CN115851660A - Application of protein OsPIS and coding gene in improving plant stress resistance - Google Patents
Application of protein OsPIS and coding gene in improving plant stress resistance Download PDFInfo
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Abstract
The invention discloses application of protein OsPIS and a coding gene in improving plant stress resistance. The invention screens a protein, which is a protein with an amino acid sequence shown as SEQ ID NO.1 or a fusion protein obtained by connecting protein labels at the N end or/and the C end of the amino acid sequence shown as SEQ ID NO. 1. The invention clones the OsPIS protein and the coding gene thereof, and introduces the coding gene of the OsPIS protein into rice, thereby obviously improving the drought resistance and salt tolerance of rice plants. The protein and the coding gene thereof have important application value for cultivating stress-resistant plant varieties, thereby having important significance for improving the crop yield; the invention has wide application space and market prospect in the agricultural field.
Description
Technical Field
The invention relates to a plant stress resistance related protein OsPIS, a coding gene and application thereof, in particular to the protein OsPIS, and related biological materials and application thereof.
Background
Rice (1)Oryza sativaL.) is one of the main food crops in the world, nearly half of the population on the earth eats rice, and a large amount of fresh water resources are consumed for planting the rice. Water resource shortage is a serious ecological problem that currently restricts the development of global agricultural production. Drought is a major limiting factor affecting food safety worldwide for a long time, and as global air temperature rises, arid and semi-arid land areas are increasing year by year. The area of the Chinese arid and semi-arid cultivated land accounts for about 51 percent of the total cultivated land area, and is nearly 2.5 multiplied by 10 every year 6 hm 2 The cultivated land is affected by drought to different extents. At present, with the global warming and the disruption of ecological balance, the phenomenon of water resource shortage is more serious. The normal growth and development and the high yield of the crops must be realizedSufficient moisture provides a safeguard. Therefore, drought, high salt environment and the like are the most important abiotic stress factors affecting crop yield, especially the traditional rice production will face a serious challenge, and the main way of improving the water utilization efficiency of rice is to improve the drought resistance and salt tolerance of the rice.
Phospholipids are not only the major structural components of cell membranes, but also act as signaling molecules regulating plant growth, development and response to external environmental stimuli. Phosphatidylinositol (PI) is a major phospholipid in eukaryotes, accounts for 15 to 20% of the total phospholipid in eukaryotes, is not only a basic component of cell membranes, but also is involved in the process of anchoring specific proteins on cell membranes, and is also a precursor substance of important signal molecules in cells. The PI signaling pathway begins with the reaction of inositol and CDP-diacylglycerol (CDP-DAG) to form PI catalyzed by PI synthase (PIS). PI is catalyzed by phosphatidylinositol-4-kinase (PI 4K) to form phosphatidylinositol-4-phosphate (PIP), PIP is phosphorylated by phosphatidylinositol 4-phosphate 5-kinase (PIP 5K) to form phosphatidylinositol-4,5-diphosphate (PIP) 2 ）。PIP 2 Two second messenger substances inositol-1,4,5-triphosphate (IP 3) and Diacylglycerol (DAG) are produced under the catalysis of phosphoinositide-specific phospholipase C (PLC). Water-soluble IP 3 Ca diffusing into the cytoplasm to promote intracellular storage 2+ Release intracellular Ca 2+ The concentration is increased, and a series of physiological and biochemical reactions of the plant responding to the external environment stimulation are adjusted. DAG can be rapidly catalyzed by diacylglycerol kinase (DGK) to form Phosphatidic Acid (PA). PA can also be hydrolyzed PI, PIP and PIP directly by phospholipase D (PLD) 2 And forming an isophospholipid structure. More and more experiments have shown that PA is also an important second messenger in plants involved in regulating the response of plant cells to external signal stimuli.
In the long-term evolution process, plants develop a series of salt-tolerant and drought-resistant mechanisms. With the rapid development of molecular biology, the physiological biochemical mechanism of plant salt tolerance, drought resistance and physiological biochemical mechanism is increasingly clear, so that the cloning of the gene related to the salt tolerance, drought resistance and the physiological biochemical mechanism of the plant becomes possible. Enhancing the salt-tolerant and drought-resistant physiology of plantsThe research proves the life activity rule of the plant under the stress and artificially regulates and clones the rice phosphatidylinositol synthase geneOsPISThe method utilizes the genetic engineering technology to improve the salt tolerance and drought resistance of plants and culture excellent varieties with adverse environmental resistance so as to improve the yield and quality of crops, and has important significance for obtaining agricultural high and stable yield.
Disclosure of Invention
The invention aims to solve the technical problem of how to effectively improve the drought resistance and salt tolerance of plants.
To solve the above technical problems, the present invention provides a protein, named OsPIS protein or protein OsPIS, derived from rice (A)Oryza sativaL.), is a protein shown as any one of the following (a 1) or (a 2) or (a 3):
(a1) Protein with amino acid sequence shown as SEQ ID NO. 1;
(a2) The fusion protein is obtained by connecting protein labels at the N end or/and the C end of the amino acid sequence shown in SEQ ID NO. 1;
(a3) And (b) a protein which is obtained by substituting and/or deleting and/or adding one or more amino acid residues in the amino acid sequence shown in SEQ ID NO.1, has more than 90% of identity with the protein shown in (a 1) and has the same function.
Wherein, SEQ ID NO.1 consists of 223 amino acid residues.
The protein can be artificially synthesized, or can be obtained by synthesizing the coding gene and then carrying out biological expression.
Among the above proteins, 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, tracing and/or purification of the target protein. The protein tag may be a Flag tag, a His tag, an MBP tag, an HA tag, a myc tag, a GST tag, and/or a SUMO tag, among others.
In the above proteins, identity refers to the identity of amino acid sequences. Amino acid sequence identity can be determined using homology search sites on the Internet, such as the BLAST web page of the NCBI home web site. For example, in the advanced BLAST2.1, by using blastp as a program, setting the value of Expect to 10, setting all filters to OFF, using BLOSUM62 as a Matrix, setting Gap existence cost, per residual Gap cost, and Lambda ratio to 11,1 and 0.85 (default values), respectively, and performing a calculation by searching for the identity of a pair of amino acid sequences, a value (%) of the identity can be obtained.
In the above protein, the 90% or more identity may be at least 91%, 92%, 95%, 96%, 98%, 99% or 100% identity.
The related biological materials of the protein OsPIS also belong to the protection scope of the invention. The relevant biomaterial described above is any one of the following (c 1) to (c 10):
(c1) A nucleic acid molecule encoding the protein OsPIS;
(c2) An expression cassette comprising the nucleic acid molecule of (c 1);
(c3) A recombinant vector comprising the nucleic acid molecule of (c 1), or a recombinant vector comprising the expression cassette of (c 2);
(c4) A recombinant microorganism comprising (c 1) said nucleic acid molecule, or a recombinant microorganism comprising (c 2) said expression cassette, or a recombinant microorganism comprising (c 3) said recombinant vector;
(c5) A transgenic plant cell line comprising the nucleic acid molecule of (c 1), or a transgenic plant cell line comprising the expression cassette of (c 2), or a transgenic plant cell line comprising the recombinant vector of (c 3);
(c6) A transgenic plant tissue comprising (c 1) said nucleic acid molecule, or a transgenic plant tissue comprising (c 2) said expression cassette, or a transgenic plant tissue comprising (c 3) said recombinant vector;
(c7) A transgenic plant organ containing (c 1) said nucleic acid molecule, or a transgenic plant organ containing (c 2) said expression cassette, or a transgenic plant organ containing (c 3) said recombinant vector;
(c8) A transgenic plant containing the nucleic acid molecule of (c 1), or a transgenic plant containing the expression cassette of (c 2), or a transgenic plant containing the recombinant vector of (c 3);
(c9) A tissue culture produced from regenerable cells of said transgenic plant of (c 8);
(c10) Protoplasts produced from the tissue culture of (c 9).
Wherein the nucleic acid molecule may be DNA, such as cDNA, genomic DNA or recombinant DNA; the nucleic acid molecule may also be RNA, such as mRNA or hnRNA, etc.
In the above-mentioned related biological material, the nucleic acid molecule (c 1) encoding the protein OsPIS may specifically be any of the following (d 1), (d 2) or (d 3)
(d1) DNA molecule shown in SEQ ID NO. 2;
(d2) The coding sequence is DNA molecule shown in SEQ ID NO. 2;
(d3) A DNA molecule which hybridizes with the DNA molecule defined in (d 1) or (d 2) under stringent conditions and encodes the protein OsPIS.
Wherein, SEQ ID NO.2 consists of 672 nucleotides, the Open Reading Frame (ORF) thereof is 1 st to 672 th from the 5' end, and the encoded amino acid sequence is the protein shown as SEQ ID NO. 1.
The stringent conditions are hybridization and washing at 68 ℃ for 2 times, 5min each, in a solution of 2 XSSC, 0.1% SDS, and hybridization and washing at 68 ℃ for 2 times, 15min each, in a solution of 0.5 XSSC, 0.1% SDS.
The invention also discloses an expression cassette, a recombinant expression vector, a transgenic cell line or a recombinant bacterium containing the coding gene of the protein related to the plant stress resistance.
In the above-mentioned related biological materials, the expression cassette described in (c 2) refers to a DNA capable of expressing the protein OsPIS in the host cell, and the DNA may include not only a promoter for initiating the transcription of the OsPIS gene but also a terminator for terminating the transcription of the OsPIS gene.
In the above-mentioned related biological materials, the recombinant vector of (c 3) may contain a DNA molecule for encoding protein OsPIS shown in SEQ ID NO. 2.
The plant expression vector can be used for constructing a recombinant vector containing the OsPIS coding gene expression cassette. The plant expression vector can be a Gateway system vector or a binary agrobacterium vector and the like, such as pGWB411, pGWB412, pGWB405, pBin438, pCAMBIA1300, pCAMBIA1302, pCAMBIA2300, pCAMBIA2301, pCAMBIA1301, pBI121, pCAMBIA1391-Xa or pCAMBIA1391-Xb and the like. When the OsPIS is used for constructing a recombinant vector, any one of enhanced, constitutive, tissue-specific or inducible promoters, such as cauliflower mosaic virus (CAMV) 35S promoter, ubiquitin gene Ubiqutin promoter (pUbi) and the like, can be added in front of the transcription initiation nucleotide, and can be used independently or combined with other plant promoters; in addition, when the gene of the present invention is used to construct plant expression vectors, enhancers, including translational or transcriptional enhancers, may be used, and these enhancer regions may be ATG initiation codon or initiation codon of adjacent regions, etc., but must be in the same reading frame as the coding sequence to ensure proper translation of the entire sequence. The translational control signals and initiation codons are widely derived, either naturally or synthetically. The translation initiation region may be derived from a transcription initiation region or a structural gene.
In a specific embodiment of the invention, the recombinant expression vector is obtained by inserting the coding gene between multiple cloning sites of the vector pCBGUS;
the vector pCBGUS is obtained by a method comprising the following steps:
(1) Passing the pCAMBIA1301 vector throughHindIII andEcoRi, double enzyme digestion, and recovery of a large carrier fragment;
(2) The pBI121 vector is subjected toHindIII andEcoRi double digestion, recovery comprisinggusAA fragment of a gene;
(3) Combining the vector large fragment recovered in step (1) with the vector containing fragment recovered in step (2)gusAThe gene segments are connected to obtain the recombinant vector pCBGUS.
The pCAMBIA1301 vector is purchased from CAMBIA corporation; the pBI121 vector was purchased from Clontech.
In the above-mentioned related biological materials, (c 4) the recombinant microorganism may be specifically yeast, bacteria, algae and fungi.
In the above-mentioned related biological material, (c 7) the transgenic plant organ may be a root, stem, leaf, flower, fruit and seed of the transgenic plant.
In the above-mentioned related biological materials, (c 9) the tissue culture may be derived from roots, stems, leaves, flowers, fruits, seeds, pollen, embryos and anthers.
In the related biological material, the transgenic plant cell line, the transgenic plant tissue and the transgenic plant organ do not comprise propagation materials.
The invention also provides application of the protein OsPIS or the related biological material thereof in any one of the following (b 1) - (b 22):
(b1) Improving the salt tolerance of plants;
(b2) Preparing a product for improving the salt tolerance of plants;
(b3) Improving the drought resistance of the plants;
(b4) Preparing a product for improving the drought resistance of plants;
(b5) Improving the rooting condition of the plants under drought and/or salt stress conditions;
(b6) Preparing a product for improving the rooting condition of plants under drought and/or salt stress conditions;
(b7) Increasing the growth vigor of the plant under drought and/or salt stress conditions;
(b8) Preparing a product of the growth vigor of the plant under drought and/or salt stress conditions;
(b9) Preparing the survival rate of the plants under the drought and/or salt stress condition;
(b10) Preparing a product that increases the survival rate of plants under drought and/or salt stress conditions;
(b11) Increasing the abscisic acid content of plants under drought and/or salt stress conditions;
(b12) Preparing a product for improving the content of the plant abscisic acid under the drought and/or salt stress condition;
(b13) Increasing the proline content of plants under drought and/or salt stress conditions;
(b14) Preparing a product for improving the proline content of plants under drought and/or salt stress conditions;
(b15) DescendPlant H under conditions of low drought and/or salt stress 2 O 2 Content (c);
(b16) Preparation of plants H for reducing drought and/or salt stress conditions 2 O 2 Content of the product
(b17) Reducing the malondialdehyde content of plants under drought and/or salt stress conditions;
(b18) Preparing a product for reducing the malondialdehyde content of a plant under drought and/or salt stress conditions;
(b19) Increasing plant SOD activity under drought and/or salt stress conditions;
(b20) Preparing a product for improving the SOD activity of plants under drought and/or salt stress conditions;
(b21) Increasing plant POD activity under drought and/or salt stress conditions;
(b22) Products are prepared that increase plant POD activity under drought and/or salt stress conditions.
The application of the protein OsPIS or the related biological materials thereof in plant breeding is also within the protection scope of the invention.
Among the above applications, the plant breeding application may be specifically that containing the protein OsPIS or the related biological material (e.g., protein OsPIS-encoding gene)OsPIS) The plant of (2) is crossed with other plants to carry out plant breeding.
The invention further provides a method for cultivating the transgenic plant with high drought resistance and salt tolerance.
The method for cultivating the transgenic plant with high drought resistance and salt tolerance comprises the steps of improving the expression quantity of the coding gene of protein OsPIS in a target plant and/or the content of the protein OsPIS and/or the activity of the protein OsPIS to obtain the transgenic plant; the drought resistance and salt tolerance of the transgenic plant are higher than those of the target plant.
In the above method, the method for increasing the expression level of the gene encoding the protein OsPIS and/or the content of the protein OsPIS and/or the activity of the protein OsPIS in the plant of interest is to express or overexpress the protein OsPIS in the plant of interest.
In the above method, the expression or overexpression is carried out by introducing a gene encoding the protein OsPIS into a target plant.
In the above method, the gene encoding protein OsPIS can be introduced into a target plant by a plant expression vector carrying the OsPIS gene of the present invention. The plant expression vector carrying the gene OsPIS of the present invention can transform plant cells or tissues by using conventional biological methods such as Ti plasmid, ri plasmid, plant virus vector, direct DNA transformation, microinjection, conductance, agrobacterium mediation, etc., and culture the transformed plant cells or tissues into plants.
In the method, the nucleotide sequence of the coding gene of the protein OsPIS is a DNA molecule shown in SEQ ID NO. 2.
In a specific embodiment of the present invention, the plant expression vector carrying the gene OsPIS of the present invention may be pCAMBIA1301-OsPIS. Specifically, pCAMBIA1301-OsPIS utilizes restriction enzymeHindIII andEcoRi is obtained by inserting DNA molecule shown in SEQ ID NO.2 into pCAMBIA1301 vector.
In the method, the drought resistance and the salt tolerance are mainly embodied in that the root number, the root length, the fresh weight and the survival rate of the plant are improved, the ABA content is improved, the proline content is improved, the SOD activity is improved, the POD activity is improved, and the H is reduced 2 O 2 Content and reduction of malondialdehyde content.
In the present invention, the plant is any one of the following (e 1) to (e 5):
(e1) A dicotyledonous plant;
(e2) A monocot plant;
(e3) A gramineous plant;
(e4) Plants of the genus Oryza;
(e5) Rice (1)Oryza sativa）。
Compared with the prior art, the invention has the beneficial effects that:
the invention providesOsPISThe protein coded by the gene can improve the stress resistance of plants: over-expressionOsPISThe gene can improve the drought resistance and salt tolerance of rice and interfere expressionOsPISThe gene can reduce the drought resistance and salt tolerance of rice. From the test resultsIn the NaCl stress, the transgenic plant shows a good growth state, and the seedling length and the fresh weight of the over-expression transgenic rice material are respectively increased by 102-124% and 159-189% compared with the wild WT material; under the stress of mannitol, the seedling length and the fresh weight of the overexpression transgenic rice material are respectively increased by 91-103% and 145-176% compared with the wild WT material; the survival rate of the over-expressed transgenic rice is obviously higher than that of wild plants, is respectively increased by 1090-1211% and 1071-1244% compared with that of the wild plants, and expresses very strong salt tolerance and drought resistance; specifically, the transgenic rice material is over-expressed, the ABA content, the proline content, the SOD activity and the POD activity are increased, and the malondialdehyde content and the H content are reduced 2 O 2 And (4) content. Therefore, the protein and the coding gene thereof have important application value for cultivating stress-resistant plant varieties, thereby having important significance for improving the crop yield and having wide application space and market prospect in the agricultural field.
Drawings
FIG. 1 the present inventionOsPISAnalysis of adversity stress expression of the gene in Yangjing 805 rice.
FIG. 2 the present inventionOsPISPCR detection result diagram of gene over-expression rice strain.
FIG. 3 the present inventionOsPISThe gene is expressed in over-expressed rice lines and wild rice plants.
FIG. 4 the present inventionOsPISGrowth and rooting conditions of the transgenic rice plants on an MS culture medium containing 200 mM NaCl and 200 mM mannitol are shown, wherein WT is a wild rice plant, and OE4, OE5 and OE7 are overexpression transgenic rice plants.
FIG. 5 the present inventionOsPISThe salt tolerance and drought resistance potted plant identification of the gene transgenic rice plant is that WT is a wild rice plant, and OE4, OE5 and OE7 are over-expression transgenic rice plants.
FIG. 6 the present inventionOsPISThe stress resistance physiological and biochemical indexes of the transgenic rice plants are determined, wherein WT is a wild rice plant, and OE4, OE5 and OE7 are over-expression transgenic rice plants.
Detailed Description
The invention will now be further illustrated, but is not limited, by the following specific examples.
In the following examples, the test materials and sources used include:
rice (1)Oryza sativa) Yangjing 805 and Zhonghua 11 are the varieties of the Chinese medicinal herbs and are preserved in laboratories of plant production and processing practice education centers of Jiangsu province of the institute of Huaiyin industry and science and food engineering.
Escherichia coli (Escherichia coli) DH5 alpha is preserved by laboratories of plant production and processing practice education center of Jiangsu province of Huaiyin institute of Industrial and science of Huaiyin and food engineering institute. Cloning vector PMD-18-Simple T, various restriction enzymes, taq polymerase, ligase, dNTP, 10 XPCR buffer and DNA marker were purchased from Bao bioengineering Dai Lian Limited. All chemicals were purchased from sigma chemical, usa and from shanghai medicinal chemicals.
The general Molecular biology procedures of the present invention are described in detail in Molecular cloning, 2nd ed, cold Spring Harbor Laboratory Press, 1989.
Conventional genetic manipulations In the examples described below were performed with reference to the Molecular cloning literature [ Sambook J, fress EF, manndes T et al In: molecular cloning. 2nd ed. Cold Spring Harbor Laboratory Press, 1989 ].
1/2 Hoagland nutrient solutions are described in the following references [ Feibing Wang, weili Kong, gary Wong, life Fu, rihe Peng, zhenjun Li, quanhong Yao.AtMYB12 regulates flavonoids accumulation and abiotic stress tolerance in transgenic Arabidopsis thaliana. Molecular Genetics and Genomics, 2016, 291:1545-1559】。
Example 1 obtaining of protein related to stress resistance of Rice and Gene encoding the same
1. Experimental materials
The Rice variety ` japonica 805 ` Plant leaf material was removed and quick frozen for storage at-80 ℃ by reference to the method of Jan et al (2013) [ Asad Jan, kyonoshin Maruyama, daisuke Todaka, satoshi Kidokoro, mitsuru Abo, etsuro Yoshimura, kazuo Shinozaki, kazuo Nakashima and Kazuko Yamaguchi-Shinozaki, osTZF1, a CCCH-Tandem Zincin Finger Protein, consersDelayed Senesence and Stress in Rice by regulating Stress-Related genes, plant Physiology, 2013, 161.
Leaf Total RNA extraction and purification
Taking a seedling of Yangjing 805', spreading leaves about 2.0 g, grinding the seedling into powder in liquid nitrogen, adding the powder into a10 mL centrifuge tube, and extracting the total RNA of the tuberous root of the sweet potato by using an Aplygen plant RNA extraction kit (Aplygen Technologies Inc, beijing), wherein the kit comprises: plant RNA Reagent, plant tissue cracking, RNA separation, removal of Plant polysaccharides and polyphenols; extraction Reagent, organic Extraction to remove protein, DNA, polysaccharide and polyphenol; plant RNA Aid, removing Plant polysaccharide polyphenols and secondary metabolites. mRNA was purified from total RNA using the QIAGEN Oligotex Mini mRNA Kit (QIAGEN, gmbH, germany). And finally, taking 1 mu L of the DNA fragment to carry out electrophoresis on 1.2% agarose gel to detect the integrity of the DNA fragment, taking another 2 mu L of the DNA fragment to dilute the DNA fragment to 500 mu L, detecting the quality (OD 260) and the purity (OD 260/OD 280) of the DNA fragment by using an ultraviolet spectrophotometer, and extracting the total RNA of the leaves of the Yangjing 805 seedling with non-denaturing gel agarose gel electrophoresis to detect that 28S and 18S bands are clear and the brightness ratio of the two bands is 1.5-2: 1, which shows that the total RNA is not degraded, and the purified mRNA meets the experimental requirements and can be used for cloning the total length of the OsPIS protein cDNA of rice.
Full Length cloning of protein cDNA
To be provided withOsPISDesigning a primer for the gene cDNA sequence to clone the full length of the OsPIS protein cDNA.
The primer sequences are as follows:
OsPIS-GC-F：5’-ATGGCACAACCTTCTTCTAAGAAGA-3’
OsPIS-GC-R：5’-TCACTTGCCGCGCTTCAAATCA-3’
the total RNA of the unfolded leaf of the seedling of Yangjing 805 is reversely transcribed by Oligo (dT) to be used as a template, high fidelity Fastpfu enzyme is used for PCR amplification, the PCR condition is 95 ℃ for 1min, then 95 ℃ for 20s, 53 ℃ for 20s and 72 ℃ for 1min, 36 cycles are carried out, and finally 72 ℃ is extended for 5min. Detecting the PCR amplification product by agarose gel electrophoresis to obtain an amplification fragment with the length of 690 bp.
The results of the above-mentioned steps are combined,the target cDNA sequence is obtained, and the nucleotide sequence is shown as the sequence SEQ ID NO 1 in the sequence table. The sequence SEQ ID NO 1 in the sequence table consists of 672 bases, the 1 st to 672 th bases from the 5' end are an open reading frame thereof, and the protein with an amino acid residue sequence shown by the sequence SEQ ID NO 2 in the sequence table is coded. The sequence SEQ ID NO 2 of the sequence table consists of 223 amino acid residues. This gene was namedOsPISThe protein encoded by the protein is named OsPIS.
Example 2OsPISAnalysis of stress expression of genes
1. Stress management
Surface sterilizing the plump seeds of Yangjing 805' with 1% sodium hypochlorite for 20 min, washing with distilled water for 6 times, soaking in distilled water for 24-36 h, placing on wet gauze, and accelerating germination at 32 deg.C to about 2 d. Sowing the seeds with consistent germination on a foam plastic pore plate adhered with gauze, carrying out liquid culture, and starting the following stress treatment after the seeds grow normally to 4 w.
And (3) mannitol treatment: transferring the rice seedlings from the nutrient solution to a solution containing 200 mM mannitol, and taking the roots and leaves of the rice at 0, 3, 6, 12, 24 and 48 h respectively;
PEG treatment: the treatment method is the same as that of mannitol, the concentration of PEG6000 solution is 20%, and the roots and leaves of the paddy rice are respectively 0, 3, 6, 12, 24 and 48 h;
NaCl treatment: the treatment method is the same as that of mannitol, the concentration of NaCl solution is 200 mM, and the roots and leaves of the paddy rice are respectively taken at 0, 3, 6, 12, 24, 48 h;
ABA treatment: the treatment method is the same as mannitol, the concentration of the ABA solution is 100 mu M, and the roots and leaves of the rice are respectively extracted from 0, 3, 6, 12, 24 and 48 h;
and (4) comparison treatment: the roots and leaves of seedlings without any treatment were taken directly as controls (0 h).
All samples were frozen immediately after sampling in liquid nitrogen and stored at-80 ℃.
Adversity stress qRT-PCR analysis
Respectively extracting total RNA of each treated root and leaf in the steps, carrying out reverse transcription to obtain cDNA, carrying out qRT-PCR analysis, and identifyingOsPISGene expression in rice after different stress treatmentThe characteristics are achieved.OsActinThe gene is internal reference:OsActin-F:5'-TTATGGTTGGGATGGGACA-3' andOsActin-R：5’-AGCACGGCTTGAATAGCG-3’；OsPISthe primer sequence is as follows:OsPIS-F:5'-TGTTCGCCGATGAGAAGTCA-3' andOsPIS-R：5’-GTTTCAACGCCCAACCAACT-3’。
the results are shown in figure 1 of the drawings,OsPISthe gene can be induced and expressed by mannitol, PEG6000, naCl and ABA, which shows thatOsPISThe gene is related to the salt tolerance and drought resistance of plants.
Example 3OsPISConstruction of gene overexpression vector and obtaining of overexpression rice plant
1. OsPISConstruction of Gene overexpression vectors
The DNA fragment containing the nucleotide shown in SEQ ID NO 1 of the sequence table which is sequenced and identified correctly in example 1 is usedBamHI andSaci double digestion, recovery of the DNA fragment on a 1% agarose gel, passage through T 4 DNA ligase to be recoveredOsPISThe gene fragment is connected with plasmid pYPx245 containing double 35S promoter, and the rice containing the gene fragment is obtained by enzyme digestion identification and sequence analysis and determinationOsPISRecombinant plasmid AH128 of the gene. The expression vector further comprisesgusAA reporter gene and an intron-containing kanamycin resistance marker gene.
Over-expressionOsPISGene-transformed rice
The rice to be constructedOsPISOverexpression vector pCAMBIA1301-OsPISThe rice is transformed by the following specific method:
(1) Preparation of Agrobacterium
A step (a): pCAMBIA1301-OsPISAgrobacterium tumefaciens EHA105 strain (Biovector Co., LTD) was transformed by electroporation to obtain a recombinant strain containing pCAMBIA1301-OsPISThe recombinant Agrobacterium A of (1) was named EHA105/pCB-OsPISAnd plated on a plate containing kanamycin resistance to select transformants.
A step (b): a single bacterium of Agrobacterium was picked and inoculated into 5mL of LB liquid medium (rifampicin 50. Mu.g/mL, chloramphenicol 100. Mu.g/mL), and cultured at 28 ℃ and 250rpm for 20 hours.
A step (c): the 1 mL bacterial strain is transferred into 20-30 mL LB liquid medium (rifampicin 50. Mu.g/mL, chloramphenicol 100. Mu.g/mL), cultured at 28 ℃ and 250rpm for about 12h, and the OD 600 is determined to be about 1.5.
Step (d): the cells were collected by centrifugation at 8000rpm,4 ℃ for 10min, resuspended in Agrobacterium transformation permeate (5% sucrose, 0.05% Silwet L-77) and diluted to OD 600. Apprxeq.0.8.
(2) Acquisition of mature embryo callus of rice
Step (a): removing glumes of No. 11 seeds of the mature rice variety, and disinfecting for 1-2 min by using 70% alcohol;
step (b): then soaking with 20% sodium hypochlorite for 30-40 min, washing with sterile distilled water for 4 times, transferring the seeds onto sterilized filter paper, blotting surface water, and inoculating on NB induction culture medium;
step (c): after dark culture 7-10 d, when scutellum is enlarged and endosperm is softened, embryo and bud are removed, and the peeled embryogenic callus is transferred to NB subculture medium for about 3 w subculture once, and can be used as receptor for transformation after 2-3 subcultures.
(3) Agrobacterium mediated transformation of rice callus
Step (a): selecting good embryogenic callus, and soaking in the staining solution for 30 min;
step (b): taking out the callus, removing the redundant bacteria liquid by using sterile filter paper, and then placing the callus on an NB co-culture medium for culturing until the bacterial colony just appears (about 2-3 d);
step (c): shaking and washing with sterile water for 3-4 times until the supernatant is completely clean, and shaking and washing with 500 mg/L cefmenomycin solution for 40min;
step (d): taking out the callus, putting the callus into a sterile culture dish only provided with filter paper, air-drying the callus at 0.4 m/s for 4 h, and transferring the callus into an NB screening culture medium for two screening rounds (each round is 3-4 w);
a step (e): pre-differentiating the resistant callus by 2-3 w, and then transferring the resistant callus to a differentiation culture medium to culture 2-3 w by illumination;
step (f): transferring the young buds to a strong seedling culture medium for culturing about 30 d when the young buds grow to about 1 cm;
step (g): removing the sealing film, hardening seedling, culturing to about 1 w, and transplanting to soil.
Over-expressionOsPISPCR detection of gene rice plant
(1) Test method
Extraction of T by CTAB method 2 Genomic DNA of rice transgenic plants and wild-type plants. PCR detection by conventional method, and the used methodhptThe gene II primers are as follows: primer 1: 5'-ACAGCGTCTCCGACCTGATGCA-3' and Primer2:5'-AGTCAATGACCGCTGTTATGCG-3'. To a 0.2 mL Eppendorf centrifuge tube were added 2. Mu.L of 10 XPCR buffer, 1. Mu.L of 4dNTP (10 mol/L), 1. Mu.L of each primer (10. Mu. Mol/L), 2. Mu.L of template DNA (50 ng/uL), 0.25. Mu.L of Taq DNA polymerase, and ddH 2 O to a total volume of 20. Mu.L. The reaction procedure was pre-denaturation at 94 ℃ for 5min, denaturation at 94 ℃ for 30 s, renaturation at 55 ℃ for 30 s, and extension at 72 ℃ for 2 min for 35 cycles.
(2) Test results
The results of the electrophoretic detection amplification are shown in FIG. 2 [ FIG. 2, lane M: marker; lane W: water; lane P: positive control (recombinant plasmid pCAMBIA1301-OsPIS) (ii) a Lane WT: wild type rice plants; lanes OE1-OE11: for the transformation of pCAMBIA1301-OsPISOver-expression of rice transgenic plant of (1). As can be seen from the figure, the pCAMBIA1301-OsPISThe rice pseudotransgenic plant and the positive control amplify a target strip of 591 bp, which indicates thatOsPISThe gene is integrated into the genome of the rice, and the regenerated plants are proved to be transgenic plants; wild-type rice plants did not have the target band of 591 bp amplified. Transgenic plants were subsequently analyzed for function.
Over-expressionOsPISqRT-PCR detection of gene rice plant
(1) Test method
Extraction of Positive overexpressionOsPISAnd carrying out reverse transcription on the total RNA of the rice strain to obtain cDNA, and carrying out qRT-PCR analysis by taking the non-transformed rice wild type as a control.OsActinThe gene is internal reference:OsActin-F:5'-TTATGGTTGGGATGGGACA-3' andOsActin-R：5’-AGCACGGCTTGAATAGCG-3’；OsPISthe primer sequence is as follows:OsPIS-F:5'-TGTTCGCCGATGAGAAGTCA-3' andOsPIS-R：5’-GTTTCAACGCCCAACCAACT -3’。
(2) Test results
The results are shown in FIG. 3, where WT is a wild-type rice plant and OE1-OE11 are all positive transgenic over-expressedOsPISRice plant, showOsPISThe transgenic rice plants have different degrees of expression, and overexpression strains OE4, OE5 and OE7 with the highest expression quantity are selected for subsequent analysis.
Example 4OsPISIdentification of drought resistance and salt tolerance of gene transgenic rice plant
1. In-vitro identification of drought resistance and salt tolerance of transgenic rice plants
(1) Test method
Disinfecting seeds of over-expression rice materials and wild type materials, sowing the seeds on an MS solid flat plate, selecting the seeds with consistent germination states after the seeds germinate 2-3 d, respectively sowing the seeds on different medium finger tube culture media of MS, MS + NaCl (200 mM) and MS + mannitol (200 mM), and after the seedlings grow to 7-10 d, carrying out photography and growth vigor statistics on the differences of the seedlings treated differently, wherein the statistics comprises the seedling length and fresh weight data.
(2) Test results
The results show that under the conditions of salt stress and mannitol treatment, the results are shown in figure 4, and the over-expressed rice material and the wild type material are both subjected to the conditions of salt stress and mannitol stress, so that the plants become small; however, compared with the wild type WT, the over-expressed rice material has a relatively good growth state, and growth potential data statistics show that under salt stress, the seedling length and the fresh weight of the over-expressed rice material are respectively increased by 102-124% and 159-189% compared with the wild type WT material; under the stress of mannitol, the seedling length and the fresh weight of the over-expressed rice material are respectively increased by 91-103% and 145-176% compared with the wild WT material; indicating overexpressionOsPISThe gene can obviously improve the salt tolerance and drought resistance of transgenic rice plants.
Drought resistance and salt tolerance potted plant identification of transgenic rice plant
(1) Test method
To verify the salt tolerance and drought resistance of transgenic rice material, homozygous T is used 2 Sterilizing the surfaces of over-expression rice material and wild rice seed,accelerating germination with purified water, inoculating on MS culture medium, and growing to about 3-4 d. And (3) selecting the seedlings with consistent growth vigor, and planting the seedlings in nutrient soil: in vermiculite =1:2, watering is carried out every day, and salt and drought stress treatment is started after the plants grow to 4 w. Irrigate 1 time each 2 d with 1/2 Hoagland nutrient solution containing 200 mM NaCl, 200 mL each, treat 4 w, observe its phenotype, take photographs and investigate its survival rate; after drought treatment 6 w, their phenotypes were observed, photographed and investigated for survival. The following calculation approaches relating to improved survival are: (survival of over-expressed plants-survival of wild type plants) 100%/survival of wild type plants.
(2) Test results
The results show that after the salt stress treatment condition of 4 w or the drought stress treatment of 6 w, the results are shown in fig. 5, the growth state of the transgenic plant is obviously superior to that of the wild plant, the survival rate of the transgenic plant is obviously higher than that of the wild plant, and the survival rate is respectively improved by 1090-1211% and 1071-1244% compared with that of the wild plant; indicating overexpressionOsPISThe gene can obviously improve the salt tolerance and drought resistance of transgenic rice plants.
Example 5OsPISDetermination of stress-resistant physiological and biochemical indexes of genetically modified rice plants
1. Determination of abscisic acid content
(1) Test method
Abscisic acid (ABA) plays an important role in plant stress response. The ABA can improve the salt tolerance of plants, relieve the osmotic stress and the ionic stress caused by excessive salt, maintain the water balance, induce the large accumulation of proline which is a plant osmotic regulator substance, maintain the stability of a cell membrane structure and improve the activity of protective enzymes. When drought is stressed, the ABA can obviously reduce the water evaporation of leaves, reduce the permeability of leaf cell membranes, increase the content of soluble protein in the leaf cells, induce the formation of protective enzyme of a biological membrane system, reduce the peroxidation degree of membrane lipid, enhance the oxidation resistance and improve the drought resistance of plants. Therefore, the ABA can be used as a biochemical index of the stress resistance of plants.
Assay references [ Shang Gao, li Yuan, hong Zhai, chenglong Liu, shaozhen He, qingchang Liu. Transgenic sweetpotato plants expressing an LOS5Gene are tall to salt Plant Cell, tissue and Organ Culture, 2011, 107: 205-213. ABA content of rice plants is detected. The rice plants are rice plants treated with 2 w, rice plants subjected to salt stress of 2 w and rice plants subjected to drought stress of 4 w in the blank control. The experiment was repeated three times and the results averaged.
(2) Test results
The test results of ABA content determination of rice plants are shown in A in FIG. 6 (Normal is blank control, salt stress is Salt stress, and dry stress is Drought stress). The result shows that the ABA content of over-expressed rice OE4 plants, OE5 plants and OE7 plants is obviously higher than that of wild rice plants.
Determination of proline content
(1) Test method
Under normal conditions, the content of free proline in plants is low, but when the plants are stressed by drought, salt and the like, a large amount of free amino acid is accumulated, and the accumulation index is related to the stress resistance of the plants. Therefore, proline can be used as a biochemical index of plant stress resistance.
Assay references [ Feibing Wang, weili Kong, gary Wong, life Fu, rihe Peng, zhenjun Li, quanhong Yao.AtMYB12 regulates flavonoids accumulation and abiotic stresstolerance in transgenic Arabidopsis thalianaMolecular Genetics and Genomics, 2016, 291, 1545-1559 ] rice plants were tested for proline content. The rice plants are rice plants treated with 2 w, rice plants subjected to salt stress of 2 w and rice plants subjected to drought stress of 4 w in the blank control. The experiment was repeated three times and the results averaged.
(2) Test results
The results of the proline content determination experiments for rice plants are shown in B in FIG. 6 (Normal is blank control, salt stress is Salt stress, and dry stress is Drought stress). The results show that the proline content of transgenic rice OE4 plants, OE5 plants and OE7 plants is obviously higher than that of wild rice plants.
22 Determination of content
(1) Test method
When plants are in stress or aging, the metabolism of active oxygen in vivo is enhanced to increase H 2 O 2 Accumulation occurs. H 2 O 2 Can directly or indirectly oxidize intracellular biomacromolecules such as nucleic acid, protein and the like, and damage cell membranes, thereby accelerating the aging and disintegration of cells. Thus, H 2 O 2 The higher the content of (a), the greater the degree to which the plant suffers stress injury.
Assay references [ Feibing Wang, weili Kong, gary Wong, life Fu, rihe Peng, zhenjun Li, quanhong Yao.AtMYB12 regulates flavonoids accumulation and abiotic stresstolerance in transgenic Arabidopsis thalianaMolecular Genetics and Genomics, 2016, 291 1545-1559 ] rice plants were examined for H 2 O 2 And (4) content. The rice plants were rice plants treated with 2 w, salt stressed 2 w, drought stressed 4 w in the blank control. The experiment was repeated three times and the results averaged.
(2) Test results
Rice plant H 2 O 2 Results of the assay are shown in FIG. 6C (Normal is blank, salt stress, and Drought stress). The results show that the transgenic rice plants OE2, OE4 and OE7 are H 2 O 2 The content is obviously lower than that of wild rice plants.
Determination of content
(1) Test method
The organ of the plant is aged or damaged under the stress, membrane lipid peroxidation usually occurs, and Malondialdehyde (MDA) is the final decomposition product of the membrane lipid peroxidation, and the content of the Malondialdehyde (MDA) can reflect the degree of the plant suffering from the stress injury, namely the higher the content of the MDA, the greater the degree of the plant suffering from the stress injury.
Assay references [ Feibing Wang, weili Kong, gary Wong, life Fu, rihe Peng, zhenjun Li, quanhong Yao.AtMYB12 regulates flavonoids accumulation and abiotic stresstolerance in transgenic Arabidopsis thaliana. Molecular Genetics and Genomics, 2016, 291:1545-1559 ] rice plants were tested for their MDA content. The rice plants are rice plants treated with 2 w, rice plants subjected to salt stress of 2 w and rice plants subjected to drought stress of 4 w in the blank control. The experiment was repeated three times and the results averaged.
(2) Test results
The results of the rice plant MDA assay are shown in FIG. 6D (Normal is blank, salt stress is Salt stress, and Drought stress is Drought stress). Results show that the MDA content of transgenic rice OE4 plants, OE5 plants and OE7 plants is obviously lower than that of wild rice plants.
Activity assay
(1) Test method
The activity of superoxide dismutase (SOD) can be used as a physiological and biochemical index of plant stress resistance. The lower the activity of SOD, the greater the degree of stress injury suffered by the plant.
Assay references [ Feibing Wang, weili Kong, gary Wong, life Fu, rihe Peng, zhenjun Li, quanhong Yao.AtMYB12 regulates flavonoids accumulation and abiotic stresstolerance in transgenic Arabidopsis thalianaMolecular Genetics and Genomics, 2016, 291, 1545-1559 ] SOD activity of rice plants was examined. The rice plants are rice plants treated with 2 w, rice plants subjected to salt stress of 2 w and rice plants subjected to drought stress of 4 w in the blank control. The experiment was repeated three times and the results averaged.
(2) Test results
The results of the SOD activity assay for rice plants are shown in E in FIG. 6 (Normal is blank control, salt stress is Salt stress, and dry stress is Drought stress). The results show that the SOD activity of transgenic rice OE4 plants, OE5 plants and OE7 plants is obviously higher than that of wild rice plants.
Activity assay
(1) Test method
Peroxidase (POD) activity can be used as a physiological and biochemical indicator of plant stress resistance. The lower the activity of POD, the greater the degree to which the plant suffers stress injury.
Assay references [ Feibing Wang, weili Kong, gary Wong, life Fu, rihe Peng, zhenjun Li, quanhong Yao.AtMYB12 regulates flavonoids accumulation and abiotic stresstolerance in transgenic Arabidopsis thalianaMolecular Genetics and Genomics, 2016, 291-1545-1559 ] rice plants were examined for POD activity. The rice plants are rice plants treated with 2 w, rice plants subjected to salt stress of 2 w and rice plants subjected to drought stress of 4 w in the blank control. The experiment was repeated three times and the results averaged.
(2) Test results
The results of the POD activity assay for rice plants are shown in FIG. 6 as F (Normal is blank, salt stress is Salt stress, and dry stress is Drought stress). The result shows that the POD activity of transgenic rice plants OE4, OE5 and OE7 is obviously higher than that of wild rice plants.
The measurement result of physiological and biochemical indexes shows that the over-expression is performedOsPISThe gene can obviously improve the salt tolerance and drought resistance of transgenic rice plants.
Claims (10)
1. The protein is a protein represented by any one of the following (a 1), (a 2) or (a 3):
(a1) Protein with amino acid sequence shown as SEQ ID NO. 1;
(a2) The N end or/and the C end of the amino acid sequence shown in SEQ ID NO.1 is connected with a protein label to obtain a fusion protein;
(a3) And (b) a protein which is obtained by substituting and/or deleting and/or adding one or more amino acid residues in the amino acid sequence shown in SEQ ID NO.1, has more than 90% of identity with the protein shown in (a 1) and has the same function.
2. The protein-related biomaterial of claim 1, wherein: the related biomaterial is any one of the following (c 1) - (c 10):
(c1) A nucleic acid molecule encoding the protein of claim 1;
(c2) An expression cassette comprising the nucleic acid molecule of (c 1);
(c3) A recombinant vector comprising the nucleic acid molecule of (c 1), or a recombinant vector comprising the expression cassette of (c 2);
(c4) A recombinant microorganism comprising (c 1) said nucleic acid molecule, or a recombinant microorganism comprising (c 2) said expression cassette, or a recombinant microorganism comprising (c 3) said recombinant vector;
(c5) A transgenic plant cell line comprising (c 1) said nucleic acid molecule, or a transgenic plant cell line comprising (c 2) said expression cassette, or a transgenic plant cell line comprising (c 3) said recombinant vector;
(c6) A transgenic plant tissue comprising (c 1) said nucleic acid molecule, or a transgenic plant tissue comprising (c 2) said expression cassette, or a transgenic plant tissue comprising (c 3) said recombinant vector;
(c7) A transgenic plant organ containing (c 1) said nucleic acid molecule, or a transgenic plant organ containing (c 2) said expression cassette, or a transgenic plant organ containing (c 3) said recombinant vector;
(c8) A transgenic plant containing the nucleic acid molecule of (c 1), or a transgenic plant containing the expression cassette of (c 2), or a transgenic plant containing the recombinant vector of (c 3);
(c9) A tissue culture produced from regenerable cells of said transgenic plant of (c 8);
(c10) Protoplasts produced from the tissue culture of (c 9).
3. The related biological material according to claim 2, wherein: (c1) Wherein the nucleic acid molecule encoding the protein of claim 1 is represented by any one of (d 1), (d 2) and (d 3):
(d1) DNA molecule shown in SEQ ID NO. 2;
(d2) The coding sequence is DNA molecule shown in SEQ ID NO. 2;
(d3) A DNA molecule which hybridizes under stringent conditions to the DNA molecule defined in (d 1) or (d 2) and which encodes the protein as claimed in claim 1.
4. Use of the protein of claim 1 or the related biomaterial of claim 2 or 3 in any one of:
(b1) Improving the salt tolerance of the plants;
(b2) Preparing a product for improving the salt tolerance of plants;
(b3) Improving the drought resistance of the plants;
(b4) Preparing a product for improving the drought resistance of plants;
(b5) Improving the rooting condition of the plants under drought and/or salt stress conditions;
(b6) Preparing a product for improving the rooting condition of plants under drought and/or salt stress conditions;
(b7) Increasing the growth vigor of the plant under drought and/or salt stress conditions;
(b8) Preparing a product of the growth vigor of the plant under drought and/or salt stress conditions;
(b9) Preparing the survival rate of the plants under the drought and/or salt stress condition;
(b10) Preparing a product that increases the survival rate of plants under drought and/or salt stress conditions;
(b11) Increasing the abscisic acid content of plants under drought and/or salt stress conditions;
(b12) Preparing a product for improving the content of the plant abscisic acid under the drought and/or salt stress condition;
(b13) Increasing the proline content of plants under drought and/or salt stress conditions;
(b14) Preparing a product for improving the proline content of plants under drought and/or salt stress conditions;
(b15) Reduction of plant H under drought and/or salt stress conditions 2 O 2 Content (c);
(b16) Preparation of plants H for reducing drought and/or salt stress conditions 2 O 2 Products of content
(b17) Reducing the malondialdehyde content of plants under drought and/or salt stress conditions;
(b18) Preparing a product for reducing the malondialdehyde content of a plant under drought and/or salt stress conditions;
(b19) Improving the SOD activity of plants under drought and/or salt stress conditions;
(b20) Preparing a product for improving the SOD activity of plants under drought and/or salt stress conditions;
(b21) Increasing plant POD activity under drought and/or salt stress conditions;
(b22) Products are prepared that increase plant POD activity under drought and/or salt stress conditions.
5. Use of a protein as defined in claim 1 or a related biological material as defined in claim 2 or 3 in plant breeding.
6. A method for cultivating transgenic plants with high drought resistance and salt tolerance is characterized in that: the method comprises increasing the expression level of a gene encoding the protein of claim 1 and/or the content of the protein and/or the activity of the protein in a target plant to obtain a transgenic plant; the drought resistance and salt tolerance of the transgenic plant are higher than those of the target plant.
7. The method of claim 6, wherein: the method for improving the expression amount of the gene coding for the protein of claim 1 and/or the content of the protein and/or the activity of the protein in a target plant is to express or over-express the protein of claim 1 in the target plant.
8. The method of claim 7, wherein: the method for expression or overexpression is to introduce a gene encoding the protein of claim 1 into a target plant.
9. The method of claim 8, wherein: the nucleotide sequence of the gene encoding the protein of claim 1 is a DNA molecule represented by SEQ ID NO. 2.
10. The use according to claim 4 or 5, or the method according to any one of claims 6 to 9, wherein: the plant is any one of the following (e 1) to (e 5):
(e1) A dicotyledonous plant;
(e2) A monocot plant;
(e3) A gramineous plant;
(e4) Plants of the genus Oryza;
(e5) A rice plant.
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Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN117305266A (en) * | 2023-03-10 | 2023-12-29 | 苏州健雄职业技术学院 | Gene OsBDG1 related to rice stress resistance and application of encoding protein thereof |
| CN117305266B (en) * | 2023-03-10 | 2024-05-03 | 苏州健雄职业技术学院 | Gene OsBDG1 related to rice stress resistance and application of coded protein thereof |
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| Publication number | Priority date | Publication date | Assignee | Title |
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| CN117305266A (en) * | 2023-03-10 | 2023-12-29 | 苏州健雄职业技术学院 | Gene OsBDG1 related to rice stress resistance and application of encoding protein thereof |
| CN117305266B (en) * | 2023-03-10 | 2024-05-03 | 苏州健雄职业技术学院 | Gene OsBDG1 related to rice stress resistance and application of coded protein thereof |
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