CN116121298B - Application of Inhibiting the Expression of HSRP1 Gene in Improving Heat Resistance of Plants - Google Patents

Abstract

The invention discloses a method for inhibitingHSRP1Use of expression of genes for improving heat resistance of plantsHSRP1The gene number of the gene AT the TAIR official website is AT4G37930. The invention obtains arabidopsis through plant genome cloningHSRP1Genes, constructHSRP1Gene CRISPR-Cas9 gene knockout vector obtained by using agrobacterium-mediated methodHSRP1A gene knockout transgenic line. The inventor discloses through a culture medium heat stress simulation experimentHSRP1The gene can provide gene resources for crop heat-resistant molecular breeding. Can enable the plant to survive and grow in a temperature range beyond the original adaptation range, and provides a new way for molecular breeding and genetic modification of the plant.

Description

Application of Inhibiting the Expression of HSRP1 Gene in Improving Heat Resistance of Plants

technical field

The invention belongs to the field of biotechnology, specifically relates to the application of HSRP1 gene in plant heat resistance, and more specifically relates to the application of inhibiting the expression of HSRP1 gene in improving plant heat resistance.

Background technique

With the intensification of the greenhouse effect and global warming, high temperature and disastrous climate has become a global concern and a severe challenge for many agricultural areas. The high temperature and disastrous climate seriously inhibits the growth and development of crops, and leads to a decline in the yield and quality of crops. High temperature stress damages the vegetative tissue of plants, affects plant seed germination, reproductive development, photosynthesis, stability of plasma membrane and plant water content, and ultimately leads to crop yield reduction. Usually, under high temperature stress, plants show delayed seed germination, deformed stigma, abnormal pollen development, leaf wilting, and in severe cases, plants dry out and die. At present, global warming has posed a serious threat to the production of crops, and it is showing an increasing trend year by year. Understanding the adaptation mechanism of plants to high temperature stress is conducive to the scientific selection and breeding of heat-tolerant crops, which can effectively increase crop yield and reduce the increasing global food pressure.

SHMT is a pyridoxal 5-phosphate-dependent enzyme that catalyzes the reversible conversion of serine to glycine in a tetrahydrofolate-dependent or -independent manner. The conversion of serine to glycine provides C1 units for a series of important biosynthetic processes, such as the synthesis of methionine, thymidylate and purine, indicating its importance in DNA biogenesis and cellular methylation reactions. In plants, the other direction of the SHMT reaction: the conversion of glycine to serine is also indispensable, which is an indispensable step in the photorespiratory pathway. The SHMT protein family is very conserved in evolution, but the number of genes encoding SHMT varies in different species. In Arabidopsis, seven different SHMT proteins are located in different cellular regions: mitochondria, cytoplasm, chloroplast and nucleus, and their functions are somewhat different. According to the literature, SHMT proteins are widely involved in one-carbon metabolism, biotic stress and abiotic stress, etc., but their functions in participating in high temperature stress have not yet been clearly studied.

Contents of the invention

The object of the present invention is to provide the application of HSRP1 gene in plant heat resistance, and improve the heat resistance of plant by inhibiting the expression of HSRP1 gene.

In order to achieve the above object, the technical scheme adopted in the present invention is summarized as follows:

Based on the whole genome sequencing of Arabidopsis thaliana published on the official website of TAIR (http://www.arabidopsis.org/), the nucleotide sequence information of the Arabidopsis HSRP1 gene (serine hydroxymethyltransferase, sequence number: AT4G37930) was obtained . The nucleotide sequence of the HSRP1 gene is shown in SEQ ID NO.1. The nucleotide sequence of the HSRP1 gene coding frame is 1554 bp in length and consists of 517 amino acids, and its sequence can be obtained through the official website of TAIR.

The present invention also constructs a series of plant expression vectors, the expression vectors, recombinant vectors or transgenic plant lines containing the above genes and the function of the host cells containing the vectors in improving the heat resistance of plants also fall within the protection scope of the present invention .

The function of the gene protected in the present invention not only includes the above-mentioned HSRP1 gene, but also includes a higher homology with the HSRP1 gene (such as a homology higher than 80%; more preferably higher than 90%; more preferably higher than 95%; more preferably The function of the homologous genes in thermotolerance.

The present invention constructed a CRISPR-Cas9 gene knockout vector according to the CDS gene sequence of HSRP1 , obtained HSRP1 gene knockout strains through Agrobacterium infection, and analyzed wild-type Col-0 and HSRP1 gene knockout transgenic lines under high temperature stress biological functions, thus providing genetic resources for molecular breeding of heat-resistant crops.

The biological function of the HSRP1 gene disclosed in the present invention in plant heat resistance is specifically manifested in that after high temperature stress treatment, the seedling survival rate of the HSRP1 gene knockout mutant is significantly higher than that of the wild type.

The above applications are concluded by simulating high temperature stress experiments in culture medium.

According to its function, heat-resistant plants can be obtained by transgenic means. Specifically, the HSRP1 gene can be knocked out or knocked down in the target plant to obtain a transgenic plant whose heat resistance is higher than that of the target plant.

As an embodiment of the present invention, polynucleotides are cloned into CRISRP vectors by conventional methods, and the recombinant vectors with foreign genes are introduced into plant cells that can express HSRP1 protein, so that the Deletion of HSRP1 protein in plant cells. HSRP1 gene-deleted mutant plants can be obtained by regenerating the plant cells into plants. And use Agrobacterium transformation method to transfer the recombinant plasmid into plants.

In order to improve the excellent traits of the plants, the present invention also protects a new plant breeding method, by inhibiting the expression of the HSRP1 gene in the target plants, to obtain plants with stronger heat resistance than the target plants;

The implementation of "inhibiting the expression of the HSRP1 gene in the target plant" can be as follows (1) or (2) or (3):

(1) Knock out or knock down the HSRP1 gene in the target plant;

(2) Introducing a silencer;

(3) Other common methods in this field.

Wherein, the target plant of the present invention is Arabidopsis thaliana.

The target gene, also known as the target gene, is used in gene engineering design and operation to recombine genes, change the traits of recipient cells and obtain expected expression products. It can be from the organism itself or from a different organism.

In the present invention, there is no particular limitation on the plants applicable to the present invention, as long as they are suitable for gene transformation operations, such as various crops, floral plants, or forestry plants. The plant may be, for example (not limited to): dicotyledonous plants, monocotyledonous plants or gymnosperms.

As a preferred mode, the "plant" includes, but is not limited to: Arabidopsis thaliana, and any gene that has this gene or is homologous to it is applicable.

The "plant" mentioned in the present invention includes the whole plant, its parent and progeny plants and different parts of the plant, including seeds, fruits, buds, stems, leaves, roots (including tubers), flowers, tissues and organs. Different parts have our target gene or nucleic acid. The "plant" mentioned here also includes plant cells, suspension cultures, callus tissue, embryos, meristematic regions, gametophytes, sporophytes, pollen, and microspores, where each of the aforementioned objects contains the gene/nucleic acid of interest .

The invention includes any plant cell, or any plant obtained or obtainable by a method therein, and all plant parts and propagules thereof. This patent also covers transfected cells, tissues, organs or whole plants obtained by any of the aforementioned methods. The only requirement is that the offspring exhibit the same genotypic or phenotypic characteristics, and the offspring obtained using the method of this patent have the same characteristics.

The invention also extends to harvestable parts of plants as described above, but not limited to seeds, leaves, fruits, flowers, stems, roots, rhizomes, tubers and bulbs. It further relates to other derivatives of the harvested plants, such as dry granules or powders, oils, fats and fatty acids, starches or proteins. The invention also relates to foods or food additives obtained from related plants.

Advantages of the present invention:

(1) The present invention obtains the Arabidopsis thaliana HSRP1 gene by cloning the plant genome, constructs the HSRP1 gene knockout vector, and obtains the HSRP1 gene knockout transgenic line by using the method mediated by Agrobacterium. The inventor revealed the role of HSRP1 in plant heat resistance through high temperature stress simulation experiments in the medium, and provided genetic resources for crop heat resistance molecular breeding.

(2) Heat-resistant plants can be obtained by transgenic means. Specifically, the HSRP1 gene of the target plant can be knocked out to obtain a transgenic plant. A new way.

Description of drawings

Figure 1 is the qRT-PCR identification of HSRP1 gene expression in the HSRP1 knockout transgenic line ( hsrp1-91 ) and wild type; in the figure, the HSRP1 gene expression in the HSRP1 knockout transgenic line ( hsrp1-91 ) was significantly lower than Wild type.

Figure 2 is a diagram of the sequencing results of HSRP1 gene amplification products; the G base in the small box in the figure is a single base insertion generated after the genome is edited.

Figure 3 is a statistical graph of the survival rate of the HSRP1 gene knockout transgenic line ( hsrp1-91 ) and wild-type materials after being treated at 42°C for 2 hours, and then recovered at 22°C for 5 days. In the figure, the HSRP1 gene knockout transgenic line ( hsrp1 -91) had a significantly higher survival rate than wild type.

Fig. 4 is a phenotype diagram of the HSRP1 gene knockout transgenic line ( hsrp1-91 ) and the wild-type material after being treated at 42°C for 2 hours and recovered at 22°C for 5 days.

Implementation

The present invention will be described in detail through specific examples below. These embodiments are provided for a more thorough understanding of the present invention and to fully convey the scope of the present invention to those skilled in the art.

Unless otherwise specified, the technical means used in the embodiments are conventional means well known to those skilled in the art. The test methods in the following examples are conventional methods unless otherwise specified. Unless otherwise specified, the reagents and materials used can be obtained through commercial channels.

Unless otherwise defined, all professional and scientific terms used herein have the same meanings as commonly understood by those skilled in the art. In addition, any methods and materials similar or equivalent to those described can also be applied in the present invention. The preferred implementation methods and materials described herein are for demonstration purposes only.

The practice of the present invention will employ, unless otherwise indicated, conventional techniques of botany, microbiology, tissue culture, molecular biology, chemistry, biochemistry, recombinant DNA and bioinformatics, techniques apparent to those skilled in the art. These techniques have been fully explained in the published literature. In addition, the DNA extraction, phylogenetic tree construction, gene editing method, gene editing vector construction, gene editing plant acquisition and other methods used in the present invention, except for the following Except for the method adopted in the embodiment, it can be realized by adopting the methods already disclosed in the existing documents.

As used herein, the terms "nucleic acid", "nucleic acid sequence", "nucleotide", "nucleic acid molecule" or "polynucleotide" are meant to include isolated DNA molecules (e.g., cDNA or genomic DNA), RNA Molecule (eg, messenger RNA), natural type, mutant type, synthetic DNA or RNA molecule, DNA or RNA molecule composed of nucleotide analogs, single-stranded or double-stranded structure. These nucleic acids or polynucleotides include gene coding sequences, antisense sequences and regulatory sequences of non-coding regions, but are not limited thereto. 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 the genomic sequence, and/or include the coding sequence in the cDNA, and/or include the cDNA and its regulatory sequences. In particular embodiments, eg in relation to an isolated nucleic acid sequence, it is preferentially assumed that it is cDNA.

In addition, for a more intuitive understanding of the technical solution of the present invention, some technical terms involved in the present invention are explained as follows:

"Mutant" refers to a mutated individual having phenotypic characteristics different from those of the wild type.

"Expression vector" refers to a vector that adds expression elements (such as promoter, RBS, terminator, etc.) on the basis of the basic skeleton of the cloning vector to enable the expression of the target gene.

In the present invention, the HSRP1 gene knockout homozygous mutant strain ( hsrp1-91 ) is obtained through the CRISPR-Cas9 gene knockout technology and the method mediated by Agrobacterium to obtain the HSRP1 gene knockout transgenic line. In the present invention, it is also verified functionally with wild-type plants under high temperature stress.

1. Gene source and isolation:

Based on the Arabidopsis whole genome sequencing published on the TAIR official website (http://www.arabidopsis.org/), the nucleotide sequence information of the Arabidopsis intermediary factor HSRP1 was obtained. The nucleotide sequence of the HSRP1 gene is shown in SEQ ID NO.1. The nucleotide sequence of the coding frame of HSRP1 gene (AT4G37930) is 1554 bp in length, consists of 517 amino acids, and has a molecular weight of about 57.40kD. Use the CRISPR-P v2.0 website to design gene target primers:

F: 5'-GACAGCTTAACGCACCTTTAGAGGGTTTTAGAGCTAGAAATAGCAAGTTAA-3';

R: 5'- CCCTCTAAAGGTGCGTTAAGCTGTCAATCACTACTTCGACTCTAGCTG -3' ; the pHSE401 vector was used as a template, and 2 x PLANTA MAX MASTER MIX high-fidelity enzyme amplification (Vazyme) was used, and the annealing temperature was 55°C. The recovered fragment is subjected to homologous recombination with the vector, and finally the gene knockout vector is obtained.

2. Functional identification of HSRP1 gene

In order to study the role of HSRP1 gene in plant heat tolerance, its function was analyzed by comparing the Arabidopsis gene knockout mutant ( hsrp1-91 ) and wild type.

2.1 Construction of HSRP1 gene knockout mutant materials

The pHSE401 vector was selected for the construction of HSRP1 knockout strains. The method is as follows: for the gene knockout strain, the pHSE401 gene knockout vector was constructed according to the CDS gene sequence of the HSRP1 gene, and transformed into Escherichia coli and Agrobacterium.

2.2 Screening of HSRP1 knockout transgene-positive strains

Transform the correctly constructed recombinant plasmid into Agrobacterium GV3101 by freezing and thawing with liquid nitrogen, shake the bacteria overnight, transfer to OD=0.8, resuspend in transgenic buffer, infect Col-0 Arabidopsis inflorescence, avoid After 16 hours of light, the seeds were cultivated in a long-day light incubator, and the harvested seeds were grown in the soil until the true leaves grew, and sprayed with a solution of 25 µg/mL Basta for screening. The screening process was as follows: the seeds were sown in nutrient soil, and cultivated in a light incubator at 22°C and 70% relative humidity at 16 h/8 h light and dark. Wild-type Arabidopsis was used as a control, sprayed with a solution of Basta at a concentration of 25 µg/mL, and seedlings resistant to Basta were screened out. The protein was extracted after the true leaves unfolded, and the positive plants were identified by western blotting (WB). The seeds were harvested, multiplied and identified until the T3 generation was homozygous.

The qRT-PCR gene expression was detected on the harvested homozygous seeds (Fig. 1). Compared with Col-0, the gene knockout homozygous mutant ( hsrp1 -91) had relatively lower expression.

2.3 Homozygous detection of HSRP1 knockout mutants

Primers located at about 300 bp before and after the HSRP1 target site were designed, HSRP1 -CAS91-CX-F: GCCCAGTGAAGCTGTTGATG and SHMT1-CAS91-CX-R: GTTAGTCATGACAGACCCAAC. The genomic DNA near the target is amplified and then sequenced. Homozygous identification was performed by sequencing (Figure 2). As shown in the figure, the HSRP1 gene knockout homozygous mutant strain ( hsrp1-91 ) was inserted in a single base, resulting in the premature appearance of the stop codon and premature termination of protein translation.

2.4 Phenotype analysis of HSRP1 knockout mutants

To analyze the sensitivity of seedlings of wild-type Arabidopsis line (Col-0) and homozygous HSRP1 knockout mutant ( hsrp1-91 ) to high temperature. The above strains were subjected to high-temperature stress treatment, as follows:

In order to count the seedling survival rate after high temperature treatment, we used 42°C high temperature to treat Arabidopsis. The experimental treatment of survival rate was as follows: Arabidopsis seeds were inoculated on 1/2 MS medium, vernalized at 4°C in the dark for three days after inoculation, and then moved to a temperature of 22°C, humidity of 60%, and light intensity of 100 μmol.m ^-2 .s ^-1 were grown under full sun for 9 days and then treated with high temperature for 2 hours. After the treatment, it was placed at 22°C for recovery, and the survival of the seedlings was counted after 5 days of recovery.

After the high temperature treatment, the recovery was carried out at 22°C, and the survival rate of the seedlings was counted after 5 days of recovery, and the survival rate data obtained are shown in Figure 3. The results showed that the survival rate of Arabidopsis thaliana mutant ( hsrp1-91 ) after HSRP1 gene knockout was significantly higher than that of wild type (Col-0) after high temperature treatment. This indicates that in Arabidopsis, down-regulated expression of the HSRP1 gene seriously affects its heat tolerance, and knocking out the HSRP1 gene can improve the heat tolerance of Arabidopsis plants.

After inoculation, the growth phenotype of the HSRP1 gene knockout mutant recovered for 5 days after high temperature treatment is shown in FIG. 4 . The results showed that the survival rate of Arabidopsis thaliana after HSRP1 gene knockout was significantly higher than that of wild type (Col-0) after high temperature treatment. This indicates that in Arabidopsis, down-regulated expression of the HSRP1 gene seriously affects its heat tolerance, and knocking out the HSRP1 gene can improve the heat tolerance of Arabidopsis plants.

The above experimental data show that in Arabidopsis, inhibiting the expression of the HSRP1 gene can significantly improve the high temperature stress tolerance of Arabidopsis plants.

The embodiments described above are only preferred embodiments of the present invention, and are only used to explain the present invention, not to limit the implementation scope of the present invention. Technical content, other implementation modes can be easily made through replacement or change, so all changes and improvements made on the principle of the present invention should be included in the patent scope of the present invention.

Claims (3)

1. The application of suppressing the expression of HSRP1 gene in improving the heat resistance of Arabidopsis thaliana, is characterized in that, the nucleotide sequence of described HSRP1 gene is as shown in SEQ ID NO.1, and the mode of suppressing HSRP1 gene expression is by constructing HSRP1 gene knockout vector to obtain heat-resistant transgenic plants. 2. The application according to claim 1, characterized in that the heat resistance is high temperature resistance, and the high temperature is 42°C. 3. The application according to claim 2, characterized in that the heat resistance is as follows: under high temperature stress, the survival rate of the HSRP1 gene knockout mutant is higher than that of the wild type.

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