CN116121298B - Application of inhibiting expression of HSRP1 gene in improving heat resistance of plants - Google Patents
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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
Technical Field
The invention belongs to the field of biotechnology, in particular toHSRP1Use of genes in plant thermotolerance, more particularly in inhibition HSRP1The application of gene expression in improving plant heat resistance.
Background
As the greenhouse effect increases, global climate warms, and high Wen Zaihai climates have become a global concern, a serious challenge for many agricultural areas. High Wen Zaihai climates severely inhibit crop growth and development and result in reduced crop yield and quality. High temperature stress damages the plant's vegetative tissues, affects the plant's seed germination, reproductive development, photosynthesis, plasma membrane stabilization, and plant water content, ultimately resulting in crop yield loss. In general, under high temperature stress, plants exhibit delayed germination of seeds, stigma malformation, pollen dysplasia, leaf wilting, and, in severe cases, cause death of plants by desiccation. Global warming has now posed a serious threat to crop production and has shown a growing trend year by year. Knowing the plant high temperature stress adaptability mechanism is beneficial to scientifically breeding heat-resistant crops, thereby effectively improving the crop yield and relieving the increasing grain pressure worldwide.
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 serine to glycine reaction provides a C1 unit for a range of important biosynthetic processes, such as methionine, thymidylate and purine synthesis, indicating its importance in DNA biogenesis and cellular methylation reactions. Whereas in plants, the other direction of the SHMT reaction: glycine is also an indispensable step in the light respiratory pathway, and conversion of glycine to serine is also an indispensable step. The SHMT protein family is very evolutionarily conserved, but the number of genes encoding SHMT will vary among species. In arabidopsis, 7 different SHMT proteins are localized to different cellular regions: mitochondria, cytoplasm, chloroplasts and nuclei have a certain difference in their functions. SHMT proteins are widely involved in one-carbon metabolism, biotic and abiotic stress, etc. according to literature, but the function of participating in high temperature stress has not been clearly studied.
Disclosure of Invention
The invention aims to provideHSRP1Use of genes in plant thermotolerance by inhibitionHSRP1Expression of the gene improves the heat resistance of the plant.
In order to achieve the above purpose, the technical scheme adopted by the invention is summarized as follows:
arabidopsis thaliana was obtained based on the published whole genome sequencing of Arabidopsis thaliana from the TAIR official website (http:// www.arabidopsis.org /)HSRP1Nucleotide sequence information of the gene (serine hydroxymethyl transferase, SEQ ID NO: AT4G 37930). The saidHSRP1The nucleotide sequence of the gene is shown as SEQ ID NO. 1.HSRP1The length of the gene coding frame nucleotide sequence is 1554 and bp, and 517 ammonia are usedThe amino acid composition and the sequence thereof can be obtained through inquiry of a TAIR official website.
The invention also constructs a series of plant expression vectors, recombinant vectors or transgenic plant systems containing the genes, and the functions of host cells containing the vectors in improving the heat resistance of plants also fall into the protection scope of the invention.
The functions of the gene protected by the present invention include not only the aboveHSRP1Genes, also includingHSRP1Genes have the function of homology genes with higher homology (homology higher than 80%, more preferably higher than 90%, still more preferably higher than 95%, still more preferably higher than 98%) in terms of thermostability.
The invention is based on HSRP1CRISPR-Cas9 gene knockout vector is constructed by CDS gene sequence of (2), and the CRISPR-Cas9 gene knockout vector is obtained through agrobacterium infection HSRP1Gene knockout lines, wild type Col-0 and were analyzedHSRP1The gene knockout transgenic line has biological functions in high temperature stress, so as to provide gene resources for crop heat-resistant molecular breeding.
The invention discloses HSRP1The biological function of the gene in plant heat resistance is specifically expressed in the following steps: after the high-temperature stress treatment, the mixture is subjected to a high-temperature stress treatment, HSRP1the survival rate of seedlings of the gene knockout mutant strain is obviously higher than that of wild type.
The above application was concluded by simulating high temperature stress experiments in culture medium.
According to its function, a heat-resistant plant can be obtained by means of transgenesis, in particular, by combiningHSRP1The gene is knocked out or knocked down in the target plant to obtain transgenic plant with heat resistance higher than that of the target plant.
As one embodiment of the present invention, a polynucleotide is cloned into a CRISPRP vector by a conventional method, and the recombinant vector having a foreign gene is introduced into a plant cell capable of expressing an HSRP1 protein, thereby allowing the plant cell to be transformed with the recombinant vectorHSRP1 protein is deleted. Can be obtained by regenerating said plant cells into plantsHSRP1A mutant plant with a deletion of a gene. And transferring the recombinant plasmid into plants by using an agrobacterium transformation method.
In order to improve the excellent properties of plants, the present invention also provides a novel plant breeding method by inhibiting the growth of plants of interestHSRP1The expression of the gene can obtain a plant with heat resistance stronger than that of the target plant;
"inhibition of plants of interestHSRP1The expression "of the gene may be achieved as follows (1) or (2) or (3):
(1) Will beHSRP1Knocking out or knocking down the gene in a target plant;
(2) Introducing a silencer;
(3) Other methods are common in the art.
Wherein the plant of interest of the present invention is Arabidopsis thaliana.
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:
(1) The invention obtains arabidopsis through plant genome cloningHSRP1Genes, constructHSRP1Gene knockout vector obtained by agrobacterium-mediated methodHSRP1A gene knockout transgenic line. The inventor discloses through a high temperature stress simulation experiment in a culture mediumHSRP1The gene can provide gene resources for crop heat-resistant molecular breeding.
(2) Plants which are heat-resistant can be obtained by means of transgenesis, in particular, by introducing plants of interestHSRP1The gene is knocked out to obtain a transgenic plant, the heat resistance of the plant is higher than that of a target plant, and a new way is provided for heat-resistant breeding of the plant.
Drawings
FIG. 1 is a schematic diagram of a conventional gas turbineHSRP1Transgenic line with knocked out genehsrp1-91) and wild typeHSRP1qRT-PCR identification of gene expression quantity; in the drawing the view of the figure,HSRP1transgenic line with knocked out genehsrp1-91)HSRP1The gene expression level is significantly lower than that of the wild type.
FIG. 2 is a schematic diagram of a conventional deviceHSRP1Sequencing result graphs of gene amplification products; the G bases in the small boxes in the figure are single base insertions resulting from the genome being edited.
FIG. 3 is a schematic view ofHSRP1Transgenic line with knocked out genehsrp1-91) and wild-type material are treated at a high temperature of 42℃for 2 hoursAfter that, a statistical graph of survival after recovery for 5 days at 22℃is shown, in which,HSRP1transgenic line with knocked out genehsrp1-91) survival rate is significantly higher than wild type.
FIG. 4 is a diagram ofHSRP1Transgenic line with knocked out genehsrp1-91) and wild-type material recovered a phenotype profile after 5 days at 22 ℃ after 2 hours of high temperature treatment at 42 ℃.
Description of the embodiments
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.
In addition, in order to more intuitively understand the technical scheme of the present invention, some terms related to the present invention are explained as follows:
"mutant" refers to a mutant individual having a phenotype that differs from the wild type.
The expression vector refers to a vector which is formed by adding expression elements (such as a promoter, RBS, terminator and the like) on the basis of the basic skeleton of a cloning vector so that a target gene can be expressed.
In the present inventionHSRP1Knockout of homozygous mutant strainhsrp1-91) obtained by means of agrobacterium-mediated methods by CRISPR-Cas9 gene knockout technologyHSRP1A gene knockout transgenic line. The invention also performs functional verification on the wild plant and the wild plant under high temperature stress.
1. Gene source and isolation:
arabidopsis thaliana mediating factors are obtained based on the Arabidopsis thaliana genome sequencing published by the TAIR official website (http:// www.arabidopsis.org /)HSRP1Nucleotide sequence information of (a) is provided. The saidHSRP1The nucleotide sequence of the gene is shown as SEQ ID NO. 1.HSRP1The coding frame nucleotide sequence of the gene (AT 4G 37930) is 1554 bp in length and consists of 517 amino acids, and the molecular weight of the gene is about 57.40kD. Gene target primers were designed using CRISPR-P v2.0 website:
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 ℃. And (3) recovering the fragment and carrying out homologous recombination on the fragment and the vector, and finally obtaining the gene knockout vector.
2. HSRP1Functional identification of genes
To study HSRP1The effect of the gene in plant heat resistance is achieved by knocking out the mutant strain from Arabidopsis thalianahsrp1-91) and wild type comparison analysis of their function.
2.1 HSRP1Construction of Gene knockout mutant Material
HSRP1The gene knockout strain was constructed by selecting pHSE401 vector. The method comprises the following steps: gene knockout lines are based onHSRP1The CDS gene sequence of the gene constructs a pHSE401 gene knockout vector and converts escherichia coli and agrobacterium.
2.2 HSRP1Screening of Gene knockout transgenic Positive strains
Transferring the constructed correct recombinant plasmid into agrobacterium GV3101 by utilizing a liquid nitrogen freeze thawing method, shaking overnight, transferring and shaking to OD=0.8, re-suspending by using a transgenic buffer solution, infecting the inflorescence of Col-0 arabidopsis thaliana, keeping away from light for 16 hours, culturing and collecting seeds in a long-day illumination incubator, growing the harvested seeds in soil until true leaves grow out, and spraying and screening by using a solution with the concentration of 25 mug/mL Basta. The screening process is as follows: seeds were sown in nutrient soil and cultured in 16 h/8 h light dark, 22 ℃,70% relative humidity light incubator. And (3) taking wild arabidopsis thaliana as a control, spraying a solution with the concentration of 25 mu g/mL of Basta, and screening out Basta-resistant seedlings. Protein was extracted after true leaf expansion, and positive plants were identified using immunoblotting (WB). And harvesting seeds, and carrying out propagation identification until the T3 generation is homozygous.
The qRT-PCR gene expression level of the harvested homozygous seeds is detected (figure 1), and compared with Col-0, the homozygous mutant strain is knocked outhsrp1-91) the expression level is relatively low.
2.3 HSRP1Homozygous detection of gene knockout mutants
The design is located inHSRP1Primers at about 300 bp before and after the target,HSRP1CAS91-CX-F: GCCCAGTGAAGCTGTTGATG and SHMT1-CAS91-CX-R: GTTAGTCATGACAGACCCAAC. Genomic DNA near the target is amplified and sequenced. Homozygosity was identified by sequencing (fig. 2). As shown in the drawing, the liquid crystal display device, HSRP1knockout of homozygous mutant strainhsrp1-91) the premature occurrence of the stop codon due to single base insertion and premature termination of protein translation.
2.4 HSRP1Phenotype analysis of knockout mutants
For analysis of wild type Arabidopsis line (Col-0),HSRP1Knockout of homozygous mutanthsrp1-91) sensitivity of seedlings to high temperatures. The strain is subjected to high-temperature stress treatment, and the method is as follows:
to count the survival rate of seedlings after the high temperature treatment, we used a high temperature of 42℃to treat Arabidopsis thaliana. The survival rate experiment was processed as follows: inoculating Arabidopsis seeds on 1/2 MS culture medium, vernalizing at 4deg.C in dark for three days, and transferring to a temperature of 22deg.C, humidity of 60%, and illumination intensity of 100 μmol.m -2 .s -1 High temperature treatment is carried out after the growth for 9 days under full sunlight, and the treatment time is 2 h. And (5) after treatment, placing the seedlings at 22 ℃ for recovery, and counting the survival condition of the seedlings after recovery for 5 days.
Recovery was performed at 22 ℃ after high temperature treatment, and statistical of the survival rate of seedlings was performed after recovery for 5 days, and the obtained survival rate data is shown in fig. 3. The results show that the data obtained from the above-mentioned method,HSRP1arabidopsis mutant strain after gene knockouthsrp1-91) the survival rate after high temperature treatment is significantly higher than that of the wild type (Col-0). This indicates that, in Arabidopsis thaliana,HSRP1down-regulated expression of genes severely affects their tolerance to heat, knockdown HSRP1The gene can improve the heat resistance of the Arabidopsis plant.
After the inoculation, the seed is inoculated,HSRP1the phenotype of the gene knockout mutant recovered for 5 days after the high temperature treatment is shown in FIG. 4. The results show that the data obtained from the above-mentioned method,HSRP1the survival rate of the arabidopsis after gene knockout is significantly higher than that of the wild type (Col-0) after high temperature treatment. This indicates that, in Arabidopsis thaliana,HSRP1down-regulated expression of genes severely affects their tolerance to heat, knockdownHSRP1Gene can be usedImproving the heat resistance of the Arabidopsis plants.
The experimental data show that in Arabidopsis thaliana, inhibition is achievedHSRP1The expression of the gene can obviously improve the high temperature stress resistance of the arabidopsis plant.
The above-mentioned embodiments are merely preferred embodiments of the present invention, which are not intended to limit the scope of the present invention, and other embodiments can be easily made by those skilled in the art through substitution or modification according to the technical disclosure in the present specification, so that all changes and modifications made in the principle of the present invention shall be included in the scope of the present invention.
Claims (3)
1. Inhibition ofHSRP1Use of gene expression for improving heat resistance of Arabidopsis thaliana, characterized in thatHSRP1The nucleotide sequence of the gene is shown as SEQ ID NO.1, and inhibition is carried outHSRP1The gene expression is achieved by constructingHSRP1And (3) knocking out the vector to obtain a heat-resistant transgenic plant.
2. The use according to claim 1, wherein the heat resistance is high temperature resistance and the high temperature is 42 ℃.
3. The use according to claim 2, characterized in that said heat resistance is expressed as: under the condition of high-temperature stress,HSRP1the survival rate of the gene knockout mutant strain is higher than that of the wild type.
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Citations (2)
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|---|---|---|---|---|
| JPH025861A (en) * | 1988-06-25 | 1990-01-10 | Nippon Zeon Co Ltd | Novel thermophile serine hydroxymethyl transferase |
| CN101955955A (en) * | 2010-09-17 | 2011-01-26 | 山东农业大学 | Application of Arabidopsis gene AtSDH in regulating plant stress resistance |
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| MX2008000118A (en) * | 2005-07-18 | 2008-03-24 | Basf Plant Science Gmbh | Yield increase in plants overexpressing the shsrp genes. |
| WO2010046221A1 (en) * | 2008-10-23 | 2010-04-29 | Basf Plant Science Gmbh | Plants with increased yield (nue) |
| KR20120125581A (en) * | 2011-05-06 | 2012-11-16 | 경상대학교산학협력단 | SUMO-modified proteins in Arabidopsis resistant to heat stress |
| CN102242135B (en) * | 2011-05-11 | 2013-11-06 | 浙江省农业科学院 | Rice serine hydroxymethyl transferase protein and function of coding gene thereof |
| CN103173424A (en) * | 2013-04-16 | 2013-06-26 | 中国农业科学院生物技术研究所 | RPRP (Rice Photorespiration Related Protein)-1 capable of increasing photosynthetic efficiency of plants as well as coding gene and application of RPRP-1 |
| CA2975486A1 (en) * | 2017-08-04 | 2019-02-04 | Rutgers, The State University Of New Jersey | Compositions and methods comprising endophytic bacterium for application to target plants to increase plant growth, and increase resistance to abiotic and biotic stressors |
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| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| JPH025861A (en) * | 1988-06-25 | 1990-01-10 | Nippon Zeon Co Ltd | Novel thermophile serine hydroxymethyl transferase |
| CN101955955A (en) * | 2010-09-17 | 2011-01-26 | 山东农业大学 | Application of Arabidopsis gene AtSDH in regulating plant stress resistance |
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