obtain strips of about 1000bp through amplification, the results are shown in figure 2, and the other transgenic plants are transgenic positive plants containing target strips except the plants with the numbers of 9 and 14 which are not amplified to obtain the target strips; no-load control CK plants contained no target band.

2.2 fruit ripening cycle Observation and fruit index analysis

Will identify T ₀ And (3) transferring empty carrier plants and positive plants of the SnRK2.1 gene to nutrient soil: vermiculite: the turf is 1: 1:1, culturing at 25 ℃, wherein the photoperiod is 12h/12h, observing the fruit ripening period and measuring the index after the plants enter the flowering and fruiting period, and the result is shown in figures 3 and 4.

The observation of the fruit ripening period shows that T grows to 127d ₀ The positive plants transformed with the SnRK2.1 gene have bloomed and fruited (OE in figure 3), the plants transformed with the empty vector (CK in figure 3) have not bloomed, and the SnRK2.1 gene can promote the plant ripening and shorten the ripening period of strawberry fruits. The statistical analysis result of the fruit index shows that T is reached when the fruit grows to 21 days after flowers ₀ The sugar content of the trans-SnRK2.1 gene positive plant (shown as OE in figure 4) and the trans-empty vector plant (shown as CK in figure 4) have no significant difference; at 23d post-anthesis, the sugar content of the OE plants is significantly increased compared to CK plants, with a fructose and glucose content OE significantly higher than that of CK plants, P<0.05, the content of sucrose OE plant is remarkably higher than that of CK plant, P<0.01, specifically, the fructose content is improved by 7.87 percent, the glucose content is improved by 9.84 percent, and the sucrose content is improved by 18.61 percent. (shown in FIG. 4).

The sugar content determination method comprises the following steps:

2.2.1 fructose, glucose and sucrose content determination

The determination was done using HPLC method at vegetable research center, agroforestry academy of sciences, Beijing.

Specifically, about 0.5g of a fruit sample was put in a mortar, and 5ml of 80% ethanol was added thereto, and ground, and extracted in a water bath at 80 ℃ for 30min (while shaking several times). Centrifuging at 4000g for 10min, collecting supernatant, and repeating for 3 times. Mixing the supernatants, oven drying at 40 deg.C, adding 1ml deionized water for dissolving, filtering with 0.22 μm needle filter, and measuring fructose, glucose, and sucrose. In the HPLC analysis, fructose, glucose and sucrose are respectively used as standard substances to carry out qualitative analysis according to the retention time of the standard substances and quantitative analysis by adopting a standard curve method (external standard method). HPLC analysis adopts Dikma polyamine HILIC 250 x 4.6mm,5 μm chromatographic column, mobile phase is 75% acetonitrile water solution, detector is SHIMADZU, RID-10A differential refractometer, flow rate is 1.0ml/min, detection temperature is 35 deg.C, and sample amount is 10 μ L.

The results show that the SnRK2.1 gene plays an important role in the ripening cycle and quality formation of strawberry fruits, and can be applied to the regulation and control of the ripening cycle and quality improvement of the strawberry fruits in practical production.

Thirdly, cloning of SnRK2.1 gene, construction of pH7WG2D-SnRK2.1, SnRK2.1 gene expression strain EHA105/pH7WG2D-SnRK2.1 and empty vector transformation strain EHA105/pH7WG2D

Taking strawberry diploid variety Dibosco (Fragaria vesca, cv. Dibosco) fruits as experimental materials, extracting total RNA by using an Omega RNA extraction kit, and reversing by using M-MLV reverse transcriptase to obtain cDNA. Primers P1 and P2 were designed based on the coding region sequence of SnRK2.1 gene. PCR amplification was performed using the cDNA obtained by reverse transcription as a template, and primers P1 and P2. The primer sequences are as follows:

P1:5’-AAAAAGCAGGCTCAATGGAGCAGAAACTCATCTCTGAAGAGGATCTGGAGAGGTATGAGATAGTGAA-3’

P2:5’-AGAAAGCTGGGTCAACGCACATACAAAATCACCAC-3’

and (3) PCR reaction system:

the PCR reaction conditions are as follows: 5min at 98 ℃; 10s at 98 ℃, 30s at 56 ℃, 40s at 72 ℃ and 30 cycles; 5min at 72 ℃.

The PCR product was subjected to 1% agarose gel electrophoresis, and the band of about 1kb was excised. The fragment was recovered using the agarose gel recovery kit for Tiangen. Taking the fragment as a template, carrying out second round PCR amplification by using primers P3 and P4, carrying out electrophoresis detection and recovery in the same system, wherein the sequences of the primers P3 and P4 are as follows:

P3：5’-GGGGACAAGTTTGTACAAAAAAGCAGGCT-3’

P4：5’-GGGGACCACTTTGTACAAGAAAGCTGGGT-3’

the recovered product after two rounds of PCR amplification was subjected to the BP reaction (invitrogen, 11789-:

adding 1 mul of protease K after 1h at 25 ℃, terminating the reaction at 37 ℃ for 10min, and converting the product after the BP reaction into DH5 alpha by the following conversion method: adding the product into 50 mul DH5 alpha colibacillus competence, placing on ice for 30min, thermally shocking at 42 ℃ for 45s, immediately cooling on ice for 2min, adding 500 mul non-resistant LB, centrifuging at 37 ℃ for 1h at 180rpm and 7000rpm for 3min, sucking 400 mul supernatant, re-suspending the thallus, coating on an LB solid culture medium containing 50mg/L kanamycin by using a coater, culturing overnight at 37 ℃, picking out a single clone in an LB liquid culture medium containing 50mg/L kanamycin, culturing in a shaking culture apparatus at 37 ℃ for 8h, carrying out bacterium liquid PCR identification, and selecting a positive clone for sequencing. The plasmid (recombinant plasmid pDONR221-SnRK2.1 containing the SnRK2.1 gene with the coding sequence of the coding chain being SEQ ID No.2 in the sequence table) with correct sequencing is subjected to LR reaction (invitrogen,11791-020) with a pH7WG2D,1 plasmid (VIB-UGENT CENTER FOR PLANT SYSTEMS BIOLOGY, Vector ID:1-12) with the Vector diagram being shown in FIG. 1 to obtain the recombinant plasmid which contains the coding sequence of the coding chain being the SnRK2.1 gene with the coding sequence of SEQ ID No.2 in the sequence table and can express the SnRK2.1 gene and is named as pH7WG 2D-SnRK2.1. The coding amino acid sequence of the SnRK2.1 gene is protein SnRK2.1 of SEQ ID No.1 in a sequence table.

The system is as follows:

after 1h at 25 ℃ 1. mu.l of protease K was added and the reaction was stopped at 37 ℃ for 10 min. Transforming the product into escherichia coli competence DH5 alpha, coating the product on LB solid culture medium containing 50mg/L spectinomycin resistance, picking out spots and shaking little after overnight culture at 37 ℃, carrying out PCR identification by using primers P1 and P2, selecting positive clone (the size of the obtained PCR product is 1014bp) to shake big, extracting plasmid by using a plasmid miniprep kit, and transforming agrobacterium-sensitive EHA 105. The transformation method comprises the following steps: mu.g of DNA with pH7WG2D-SnRK2.1, adding 100. mu.l of EHA105 Agrobacterium tumefaciens competence, mixing gently, and standing on ice for 30 min; quick freezing with liquid nitrogen for 1min, and water bathing at 37 deg.C for 5 min; adding 800 μ l of LB liquid culture medium without resistance, shaking and culturing at 28 deg.C and 180rpm for 4 h; centrifuging at 5000rpm for 8min to collect thallus, coating 100 μ L of thallus on a plate containing 25mg/L rifampicin and 50mg/L spectinomycin, culturing at 28 ℃ for 2d, selecting a single colony, shaking slightly, performing PCR identification on the thallus by using primers P1 and P2, selecting positive clone (the size of a PCR product is 1014bp), shaking greatly to saturate and preserving the thallus, and obtaining agrobacterium of a recombinant vector successfully transformed with the SnRK2 RK.1 gene expression strain or EHA105/pH7WG 2D-SnRK2.1.

Meanwhile, the plasmid (empty vector) with pH7WG2D and 1 is transferred into agrobacterium EHA105 to obtain empty vector transformation strain EHA105/pH7WG2D, and the positive monoclonal colony growing on a plate containing 25mg/L rifampicin and 50mg/L spectinomycin is selected to be saturated in large shaking and to preserve the strain (the empty vector for short) and is used as a control for the phenotype identification of the transgenic plant.

Sequence listing

<110> university of agriculture in China

Application of <120> strawberry SnRK2.1 gene in regulation and control of strawberry fruit ripening and quality formation

<130> GNCSQ201840

<160> 2

<170> PatentIn version 3.5

<210> 1

<211> 337

<212> PRT

<213> strawberry (Fragaria visco)

<400> 1

Met Glu Arg Tyr Glu Ile Val Lys Asp Ile Gly Ser Gly Asn Phe Gly

1 5 10 15

Val Ala Lys Leu Val Lys Asp Lys Trp Ser Gly Glu Leu Tyr Ala Ile

20 25 30

Lys Phe Ile Glu Arg Gly Gln Lys Ile Asp Glu His Val Gln Arg Glu

35 40 45

Ile Met Asn His Arg Ser Leu Lys His Pro Asn Ile Ile Arg Phe Lys

50 55 60

Glu Val Leu Leu Thr Gln Thr Asp Leu Ala Ile Val Met Glu Tyr Ala

65 70 75 80

Ala Gly Gly Glu Leu Phe Glu Arg Ile Cys Asn Ala Gly Arg Phe Ser

85 90 95

Glu Asp Glu Ala Arg Phe Phe Phe Gln Gln Leu Ile Ser Gly Val Ser

100 105 110

Tyr Cys His Ser Met Gln Ile Cys His Arg Asp Leu Lys Leu Glu Asn

115 120 125

Thr Leu Leu Asp Ser Ser Ser Ala Pro Arg Leu Lys Ile Cys Asp Phe

130 135 140

Gly Tyr Ser Lys Ser Ser Val Leu His Ser Gln Pro Lys Ser Thr Val

145 150 155 160

Gly Thr Pro Ala Tyr Ile Ala Pro Glu Val Leu Ser Lys Lys Glu Tyr

165 170 175

Asp Gly Lys Ile Ala Asp Val Trp Ser Cys Gly Val Thr Leu Tyr Val

180 185 190

Met Leu Val Gly Ala Tyr Pro Phe Glu Asp Pro Glu Asp Pro Arg Asn

195 200 205

Phe Arg Lys Thr Leu Gln Arg Ile Leu Ser Val Ser Tyr Ser Ile Pro

210 215 220

Asp Tyr Val Arg Val Ser Arg Glu Cys Thr His Leu Leu Ser Arg Ile

225 230 235 240

Phe Val Ala Asn Pro Glu Lys Arg Ile Thr Ile Pro Glu Ile Lys Gln

245 250 255

His Pro Trp Phe Leu Lys Asn Leu Pro Ser Glu Phe Met Asp Glu Asp

260 265 270

Asp Met Gln Ile Gly Glu Val Gln Lys Asn Glu Ile Ser Gln Ser Val

275 280 285

Glu Asp Ile Val Ser Ile Ile Gln Glu Ala Arg Lys Leu Gly Asp Gly

290 295 300

Ile Lys Val Gly His Phe Leu Gly Ser Met Asp Leu Asp Glu Ile Asp

305 310 315 320

Asp Ala Asp Ile Asp Asp Ile Glu Thr Ser Gly Asp Phe Val Cys Ala

325 330 335

Leu

<210> 2

<211> 1014

<212> DNA

<213> strawberry (Fragaria visco)

<400> 2

atggagaggt atgagatagt gaaagatatt ggttctggga actttggtgt tgcaaaattg 60

gtgaaggaca aatggagtgg tgagctctat gccatcaagt tcattgagag aggccagaag 120

attgatgagc atgtacagag agagatcatg aaccacaggt cactgaagca tccaaatatc 180

ataagattta aggaggtctt gttaacacaa accgatttag cgattgtcat ggaatatgca 240

gcaggaggag aactttttga aagaatatgc aatgctggta gattcagtga agatgaggcc 300

agatttttct tccagcagct gatttcagga gtcagttact gtcattcaat gcaaatctgt 360

cacagagatc ttaaactgga gaacacactt ctagattcaa gctcagctcc acgtctcaaa 420

atatgtgatt tcggttattc caagtcatct gtgctgcatt cacaacccaa atcaactgta 480

ggaacacctg cctatattgc accagaagtc ctgtcaaaaa aagagtatga tgggaagata 540

gcagatgttt ggtcttgtgg ggttacccta tatgtgatgc tggttggtgc ttatcctttt 600

gaagatccag aagatcccag aaatttcagg aagacacttc agcgaattct cagcgtcagc 660

tattcaattc ctgactatgt acgcgtttcc agggaatgta cacatcttct ttctcgaatt 720

ttcgttgcta accccgaaaa gagaattaca atcccagaga taaagcagca cccctggttt 780

ctgaaaaatt taccatcgga attcatggat gaagatgaca tgcagattgg tgaggttcag 840

aaaaatgaga tttcacaaag tgttgaagat atagtgtcca ttattcaaga ggctcgaaaa 900

cttggtgatg gcatcaaagt tggacatttt cttgggagca tggaccttga tgagatagat 960

gatgccgaca tcgatgatat agaaactagt ggtgattttg tatgtgcgtt gtga 1014

Claims

1. Any of the following uses of the protein:

p1, the use of the protein for increasing the sucrose, glucose and/or fructose content of a plant fruit;

p2, the application of the protein in preparing products for increasing the content of sucrose, glucose and/or fructose in plant fruits;

p3, the use of said protein in shortening the ripening cycle of the fruit of plants;

p4 and application of the protein in preparing products for shortening the mature period of plant fruits;

the amino acid sequence of the protein is shown as SEQ ID No.1 in a sequence table;

the plant is strawberry.

2. Use of a biological material related to a protein according to claim 1 in any of the following applications:

q1, use of the biomaterial for increasing the sucrose, glucose and/or fructose content of a fruit;

q2, the application of the biological material in preparing products for improving the content of sucrose, glucose and/or fructose in plant fruits;

q3, use of the biomaterial in shortening the ripening cycle of plant fruits;

q4, and application of the biological material in preparation of products for shortening the mature period of plant fruits;

the biomaterial is any one of the following B1) to B7):

B1) a nucleic acid molecule encoding the protein of claim 1;

B2) an expression cassette comprising the nucleic acid molecule of B1);

B3) a recombinant vector containing the nucleic acid molecule according to B1) or a recombinant vector containing the expression cassette according to B2);

B5) a transgenic plant cell line comprising B1) the nucleic acid molecule or a transgenic plant cell line comprising B2) the expression cassette;

B6) transgenic plant tissue comprising the nucleic acid molecule according to B1) or transgenic plant tissue comprising the expression cassette according to B2);

the plant is strawberry.

3. Use according to claim 2, characterized in that: B1) the coding sequence of the nucleic acid molecule as a coding chain is a DNA molecule shown by SEQ ID No.2 in a sequence table.

4. Use according to claim 2, characterized in that: B1) the nucleic acid molecule is a DNA molecule with nucleotide shown in SEQ ID No.2 in a sequence table.