CN115851660A - Application of protein OsPIS and coding gene in improving plant stress resistance - Google Patents

Abstract

The invention discloses the application of the protein OsPIS and the coding gene in improving the stress resistance of plants. The present invention screens a protein, which is a protein whose amino acid sequence is shown in SEQ ID NO.1 or a fusion protein obtained by linking protein tags at the N-terminal or/and C-terminal of the amino acid sequence shown in SEQ ID NO.1. The invention clones the OsPIS protein and its encoding gene, and introduces the encoding gene of the OsPIS protein into rice, so as to significantly improve the drought resistance and salt tolerance of rice plants. The protein and its encoding gene of the present invention have important application value for cultivating stress-resistant plant varieties, and thus have great significance for improving crop yield; the present invention will have broad application space and market prospect in the field of agriculture.

Description

Application of Protein OsPIS and Its Encoding Gene in Improving Plant Stress Resistance

technical field

The invention relates to a plant stress resistance-related protein OsPIS and its coding gene and application, in particular to the protein OsPIS and its related biological materials and application.

Background technique

Rice ( Oryza sativa L.) is one of the main food crops in the world. Nearly half of the world's population lives on rice. Planting rice requires a lot of freshwater resources. The shortage of water resources is a serious ecological problem restricting the development of global agricultural production. Drought has long been the main limiting factor affecting food security around the world. With the increase of global temperature, the area of arid and semi-arid land is increasing year by year. The area of arid and semi-arid cultivated land in China accounts for about 51% of the total cultivated land area, and nearly 2.5×10 ⁶ hm ² of cultivated land are affected by drought to varying degrees every year. At present, with the warming of the global climate and the destruction of the ecological balance, the phenomenon of water shortage becomes more serious. The normal growth and development and high yield of crops must have sufficient water to provide protection. Therefore, drought and high-salt environment are the most important abiotic stress factors affecting crop yield, especially the production of traditional rice will face severe challenges, and the main way to improve rice water use efficiency is to improve its drought resistance and salt tolerance .

Phospholipids are not only the main structural components of cell membranes, but also act as signaling molecules to regulate plant growth, development and responses to external environmental stimuli. Phosphatidylinositol (PI) is a major phospholipid in eukaryotes, accounting for 15-20% of the total phospholipids in eukaryotes. It is not only the basic component of the cell membrane, but also participates in the anchoring of specific proteins in the cell membrane It is also a precursor of important signaling 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 (PI4K) to form phosphatidylinositol-4-phosphate (PIP), which is phosphorylated by phosphatidylinositol 4-phosphate 5-kinase (PIP5K) to form phosphatidylinositol Inositol-4,5-bisphosphate (PIP ₂ ). PIP ₂ is catalyzed by phosphoinositide-specific phospholipase C (PLC) to generate two second messenger substances, inositol-1,4,5-triphosphate (IP3) and diacylglycerol (DAG). Water-soluble IP ₃ diffuses into the cytoplasm to promote the release of Ca ²⁺ stored in the cell, increase the concentration of Ca ²⁺ in the cell, and regulate a series of physiological and biochemical reactions of plants in response to external environmental stimuli. Under the action of diacylglycerol kinase (DGK), DAG can be rapidly catalyzed to form phosphatidic acid (PA). PA can also be directly formed by the hydrolysis of phospholipid structures such as PI, PIP and PIP ₂ by phospholipase D (PLD). More and more experiments have shown that PA is also an important second messenger in plants and participates in regulating the response of plant cells to external signal stimuli.

During the long-term evolution process, plants have developed a series of salt-tolerant and drought-resistant mechanisms. With the rapid development of molecular biology, the physiological and biochemical mechanisms of plant salt tolerance and drought resistance have become increasingly clear, making it possible to clone genes related to plant salt tolerance and drought resistance. Strengthen the research on plant salt-tolerance and drought-resistance physiology, ascertain the life activities of plants under adversity and regulate them artificially, clone the rice phosphatidylinositol synthase gene OsPIS , use genetic engineering technology to improve plant salt-tolerance and drought resistance, and cultivate plants with Excellent varieties resistant to adverse environmental traits to improve crop yield and quality are of great significance for obtaining high and stable agricultural yields.

Contents of the invention

The technical problem to be solved by the invention is how to effectively improve the drought resistance and salt tolerance of plants.

To solve the above technical problems, the present invention firstly provides a protein named as OsPIS protein or protein OsPIS, which is derived from rice ( Oryza sativa L.), as shown in any of the following (a1) or (a2) or (a3) protein:

(a1) A protein whose amino acid sequence is shown in SEQ ID NO.1;

(a2) A fusion protein obtained by linking protein tags at the N-terminal or/and C-terminal of the amino acid sequence shown in SEQ ID NO.1;

(a3) Substitution and/or deletion and/or addition of one or several amino acid residues to the amino acid sequence shown in SEQ ID NO.1 has more than 90% identity with the protein shown in (a1) and proteins with the same function.

Among them, SEQ ID NO.1 consists of 223 amino acid residues.

The above-mentioned proteins can be synthesized artificially, or their coding genes can be synthesized first, and then biologically expressed.

Among the above-mentioned proteins, a protein-tag refers to a polypeptide or protein that is fused and expressed with a target protein using DNA in vitro recombination technology, so as to facilitate the expression, detection, tracking and/or purification of the target protein. The protein tag can be Flag tag, His tag, MBP tag, HA tag, myc tag, GST tag and/or SUMO tag, etc.

In the above-mentioned proteins, the identity refers to the identity of amino acid sequences. Amino acid sequence identities can be determined using homology search sites on the Internet, such as the BLAST webpage of the NCBI homepage. For example, in advanced BLAST2.1, by using blastp as the program, set the Expect value to 10, set all Filters to OFF, use BLOSUM62 as Matrix, and set Gap existence cost, Per residue gap cost and Lambda ratio to 11, 1 and 0.85 (the default value) and search for the identity of a pair of amino acid sequences to calculate, and then the value (%) of the identity can be obtained.

Among the above proteins, the above 90% identity may be at least 91%, 92%, 95%, 96%, 98%, 99% or 100% identity.

Biological materials related to the above-mentioned protein OsPIS also belong to the protection scope of the present invention. The above-mentioned relevant biological materials are any of the following (c1)-(c10):

(c1) a nucleic acid molecule encoding the protein OsPIS;

(c2) an expression cassette comprising the nucleic acid molecule of (c1);

(c3) A recombinant vector containing the nucleic acid molecule described in (c1), or a recombinant vector containing the expression cassette described in (c2);

(c4) A recombinant microorganism containing the nucleic acid molecule described in (c1), or a recombinant microorganism containing the expression cassette described in (c2), or a recombinant microorganism containing the recombinant vector described in (c3);

(c5) a transgenic plant cell line containing the nucleic acid molecule described in (c1), or a transgenic plant cell line containing the expression cassette described in (c2), or a transgenic plant cell line containing the recombinant vector described in (c3);

(c6) Transgenic plant tissue containing the nucleic acid molecule described in (c1), or a transgenic plant tissue containing the expression cassette described in (c2), or a transgenic plant tissue containing the recombinant vector described in (c3);

(c7) Transgenic plant organs containing the nucleic acid molecules described in (c1), or transgenic plant organs containing the expression cassettes described in (c2), or transgenic plant organs containing the recombinant vectors described in (c3);

(c8) Transgenic plants containing the nucleic acid molecules described in (c1), or transgenic plants containing the expression cassettes described in (c2), or transgenic plants containing the recombinant vectors described in (c3);

(c9) tissue culture produced from the regenerative cells of the transgenic plant described in (c8);

(c10) Protoplasts produced from the tissue culture of (c9).

Wherein, the nucleic acid molecule can be DNA, such as cDNA, genomic DNA or recombinant DNA; the nucleic acid molecule can also be RNA, such as mRNA or hnRNA.

Among the above-mentioned related biological materials, (c1) the nucleic acid molecule encoding the protein OsPIS can specifically be any of the following (d1) or (d2) or (d3)

(d1) DNA molecule shown in SEQ ID NO.2;

(d2) The coding sequence is the DNA molecule shown in SEQ ID NO.2;

(d3) A DNA molecule that hybridizes to the DNA molecule defined in (d1) or (d2) under stringent conditions and encodes the protein OsPIS.

Among them, SEQ ID NO.2 consists of 672 nucleotides, its open reading frame (ORF) is from the 1st to 672nd position from the 5' end, and encodes a protein whose amino acid sequence is shown in SEQ ID NO.1.

The stringent condition is to hybridize at 68°C in a solution of 2×SSC and 0.1% SDS and wash the membrane twice for 5 min each time, and to hybridize at 68°C in a solution of 0.5×SSC and 0.1% SDS And wash the membrane twice, 15min each time.

Expression cassettes, recombinant expression vectors, transgenic cell lines or recombinant bacteria containing the genes encoding the proteins related to plant stress resistance also belong to the protection scope of the present invention.

In the above-mentioned related biological materials, the expression cassette described in (c2) refers to the DNA capable of expressing the protein OsPIS in the host cell, and the DNA may include not only a promoter that initiates the transcription of the OsPIS gene, but also a terminator that terminates the transcription of the OsPIS gene .

Among the above-mentioned related biological materials, (c3) the recombinant vector may contain the DNA molecule for encoding the protein OsPIS shown in SEQ ID NO.2.

A plant expression vector can be used to construct a recombinant vector containing the expression cassette of the gene encoding OsPIS. The plant expression vector can be a Gateway system vector or a binary Agrobacterium vector, such as pGWB411, pGWB412, pGWB405, pBin438, pCAMBIA1300, pCAMBIA1302, pCAMBIA2300, pCAMBIA2301, pCAMBIA1301, pBI121, pCAMBIA1391-Xa or pCAMBIA1391-Xb, etc. When using OsPIS to construct a recombinant vector, any enhanced, constitutive, tissue-specific or inducible promoter can be added before its transcription initiation nucleotide, such as cauliflower mosaic virus (CAMV) 35S promoter, pan- Biotin gene Ubiqutin promoter (pUbi), etc., they can be used alone or in combination with other plant promoters; in addition, when using the gene of the present invention to construct a plant expression vector, enhancers, including translation enhancers or transcription enhancers, can also be used Enhancers, these enhancer regions can be ATG start codons or adjacent region start codons, etc., but must be the same as the reading frame of the coding sequence to ensure correct translation of the entire sequence. The sources of the translation control signals and initiation codons are extensive and can be natural or synthetic. The translation initiation region can be from a transcription initiation region or a structural gene.

In a specific embodiment of the present invention, the recombinant expression vector is a recombinant expression vector obtained by inserting the coding gene between the multiple cloning sites of the vector pCBGUS;

The vector pCBGUS is obtained by a method comprising the following steps:

(1) The pCAMBIA1301 vector was digested with Hind III and EcoR I, and the large fragment of the vector was recovered;

(2) The pBI121 vector was digested with Hind III and EcoR I, and the fragment containing the gusA gene was recovered;

(3) Ligate the large fragment of the vector recovered in step (1) with the fragment containing the gusA gene recovered in step (2) to obtain the recombinant vector pCBGUS.

The pCAMBIA1301 vector was purchased from CAMBIA Company; the pBI121 vector was purchased from Clontech Company.

Among the above-mentioned related biological materials, the recombinant microorganisms mentioned in (c4) can specifically be yeast, bacteria, algae and fungi.

In the above-mentioned related biological materials, (c7) the transgenic plant organs can be roots, stems, leaves, flowers, fruits and seeds of transgenic plants.

Among the above-mentioned related biological materials, the tissue culture mentioned in (c9) can be derived from roots, stems, leaves, flowers, fruits, seeds, pollen, embryos and anthers.

Among the above-mentioned related biological materials, the transgenic plant cell lines, transgenic plant tissues and transgenic plant organs do not include propagation materials.

The present invention also provides the application of the above-mentioned protein OsPIS or its related biological materials in any of the following (b1)-(b22):

(b1) Improve plant salt tolerance;

(b2) preparation of products that increase the salt tolerance of plants;

(b3) Improve plant drought resistance;

(b4) preparation of products that increase the drought resistance of plants;

(b5) improving rooting of plants under conditions of drought and/or salt stress;

(b6) preparation of a product that improves the rooting of plants under conditions of drought and/or salt stress;

(b7) Improving the growth of plants under conditions of drought and/or salt stress;

(b8) Preparation of products of growth of plants under conditions of drought and/or salt stress;

(b9) prepare the survival rate of plants under drought and/or salt stress conditions;

(b10) Preparation of products that increase the survival of plants under conditions of drought and/or salt stress;

(b11) increasing plant abscisic acid content under conditions of drought and/or salt stress;

(b12) Preparation of products that increase the abscisic acid content of plants under conditions of drought and/or salt stress;

(b13) increasing proline content in plants under conditions of drought and/or salt stress;

(b14) Preparation of products for increasing the proline content of plants under conditions of drought and/or salt stress;

(b15) Reducing plant _H2O2 content under conditions of drought and/or salt stress _;

(b16) Preparation _of products for reducing the _H2O2 content of plants under conditions of drought and/or salt stress

(b17) Reduction of malondialdehyde content in plants under conditions of drought and/or salt stress;

(b18) Preparation of products for reducing malondialdehyde content in plants under conditions of drought and/or salt stress;

(b19) improving plant SOD activity under conditions of drought and/or salt stress;

(b20) preparing a product that improves plant SOD activity under conditions of drought and/or salt stress;

(b21) increasing plant POD activity under conditions of drought and/or salt stress;

(b22) Preparation of a product that improves plant POD activity under conditions of drought and/or salt stress.

The application of the above-mentioned protein OsPIS or its related biological materials in plant breeding is also within the protection scope of the present invention.

Among the above applications, the application in plant breeding can specifically be the crossing of plants containing the protein OsPIS or the related biological material (such as the gene OsPIS encoding the protein OsPIS ) with other plants for plant breeding.

The invention further provides a method for cultivating transgenic plants with high drought resistance and salt tolerance.

The method for cultivating transgenic plants with high drought resistance and salt tolerance of the present invention comprises 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 target plant 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 target plant is to express or overexpress the protein OsPIS in the target plant.

In the above method, the expression or overexpression method is to introduce the gene encoding the protein OsPIS into the target plant.

In the above method, the gene encoding the protein OsPIS can be introduced into the target plant through the 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 conventional biological methods such as Ti plasmid, Ri plasmid, plant virus vector, direct DNA transformation, microinjection, conductance, Agrobacterium-mediated, and transform Plant cells or tissues grown into plants.

In the above method, the nucleotide sequence of the gene encoding the protein OsPIS is the 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 is obtained by using restriction endonucleases Hind III and EcoR I to insert the DNA molecule shown in SEQ ID NO.2 into the pCAMBIA1301 vector.

In the above method, the high drought resistance and salt tolerance are mainly reflected in increasing the root number, root length, fresh weight and survival rate of plants, increasing ABA content, increasing proline content, increasing SOD activity, increasing POD activity, reducing H ₂ O ₂ content and reduced MDA content.

In the present invention, the plant is any one of the following (e1) to (e5):

(e1) dicotyledonous plants;

(e2) Monocots;

(e3) Poaceae;

(e4) Plants of the genus Oryza;

(e5) Rice ( Oryza sativa ).

Compared with prior art, the beneficial effect of the present invention is:

The protein encoded by the OsPIS gene provided by the invention can improve the stress resistance of plants: overexpressing the OsPIS gene can improve the drought resistance and salt tolerance of rice, and interfering with the expression of the OsPIS gene can reduce the drought resistance and salt tolerance of rice. From the test results, under NaCl stress, the transgenic plants showed a good growth state, and the seedling length and fresh weight of the overexpressed transgenic rice material were increased by 102-124% and 159-189% respectively compared with the wild-type WT material; Under mannitol stress, the seedling length and fresh weight of overexpressed transgenic rice plants increased by 91-103% and 145-176% respectively compared with wild-type WT plants; the survival rate of overexpressed transgenic rice plants was significantly higher than that of wild-type plants, Compared with the wild-type plants, they increased by 1090-1211% and 1071-1244%, respectively, and expressed strong salt tolerance and drought resistance; specifically, the overexpression of transgenic rice materials increased ABA content, proline content, and SOD activity , POD activity, reduced malondialdehyde content and H ₂ O ₂ content. Therefore, the protein and its coding gene of the present invention have important application value for cultivating stress-resistant plant varieties, thereby improving the yield of crops, and have broad application space and market prospects in the agricultural field.

Description of drawings

Fig. 1 Analysis of adversity stress expression of OsPIS gene of the present invention in rice variety Yangjing 805.

Fig. 2 is a diagram of the PCR detection results of the OsPIS gene overexpressed rice line of the present invention.

Fig. 3 Expression of the OsPIS gene of the present invention in overexpressed rice lines and wild-type rice plants.

Figure 4 shows the growth and rooting of OsPIS gene transgenic rice plants of the present invention on MS medium of 200 mM NaCl and 200 mM mannitol, WT is wild type rice plants, OE4, OE5 and OE7 are overexpression transgenic rice plants.

Figure 5 Pot identification of salt tolerance and drought resistance of OsPIS gene transgenic rice plants of the present invention, WT is wild type rice plants, OE4, OE5 and OE7 are overexpression transgenic rice plants.

Fig. 6 Determination of stress resistance physiological and biochemical indexes of OsPIS gene transgenic rice plants of the present invention, WT is wild type rice plants, OE4, OE5 and OE7 are overexpression transgenic rice plants.

Detailed ways

The present invention will be further described below in conjunction with specific examples, but the present invention is not limited.

In the following examples, used test materials and sources thereof include:

Rice ( Oryza sativa ) cultivars Yangjing 805 and Zhonghua 11 were preserved by the Laboratory of Jiangsu Plant Production and Processing Practice Education Center, College of Life Science and Food Engineering, Huaiyin Institute of Technology.

Escherichia coli ( Escherichia coli ) DH5α was preserved by the Laboratory of Jiangsu Plant Production and Processing Practice Education Center, School of Life Science and Food Engineering, Huaiyin Institute of Technology. The cloning vector PMD-18-Simple T, various restriction enzymes, Taq polymerase, ligase, dNTP, 10×PCR buffer and DNA marker were purchased from Treasure Bioengineering Dalian Co., Ltd. All chemical reagents were purchased from Sigma Chemical Company, USA and Sinopharm Chemical Reagent Company, Shanghai.

For the conventional molecular biology operations in the present invention, refer to "Molecular Cloning" [Molecular Cloning. 2nded. Cold Spring Harbor Laboratory Press, 1989].

The conventional gene manipulations in the following examples were carried out with reference to molecular cloning literature [Sambook J, Frets EF, Mannsdes T et al. In: Molecular Cloning. 2nd ed. Cold Spring Harbor Laboratory Press, 1989].

The 1/2 Hoagland nutrient solution is recorded in the following literature [Feibing Wang, Weili Kong, Gary Wong, Lifeng Fu, Rihe Peng, Zhenjun Li, Quanhong Yao. AtMYB12 regulates flavonoidsaccumulation and abiotic stress tolerance in transgenic Arabidopsis thaliana .Molecular Genetics and Genomics, 2016, 291:1545-1559].

Example 1 Acquisition of Rice Stress Resistance-Related Proteins and Their Encoding Genes

1. Experimental materials

Referring to Jan et al. (2013) [Asad Jan, Kyonoshin Maruyama, Daisuke Todaka, Satoshi Kidokoro, Mitsuru Abo, Etsuro Yoshimura, Kazuo Shinozaki, Kazuo Nakashima and Kazuko Yamaguchi-Shinozaki. byRegulating Stress-Related Genes. Plant Physiology, 2013, 161:1202-1216], the rice variety 'Yangjing 805' plant leaf material was removed, quick-frozen in liquid nitrogen, and stored at -80°C.

Total RNA extraction and purification from leaves

Take about 2.0 g of the expanded leaves of 'Yangjing 805' seedlings, grind them into powder in liquid nitrogen, put them into a 10 mL centrifuge tube, and use the Applygen Plant RNA Extraction Kit (Applygen Technologies Inc, Beijing) to extract total RNA from sweet potato tubers, kit Including: Plant RNA Reagent, plant tissue lysis, RNA isolation, removal of plant polysaccharides and polyphenols; Extraction Reagent, organic extraction to remove protein, DNA, polysaccharides and polyphenols; Plant RNA Aid, removal of plant polysaccharides polyphenols and secondary metabolism product. mRNA was purified from total RNA using the QIAGEN Oligotex Mini mRNA Kit (QIAGEN, GmbH, Germany). Finally, 1 μL was tested for its integrity by electrophoresis on 1.2% agarose gel, another 2 μL was diluted to 500 μL, and the quality (OD260) and purity (OD260/OD280) of the extracted 'Yang The total RNA of japonica 805' seedling leaves was detected by non-denaturing agarose gel electrophoresis, and the 28S and 18S bands were clear, and the brightness ratio of the two was 1.5-2:1, indicating that the total RNA was not degraded, and the purified mRNA met the experimental requirements , which can be used to clone the full-length cDNA of rice OsPIS protein.

Full-length cloning of protein cDNA

The primers were designed according to the cDNA sequence of OsPIS gene to carry out the full-length cloning of 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 leaves of 'Yangjing 805' seedlings was reverse-transcribed with Oligo (dT) as a template, and high-fidelity FastPfu enzyme was used for PCR amplification. and 72°C for 1 min for 36 cycles, with a final extension at 72°C for 5 min. The PCR amplification product was detected by agarose gel electrophoresis, and an amplified fragment with a length of 690 bp was obtained.

Based on the results of the above steps, the target cDNA sequence was obtained, the nucleotide sequence of which is shown in the sequence SEQ ID NO 1 in the sequence listing. The sequence SEQ ID NO 1 in the sequence listing is composed of 672 bases, from the 1st to the 672nd base at the 5' end is its open reading frame, encoding the amino acid residues shown in the sequence SEQ ID NO 2 in the sequence listing sequence of proteins. The sequence SEQ ID NO 2 in the sequence listing consists of 223 amino acid residues. The gene was named OsPIS , and the protein encoded by it was named OsPIS.

Example 2 Analysis of the expression of the adversity stress of the OsPIS gene

1. Adversity stress treatment

The plump seeds of ‘Yangjing 805’ were surface-sterilized with 1% sodium hypochlorite for 20 min, washed 6 times with distilled water, then soaked in distilled water for 24-36 h, placed on wet gauze, and germinated at 32 °C for about 2 days. Seeds that germinated uniformly were sown on foamed plastic orifice plates with gauze for liquid culture. After 4 weeks of normal growth, the following stress treatments were started.

Mannitol treatment: rice seedlings were transferred from the nutrient solution to a solution containing 200 mM mannitol, and the roots and leaves of rice were harvested 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 rice are taken at 0, 3, 6, 12, 24, and 48 hours respectively;

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 rice are taken at 0, 3, 6, 12, 24, and 48 h respectively;

ABA treatment: the treatment method is the same as that of mannitol, the concentration of ABA solution is 100 μM, and the roots and leaves of rice are taken at 0, 3, 6, 12, 24, and 48 hours respectively;

Control treatment: The roots and leaves of seedlings without any treatment were directly taken as the control (0 h).

All samples were immediately frozen in liquid nitrogen and stored at -80°C.

Adversity stress qRT-PCR analysis

The root and leaf total RNA of each treatment in the above steps were extracted respectively, cDNA was obtained by reverse transcription, and qRT-PCR analysis was performed to identify the expression characteristics of the OsPIS gene after different stress treatments in rice. OsActin gene as internal reference: OsActin -F: 5'-TTATGGTTGGGATGGGACA-3' and OsActin -R: 5'-AGCACGGCTTGAATAGCG-3'; OsPIS primer sequence is: OsPIS -F: 5'-TGTTCGCCGATGAGAAGTCA-3' and OsPIS -R: 5'-GTTTCAACGCCCAACCAACT-3'.

The results are shown in Figure 1, the expression of OsPIS gene can be induced by mannitol, PEG6000, NaCl and ABA, indicating that OsPIS gene is related to plant salt tolerance and drought resistance.

Example 3 Construction of OsPIS Gene Overexpression Vector and Obtainment of Overexpressed Rice Plants

1. Construction of OsPIS gene overexpression vector

The DNA fragment identified correctly by sequencing in Example 1 containing the nucleotide shown in SEQ ID NO 1 in the sequence table was double digested with Bam HI and Sac I, and the DNA fragment was recovered with 1% agarose gel, passed through _T4DNA The recovered OsPIS gene fragment was connected with the pYPx245 plasmid containing double 35S promoters with ligase, and the recombinant plasmid AH128 containing the rice OsPIS gene was obtained through enzyme digestion identification and sequence analysis. The expression vector also contains a gusA reporter gene and a kanamycin resistance marker gene with an intron.

Transformation of rice with overexpression of OsPIS gene

The overexpression vector pCAMBIA1301- OsPIS of the rice OsPIS gene that will be constructed is transformed into rice, and the specific method is as follows:

(1) Preparation of Agrobacterium

Step (a): Transform pCAMBIA1301- OsPIS into Agrobacterium tumefaciens EHA105 strain (BiovectorCo., LTD) by electric shock method to obtain recombinant Agrobacterium A containing pCAMBIA1301- OsPIS , and name the recombinant Agrobacterium A as EHA105/pCB- OsPIS , Transformants were screened on plates containing kanamycin resistance.

Step (b): Pick Agrobacterium Amononas and inoculate it in 5 mL of LB liquid medium (rifampicin 50 μg/mL, chloramphenicol 100 μg/mL), and incubate at 28°C and 250 rpm for 20 hours.

Step (c): Transfer 1 mL of bacterial liquid into 20-30 mL of LB liquid medium (rifampicin 50 μg/mL, chloramphenicol 100 μg/mL), culture at 28°C, 250 rpm for about 12 hours, measure OD 600 ≈1.5.

Step (d): Collect the cells by centrifugation at 8000rpm, 4°C, 10min, resuspend in Agrobacterium transformation permeate solution (5% sucrose, 0.05% Silwet L-77) and dilute to OD 600 ≈ 0.8.

(2) Acquisition of mature rice embryo callus

Step (a): remove the chaff from the seeds of mature rice variety Zhonghua No. 11, and disinfect with 70% alcohol for 1-2 minutes;

Step (b): Then soak in 20% sodium hypochlorite for 30-40 min, rinse 4 times with sterile distilled water, transfer the seeds to sterilized filter paper to blot the surface moisture, and then inoculate on NB induction medium;

Step (c): After 7-10 days of dark culture, when the scutellum expands and the endosperm becomes soft, remove the embryo and bud, transfer the stripped embryogenic callus to NB subculture medium, and subculture for about 3 w Once, after 2-3 subcultures, it can be used as a recipient for transformation.

(3) Agrostalk-mediated transformation of rice callus

Step (a): Select a good embryogenic callus and put it in the above infection solution, soak for 30 min;

Step (b): Take out the callus, absorb the excess bacterial solution with sterile filter paper, and then culture it on NB co-cultivation medium until colonies appear (about 2-3 days);

Step (c): Shake and wash with sterile water 3-4 times until the supernatant is completely clean, then shake and wash with 500 mg/L cephalosporin solution for 40 minutes;

Step (d): Take out the callus, put it in a sterile petri dish with filter paper only, air-dry it at 0.4 m/s for 4 h, transfer to NB screening medium for two rounds of screening (3-4 w per round);

Step (e): pre-differentiate the resistant calli for 2-3 w, and then transfer to the differentiation medium for light culture for 2-3 w;

Step (f): When the young shoots grow to about 1 cm, they are transferred to the strong seedling medium and cultivated for about 30 days;

Step (g): Remove the sealing film and cultivate the seedlings for about 1 w, then transplant them into the soil.

PCR detection of rice plants overexpressing OsPIS gene

(1) Test method

Genomic DNA of T ₂ rice transgenic plants and wild-type plants was extracted by CTAB method. PCR detection was performed by conventional methods, and the hpt II gene primers used were: Primer 1: 5'-ACAGCGTCTCCGACCTGATGCA-3' and Primer 2: 5'-AGTCAATGACCGCTGTTATGCG-3'. In a 0.2 mL Eppendorf centrifuge tube, add 2 μL of 10×PCR buffer, 1 μL of 4dNTP (10 mol/L), 1 μL of both primers (10 μmol/L), 2 μL of template DNA (50ng/uL), 0.25 μL of Taq DNA polymerase, and add ddH ₂ O to a total volume of 20 μL. The reaction program was pre-denaturation at 94°C for 5 min, denaturation at 94°C for 30 s, refolding at 55°C for 30 s, and extension at 72°C for 2 min, a total of 35 cycles.

(2) Test results

The amplification results of electrophoresis detection are shown in Figure 2 [in Figure 2, lane M: Maker; lane W: water; lane P: positive control (recombinant plasmid pCAMBIA1301- OsPIS ); lane WT: wild-type rice plant; lane OE1-OE11: for Transgenic rice plants overexpressing pCAMBIA1301- OsPIS were transformed]. It can be seen from the figure that a target band of 591 bp was amplified from the pseudo-transgenic rice plants transformed with pCAMBIA1301- OsPIS and the positive control, indicating that the OsPIS gene had been integrated into the rice genome, and proved that these regenerated plants were transgenic plants; wild-type rice The plant did not amplify the target band of 591 bp. Transgenic plants for subsequent functional analysis.

qRT-PCR detection of rice plants overexpressing OsPIS gene

(1) Test method

The total RNA of rice lines with positive overexpression of OsPIS was extracted, reverse-transcribed to obtain cDNA, and analyzed by qRT-PCR. The untransformed rice wild type was used as a control. OsActin gene as internal reference: OsActin -F: 5'-TTATGGTTGGGATGGGACA-3' and OsActin -R: 5'-AGCACGGCTTGAATAGCG-3'; OsPIS primer sequence is: OsPIS -F: 5'-TGTTCGCCGATGAGAAGTCA-3' and OsPIS -R: 5'-GTTTCAACGCCCAACCAACT-3'.

(2) Test results

The results are shown in Figure 3. WT is a wild-type rice plant, and OE1-OE11 are all positive trans-expressed OsPIS rice plants, indicating that OsPIS is expressed to varying degrees in transgenic rice plants, and the over-expression line OE4 with the highest expression level was selected. , OE5 and OE7 were used for subsequent analysis.

Example 4 Identification of drought resistance and salt tolerance of OsPIS gene transgenic rice plants

1. In vitro identification of drought resistance and salt tolerance of transgenic rice plants

(1) Test method

The seeds of the overexpressed rice material and the wild-type material were sterilized, and the seeds were sown on MS solid plates. After the seeds germinated for 2-3 days, the seeds with the same germination state were selected and sowed on MS, MS+NaCl (200 mM) and MS+ On different middle finger tube media with mannitol (200mM), after the seedlings grew for 7-10 days, the growth vigor of the seedlings with different treatments began to show differences, and the photos and growth vigor statistics were taken, including the data of seedling length and fresh weight.

(2) Test results

The results show that under salt stress and mannitol treatment conditions, the results are shown in Figure 4. Both the overexpressed rice material and the wild-type material have smaller plants due to the presence of salt stress and mannitol stress; but the overexpressed rice material and the wild-type material Compared with WT, the growth state is relatively better, and the statistics of growth potential show that under salt stress, the seedling length and fresh weight of overexpressed rice materials are 102-124% and 159-189% higher than those of wild-type WT materials; Under mannitol stress, the seedling length and fresh weight of the overexpressed rice material were increased by 91-103% and 145-176%, respectively, compared with the wild-type WT material; indicating that overexpressing the OsPIS gene significantly improved the salt tolerance and drought resistance of the transgenic rice plants .

Pot identification of drought resistance and salt tolerance of transgenic rice plants

(1) Test method

In order to verify the salt tolerance and drought resistance of transgenic rice materials, the surface of homozygous _T2 overexpressed rice materials and wild-type rice seeds were sterilized, germinated with pure water, inoculated on MS medium, and grown for about 3-4 days. Select seedlings with consistent growth and plant them in nutrient soil with nutrient soil: vermiculite = 1:2, pay attention to watering every day, and start salt and drought stress treatment after the plants grow to 4 weeks. Irrigate with 1/2 Hoagland nutrient solution containing 200 mM NaCl once every 2 days, 200 mL each time, treat for 4 w, observe its phenotype, take pictures and investigate its survival rate; after 6 w of drought treatment, Observe their phenotype, take pictures and investigate their survival rate. The following calculation method related to the improvement of survival rate is: (survival rate of overexpressed plants-survival rate of wild-type plants)*100%/survival rate of wild-type plants.

(2) Test results

The results showed that after 4 w of salt stress treatment or 6 w of drought stress treatment, the results are shown in Figure 5. The growth status of transgenic plants was significantly better than that of wild type plants, and the survival rate of transgenic plants was significantly higher than that of wild type plants, and was significantly higher than that of wild type plants. Compared with the plants, the increase was 1090-1211% and 1071-1244%, respectively; it indicated that the overexpression of the OsPIS gene significantly improved the salt tolerance and drought resistance of the transgenic rice plants.

Example 5 Determination of Stress Resistance Physiological and Biochemical Indexes of OsPIS Gene Transgenic Rice Plants

1. Determination of Abscisic Acid Content

(1) Test method

Abscisic acid (ABA) plays an important role in plant stress response. ABA can improve the salt tolerance of plants, relieve osmotic stress and ion stress caused by excessive salinity, maintain water balance, induce the accumulation of proline, a plant osmotic regulator substance, maintain the stability of cell membrane structure, and increase the activity of protective enzymes. Under drought stress, ABA can significantly reduce leaf water evaporation, reduce leaf cell membrane permeability, increase leaf cell soluble protein content, induce the formation of biofilm system protective enzymes, reduce membrane lipid peroxidation, enhance antioxidant capacity, and improve plant drought resistance sex. Therefore, ABA can be used as a biochemical indicator of plant stress resistance.

Determination method reference [Shang Gao, Li Yuan, Hong Zhai, Chenglong Liu, Shaozhen He, Qingchang Liu. Transgenic sweetpotato plants expressing an LOS5 gene are tolerant to salt stress. Plant Cell, Tissue and Organ Culture, 2011,107: 205- 213] to detect the ABA content of rice plants. The rice plants were the rice plants treated for 2 w in the blank control, the rice plants under the salt stress for 2 w, and the rice plants under the drought stress for 4 w. The experiment was repeated three times, and the results were averaged.

(2) Test results

The experimental results of ABA content determination in rice plants are shown in A in Figure 6 (Normal is blank control, Salt stress is salt stress, and Drought stress is drought stress). The results showed that the ABA content of overexpressed rice OE4 plants, OE5 plants and OE7 plants was significantly higher than that of wild-type rice plants.

Determination of proline content

(1) Test method

Under normal conditions, the free proline content of plants is very low, but when encountering drought, salt and other stresses, free amino acids will accumulate in large quantities, and the accumulation index is related to the stress resistance of plants. Therefore, proline can be used as a biochemical indicator of plant stress resistance.

Determination method reference [Feibing Wang, Weili Kong, Gary Wong, Lifeng Fu, RihePeng, Zhenjun Li, Quanhong Yao. AtMYB12 regulates flavonoids accumulation and abiotic stress tolerance in transgenic Arabidopsis thaliana . MolecularGenetics and Genomics, 2016, 291:15945- Proline content of rice plants. The rice plants were the rice plants treated for 2 w in the blank control, the rice plants under the salt stress for 2 w, and the rice plants under the drought stress for 4 w. The experiment was repeated three times, and the results were averaged.

(2) Test results

The experimental results of proline content determination in rice plants are shown in Figure 6 B (Normal is blank control, Salt stress is salt stress, and Drought stress is drought stress). The results showed that the proline content of transgenic rice OE4 plants, OE5 plants and OE7 plants was significantly higher than that of wild-type rice plants.

₂₂ content determination

(1) Test method

When plants are under adversity or aging, H ₂ O ₂ accumulates due to the enhanced metabolism of active oxygen in the body. H ₂ O ₂ can directly or indirectly oxidize biological macromolecules such as nucleic acid and protein in cells, and damage cell membranes, thereby accelerating cell aging and disintegration. Therefore, the higher the H ₂ O ₂ content, the greater the degree of stress damage to plants.

Determination method reference [Feibing Wang, Weili Kong, Gary Wong, Lifeng Fu, RihePeng, Zhenjun Li, Quanhong Yao. AtMYB12 regulates flavonoids accumulation and abiotic stress tolerance in transgenic Arabidopsis thaliana . MolecularGenetics and Genomics, 2016, 291:15945- _H2O2 content _of rice plants. The rice plants were the rice plants treated for 2 w in the blank control, the rice plants under the salt stress for 2 w, and the rice plants under the drought stress for 4 w. The experiment was repeated three times, and the results were averaged.

(2) Test results

The experimental results of measuring H ₂ O ₂ content in rice plants are shown in Figure 6 C (Normal is blank control, Salt stress is salt stress, and Drought stress is drought stress). The results showed that the H ₂ O ₂ content of transgenic rice OE2 plants, OE4 plants and OE7 plants was significantly lower than that of wild-type rice plants.

Assay

(1) Test method

Membrane lipid peroxidation often occurs when plant organs are aging or damaged under adversity. Malondialdehyde (MDA) is the final decomposition product of membrane lipid peroxidation, and its content can reflect the degree of damage to plants by adversity. The higher the degree, the greater the degree of damage to the plant suffered from adversity.

Determination method reference [Feibing Wang, Weili Kong, Gary Wong, Lifeng Fu, RihePeng, Zhenjun Li, Quanhong Yao. AtMYB12 regulates flavonoids accumulation and abiotic stress tolerance in transgenic Arabidopsis thaliana . MolecularGenetics and Genomics, 2016, 291:15945- MDA content of rice plants. The rice plants were the rice plants treated for 2 w in the blank control, the rice plants under the salt stress for 2 w, and the rice plants under the drought stress for 4 w. The experiment was repeated three times, and the results were averaged.

(2) Test results

The experimental results of MDA content determination in rice plants are shown in D in Figure 6 (Normal is blank control, Salt stress is salt stress, and Drought stress is drought stress). The results showed that the MDA content of transgenic rice OE4 plants, OE5 plants and OE7 plants was significantly lower than that of wild-type rice plants.

activity assay

(1) Test method

The activity of superoxide dismutase (SOD) can be used as a physiological and biochemical indicator of plant stress resistance. The lower the activity of SOD, the greater the degree of stress damage to plants.

Determination method reference [Feibing Wang, Weili Kong, Gary Wong, Lifeng Fu, RihePeng, Zhenjun Li, Quanhong Yao. AtMYB12 regulates flavonoids accumulation and abiotic stress tolerance in transgenic Arabidopsis thaliana . MolecularGenetics and Genomics, 2016, 291:15945- SOD activity of rice plants. The rice plants were the rice plants treated for 2 w in the blank control, the rice plants under the salt stress for 2 w, and the rice plants under the drought stress for 4 w. The experiment was repeated three times, and the results were averaged.

(2) Test results

The results of the SOD activity assay of rice plants are shown in E in Figure 6 (Normal is blank control, Salt stress is salt stress, and Drought stress is drought stress). The results showed that the SOD activity of transgenic rice OE4 plants, OE5 plants and OE7 plants was significantly higher than that of wild-type 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 of stress damage to plants.

Determination method reference [Feibing Wang, Weili Kong, Gary Wong, Lifeng Fu, RihePeng, Zhenjun Li, Quanhong Yao. AtMYB12 regulates flavonoids accumulation and abiotic stress tolerance in transgenic Arabidopsis thaliana . MolecularGenetics and Genomics, 2016, 291:15945- POD activity of rice plants. The rice plants were the rice plants treated for 2 w in the blank control, the rice plants under the salt stress for 2 w, and the rice plants under the drought stress for 4 w. The experiment was repeated three times, and the results were averaged.

(2) Test results

The results of the POD activity assay of rice plants are shown in Figure 6 F (Normal is blank control, Salt stress is salt stress, and Drought stress is drought stress). The results showed that the POD activity of transgenic rice OE4 plants, OE5 plants and OE7 plants was significantly higher than that of wild-type rice plants.

The results of the determination of physiological and biochemical indexes showed that the overexpression of OsPIS gene significantly improved the salt tolerance and drought resistance of the transgenic rice plants.

Claims (10)

1. Protein, which is the protein shown in any of the following (a1) or (a2) or (a3): (a1) A protein whose amino acid sequence is shown in SEQ ID NO.1; (a2) A fusion protein obtained by linking protein tags at the N-terminal or/and C-terminal of the amino acid sequence shown in SEQ ID NO.1; (a3) Substitution and/or deletion and/or addition of one or several amino acid residues to the amino acid sequence shown in SEQ ID NO.1 has more than 90% identity with the protein shown in (a1) and proteins with the same function. 2. The protein-related biological material according to claim 1, characterized in that: the related biological material is any one of the following (c1) to (c10): (c1) a nucleic acid molecule encoding the protein of claim 1; (c2) an expression cassette comprising the nucleic acid molecule of (c1); (c3) A recombinant vector containing the nucleic acid molecule described in (c1), or a recombinant vector containing the expression cassette described in (c2); (c4) A recombinant microorganism containing the nucleic acid molecule described in (c1), or a recombinant microorganism containing the expression cassette described in (c2), or a recombinant microorganism containing the recombinant vector described in (c3); (c5) a transgenic plant cell line containing the nucleic acid molecule described in (c1), or a transgenic plant cell line containing the expression cassette described in (c2), or a transgenic plant cell line containing the recombinant vector described in (c3); (c6) Transgenic plant tissue containing the nucleic acid molecule described in (c1), or a transgenic plant tissue containing the expression cassette described in (c2), or a transgenic plant tissue containing the recombinant vector described in (c3); (c7) Transgenic plant organs containing the nucleic acid molecules described in (c1), or transgenic plant organs containing the expression cassettes described in (c2), or transgenic plant organs containing the recombinant vectors described in (c3); (c8) Transgenic plants containing the nucleic acid molecules described in (c1), or transgenic plants containing the expression cassettes described in (c2), or transgenic plants containing the recombinant vectors described in (c3); (c9) tissue culture produced from the regenerative cells of the transgenic plant described in (c8); (c10) Protoplasts produced from the tissue culture of (c9). 3. The related biological material according to claim 2, characterized in that: (c1) the nucleic acid molecule encoding the protein described in claim 1 is any of the following (d1) or (d2) or (d3) : (d1) DNA molecule shown in SEQ ID NO.2; (d2) The coding sequence is the DNA molecule shown in SEQ ID NO.2; (d3) A DNA molecule that hybridizes to the DNA molecule defined in (d1) or (d2) under stringent conditions and encodes the protein described in claim 1. 4. The protein of claim 1 or the related biological material of claim 2 or 3 in any of the following applications: (b1) Improve plant salt tolerance; (b2) preparation of products that increase the salt tolerance of plants; (b3) Improve plant drought resistance; (b4) preparation of products that increase the drought resistance of plants; (b5) improving rooting of plants under conditions of drought and/or salt stress; (b6) preparation of a product that improves the rooting of plants under conditions of drought and/or salt stress; (b7) Improving the growth of plants under conditions of drought and/or salt stress; (b8) Preparation of products of growth of plants under conditions of drought and/or salt stress; (b9) prepare the survival rate of plants under drought and/or salt stress conditions; (b10) Preparation of products that increase the survival of plants under conditions of drought and/or salt stress; (b11) increasing plant abscisic acid content under conditions of drought and/or salt stress; (b12) Preparation of products that increase the abscisic acid content of plants under conditions of drought and/or salt stress; (b13) increasing proline content in plants under conditions of drought and/or salt stress; (b14) Preparation of products for increasing the proline content of plants under conditions of drought and/or salt stress; (b15) Reducing plant _H2O2 content under conditions of drought and/or salt stress _; (b16) Preparation _of products for reducing the _H2O2 content of plants under conditions of drought and/or salt stress (b17) Reduction of malondialdehyde content in plants under conditions of drought and/or salt stress; (b18) Preparation of products for reducing malondialdehyde content in plants under conditions of drought and/or salt stress; (b19) improving plant SOD activity under conditions of drought and/or salt stress; (b20) preparing a product that improves plant SOD activity under conditions of drought and/or salt stress; (b21) increasing plant POD activity under conditions of drought and/or salt stress; (b22) Preparation of a product that improves plant POD activity under conditions of drought and/or salt stress. 5. Use of a protein as claimed in claim 1 or a related biological material as claimed in claim 2 or 3 in plant breeding. 6. A method for cultivating drought-resistant and high-salt-tolerant transgenic plants, characterized in that: the method comprises increasing the expression of the gene encoding the protein of claim 1 in the target plant and/or the content of the protein and/or the activity of the protein to obtain a transgenic plant; the drought resistance and salt tolerance of the transgenic plant are higher than that of the target plant. 7. The method according to claim 6, characterized in that: the method of improving the expression of the gene encoding the protein of claim 1 in the target plant and/or the content of the protein and/or the activity of the protein The method is to express or overexpress the protein of claim 1 in the target plant. 8. The method according to claim 7, characterized in that: the expression or overexpression method is to introduce the coding gene of the protein according to claim 1 into the target plant. 9. The method according to claim 8, characterized in that: the nucleotide sequence of the gene encoding the protein according to claim 1 is the DNA molecule shown in SEQ ID NO.2. 10. The application according to claim 4 or 5, or the method according to any one of claims 6-9, characterized in that: the plant is any one of the following (e1) to (e5): (e1) dicotyledonous plants; (e2) Monocots; (e3) Poaceae; (e4) Plants of the genus Oryza; (e5) Rice.

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