CN118374542B

CN118374542B - Application of CRY1 gene in negative regulation of stress tolerance of plant endoplasmic reticulum

Info

Publication number: CN118374542B
Application number: CN202410797284.5A
Authority: CN
Inventors: 李志远; 宁昕; 徐秀美; 吕丹丹; 陈立超; 孙扬; 石艳云; 王珂; 张立新
Original assignee: Sanya Research Institute Of Henan University; Henan University
Current assignee: Sanya Research Institute Of Henan University; Henan University
Priority date: 2024-06-20
Filing date: 2024-06-20
Publication date: 2024-10-15
Anticipated expiration: 2044-06-20
Also published as: CN118374542A

Abstract

The invention provides an application of CRY1 (CRYPTOCHROME 1) genes in negative regulation of endoplasmic reticulum stress tolerance of plants, wherein the nucleotide sequence of the CRY1 genes is shown as SEQ ID NO.1, the negative regulation is shown in that the tolerance of plants which overexpress the CRY1 genes to endoplasmic reticulum stress is reduced, and the tolerance of plants which reduce the expression of the CRY1 genes to endoplasmic reticulum stress is improved, so the invention provides a novel method for improving the endoplasmic reticulum stress tolerance of plants, and the tolerance of plants to endoplasmic reticulum stress can be improved by reducing the expression of the CRY1 genes in target plants or reducing the activity of CRY1 proteins in target plants, thereby providing a novel gene resource for molecular breeding and genetic modification of plants.

Description

Application of CRY1 gene in negative regulation of stress tolerance of plant endoplasmic reticulum

Technical Field

The invention belongs to the technical field of biology, and particularly relates to an application of CRY1 genes in negative regulation of plant endoplasmic reticulum stress tolerance.

Background

Endoplasmic Reticulum (ER) stress is an important stress commonly seen in many eukaryotic organisms. Endoplasmic reticulum stress can be induced by biological and non-biological factors such as pathogen infection, endoplasmic reticulum stress, high salinity, injury, and endoplasmic reticulum stress inducers, and results in accumulation of misfolded proteins in the endoplasmic reticulum and accumulation of unfolded proteins in the endoplasmic reticulum. To relieve endoplasmic reticulum stress, the levels of protein folding partners and endoplasmic reticulum-related protein degradation mechanisms are increased, which is called an unfolded protein response. In this process, various membrane-associated transcription factors transmit stress signals into the nucleus and up-regulate genes encoding components of the folding mechanism of endoplasmic reticulum proteins, including endoluminal binding proteins (BIPs), calmodulin (CNX), etc., which help clear misfolded proteins. In animal and plant cells, programmed cell death is induced when unfolded protein responses fail to cope with endoplasmic reticulum stress.

CRYPTOCHROME (CRY) is a yellow element type blue photoreceptor, and CRY1 plays an important role in regulating blue light to inhibit hypocotyl elongation, while CRY2 mainly inhibits hypocotyl growth under low intensity blue light in the current report. CRYs are structurally divided into an N-terminal region associated with a photolytic enzyme and a C-terminal extension region. Current studies have demonstrated that both the C-terminal region CCT1 of the CRY1 protein and the C-terminal region CCT2 of the CRY2 protein of arabidopsis have photosensitive properties and are involved in the response to light. Up to now, no studies have shown that CRY1 gene is associated with tolerance of plants to endoplasmic reticulum stress.

Disclosure of Invention

The invention aims to provide an application of CRY1 genes in negative regulation of plant endoplasmic reticulum stress tolerance.

In order to achieve the above purpose, the technical scheme adopted by the invention is summarized as follows:

Nucleotide sequence information of the Arabidopsis CRY1 gene (i.e., AT4G 08920) was obtained based on the Arabidopsis whole genome sequencing published by the Arabidopsis Tair functional network (https:// www.arabidopsis.org /). The CRY1 gene coding frame nucleotide sequence has the length of 2046 bp and is composed of 681 amino acids, and the sequence can be obtained through the inquiry of an Arabidopsis Tair functional network.

The invention also constructs a series of plant expression and knockout vectors, and expression vectors, gene editing vectors or transgenic plant lines containing the genes and host cells containing the vectors also fall into the protection scope of the invention in the aspect of improving the stress tolerance of the endoplasmic reticulum of the plants.

The functions of the genes protected by the invention not only include the CRY1 genes, but also include the functions of homologous genes with higher homology (such as homology higher than 80%, more preferably higher than 90%, still more preferably higher than 95%, still more preferably higher than 98%) with CRY1 genes in terms of endoplasmic reticulum stress tolerance.

According to the invention, a 35S-started pCAMBIA1300 over-expression vector and a pHEC gene editing vector of an EC1.2p promoter are constructed according to a CDS gene sequence of CRY1, a CRY1 over-expression strain and a CRY1 knockout mutant strain are respectively obtained through agrobacterium infection, and biological functions of the CRY1 over-expression strain and a CRY1 mutant (CRY 1 knockout mutant strain) in endoplasmic reticulum stress in a seedling stage are analyzed, so that gene resources are provided for crop stress-tolerant molecular breeding.

The invention discloses a biological function of CRY1 gene in plant endoplasmic reticulum stress tolerance, which is specifically expressed in: under endoplasmic reticulum stress, CRY1 knockout mutant lines have significantly higher tolerance to endoplasmic reticulum stress than wild-type; whereas CRY1 overexpressing lines are less tolerant to endoplasmic reticulum stress than the wild-type. The above application was concluded from endoplasmic reticulum stress treatment experiments.

According to its function, plants resistant to endoplasmic reticulum stress can be obtained by means of transgenesis, in particular, transgenic plants can be obtained by knocking out the CRY1 gene, which plants have a higher tolerance to endoplasmic reticulum stress than the plants of interest.

Specifically, the target plant can be introduced by the gene editing vector. In the method, the gene editing vector may transform plant cells or tissues by using conventional biological methods such as Ti plasmid, ri plasmid, plant viral vector, direct DNA transformation, microinjection, electric conduction, agrobacterium mediation, etc., and cultivate the transformed plant tissues into plants.

In order to improve the superior traits of plants, the present invention also protects a novel plant breeding method comprising the following steps (1) or (2):

(1) Obtaining a plant with endoplasmic reticulum stress tolerance stronger than that of the target plant by reducing the activity of CRY1 protein in the target plant;

(2) Obtaining a plant with endoplasmic reticulum stress tolerance stronger than that of the target plant by reducing the expression of CRY1 gene in the target plant;

the "reducing expression of CRY1 gene in a plant of interest" may be achieved as follows (1) or (2) or (3):

(1) Knocking out CRY1 gene in target plant;

(2) Inserting a T-DNA sequence to disable expression of the gene;

(3) Other methods are common in the art.

Wherein the plant of interest of the present invention is Arabidopsis thaliana.

In addition, in order to further explore the mechanism of CRY1 for regulating endoplasmic reticulum stress, CRY1 interacting proteins were identified, proteins which can be specifically bound with CRY1 under endoplasmic reticulum stress were identified through an immunoprecipitation tandem mass spectrometry experiment, several proteins with higher scores (among which the highest score protein is BIP 1) were selected, and fusion vectors were constructed by using the full-length coding sequence of CRY1 and the N-terminal and C-terminal sequences of YFP. Two-by-two combination of the constructed vectors are used for carrying out a double-molecule fluorescence complementation experiment (BiFC), the two-by-two combination of the two vectors are transformed into arabidopsis protoplast, and then the protoplast is observed by a laser confocal microscope, so that the result shows that only the protoplast transformed with pSAT4A-CRY1-CE and pSAT4A-BIP1-NE has yellow fluorescence signals, and the protoplast transformed with pSAT4A-CRY1-CE and NE or CE and pSAT4A-BIP1-NE has no reconstructed yellow fluorescence, which indicates that the yellow fluorescence is generated by interaction of CRY1 and BIP1 rather than self-excitation, and CRY1 can interact with BIP1 in vivo.

Therefore, the invention not only finds that CRY1 can negatively regulate the tolerance of plants to endoplasmic reticulum stress, but also finds possible reasons, namely, the adaptation of plants to endoplasmic reticulum stress is regulated by affecting the protein function of BIP1 genes. Experiments show that BIP1 protein has the function of improving the stress tolerance of the endoplasmic reticulum of a plant, and when CRY1 exists, CRY1 is combined with BIP1 to limit the BIP1 protein to participate in the stress response of the endoplasmic reticulum, so that when CRY1 exists in the plant, the function of the BIP1 protein in the plant is limited, thereby leading to insufficient adaptability of the plant to the stress response of the endoplasmic reticulum, and when CRY1 is absent, the function of the BIP1 is not limited, thereby increasing the adaptability of the plant to the stress of the endoplasmic reticulum.

Genes of interest, also known as target genes, are used in genetic engineering design and manipulation to recombine genes, alter receptor cell traits and obtain desired expression products. May be of the organism itself or from a different organism.

In the present invention, the plant suitable for the present invention is not particularly limited as long as it is suitable for performing a gene transformation operation such as various crops, flower plants, forestry plants, or the like. The plant may be, for example (without limitation): dicotyledonous, monocotyledonous or gymnosperm plants.

As a preferred mode, the "plant" includes, but is not limited to: arabidopsis thaliana is suitable for use as the gene or genes homologous thereto.

As used herein, the term "plant" includes whole plants, parent and progeny plants thereof, and various parts of plants, including seeds, fruits, shoots, stems, leaves, roots (including tubers), flowers, tissues and organs, in which the gene or nucleic acid of interest is found. Reference herein to "plant" also includes plant cells, suspension cultures, callus tissue, embryos, meristematic regions, gametophytes, sporophytes, pollen and microspores, again wherein each of the foregoing comprises the gene/nucleic acid of interest.

The present invention includes any plant cell, or any plant obtained or obtainable by a method therein, as well as all plant parts and propagules thereof. The present patent also encompasses transfected cells, tissues, organs or whole plants obtained by any of the foregoing methods. The only requirement is that the sub-representations exhibit the same genotypic or phenotypic characteristics, and that the progeny obtained using the methods of this patent have the same characteristics.

The invention also extends to harvestable parts of a plant as described above, but not limited to seeds, leaves, fruits, flowers, stems, roots, rhizomes, tubers and bulbs. And further to other derivatives of the plants after harvest, such as dry granules or powders, oils, fats and fatty acids, starches or proteins. The invention also relates to a food or food additive obtained from the relevant plant.

The invention has the advantages that:

according to the invention, an arabidopsis CRY1 gene is obtained through plant genome cloning, a pCAMBIA1300 over-expression vector and a pHEC gene editing vector are constructed, a CRY1 gene over-expression strain 35S, CRY1-GFP and a CRY1 gene knockout mutant strain CRY1 are respectively obtained through agrobacterium infection, biological functions of the CRY1 gene over-expression strain and the CRY1 gene knockout mutant strain CRY1 in the seedling stage endoplasmic reticulum stress are analyzed, and the result shows that the tolerance of the CRY1 knockout mutant strain to endoplasmic reticulum stress is obviously higher than that of the wild type under the stress of endoplasmic reticulum stress inducer tunicamycin; and the tolerance of CRY1 over-expression strain to endoplasmic reticulum stress is lower than that of wild type, which reveals that CRY1 gene negatively regulates endoplasmic reticulum stress tolerance of plant, and also shows that CRY1 can interact with BIP1 in vivo, and in practical application, the tolerance of plant to endoplasmic reticulum stress can be improved by reducing the expression of CRY1 gene in target plant or reducing the activity of CRY1 protein in target plant, thus providing new gene resource for crop stress molecular breeding.

Drawings

FIG. 1 is a schematic diagram showing the sequencing of mutation sites of CRY1 gene in CRY1 mutant.

FIG. 2 shows protein expression level assays of wild-type (WT) and CRY1 transgenic overexpressing lines (35S: CRY1-GFP-1, 35S: CRY1-GFP-2, 35S: CRY1-GFP-3, 35S: CRY1-GFP-4, 35S: CRY 1-GFP-5).

FIG. 3 is a phenotypic chart of Arabidopsis WT, cry1, 35S: CRY1-GFP after 10 days of growth in different concentration gradients of TM medium (0, 150, 200 ng/mL).

FIG. 4 is a graph showing the relative fresh weight statistics of Arabidopsis WT, cry1, 35S CRY1-GFP at various concentrations of TM (150, 200 ng/mL).

FIG. 5 is a diagram showing the result of BIFC experiments on CRY1 and BIP 1.

FIG. 6 is a background verification of BIP1 related material; wherein, FIG. 6A is a diagram showing the verification result of BIP1 mutant and T-DNA insertion site, FIG. 6B is the verification of BIP gene expression in BIP1 mutant, and FIG. 6C is the Western verification result of 35S: CRY1-GFP material.

FIG. 7 is a phenotypic demonstration of endoplasmic reticulum stress for BIP 1-related materials.

Detailed Description

The present invention will be described in detail with reference to specific examples. These embodiments are provided so that this disclosure will be thorough and complete, and will fully convey the scope of the invention to those skilled in the art. The technical means used in the examples are conventional means well known to those skilled in the art unless otherwise indicated. The test methods in the following examples are conventional methods unless otherwise specified. The reagents and materials employed, unless otherwise indicated, are commercially available.

Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art. In addition, any methods and materials similar or equivalent to those described herein can be used in the present invention. The preferred methods and materials described herein are presented for illustrative purposes only.

The practice of the present invention will employ, unless otherwise indicated, conventional techniques of botanicals, microorganisms, tissue culture, molecular biology, chemistry, biochemistry, DNA recombination, and bioinformatics, which will be apparent to one of skill in the art. These techniques are fully explained in the published literature, and the methods of DNA extraction, phylogenetic tree construction, gene editing method, gene editing vector construction, gene editing plant acquisition, etc. used in the present invention can be realized by the methods disclosed in the prior art except the methods used in the examples described below.

The terms "nucleic acid", "nucleic acid sequence", "nucleotide", "nucleic acid molecule" or "polynucleotide" as used herein are meant to include isolated DNA molecules (e.g., cDNA or genomic DNA), RNA molecules (e.g., messenger RNA), natural types, mutant types, synthetic DNA or RNA molecules, DNA or RNA molecules composed of nucleotide analogs, single-or double-stranded structures. Such nucleic acids or polynucleotides include, but are not limited to, gene coding sequences, antisense sequences, and regulatory sequences of non-coding regions. These terms include a gene. "Gene" or "gene sequence" is used broadly to refer to a functional DNA nucleic acid sequence. Thus, a gene may include introns and exons in genomic sequences, and/or coding sequences in cDNA, and/or cDNA and regulatory sequences thereof. In particular embodiments, for example in relation to isolated nucleic acid sequences, it is preferred that they are cDNA.

Before describing the embodiments, in order to facilitate a person skilled in the art to understand the relevant development of the present application in detail, the following description is made with reference to the accompanying drawings, in which the principles of the present application, some experimental materials, detection methods, etc. are described:

1. Extraction and reverse transcription of total RNA of plant material

Plant material 0.2 g was rapidly ground in liquid nitrogen to an off-white powder for the experiment. 600 mu L Buffer EL is added into powder, after the powder is completely mixed uniformly, the powder is immediately centrifuged for 5min (12000 rpm), the supernatant is transferred to an adsorption column III for centrifugation for 1 min (12000 rpm), the supernatant is transferred to a new tube, 300 mu L absolute ethyl alcohol is added for adjusting the upper column environment, after the mixture is fully mixed uniformly, the mixture is added into an adsorption column V in batches, the mixture is centrifuged for 30 s (12000 rpm), and the filtrate is discarded. RWA and RWB were then added stepwise to the column V and the impurities were removed by centrifugation (RWB was washed twice). After washing impurities, a new collecting pipe is replaced by empty column centrifugation 2 min (12000 rpm), and water is added for eluting after the air drying at room temperature (high-speed centrifugation 2 min). After elution, the total RNA is put on ice for temporary storage or frozen by liquid nitrogen and then is put at-80 ℃ for preservation.

After measuring the concentration of the extracted total RNA by using Nanodrop, carrying out RNA template denaturation according to the amount of 5 mug total RNA, setting a PCR program (heating at 65 ℃ for 5min and cooling at 12 ℃ for 5 min), taking out a denatured product after the operation program is finished and cooling, and carrying out ice bath 2 min. After ice bath, GDNA WIPER Mix was added and mixed well and incubated at 42℃for 2: 2 min. RT Mix, HISCRIPTIII ENZYME MIX, oligo (dT) 20VN and RNase-free ddH ₂ O were added sequentially to the mixture. Mixing the above liquids, blowing with a pipette, and setting PCR program (25deg.C for 5min, 37deg.C for 45 min, 85deg.C for 5 s), and collecting cDNA product for subsequent experiment or preserving at-80deg.C.

2. Vector construction

The construction flow of the over-expression vector comprises the following steps:

RNA of wild type Arabidopsis materials is extracted, and the obtained total RNA is subjected to concentration measurement by using Nanodrop and then is reversely transcribed into cDNA. Simultaneously, the cDNA sequence of AT4G08920 is downloaded in an Arabidopsis thaliana functional network (https:// www.arabidopsis.org /), specific primers are designed by using Primer premier5.0, cDNA of a wild Arabidopsis thaliana material is used as a template, a PCR reaction (a PCR reaction program: 95 ℃ for 5min, 95 ℃ for 30 s,58 ℃ for 15s, 72 ℃ for 1 min for extension, 37 cycles, 72 ℃ for 5min, 12 ℃ for 5 s) is used for cloning the coding sequence of the CRY1 gene, a PCR reaction product is subjected to electrophoresis by using 1% agarose gel, a target band is recovered by using a DNA recovery purification kit of the banker, and the recovered target gene is connected with pCAMBIA1300 by using a ClonExpressUltra One Step Cloning kit of the Novain company (connection of 30min AT 55 ℃).

The construction flow of the gene editing vector comprises the following steps:

Logging in to a website http:// www.genome.arizona.edu/crispr/CRISPRsearch.html, and screening targets. The target point is provided with an enzyme cutting site (Cas 9 cutting point (3 bp away from PAM/NGG) is positioned in the enzyme cutting site); assessing off-target condition, wherein the website is (http:// www.rgenome.net/cas-offinder /); then designing a primer with a target spot and carrying out PCR amplification: PCR amplification was performed using diluted 10pCBC-DT1T2 as template. -BsF/-BsR is the normal primer concentration; -F0/-R0 diluted 20-fold; the PCR product was recovered by purification and the following cleavage-ligation system was established (Table 1):

TABLE 1 cleavage-ligation System

The over-expressed ligation product and the gene knockout PCR product were transformed into E.coli DH 5. Alpha. By heat shock transformation, and the transformed product was spread on LB plates with antibiotics, and cultured upside down at 37℃for 12h. Single colonies are picked for PCR verification, and bacterial liquid with correct strip size is sent to Shanghai biological company for sequencing to detect the construction condition. The constructed target gene is CRY1 gene, the ORF of which is 2046 bp long, 681 amino acids are encoded, the nucleotide sequence of which is shown as SEQ ID NO.1, and the amino acid sequence of which is shown as SEQ ID NO. 2.

3. Genetic material creation

The wild type of Arabidopsis thaliana is taken as a starting plant. The constructed gene editing vector is introduced into agrobacterium, the arabidopsis inflorescence is infected by the agrobacterium, and a CRY1 gene knockout mutant strain (CRY 1) is obtained through resistance screening. RNA of wild type and CRY1 mutant was extracted and reverse transcribed into cDNA, and PCR amplification of CRY1 gene was performed using specific primers using this as a template, and the PCR product was sent to Shanghai Biotechnology company for sequencing, which revealed that 4 bases were deleted on exon 1 of CRY1 gene in CRY1 mutant, which resulted in frame shift mutation of the gene (FIG. 1).

Meanwhile, a pCAMBIA1300 overexpressing vector containing a 35S promoter constructed by using the CDS sequence of the CRY1 gene was introduced into Agrobacterium, the Arabidopsis inflorescence was infected by Agrobacterium, and a CRY1 gene overexpressing strain (35S: CRY 1-GFP) was obtained by resistance screening. Placing about 0.1 g of blades of CRY1 gene overexpression material in a mortar, adding 400 mu L of whole protein extract (E buffer：125mM Tris-HCl,pH 8.8;50 mM Na₂S₂O₅;1% (w/v) SDS;10% (w/v) Glycerol), into the mortar, fully grinding, transferring into a 1.5 mL centrifuge tube, vibrating for 15min, centrifuging at room temperature (12,000 g,10 min), and sucking the supernatant into a new 1.5 mL centrifuge tube to obtain a total protein solution of the CRY1 overexpression material; If the solution is still turbid, repeating the centrifugation process, and finally collecting supernatant for Western blot. Relevant tools for preparing polyacrylamide gel are cleaned in advance, and 15% polyacrylamide gel is prepared: the upper layer is concentrated gel with concentration (acrylamide) of 4.5%, and the lower layer is separating gel with concentration (acrylamide) of 15%. The gel is prepared according to the proportion, and after the gel is solidified. 1 XSDS-PAGE buffer was prepared and pre-cooled in a refrigerator at 4 ℃. Adding a loading buffer (5 mM Tris-HCl, pH 8.8; 1.2% (w/v) SDS; 1% (w/v) Glycerol; 1% (w/v)β-Mercaptoethanol; 0.0001% (w/v) Bromophenol blue), into the total protein solution of the CRY1 overexpression material, heating at high temperature for 10: 10 min, thoroughly denaturing, adding a sample into a loading hole of the gel by using a sample loading device, and starting electrophoresis (current of 10mA per gel, time of 4-6 h). Judging the position of the target protein according to the positions of the bromoFenlan indicator and the protein Marker, and stopping electrophoresis when the target protein runs to the position of 2/3 of the gel. And preparing a transfer buffer solution, and soaking the filter paper and the nitrocellulose membrane in the transfer buffer solution in advance. Carefully remove the gel from the glass plate and also place it in the transfer buffer for soaking. The filter paper, gel, nitrocellulose membrane and filter paper are placed in sequence, a membrane transferring clamp is buckled, and membrane transferring is started (current 180 mA, time 1.5 h). At the end of transfer, the nitrocellulose membrane with protein was taken out, put into 8% skim milk, blocked at room temperature for 1h, and then the skim milk was replaced with TTBS (50 mM Tris-HCl, pH 7.4;150 mM NaCl; 0.05% (v/v) Tween-20), thereby washing the blocked nitrocellulose membrane (3 times each time 5 min). The washed nitrocellulose membrane was transferred to 1% skim milk (containing primary antibody) and incubated overnight at 4 ℃. After recovery of the primary antibody, the nitrocellulose membrane was washed with TTBS (5 total washes, 5min each). The washed nitrocellulose membrane was transferred to 1% skim milk (containing secondary antibody) and incubated at room temperature for 1 h. At the end of incubation, nitrocellulose membranes were washed with TTBS (4 total washes, 5min each). Adding a proper amount of luminous liquid on the cleaned nitrocellulose membrane, imaging by using a full-automatic chemiluminescence imager, and detecting a target strip. As shown in FIG. 2, the CRY1-GFP protein in the CRY1 gene overexpression material was stably expressed under the drive of the CaMV 35S promoter by using Arabidopsis wild-type material as a negative control.

4. Statistics of treatment results of Arabidopsis thaliana tunicamycin

We used endoplasmic reticulum stress inducers to mimic endoplasmic reticulum stress in arabidopsis. The inducer of endoplasmic reticulum stress is tunicamycin (Tunicamycin, TM) with TM concentration of 0, 150, 200 ng/mL, and Wild Type (WT), CRY1 knockout mutant (CRY 1), CRY1 overexpressing strain (35S: CRY 1-GFP) are treated.

Endoplasmic reticulum stress experiment: we inoculated WT, cry1, 35S CRY1-GFP in 1/2MS medium with TM concentrations of 0, 150, 200 ng/mL, respectively, and incubated under light, and observed for phenotype and counted for relative fresh weight. The results show that with increasing TM concentration, the growth and development of all three materials are inhibited to different degrees, but compared with the WT material, cry1 mutant material is insensitive to endoplasmic reticulum stress, and overexpressing material 35S: CRY1-GFP shows a phenotype extremely sensitive to endoplasmic reticulum stress, and the growth is severely inhibited. The result shows that the blue light receptor CRY1 is involved in regulating and controlling the stress response of the endoplasmic reticulum of Arabidopsis, and the tolerance of the plant to the stress of the endoplasmic reticulum can be effectively improved by knocking out the blue light receptor CRY1 from the plant.

5. Detection and identification of CRY1 interacting proteins

To further explore the mechanism by which CRY1 regulates endoplasmic reticulum stress, we identified CRY1 interacting proteins. Immunoprecipitation tandem mass spectrometry experiments were performed after soaking 35S:CRY1-GFP Arabidopsis materials, which were normally cultured on MS medium for 2 weeks, in H ₂ O and high concentration TM solution (1000 ng/mL) for 12H, respectively, and proteins that were likely to bind specifically to CRY1 under endoplasmic reticulum stress were identified (Table 2).

Table 2 shows the results of protein mass spectrometry after CRY1 tag vaccine co-immunoprecipitation tandem mass spectrometry

To further verify the existence of interactions between proteins in the table and CRY1, we selected several proteins with higher scores (the highest scoring protein being BIP1, a protein that plays an important role in plant response to endoplasmic reticulum stress), and constructed fusion vectors with full-length coding sequences of CRY1 and the N-terminal (NE) and C-terminal (CE) sequences of YFP. Performing BiFC experiments by using constructed vectors, jointly transforming the two combinations into Arabidopsis protoplast, and observing the protoplast by using a laser confocal microscope, wherein the result shows that only the protoplast transformed with pSAT4A-CRY1-CE and pSAT4A-BIP1-NE has yellow fluorescence signals, and the transformed pSAT4A-CRY1-CE and single NE have no reconstructed yellow fluorescence, which indicates that pSAT4A-CRY1-CE and single NE do not interact; there was also no reconstructed yellow fluorescence in protoplasts of pSAT4A-BIP1-NE alone, indicating that pSAT4A-BIP1-NE and CE alone did not interact. The above results demonstrate that the occurrence of yellow fluorescence is caused by the interaction of CRY1 and BIP1, rather than by self-activation (fig. 5). The above results indicate that CRY1 is capable of interacting with BIP1 in vivo.

6. Verification and phenotypic characterization of BIP 1-related materials

We ordered the T-DNA insertion mutant material of BIP1, and identified BIP 1-BIP 1-16 by a three-primer method to obtain homozygous mutant, but the T-DNA insertion site is in the 3' UTR region, which may not result in complete silencing of BIP1 expression. We selected partially homozygous line materials, extracted their RNAs with WT materials, reverse transcribed and subjected to qRT-PCR, and analyzed BIP1 gene transcript levels, which showed extremely significant decreases in BIP1 expression in the four selected line materials compared to WT materials (fig. 6A, B), which we used for subsequent genetic analysis. To obtain the over-expressed material of BIP1 gene, we transferred the CDS sequence of BIP1 gene into pCAMBIA1300-35S 1 FLAG vector. And transforming the constructed fusion vector into agrobacterium, infecting WT by using the agrobacterium to obtain 35S: BIP1-FLAG over-expression plant, and verifying the over-expression plant through a western blotting experiment, wherein the result shows that a stronger BIP1-FLAG fusion protein signal can be detected in the transgenic plant (figure 6C).

WT, bip1, 35S BIP1-FLAG seeds were inoculated onto 1/2MS medium and 1/2MS medium containing TM (200 ng/mL) after sterilization, grown for 10 days in a 22℃culture room, the phenotype was observed, and the relative fresh weights were counted. As a result, the three materials are phenotypically identical under normal conditions. However, under TM (200 ng/mL) stress conditions, BIP1 mutant material was more sensitive to endoplasmic reticulum stress than WT, while 35S BIP1-FLAG over-expressed material was more resistant to endoplasmic reticulum stress (FIG. 7), which also corresponds to the function of BIP1 to aid protein folding in the endoplasmic reticulum.

Therefore, we have found that CRY1 can negatively regulate the tolerance of plants to endoplasmic reticulum stress, and found possible reasons for the tolerance, by the experiments, it can be known that BIP1 protein has the function of improving the endoplasmic reticulum stress tolerance of plants, and when CRY1 exists, CRY1 limits the participation of BIP1 protein in endoplasmic reticulum stress reaction through combination with BIP1, so that when CRY1 exists in plants, the function of BIP1 protein in plants is limited, and thus the adaptability of plants to endoplasmic reticulum stress reaction is insufficient, and when CRY1 is absent, the function of BIP1 is not limited, and thus the adaptability of plants to endoplasmic reticulum stress can be increased, therefore, in practical application, the tolerance of plants to endoplasmic reticulum stress can be improved by reducing the expression of CRY1 gene in target plants or reducing the activity of CRY1 protein in target plants, and thus providing new gene resources for crop stress-resistant molecular breeding.

The foregoing description of the preferred embodiments of the invention is not intended to limit the invention to the precise form disclosed, and any such modifications, equivalents, and alternatives falling within the spirit and scope of the invention are intended to be included within the scope of the invention.

Claims

The application of CRY1 gene in negative regulation of plant endoplasmic reticulum stress tolerance, which is characterized in that the nucleotide sequence of the CRY1 gene is shown as SEQ ID NO.1, the endoplasmic reticulum stress tolerance is shown as the resistance of plants to tunicamycin, the negative regulation is shown as the decrease of the resistance of plants over-expressing CRY1 gene to endoplasmic reticulum stress, and the increase of the resistance of plants under-expressing CRY1 gene to endoplasmic reticulum stress is shown as Arabidopsis thaliana.
2. The use according to claim 1, wherein the reduction of CRY1 gene expression is by knocking out the CRY1 gene.
3. Use according to claim 2, wherein the CRY1 gene is knocked out by constructing a CRY1 Crispr-cas9 gene editing vector to obtain a CRY1 gene knockdown mutant strain having a higher endoplasmic reticulum stress tolerance than the wild type strain.
4. The use according to claim 3, wherein the constructed gene editing vector is introduced into agrobacterium with wild type arabidopsis thaliana as starting plant, the arabidopsis thaliana inflorescence is infected by agrobacterium, and the CRY1 gene knockout mutant line is obtained by resistance screening.
5. The use according to claim 1, wherein CRY1 protein is capable of interacting with BIP1 protein in vivo, said BIP1 protein being capable of increasing endoplasmic reticulum stress tolerance of plants.