CN110592087A - Application of SGR gene silencing in brassica plants - Google Patents

Abstract

The invention discloses application of SGR gene silencing in brassica plants. The invention provides an application of a substance capable of reducing the expression quantity and/or activity of SGR gene coding protein in plants in any one of the following substances: improving the storage stability of the plant; extending the shelf life of the plant; reducing the loss of dry matter during storage and/or transport of the plant; cultivating green-keeping plants; increasing the chlorophyll content of plants. The technical scheme provided by the invention provides a new direction and precious gene resources for breeding plants, particularly brassica plants.

Description

Application of SGR gene silencing in brassica plants

Technical Field

The invention relates to the technical field of biology, in particular to application of SGR gene silencing in brassica plants.

Background

SGR (static-green) gene, which encodes demagging enzyme, is a key enzyme gene in the process of plant chlorophyll degradation. Ren et al identified a leaf greening-associated gene AtNYE1 from Arabidopsis thaliana. Jiang et al screened an SGR gene-deficient mutant from Japanese japonica rice flower seedlings irradiated with cobalt-60 radiation. Subsequently, Park isolated the homologous gene OsSGR of the gene from rice (Oryza sativa). Alos et al suggested that pericarp stay green phenotype of Citrus (Citrus L.citrus orange) could be due to the expression of SGR gene being hindered. Thereafter, SGR1/NYE1 homologous genes were also found in many crops such as tomato (Solanum lycopersicum), sweet pepper (Capsicum annuum), fescue (Festuca pratensis), pea (Pisum sativum), Wuta-tsai (Brassaca campestras L. ssp. Channensis L var. rossulas Tsen et Leesavoy), banana (Muses AAA), soybean (Glycane max L Merr.).

Barry and Pandey studied 26 different degrees of green-keeping mutants of cultivated tomato, and found that these mutants were mainly caused by GF (SGR homologous gene) allele mutation, such as some site base point mutation, base insertion, base deletion, or some sequence early insertion stop codon, although these gene mutations showed different degrees of green-keeping, these genes all lost the function of GF gene and were identified as null alleles.

The discovery and utilization of functional genes related to important economic traits are effective ways for realizing the cultivation of good varieties of crops. In the history of crop breeding, all breakthrough achievements are inseparable from the utilization of key genes. In the 50's of the 20 th century, the effective use of the cyst nematode-resistant gene in the united states has enabled the total soybean production to jump the world first and remain so far; in the 60 s, the application of dwarf genes causes the 'green revolution' of world grains and the leap of grain yield; in the 70 s, the improvement of the wild sterile gene of rice by the Mr. Yuanlong Heng promoted the generation of hybrid rice in China and moved the world; in the 90s, breeding experts worldwide began to systematically research and apply important gene resources.

With the development of biotechnology, the research on gene functions helps people to better understand the nature of gene expression regulation and utilize the gene expression regulation, and meanwhile, emerging gene silencing technology is emerging continuously. Plant genetic improvement and crop breeding based on silencing techniques have achieved considerable success. The traditional method for preventing and controlling plant diseases and insect pests mainly relies on chemical reagents. However, the use of large amounts of chemical agents poses a serious threat to human health and the environment. In addition, the long-term use of the pesticide also indirectly improves the drug resistance of diseases and insects. Therefore, the search for new means for controlling diseases and pests has become one of the hot spots in recent years. Because the existing crop varieties with high-level disease and insect resistance are not discovered, the cultivation of the crop varieties with disease and insect resistance by using a hybridization method is limited to a certain extent. Studies have shown that some sRNAs from pathogens or insects can enter host cells and inhibit immune pathways within the host, while sRNAs in host cells can also be transferred to pathogens/pests to inhibit pathogen virulence. This significant finding again demonstrates the feasibility of host-induced genetic silencing (HIGS) in defending against biotic stress. Yang and the like use a 302bp sequence in a soybean (Glycine max) Mosaic virus (Mosaic virus) SMV SC 3P 3 gene as a target fragment to construct an RNAi vector and transform soybeans, and find that transgenic plants have resistance to the SMV SC3 for 3 years continuously under the field condition; compared with wild plants, the transgenic plants have improved resistance to other four SMV viruses (SC7, SC15, SC18 and SMV-R).

In addition to biotic factors, some abiotic stresses such as: cold, drought, salinity, heavy metals, etc., also have a serious impact on the growth and yield of plants. Plant response to abiotic stress is a complex molecular process based on transcriptional and post-transcriptional gene regulation of many related genes. Although the research on the genes and local regulation mechanisms related to plant stress tolerance has been broken through at present, the overall stress tolerance regulation mechanism of plants is still unclear. Studies have shown that plant tolerance to stress conditions is regulated by stress responsive genes. At present, the research idea of resisting abiotic stress of plants is relatively simple, namely identifying and regulating potential abiotic stress response genes. These potential genes are roughly classified into the following three groups. A first class of genes encoding different osmolytes; second, genes encoding ion transport and water uptake proteins; third, genes involved in the regulation of transcriptional control and signaling, such as: MYB, WRKY, DREB and other transcription factors.

The gene silencing technology is mainly used for enhancing the stress resistance of plants by interfering or silencing some negative regulatory factors of stress response. Although single overexpression or interference of the first two genes can improve the abiotic stress resistance of the plant, the stress resistance of the plant cannot be completely embodied in the transformed plant. In order to comprehensively improve the stress resistance of plants, researchers in recent years use transcription factors and related genes in signal transduction as manipulation objects, and instead, the expression of some downstream genes and physiological analysis are combined to be used as a basis for evaluating the abiotic stress resistance of transgenic plants. Hu and the like silence tomato transcription factor genes SlHB2, the salt tolerance and drought resistance of plants are improved, and a plurality of genes related to stress resistance are obviously up-regulated under normal conditions or stress conditions. YIn and the like find that hormone response elements such as abscisic acid (ABA), indoleacetic acid (IAA) and the like exist in a promoter region of tomato transcription factor gene SlMBP8, which indicates that SlMBP8 may be used as a stress response transcription factor to participate in a signal transduction process of tomato responding to abiotic stress, and after SlMBP8 is silenced, the salt resistance and drought resistance of a silenced plant are obviously improved.

Brassica (Brassica) is a genus of the Brassicaceae family. The plants of this genus include a variety of important agricultural and horticultural crops, including common vegetables such as cabbage, cabbage and mustard. This species is native to western europe, the Mediterranean region of the Ring, and temperate regions of Asia. Mainly distributed in the mediterranean region; the Chinese has 13 cultivated species, 11 varieties and 1 variant, is widely cultivated in various parts of China, is usually used for cultivating roots, bulbs or seedlings by leafy vegetables, and occupies an important position in annual supply of the vegetables. In the late period of cultivation of rhizome, head-end cabbage and leaf vegetable, and in the period of preservation and storage of leaf ball, its outer leaf is easy to age and turn yellow, resulting in loss of yield and quality reduction of product. The stay green property has very important significance for genetic improvement of brassica crop varieties and can be used as a screening marker for improved variety breeding and crossbreeding. The stay-green mutant also has great application potential in the breeding and cultivation of ornamental plant varieties.

Disclosure of Invention

The invention aims to provide application of SGR gene silencing in brassica plants.

In a first aspect, the present invention claims the use of a substance capable of reducing the expression level and/or activity of a protein coding for an SGR gene in a plant, in any one of:

(A1) improve the storage stability of the plant.

(A2) Prolonging the shelf life of the plant.

(A3) Reduce the loss of dry matter during storage and/or transport of the plant.

(A4) Reduce the weight loss rate of the plant during storage and/or transportation.

(A5) Increasing the chlorophyll content of plants.

The substance capable of reducing the expression level and/or activity of the protein encoded by the SGR gene in the plant can be any substance capable of reducing the expression level and/or activity of the protein encoded by the SGR gene in the plant. The reduction in the amount of expression and/or activity of a protein encoding an SGR gene in a plant may refer to the reduction in gene expression or product inactivation resulting from any manner of mutation silencing or interference.

Further, the substance capable of reducing the expression level and/or activity of a protein encoded by an SGR gene in a plant may be any one of the following substances:

(B1) gene editing tools for the genomic DNA sequence of the SGR gene;

the gene editing tool is a sequence specific nuclease capable of specifically cleaving a target sequence in a genomic DNA sequence encoding the SGR gene.

Wherein, the sequence-specific nuclease can be CRISPR/Cas9 nuclease, transcription activator-like effector nucleases (TALEN), Zinc Finger Nucleases (ZFN) or the like. Specific cleavage of the target fragment by the sequence-specific nuclease results in insertion, deletion and/or substitution mutations in the target fragment, thereby causing mutations in the genomic DNA sequence of the SGR gene that inhibit the expression of a normally functioning SGR protein.

(B2) RNA interference fragments against SGR gene.

(B3) A DNA fragment capable of encoding the gene editing tool of (B1) or capable of transcribing the RNA interference fragment of (B2).

(B4) An expression cassette, a recombinant vector, a recombinant microorganism or a transgenic cell line comprising the DNA fragment of step (B3).

In a second aspect, the invention claims a method of breeding a plant variety.

The method for cultivating a plant variety as claimed in the present invention is a method for cultivating a plant variety having at least one of the following traits (C1) to (C5), and may comprise the step of reducing the expression level and/or activity of a protein encoding an SGR gene in a recipient plant:

(C1) the storability is improved.

(C2) The shelf life is prolonged.

(C3) The loss of dry matter during storage and/or transport is reduced.

(C4) A reduced rate of weight loss during storage and/or transport;

(C5) chlorophyll content is increased.

Wherein said reduction in the amount of expression and/or activity of a protein encoding an SGR gene in a recipient plant may refer to reduction in expression or inactivation of the product of said SGR gene by any means of mutation silencing or interference.

Further, the reduction of the expression level and/or activity of the protein encoded by the SGR gene in the recipient plant may be any of:

(I) sexual hybridization means such as breeding process causes the expression of the encoding protein of the SGR gene to be reduced or inactivated;

(II) tissue culture means to reduce or inactivate expression of the protein encoding the SGR gene;

(III) mutagenesis means (any form of physical and chemical mutagenesis) results in reduced or inactivated expression of the protein encoding the SGR gene.

In the method, the method for decreasing the expression level and/or activity of a protein encoding an SGR gene in a recipient plant may comprise the steps of: introducing into said recipient plant a substance capable of reducing the expression level and/or activity of a protein encoding an SGR gene in a plant as described above.

In the method, the method for decreasing the expression level and/or activity of a protein encoding an SGR gene in a recipient plant may comprise the steps of: replacing the SGR gene in the recipient plant with a null allele of the SGR gene.

The null allele (null allele) is a DNA sequence of the original allelic site where the gene function disappears after gene mutation.

In the first and second aspects above, the protein encoding the SGR gene (referred to as BrNYE1 protein) may be any one of the following:

(D1) protein with an amino acid sequence of SEQ ID No. 4;

(D2) protein which is obtained by substituting and/or deleting and/or adding one or more amino acid residues to the amino acid sequence shown in SEQ ID No.4 and has the same function;

(D3) a protein having 99% or more, 95% or more, 90% or more, 85% or more, or 80% or more homology to the amino acid sequence defined in any one of (D1) to (D2) and having the same function;

(D4) a fusion protein obtained by attaching a tag to the N-terminus and/or C-terminus of the protein defined in any one of (D1) to (D3).

Further, the SGR gene (called BrNYE1 gene) can be any one of the following DNA molecules:

(E1) a DNA molecule shown as SEQ ID No.2 or SEQ ID No. 3;

(E2) a DNA molecule that hybridizes under stringent conditions to a DNA molecule defined in (E1) and encodes the BrNYE1 protein;

(E3) and (C) a DNA molecule which has more than 99%, more than 95%, more than 90%, more than 85% or more than 80% of homology with the DNA sequence limited by (E1) or (E2) and encodes the BrNYE1 protein.

Wherein, SEQ ID No.2 is the genome sequence of BrNYE1 gene; SEQ ID No.3 is the CDS sequence of the BrNYE1 gene.

In the second aspect above, the null allele of the SGR gene (referred to as the Brnye1 gene) can be any one of the following DNA molecules:

(G1) DNA molecule shown in SEQ ID No. 1;

(G2) a DNA molecule that hybridizes under stringent conditions to the DNA molecule defined in (G1) and encodes the Brnye1 protein;

(G3) a DNA molecule which has more than 99%, more than 95%, more than 90%, more than 85% or more than 80% homology with the DNA sequence limited by (G1) or (G2) and encodes the Brnye1 protein.

In each of the above proteins, the 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 genes, the stringent conditions may be as follows: 50 ℃ in 7% Sodium Dodecyl Sulfate (SDS), 0.5M Na₃PO₄Hybridization with 1mM EDTA, rinsing in 2 XSSC, 0.1% SDS at 50 ℃; also can be: 50 ℃ in 7% SDS, 0.5M Na₃PO₄Hybridization with 1mM EDTA, rinsing at 50 ℃ in 1 XSSC, 0.1% SDS; also can be: 50 ℃ in 7% SDS, 0.5M Na₃PO₄Hybridization with 1mM EDTA, rinsing in 0.5 XSSC, 0.1% SDS at 50 ℃; also can be: 50 ℃ in 7% SDS, 0.5M Na₃PO₄Hybridization with 1mM EDTA, rinsing in 0.1 XSSC, 0.1% SDS at 50 ℃; also can be: 50 ℃ in 7% SDS, 0.5M Na₃PO₄Hybridization with 1mM EDTA, rinsing in 0.1 XSSC, 0.1% SDS at 65 ℃; can also be: in a solution of 6 XSSC, 0.5% SDS at 65 ℃ and then washed once with each of 2 XSSC, 0.1% SDS and 1 XSSC, 0.1% SDS.

In the present invention, the chlorophyll content is the chlorophyll content in the leaf.

In the foregoing first and second aspects, the plant may be a dicot or a monocot.

Further, the dicotyledonous plant may be a plant of the family brassicaceae;

further, the plant is a Brassica plant (such as Chinese cabbage) or an Arabidopsis plant (such as Arabidopsis thaliana).

In a third aspect, the invention claims a method for breeding cabbage (Brassica campestris) with the characters shown in any one of (C1) - (C5).

(C1) The storability is improved.

(C2) The shelf life is prolonged.

(C3) The loss of dry matter during storage and/or transport is reduced.

(C4) Reduce the weight loss rate of the plant during storage and/or transportation.

(C5) Chlorophyll content is increased.

The method comprises the following steps (G1) or (G2):

(G1) transferring the DNA molecule shown in SEQ ID No.1 into the Chinese cabbage variety A by taking the Chinese cabbage variety A as a female parent, and culturing to obtain the target Chinese cabbage variety.

(G2) Transferring the DNA molecule shown in SEQ ID No.1 into the Chinese cabbage sterile line A by taking the Chinese cabbage sterile line A as a female parent, and culturing to obtain the target Chinese cabbage sterile line for hybridization and matching.

Further, in the step (G1), the DNA molecule shown in SEQ ID No.1 is transferred into the cabbage variety A through multiple-generation backcross, and the target cabbage variety is obtained through cultivation.

Further, in the step (G2), the Chinese cabbage sterile line A is used as a female parent, and through multi-generation backcross, the DNA molecule shown in SEQ ID No.1 is transferred into the Chinese cabbage sterile line A, and the target Chinese cabbage sterile line is obtained through cultivation and used for hybridization and pairing.

The cabbage variety A does not contain a DNA molecule shown in SEQ ID No. 1.

The Chinese cabbage sterile line A does not contain DNA molecules shown in SEQ ID No. 1. The Chinese cabbage sterile line A is a sterile line with excellent agronomic characters.

In a fourth aspect, the invention claims a method of cultivating a green-sustaining variety of green-stem vegetables.

The method specifically comprises the following steps:

(H1) using green-keeping green-mutant 'nye' of green-stem cabbages as female parent and '13A 510' as male parent to make hybridization, and using F₂Selecting plants with green-keeping phenotype as donor materials in the generation, and culturing by using free microspores to create green-keeping double haploid of the green stem vegetable;

(H2) cultivating green-keeping cytoplasmic male sterile line of green-keeping stem vegetable by taking green-keeping stem vegetable 'East 18' hybridized with Ogrua sterile source cytoplasmic male sterile line as a sterile source and green-keeping stem vegetable green-keeping mutant 'nye' as a breeding recurrent parent material;

(H3) and (4) preparing a hybridization combination by using the green-sustaining double haploid obtained in the step (H1) and the green-sustaining cytoplasmic male sterile line obtained in the step (H2) to obtain the target green stem vegetable.

In the invention, the Chinese cabbage (Brassica campestris) is a Brassica genome A cabbage vegetable.

The invention discovers that SGR gene silencing has influence on plant leaf color, plant shelf life and storage resistance for the first time, and tests prove that the gene has the function of regulating and controlling the leaf color, SGR gene silencing mutants have a stay green mutant phenotype, the chlorophyll content of aged leaves is obviously improved, the SGR gene silencing obviously prolongs the shelf life of Chinese cabbage, improves the storage resistance, and simultaneously greatly reduces the loss of dry substances in the storage or transportation process. The complementation or overexpression of the gene can reduce the chlorophyll content of the aged leaf of the mutant, and indirectly explains the application of SGR gene silencing in the aspects of shelf life, storage property and the like of brassica plants in cruciferae. Experiments prove that the cabbage Brnye1 gene can ensure that leaves are always kept green and not yellow in the later period of plant harvest, shelf life and storage and transportation, and the marketability is improved. The technical scheme provided by the invention provides a new direction and precious gene resources for breeding plants, particularly brassica plants.

Drawings

FIG. 1 is a diagram showing the results of experiments on BrNYE1 gene transfer. a is T₂Yellow phenotype of Positive transgenic plants (nye1-1, left; T)₂Transgenic plants, right); b, performing PCR verification on a positive transgenic plant (the target band is 795 bp); c, analyzing the chlorophyll content of the aged leaves of the positive transgenic plants (different lower case letters indicate that the difference is obvious); d is relative quantitative analysis of BrNYE1 gene expression in the senescent leaves of the positive transgenic plants. oxBrNYE1#1-6 shows 6 transgenic positive lines after introduction of BrNYE1 gene into Arabidopsis thaliana green-keeping mutant nye 1-1.

FIG. 2 is a comparison graph of normal temperature storage experiments of SGR gene silencing mutant and conventional varieties.

FIG. 3 is a comparison of SGR gene silencing mutant and conventional variety in cryopreservation experiments.

FIG. 4 is a chart showing the creation process and results of pakchoi. And (3) obtaining the Double Haploid (DH) of the green-sustaining Chinese cabbage by adopting a microspore culture method. a is microspore blasts; b is an embryoid; c is callus; d is a regeneration plant; e is the regeneration plant rooting; f is the obtained stay green plant.

FIG. 5 is a genetic model of CMS directed transformation.

FIG. 6 is a comparison of cytoplasmic male sterile line of Brassica oleracea with its male sterile source. a is a sterile source East 18' plant; b, a new green-sustaining sterile line CMS-nye single plant; c is the inflorescence of sterile source "East 18"; d keeping the inflorescence of the green sterile new line CMS-nye.

FIG. 7 is a graph showing the comparison between the conventional control variety "Wall 918" and the species of cabbage kept green.

FIG. 8 shows the comparison of the yield of the conventional cultivar "Wall 98" (CK) and the new Brassica campestris cultivar combination (ZH 01-15). Kg is the unit of yield.

Detailed Description

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.

Green-keeping green stalk brassica mutant 'nye' and non-green-keeping green stalk brassica variety '13 a 510': all are described in "Wang et al, Identification and fine mapping of a static-green gene (Brnye1) in pakchoi (Brassica campestis L.ssp. chips. Theroretical and Applied Genetics,2018,131: 673-.

Arabidopsis thaliana green-holding mutant nye 1-1: plant physiology 144(3) 1429-1441, available to the public from the applicant, can be used only in the experiments of the instant invention, not for any purpose.

Green stem vegetable East 18': the female sterile hybrid seed production technology of the eastern No. 18 Chinese cabbage [ J ] Jiangsu agricultural science, 41(07):138 + 139. "east No. 18" in the text can be obtained from the applicant, can be only used for the experiment of the duplication invention, and cannot be used for other purposes.

Green stem vegetable 'Wall 918': to be described in "dugaoyang 2018 creation and utilization of male sterile line of stemona and green stem vegetable [ D ]. Shenyang agriculture university" 'Wall 918' in the text, the public is available from the applicant, and can only be used in the experiment of the duplicated invention, and not for other purposes. The variety has strong heat resistance, beautiful girdling, bright green and thick leaf color, high yield, strong disease resistance and easy high yield.

pCAMIBA1300-M vector: is a plasmid obtained by modifying a multi-enzyme restriction site and an interface by taking commercial empty pCAMIBA1300 (product catalog number CAS: MLCC1244) originally purchased from a far-away organism in Wuhanbo as a framework by an applicant; specifically, a single AarI restriction enzyme recognition sequence CACCTGC is introduced into the Multiple Cloning Site (MCS) of the pCAMIBA1300 vector.

Example 1 functional verification of the cabbage BrNYE1 Gene

Cloning of BrNYE1 Gene

The SGR gene related in the invention is a green-keeping gene Brnye1 obtained by positioning and cloning in a green-keeping mutant 'nye' of green-keeping vegetables by a method of combining a second-generation sequencing technology with a traditional linkage marker in the early period of the team, and according to a Brnye1 sequence (the Brnye1 gene sequence is shown as SEQ ID No.1) of the green-keeping gene in the literature, CDS full-length amplification primers are designed by using Primer 5 software, wherein the specifically designed Primer sequences are as follows:

an upstream primer: 5' -cagtCACCTGCaaaacaacATGTGTAGTTTGTCAGCGAA-3'；

A downstream primer: 5' -cagtCACCTGCaaaatacaCTAGAGTTTCTCCGGCTTAG-3'。

Wherein, the lower case is a protective base, the underline indicates a restriction enzyme site, and the upper case indicates a gene sequence.

The cDNA of a non-chlorophytum-maintaining green stem variety '13A 510' is taken as a template, the primer is utilized for PCR amplification to obtain the allele BrNYE1 of Brnye1 gene (the genome sequence of BrNYE1 gene is shown as SEQ ID No.2, the CDS sequence is shown as SEQ ID No.3, and the coding protein is shown as SEQ ID No.4), and the nucleotide sequence of the allele is taken as a genetic complementary transformation sequence.

Second, construction of plant expression vector and transformation of Agrobacterium

Constructing an overexpression vector of the gene BrNYE1, which comprises the following specific steps:

1. amplification of target Gene

PCR reaction (50. mu.l): KOD enzyme 1. mu.l; 2 XKOD Buffer 25. mu.l; dNTP mix 10. mu.l; 1 mul of each upstream primer and downstream primer; 1 μ l of cDNA; make up to 50. mu.l with distilled water. Wherein, the upstream and downstream primers are designed in the step one; the cDNA is obtained by extracting total RNA from non-persistent green stem vegetable variety '13A 510' and performing reverse transcription.

Setting PCR reaction parameters: pre-denaturation at 98 ℃ for 10 min; denaturation at 98 ℃ for 30s, annealing at 56 ℃ for 30s, extension at 72 ℃ for 1min, and 32 cycles; 72 further extension for 15 min; 10min at 14 ℃.

After the amplification product of the target gene is detected by agarose gel electrophoresis with the concentration of 1%, the target gene is recovered by an agarose gel DNA recovery kit of TaKaRa company, and the specific operation steps are as follows: (1) preparing 1% agarose gel, and identifying and separating a target DNA band by agarose gel electrophoresis; (2) absorbing the buffer solution attached to the surface of the gel by using filter paper, and carefully cutting out a gel block containing the target gene under the irradiation of an ultraviolet lamp; (3) taking out the rubber block, weighing the mass of the rubber block, and calculating the volume of the rubber block according to the proportion of 1mg to 1 mul; (4) adding Buffer GM with the volume of 3 times of the rubber block into the rubber block; (5) heating in water bath at 50 deg.C to promote complete dissolution of the gelatin block; (6) taking out the nucleic acid purification column from the agarose gel DNA recovery kit, and placing the nucleic acid purification column on a collection tube; (7) putting the clear solution obtained in the step 5 into a purification column, centrifuging at 12000rpm for 1min, and removing the filtrate; (8) 500 μ L of Buffer WA WAs put into a nucleic acid purification column, centrifuged at 12000rpm at room temperature for 30s, and the filtrate WAs discarded; (9) taking 700 mu L of Buffer WB to a nucleic acid purification column, centrifuging at the room temperature of 12000rpm for 30s, and removing the filtrate; (10) repeating the operation of the step (8); (11) placing the nucleic acid purification column on a collection tube, and performing air separation at the room temperature of 12000rpm for 2min to remove residual liquid as far as possible; (12) discarding the collection tube, placing the nucleic acid purification column in 1.5mL EP tube, adding 30 μ L of eluent to elute DNA attached to the membrane of the nucleic acid purification column (the eluent can be preheated in a 65 deg.C constant temperature water bath kettle in advance to facilitate DNA elution), and standing at room temperature for 2 min; (13) centrifuging at 12000rpm for 2min at room temperature, and storing the recovered DNA in a low-temperature refrigerator at-20 deg.C for later use; (14) and taking a trace amount of recovered products, and detecting the recovered quality by agarose gel electrophoresis with the concentration of 1%.

2. Construction and identification of recombinant vectors

The target fragment and the no-load enzyme digestion connection system (10 μ l): pCAMIBA1300-M vector (hygromycin resistance) 2. mu.l; restriction enzyme AarI 0.5. mu.l; 3 mul of target gene (obtained by amplification in step 1); t4 Buffer 1 μ l; 0.5 of T4 ligase; make up to 10. mu.l with distilled water. Reaction conditions are as follows: water bath at constant temperature, enzyme digestion at 37 deg.C for 2h

The transformation of the recombinant plasmid was carried out as follows: (1) mixing 200. mu.L of Escherichia coli competent cell DH5a with 5. mu.L of the ligation product, and ice-cooling for 30 min; (2) quickly placing in a constant temperature water bath kettle at 42 deg.C, thermally shocking for 90s, and ice-cooling for 2 min; (3) adding 500 μ L LB liquid culture medium, mixing; (4) culturing at 37 deg.C and 200rpm for 45min to restore normal growth state of cells; (5) uniformly coating the bacterial liquid on an LB solid culture medium flat plate; (6) after 30min, the cells were incubated overnight in a 37 ℃ incubator.

The bacterial examination of the recombinant plasmid was performed as follows:

bacteria assay reaction system (20 μ l): 2 x Mix 10. mu.l; 1 mul of forward detection primer; reverse detection primer 1. mu.l; make up to 20. mu.l with distilled water.

Forward bacteria detection primer: 5'-ATGTGTAGTTTGTCAGCGAA-3', respectively;

reverse bacteria detection primer: 5'-CTAGAGTTTCTCCGGCTTAG-3' are provided.

Setting PCR reaction parameters: pre-denaturation at 95 ℃ for 5 min; denaturation at 95 ℃ for 30s, annealing at 55 ℃ for 30s, extension at 72 ℃ for 2min, and 32 cycles; 72 further extension for 15 min; 10min at 14 ℃.

Checking the bacteria correctly, shaking the bacteria, extracting the plasmid, and performing enzyme digestion verification (AarI).

The extraction of recombinant plasmid includes the following steps: picking a single clone from an LB solid medium plate, inoculating the single clone into a kanamycin-resistant LB liquid medium with the final concentration of 50 mu g/mL, and culturing at 37 ℃ overnight; (2) taking 4mL of activated bacterium liquid, centrifuging at room temperature of 10000rpm for 2min, and completely removing supernatant; (3) taking 250 mu L of Solution I reagent containing the ribonuclease A to completely resuspend the bacterial block; (4) taking 250 mu L of Solution II reagent to crack the bacterium block, and slightly reversing the bacterium block up and down for a plurality of times until the bacterium is transparent; (5) taking 350 mu L of Solution III reagent, and reversing for several times until white compact floccules are formed; (6) centrifuging at 12000rpm for 10min at room temperature, and collecting supernatant; (7) taking out the nucleic acid purification column from the kit, and placing the nucleic acid purification column on a collection tube; (8) taking the clarified supernatant obtained in the operation step (6) to a nucleic acid purification column, centrifuging at 12000rpm at room temperature for 1min, and removing the filtrate; (9) 500 μ L of Buffer WA WAs put into a nucleic acid purification column, centrifuged at 12000rpm at room temperature for 30s, and the filtrate WAs discarded; (10) taking 700 mu L of Buffer WB to a nucleic acid purification column, centrifuging at the room temperature of 12000rpm for 30s, and removing the filtrate; (11) repeating the above operation step (10); (12) placing the nucleic acid purification column on a collection tube, and performing air separation at the room temperature of 12000rpm for 2min to remove residual liquid as far as possible; (13) discarding the collecting tube, placing the nucleic acid purification column in a 1.5ml of LEP tube, adding 50 μ L of eluent to elute DNA attached to the membrane of the nucleic acid purification column (the eluent can be preheated in a 65 deg.C constant temperature water bath kettle in advance to facilitate DNA elution), and standing at room temperature for 2 min; (14) centrifuging at 12000rpm for 2min at room temperature, eluting DNA attached to nucleic acid purification column membrane, and storing at-40 deg.C in refrigerator; (15) and taking a trace amount of recovered products, and detecting the plasmid extraction quality by agarose gel electrophoresis with the concentration of 1%.

The restriction enzyme identification of the recombinant plasmid (AarI) was performed as follows:

digestion reaction system (10. mu.L system): 10 × Green Buffer 1 μ L; restriction enzyme AarI 0.5. mu.L; 3 mu L of recombinant plasmid; make up to 10. mu.L with sterile distilled water.

Incubating at 37 ℃ for 30min in an incubator, wherein the target band is a fragment of about 795bp, and the enzyme digestion product can be detected and analyzed by agarose gel electrophoresis with the concentration of 1%. And detecting to obtain a target size band, which indicates that the recombinant vector is successfully constructed.

The obtained recombinant vector is named as pCAMIBA1300M-BrNYE1, and the structure of the vector is described as a recombinant plasmid obtained by inserting the gene shown in SEQ ID No.3 into the restriction enzyme site AarI of the pCAMIBA1300-M vector.

3. The obtained recombinant plant expression vector pCAMIBA1300M-BrNYE1 is transferred into Agrobacterium tumefaciens (Agrobacterium tumefaciens) GV3101 by a freeze-thaw method.

The experiment was also set up with a control to introduce pCAMIBA1300-M empty vector into Agrobacterium tumefaciens GV 3101.

Expression of BrNYE1 gene in Arabidopsis thaliana

1. Arabidopsis thaliana planting

The arabidopsis thaliana stay green mutant nye1-1 used in the experiment is prepared by placing 100 seeds into a refrigerator at 4 ℃ for low-temperature treatment for 3-5 days, and then dibbling the seeds into a 10cm × 10cm nutrition pot, wherein the nutrition pot is filled with a substrate of turf, vermiculite and perlite which are 7:4:2, and is watered thoroughly with tap water. After the seeds are germinated, the film is lifted properly, and air is discharged. The seedlings are strong, the film is removed, and the seedlings are watered thoroughly. After the film is lifted, the water is poured once every other week. And (3) starting to pour 1/2MS nutrient solution when 6-8 true leaves grow out of the seedlings, pouring 10mL of nutrient solution and water each time until bolting and flowering.

2. Agrobacterium mediated, floral dip infestation

And (3) transferring the heterologous overexpression vector (the recombinant expression vector pCAMIBA1300M-BrNYE1 constructed in the step two) into agrobacterium by a freeze-thaw method, coating a plate (containing Kana), sealing, inversely culturing for 48h at the temperature of 28 ℃ in the dark. Picking a round large single colony in 10mL of liquid LB (Kana), shaking at 28 ℃ and 200rpm for 48h, taking 5 mu L of turbid bacterial liquid for detection, taking 1mL of qualified bacterial liquid, sending the bacterial liquid to China Dagen sequencing, taking 1mL of the bacterial liquid to propagate in 100mL of liquid LB (Kana), and subpackaging the rest in 2mL of centrifuge tubes for storage for later use. And (4) carrying out an infection experiment when the arabidopsis grows to the full bud period. Watering the arabidopsis thaliana plant with water in the first day, cutting off all the bloomes and horns and fruits which are already opened on the arabidopsis thaliana plant with small scissors in the first day, and reserving large buds to be opened. Meanwhile, 10. mu.L of turbid bacterial liquid which is propagated and shaken for 48h is taken out, and OD600 is measured by an enzyme-linked immunosorbent assay, so that the concentration of the bacterial liquid is judged. Then evenly distributing the mixture into 50mL centrifuge tubes at 12,000rpm, centrifuging for 10min, and taking care to balance; discarding the supernatant, adding 50mL 1/2MS medium (sterilized in advance, containing surfactant), mixing well, balancing, centrifuging to collect the bacteria, pouring off the supernatant, and repeating once. Diluting the recovered strain with 1/2MS liquid culture medium containing surfactant, and finishing the bacterial liquid treatment when OD600 is measured to be 0.8-1.2. And pouring the bacterial solution with qualified concentration into a sterilized large-diameter glass culture dish, completely immersing the trimmed inflorescences of the arabidopsis into the bacterial solution, uniformly rotating the culture dish back and forth, taking out after 50s, flatly placing the plants in a dark environment, and sequentially infecting 30 plants. And taking out the infected arabidopsis thaliana plant after 24h, vertically placing, culturing in a proper environment, and harvesting the individual plant at the later stage.

3. Screening and identification of transgenic plants

Respectively taking a plurality of seeds collected from each individual plant, uniformly mixing, putting into a 2mL centrifuge tube, standing in a refrigerator at 4 ℃ for 3 days, taking out, and adding ddH₂Soaking for 1h in O, and simultaneously placing the used reagent in a clean bench for ultraviolet sterilization. Soaking the seedsCentrifuging at 3,000rpm for 1min, and discarding the supernatant; adding 1mL of 1% mercuric chloride into a centrifugal tube filled with the Arabidopsis seeds on an ultraclean workbench, covering the centrifugal tube, turning the centrifugal tube up and down, centrifuging after 5min, and discarding the supernatant; then adding 1mL of 70% ethanol, reversing the mixture from top to bottom, centrifuging the mixture after 5min, and removing the supernatant; add 1mL ddH to centrifuge tube₂And O, washing upside down, and repeating for 5 times for 5min each time. The resulting seeds were treated with 500. mu.L ddH₂O suspending, sucking to MS solid culture medium (Hyg) with diameter of 9cm by a pipette, slightly rotating, spreading uniformly, covering with a cover, sealing with a sealing film, and screening at 20-25 deg.C under normal temperature for 25 d. The roots of the plants which survive in the screening culture medium are washed by tap water, the plants are transplanted into a water-permeable substrate, a layer of plastic film is covered on the substrate, the substrate is placed in an arabidopsis culture room, and the substrate is cultured in a normal-temperature illumination environment. Five days later, the film is uncovered, and the fertilizer is applied. After 10-15 true leaves grow out of the plant, respectively taking 2-3 young leaves of each individual plant, extracting genome DNA and total RNA, and carrying out PCR detection, qRT-PCR and chlorophyll content detection on the leaves.

Wherein, the primer sequences adopted for PCR detection are as follows:

a forward primer: 5'-ATGTGTAGTTTGTCAGCGAA-3', respectively;

reverse primer: 5'-CTAGAGTTTCTCCGGCTTAG-3' are provided.

The target fragment with the size of 795bp (shown as SEQ ID No.3) obtained by amplification is positive.

qRT-PCR detection is carried out, ACT2 is used as an internal reference gene (Ren et al 2007), and the adopted primer sequences are as follows:

primers for detection of the reference gene ACT 2:

a forward primer: 5'-CGCTCTTTCTTTCCAAGCTC-3', respectively;

reverse primer: 5'-AACAGCCCTGGGAGCATC-3' are provided.

Primers for detecting BrNYE1 gene:

a forward primer: 5'-GCA TCC ACC AAC GCTTC-3' are provided.

Reverse primer: 5'-GCC TAT TTG CCC ATC CTT G-3', respectively;

the method for detecting the chlorophyll content of the transgenic arabidopsis leaves comprises the following steps: selecting the plant obtained by resistance screening, taking the penultimate aged leaf, slightly modifying according to the method of Arnon (1949), and extracting chlorophyll of the fourth true leaf of the plant by using 80% (v/v) acetone ethanol solution. The absorbance was determined with a DU 800 type UV spectrophotometer (Beckman Coulter, USA) at 663, 645 and 470nm wavelength, respectively, and each measurement was repeated 3 times. The total chlorophyll content was calculated by reference to the method of Holm (1954).

The experiment was also set with Columbia wild type Arabidopsis thaliana (Col-0), non-transgenic Arabidopsis thaliana green-keeping mutant (nye1-1), and a control to transfer pCAMIBA1300-M empty vector into Arabidopsis thaliana green-keeping mutant.

The plant phenotype of the non-transgenic Arabidopsis thaliana green-keeping mutant (nye1-1) and the Arabidopsis thaliana single strain transformed with the BrNYE1 gene into nye1-1 is shown in a in FIG. 1. The result of PCR detection of BrNYE1 gene expression level of each Arabidopsis strain is shown as b in figure 1, the result of leaf chlorophyll content detection is shown as c in figure 1, and the result of qRT-PCR detection of BrNYE1 gene expression level is shown as d in figure 1. All detection indexes of the no-load control arabidopsis are basically consistent with those of Col-0, and no statistical difference exists.

The above results show that: compared with nye1-1, the leaf color of the Arabidopsis individual plant with the BrNYE1 gene transferred into nye1-1 is recovered to yellow (basically consistent with the phenotype of Columbia wild type Arabidopsis), the chlorophyll content in the aged leaves is reduced, and the leaf color accords with the expected phenotype of the transgene.

Example 2 application of Chinese cabbage BrNYE1 Gene silencing

The Chinese cabbage (Brassica campestris L.ssp. chinensis) is harvested when young and tender, and starts to yellow after 2 days at normal temperature, and is completely withered and yellow after 5 days, and the shelf life is only 3 days. In the test, an SGR gene silencing stay green mutant (green stalk vegetable stay green mutant 'nye') and a commercially available conventional variety of dwarf green (self-used number '13A 510') are used as test materials, the aging and yellowing conditions of leaves after the leaves are picked are researched, and a theoretical basis is provided for shelf life preservation.

The genetic background of the SGR gene silencing green-keeping mutant (green-keeping pedunculate cabbage green-keeping mutant 'nye') is a non-green-keeping pedunculate cabbage variety '13A 510', which is different from the non-green-keeping pedunculate cabbage variety '13A 510' only in that a BRNYE1 gene (SEQ ID No.3) in the genome of the non-green-keeping pedunculate cabbage variety '13A 510' is replaced by a Brnye1 gene (SEQ ID No.1) which is a null allele, and other sequences in the genome are completely consistent with each other.

The mutant and the control variety are planted in scientific research bases of gardening academy of Shenyang agriculture university, plants with no plant diseases and insect pests growing uniformly are selected for harvesting 40 days after sowing, the harvested plants are immediately transported back to a laboratory, old yellow leaves are removed, and the plants are spread and dried indoors for 2-4 hours. Randomly selecting 30 plants from the control variety and the stay-green mutant respectively, placing the plants in a containing box, weighing, recording and repeating for 3 times. The storage box was then packed with polyethylene perforated plastic bags (0.03mm thick with 6 holes of 1cm diameter on each side) to avoid water loss and to prevent changes in the gas conditions in the bag. Wherein 3 times of repeated control varieties and 3 times of repeated green-keeping mutant storage boxes are placed in a constant-temperature incubator, and the shelf life storage is simulated under the conditions of (20 +/-1) DEG C and humidity of 85% -95%; the control variety of 3 replicates and the green-keeping mutant containing box of 3 replicates were placed in a refrigerator and simulated for cryopreservation and transportation at (0 + -0.1) deg.C and humidity of 75% -85%. Storing at constant temperature for 7d, observing and recording the yellowing condition of the leaves once a day; cryopreserved for 30d, and observed and recorded every three days. And weighing respectively in the last day, and calculating the weight loss rate.

The results are shown in FIGS. 2 and 3. The results show that SGR gene silencing obviously prolongs the shelf life of the Chinese cabbage, improves the storage resistance, and greatly reduces the loss of dry substances in the storage or transportation process.

Example 3 application of cabbage Brnye1 gene in cultivation of green-keeping cabbage

1. Culturing with free microspore to create green-keeping double haploid

The green-keeping mutant 'nye' of green stem vegetable is selected as a female parent, and '13A 510' of which the leaves turn yellow in the aging process is selected as a male parent of the hybrid combination. From F₂Superior strains with a stay-green phenotype were selected as donor material in generations according to Zhang et al (Zhang L, Zhang Y, Gao Y, Jiang XL, Zhang MD, Wu H, Liu ZY, Feng H2016. Effects of deacetylase inhibin on microspore embryo growth and plant growth in Pakchoi (Brassica rapa ssp. chinensis L.) Scientia Horticulturae20961-66) using free microspore culture, more green-sustaining Doubled Haploids (DH) were created (fig. 4).

2. Breeding of green-keeping cytoplasmic male sterile line

The green-stem vegetable 'East 18' hybridized with the Ogrua sterile source cytoplasmic male sterile line is used as a sterile source, and the green-stem vegetable green-keeping mutant 'nye' is used as a breeding recurrent parent material to culture the cytoplasmic male sterile line. The genetic model for CMS directed transfer is shown in FIG. 5. And continuously identifying and screening the fertility of each generation of plants. After determining whether the floral organs were completely aborted, the final plant was selected as a new male sterile line, CMS-nye (FIG. 6).

3. Product ratio experiment of stay green combination

Creates an excellent variety of the cabbage with green color, and has important significance for agriculture and horticulture. A variety of combinations were prepared from 15 excellent greening DH lines created from the above-mentioned isolated microspores and the above-mentioned greening cytoplasmic male sterile line CMS-nye, and a variety ratio experiment (FIG. 7) was carried out to evaluate the yield and quality of Brassica oleracea 'Wall 918' as a control.

The results show that the biological yield of these stay-green combinations is close to "Wall 918" or slightly higher than "Wall 918" (control variety) (FIG. 8), and two stay-green combinations (ZH03, ZH02) with better comprehensive properties are screened.

<110> Shenyang agriculture university

Application of silencing of <120> SGR gene in brassica plants

<130> GNCLN190895

<160> 4

<170> Patent In version 3.5

<210> 1

<211> 1164

<212> DNA

<213> Artificial sequence

<400> 1

atgtgtagtt tgtcagcgaa catgttgtta ccgacaaagc tgaaacctgc ttattcagac 60

aaacggagta atagtctgaa ctgtctcccc gtctccaata caagatccaa gaggaagaac 120

caatcaattg ttcctgtaag gcttctttta atcatttctt ttgtctcttt atttgattta 180

ttcttggaat tacttgaaat tgttttgttt tggattcaag gaatttgagt atctactaat 240

gattcattaa atcaaagtaa agatcaaatc ttggaagttt tttttttttt aaatttggtt 300

gcagatggca agattatttg gaccggctat cttcgaatca tccaagttga aagtattgtt 360

tctaggtgtt gatgacaaga agcatccacc aacgcttcca aggacttaca ctctcactca 420

cagtgacatt actgctaaac taaccttagc tatttctctt tttttttttt tttttttttt 480

tgacagcagc tatttctcat tcaattaata attctcaggt atgtatgcgt ctatacgttt 540

ctgagaaaga acggttttag aaaatataga ggctgatgta tatagattta tgttatatac 600

agttgcaagg atgggcaaat aggctataca gagatgaagt ggtagcagaa tggaagaaag 660

ttaaagggga catgtctctt cacgtccact gccacattag cggtggccat ttcctcttgg 720

atctcttccc aaagcttaga tactacatct tttccaaaga actacctgtt gtaagtaaaa 780

atatttattt ttggtttctt ggtttcatta caaaatgttg attgtttatt ttgatatatg 840

tatcaggtgt tgaaggctat tgttcatgga gacggcaact tgttgaacaa ctatccggat 900

ttacaagaag ctcttgtttg ggtttatttc cattctaatg ccgatgagtt caacagagtt 960

gaatgttggg gtccgctttg ggaagctact tcgtctgatg gtcacaggac tcaaactctt 1020

cctcagactc ggtgcaagga tgagtgcagt tgttgtttcc cgcaggttag ctcgattccg 1080

tggtctcata gtcttagtaa cgaaggtgtt actgactacc ctgggacgca ggccgaggga 1140

atgcctaagc cggagaaact ctag 1164

<210> 2

<211> 1124

<212> DNA

<213> Artificial sequence

<400> 2

atgtgtagtt tgtcagcgaa catgttgtta ccgacaaagc tgaaacctgc ttattcagac 60

aaacggagta atagtctgaa ctgtctcccc gtctccaata caagatccaa gaggaagaac 120

caatcaattg ttcctgtaag gcttctttta atcatttctt ttgtctcttt atttgattta 180

ttcttggaat tacttgaaat tgttttgttt tggattcaag gaatttgagt atctactaat 240

gattcattaa atcaaagtaa agatcaaatc ttggaagttt tttttttttt aaatttggtt 300

gcagatggca agattatttg gaccggctat cttcgaatca tccaagttga aagtattgtt 360

tctaggtgtt gatgacaaga agcatccacc aacgcttcca aggacttaca ctctcactca 420

cagtgacatt actgctaaac taaccttagc tatttctcat tcaattaata attctcaggt 480

atgtatgcgt ctatacgttt ctgagaaaga acggttttag aaaatataga ggctgatgta 540

tatagattta tgttatatac agttgcaagg atgggcaaat aggctataca gagatgaagt 600

ggtagcagaa tggaagaaag ttaaagggga catgtctctt cacgtccact gccacattag 660

cggtggccat ttcctcttgg atctcttccc aaagcttaga tactacatct tttccaaaga 720

actacctgtt gtaagtaaaa atatttattt ttggtttctt ggtttcatta caaaatgttg 780

attgtttatt ttgatatatg tatcaggtgt tgaaggctat tgttcatgga gacggcaact 840

tgttgaacaa ctatccggat ttacaagaag ctcttgtttg ggtttatttc cattctaatg 900

ccgatgagtt caacagagtt gaatgttggg gtccgctttg ggaagctact tcgtctgatg 960

gtcacaggac tcaaactctt cctcagactc ggtgcaagga tgagtgcagt tgttgtttcc 1020

cgcaggttag ctcgattccg tggtctcata gtcttagtaa cgaaggtgtt actgactacc 1080

ctgggacgca ggccgaggga atgcctaagc cggagaaact ctag 1124

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<213> Artificial sequence

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atgtgtagtt tgtcagcgaa catgttgtta ccgacaaagc tgaaacctgc ttattcagac 60

aaacggagta atagtctgaa ctgtctcccc gtctccaata caagatccaa gaggaagaac 120

caatcaattg ttcctatggc aagattattt ggaccggcta tcttcgaatc atccaagttg 180

aaagtattgt ttctaggtgt tgatgacaag aagcatccac caacgcttcc aaggacttac 240

actctcactc acagtgacat tactgctaaa ctaaccttag ctatttctca ttcaattaat 300

aattctcagt tgcaaggatg ggcaaatagg ctatacagag atgaagtggt agcagaatgg 360

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ctcttggatc tcttcccaaa gcttagatac tacatctttt ccaaagaact acctgttgtg 480

ttgaaggcta ttgttcatgg agacggcaac ttgttgaaca actatccgga tttacaagaa 540

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ggtccgcttt gggaagctac ttcgtctgat ggtcacagga ctcaaactct tcctcagact 660

cggtgcaagg atgagtgcag ttgttgtttc ccgcaggtta gctcgattcc gtggtctcat 720

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ccggagaaac tctag 795

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<213> Artificial sequence

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Met Cys Ser Leu Ser Ala Asn Met Leu Leu Pro Thr Lys Leu Lys Pro

1 5 10 15

Ala Tyr Ser Asp Lys Arg Ser Asn Ser Leu Asn Cys Leu Pro Val Ser

20 25 30

Asn Thr Arg Ser Lys Arg Lys Asn Gln Ser Ile Val Pro Met Ala Arg

35 40 45

Leu Phe Gly Pro Ala Ile Phe Glu Ser Ser Lys Leu Lys Val Leu Phe

50 55 60

Leu Gly Val Asp Asp Lys Lys His Pro Pro Thr Leu Pro Arg Thr Tyr

65 70 75 80

Thr Leu Thr His Ser Asp Ile Thr Ala Lys Leu Thr Leu Ala Ile Ser

85 90 95

His Ser Ile Asn Asn Ser Gln Leu Gln Gly Trp Ala Asn Arg Leu Tyr

100 105 110

Arg Asp Glu Val Val Ala Glu Trp Lys Lys Val Lys Gly Asp Met Ser

115 120 125

Leu His Val His Cys His Ile Ser Gly Gly His Phe Leu Leu Asp Leu

130 135 140

Phe Pro Lys Leu Arg Tyr Tyr Ile Phe Ser Lys Glu Leu Pro Val Val

145 150 155 160

Leu Lys Ala Ile Val His Gly Asp Gly Asn Leu Leu Asn Asn Tyr Pro

165 170 175

Asp Leu Gln Glu Ala Leu Val Trp Val Tyr Phe His Ser Asn Ala Asp

180 185 190

Glu Phe Asn Arg Val Glu Cys Trp Gly Pro Leu Trp Glu Ala Thr Ser

195 200 205

Ser Asp Gly His Arg Thr Gln Thr Leu Pro Gln Thr Arg Cys Lys Asp

210 215 220

Glu Cys Ser Cys Cys Phe Pro Gln Val Ser Ser Ile Pro Trp Ser His

225 230 235 240

Ser Leu Ser Asn Glu Gly Val Thr Asp Tyr Pro Gly Thr Gln Ala Glu

245 250 255

Gly Met Pro Lys Pro Glu Lys Leu

260

Claims (10)

1. Use of a substance capable of reducing the expression level and/or activity of a protein encoded by an SGR gene in a plant, in any one of:

(A1) improving the storage stability of the plant;

(A2) extending the shelf life of the plant;

(A3) reducing the loss of dry matter during storage and/or transport of the plant;

(A4) reducing the weight loss rate of the plant in the process of storage and/or transportation;

(A5) increasing the chlorophyll content of plants.

2. Use according to claim 1, characterized in that: the substance capable of reducing the expression level and/or activity of the protein encoded by the SGR gene in the plant is any one of the following substances:

(B1) gene editing tools for the genomic DNA sequence of the SGR gene;

the gene editing tool is a sequence specific nuclease capable of specifically cleaving a target sequence in a genomic DNA sequence encoding the SGR gene;

(B2) an RNA interference fragment directed against the SGR gene;

(B3) a DNA fragment capable of encoding the gene editing tool of (B1) or capable of transcribing the RNA interference fragment of (B2);

(B4) an expression cassette, a recombinant vector, a recombinant microorganism or a transgenic cell line comprising the DNA fragment of step (B3).

3. A method for breeding a plant variety having at least one of the following traits (C1) - (C5), comprising the step of decreasing the expression level and/or activity of a protein encoding an SGR gene in a recipient plant:

(C1) the storage stability is improved;

(C2) the shelf life is prolonged;

(C3) reduced loss of dry matter during storage and/or transport;

(C4) a reduced rate of weight loss during storage and/or transport;

(C5) chlorophyll content is increased.

4. The method of claim 3, wherein: the method for reducing the expression level and/or activity of the protein coded by the SGR gene in the receptor plant comprises the following steps: introducing into said recipient plant a substance capable of reducing the expression level and/or activity of a protein encoding an SGR gene in a plant as described in claim 2; or

The method for reducing the expression level and/or activity of the protein coded by the SGR gene in the receptor plant comprises the following steps: replacing the SGR gene in the recipient plant with a null allele of the SGR gene.

5. Use or method according to any of claims 1-4, characterized in that: the encoding protein of the SGR gene is any one of the following proteins:

(D1) protein with an amino acid sequence of SEQ ID No. 4; (D2) protein which is obtained by substituting and/or deleting and/or adding one or more amino acid residues to the amino acid sequence shown in SEQ ID No.4 and has the same function;

(D3) a protein having 99% or more, 95% or more, 90% or more, 85% or more, or 80% or more homology to the amino acid sequence defined in any one of (D1) to (D2) and having the same function;

(D4) a fusion protein obtained by attaching a tag to the N-terminus and/or C-terminus of the protein defined in any one of (D1) to (D3).

6. Use or method according to any of claims 1-5, wherein: the SGR gene is any one of the following DNA molecules:

(E1) a DNA molecule shown as SEQ ID No.2 or SEQ ID No. 3;

(E2) a DNA molecule which hybridizes under stringent conditions to the DNA molecule defined in (E1) and which encodes the protein of claim 6;

(E3) a DNA molecule which has 99% or more, 95% or more, 90% or more, 85% or more or 80% or more homology with the DNA sequence defined in (E1) or (E2) and which encodes the protein of claim 6.

7. The method according to any one of claims 4-6, wherein: the invalid allele of the SGR gene is a DNA molecule shown in any one of the following genes:

(F1) DNA molecule shown in SEQ ID No. 1;

(F2) a DNA molecule which hybridizes under stringent conditions to the DNA molecule defined in (F1) and which encodes the protein of claim 8;

(F3) a DNA molecule having 99% or more, 95% or more, 90% or more, 85% or more or 80% or more homology to the DNA sequence defined in (F1) or (F2) and encoding the protein of claim 8.

8. Use or method according to any of claims 1-7, wherein: the plant is a dicotyledonous plant or a monocotyledonous plant;

further, the dicotyledonous plant is a plant of the family Brassicaceae;

further, the cruciferous plant is a brassica plant or an arabidopsis plant.

9. A method for breeding Chinese cabbage variety having at least one of the following traits (C1) - (C5):

(C1) the storability is improved.

(C2) The shelf life is prolonged.

(C3) The loss of dry matter during storage and/or transport is reduced.

(C4) A reduced rate of weight loss during storage and/or transport;

(C5) chlorophyll content is increased.

The method comprises the following steps (G1) or (G2):

(G1) transferring the DNA molecule shown in SEQ ID No.1 into the Chinese cabbage variety A by taking the Chinese cabbage variety A as a female parent, and cultivating to obtain a target Chinese cabbage variety;

(G2) transferring the DNA molecule shown in SEQ ID No.1 into the Chinese cabbage sterile line A by taking the Chinese cabbage sterile line A as a female parent, and culturing to obtain the target Chinese cabbage sterile line for hybridization and matching.

10. A method for cultivating a cabbage variety with green color comprises the following steps:

(H1) using green stalk vegetable green-keeping mutant 'nye' as female parent and green stalk vegetable '13A 510' as male parent, making hybridization, and using F₂Screening out plants with a green-keeping phenotype as donor materials in the generation to create green-keeping double haploids;

(H2) green-keeping cytoplasmic male sterile lines are cultivated by taking green-keeping stem vegetable 'East 18' hybridized with the Ogrua sterile source cytoplasmic male sterile line as a sterile source and green-keeping stem vegetable green-keeping mutant 'nye' as a breeding recurrent parent material;

(H3) and (4) preparing a hybridization combination by using the green-sustaining double haploid obtained in the step (H1) and the green-sustaining cytoplasmic male sterile line obtained in the step (H2) to obtain the target Chinese cabbage.