Protein SlRD1 and coding gene and application thereof

Abstract

The invention provides a protein SlRD1 as well as a coding gene and application thereof, and belongs to the technical field of plant genetic engineering, the amino acid sequence of the protein SlRD1 is as shown in SEQ ID NO.1, and the nucleic acid molecule for coding the protein SlRD1 is as shown in SEQ ID NO.2. The SlRD1 gene is introduced into Columbia wild-type arabidopsis thaliana to obtain SlRD1 gene-transformed arabidopsis thaliana, compared with the wild-type arabidopsis thaliana, the SlRD1 gene-transformed arabidopsis thaliana has the advantages that the resistance to drought and salt stress is obviously improved, the main root is obviously elongated, and the survival rate of the transgenic arabidopsis thaliana under salt stress is obviously higher than that of the wild-type arabidopsis thaliana; meanwhile, the sensitivity of the transgenic arabidopsis thaliana seeds to ABA is improved. The method has important research value and application value.

Description

Technical Field

[0001] The invention belongs to the technical field of plant genetic engineering, and specifically relates to protein SlRD1 and its encoding gene and application. Background Art

[0002] Tomato (Solanum lycopersicum L.) has rich nutritional value and a large market demand. It is one of the most important vegetables and horticultural crops in the world (Zhou Ming and Li Changbao, 2022). As the world's largest tomato producer and exporter, tomato has become an important vegetable crop that drives the development of my country's agricultural economy and increases farmers' income. At the same time, tomatoes have a short growth cycle, a small genome, a relatively clear genetic background, and are easy to transform, making them an important model plant for genetic research (Meissner et al., 1997). During the growth and development of tomatoes, they are often affected by a variety of adversities (such as drought, salinity, low temperature, etc.), resulting in slow seed germination, low germination rate, slow plant growth, falling flowers and fruits, etc. In severe cases, they may even cause plant death, affecting the yield and quality of tomatoes (Dong Shuchao et al., 2024). Exploring genes and molecular mechanisms related to plant resistance is of great significance for producing high-quality tomatoes under adversity.

[0003] BURP genes have only been identified in plants and are a plant-specific gene family (Phillips and Ludidiet al., 2017). The naming of the BURP gene family is based on four typical BURP members: BnBNM2 expressed during microspore embryo development in rapeseed (Boutilier et al., 1994), VfUSP in early zygotic embryo development in broad bean (Bassuner et al., 1988), and AtRD22 in Arabidopsis thaliana.

[0004] (Yamaguchi-Shinozaki and Shinozaki, 1993) and the non-catalytic β subunit LePG1β of tomato polygalacturonase isozyme 1 (Zheng et al., 1992). BURP gene family members generally contain a conserved C-terminal BURP domain and are mainly divided into BNM2-like, USP-like, RD22-like and PG1β-like subfamilies. A few species contain some unique other subfamilies (Sun et al., 2019). Studies have shown that BURP family members play an important role in plant growth and development. For example, Arabidopsis BURP protein AtUSPL1 is synthesized in the vacuole and regulates seed development (Van Son et al., 2009). OsRAFTIN1 and RA8 in rice play an important regulatory role in anther development (Jeon et al., 1999; Wang et al., 2003). SCB1 in soybean is specifically expressed in the seed coat and may be involved in regulating the differentiation of soybean seed coat thin wall cells (Batchelor et al., 2002). More and more studies have found that BURP family genes have important functions in hormone and stress response processes. AtUSPL1 of the RD22-like subfamily inhibits the drought stress response of plants through the ABA pathway (Harshavardhan et al., 2014). The expression of BURP family members in mangroves, BgBDC1-4, is induced by salt and drought stress (Banzai et al., 2001). Heterologous overexpression of soybean BURP gene SALI3-2 can improve the salt tolerance of yeast cells (Tang et al., 2007). BnBDC1, which is homologous to AtRD22, is induced by salt, osmotic and ultraviolet irradiation, as well as abscisic acid (ABA) and salicylic acid (SA) and other stresses (Yu et al., 2004). The above results indicate that BURP family genes play a certain role in plant drought and salt stress, and may be related to plant hormone signals such as ABA and SA, but the related regulatory mechanisms need further study. Summary of the invention

[0005] In view of the above problems, the present invention identified 14 BURP family members containing BURP domains from the tomato genome in the early stage, and screened SlRD1, which was significantly induced by drought and salt stress, through fluorescence quantitative PCR analysis (qRT-PCR). SlRD1 was cloned from tomato Micro-Tom, and an overexpression vector of the gene was constructed and transformed into the Columbia wild-type Arabidopsis 'col' by the inflorescence infection method. Through drought stress, salt stress treatment and ABA treatment, it was found that overexpression of SlRD1 promoted the elongation of the main root of transgenic Arabidopsis, improved the resistance of transgenic Arabidopsis to drought and salt stress, and at the same time improved the sensitivity of transgenic Arabidopsis seeds to ABA.

[0006] The technical solution adopted by the present invention is:

[0007] First, the protective protein SlRD1 of the present invention is derived from tomato Micro-Tom, and its amino acid sequence is shown in SEQ ID NO.1.

[0008] The nucleic acid molecule encoding the protein S1RD1 also falls within the protection scope of the present invention.

[0009] The nucleic acid molecule encoding protein S1RD1 is any one or more of the following sequences:

[0010] a1) the coding region is a DNA molecule as shown in SEQ ID NO.2;

[0011] a2) the nucleotide sequence is the DNA molecule shown in SEQ ID NO.2;

[0012] a3) a DNA molecule that has 75% or more identity with the nucleotide sequence defined in a1) or a2), is derived from tomato and encodes the protein S1RD1;

[0013] a4) a DNA molecule derived from tomato and encoding the protein S1RD1 that hybridizes under stringent conditions with the nucleotide sequence defined in a1) or a2).

[0014] 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.

[0015] A person skilled in the art can easily mutate the nucleotide sequence encoding the protein SlRD1 of the present invention by using known methods, such as directed evolution and point mutation. Those artificially modified nucleotides having 75% or higher identity with the nucleotide sequence of the protein SlRD1 isolated from the present invention are derived from the nucleotide sequence of the present invention and are equivalent to the sequence of the present invention as long as they encode the protein SlRD1.

[0016] The term "identity" as used herein refers to sequence similarity with a natural nucleic acid sequence. "Identity" includes nucleotide sequences that have 75% or more, or 80% or more, or 85% or more, or 90% or more, or 95% or more identity with the nucleotide sequence of the protein S1RD1 of the present invention encoding the amino acid sequence shown in SEQ ID NO.1. Identity can be evaluated by the naked eye or by computer software. Using computer software, the identity between two or more sequences can be expressed as a percentage (%), which can be used to evaluate the identity between related sequences.

[0017] An expression cassette, recombinant vector, recombinant microorganism or transgenic cell line containing any of the above-mentioned nucleic acid molecules also falls within the protection scope of the present invention.

[0018] The recombinant vector containing any of the above-mentioned nucleic acid molecules can specifically be the recombinant plasmid pBI121-SlRD1 mentioned in the examples. The recombinant plasmid pBI121-SlRD1 can be a recombinant plasmid obtained by replacing the small DNA fragment between the restriction endonuclease XbaI recognition sequence and the restriction endonuclease SmaI recognition sequence of the pBI121 plasmid with a DNA molecule having a nucleotide sequence shown in SEQ ID NO.2.

[0019] The recombinant microorganism containing any of the above-mentioned nucleic acid molecules may be a recombinant bacterium obtained by introducing a recombinant vector containing any of the above-mentioned nucleic acid molecules into a starting microorganism.

[0020] The starting microorganism may be Agrobacterium or Escherichia coli. The Agrobacterium may specifically be Agrobacterium tumefaciens GV3101.

[0021] The recombinant microorganism containing any of the above-mentioned nucleic acid molecules can specifically be GV3101 / pBI121-SlRD1 mentioned in the examples (ie, the recombinant Agrobacterium GV3101 is transformed with the recombinant plasmid pBI121-SlRD1 to obtain the recombinant Agrobacterium).

[0022] The transgenic cell lines do not include reproductive material.

[0023] Secondly, the present invention also protects the use of any of the above-mentioned proteins S1RD1, or any of the above-mentioned nucleic acid molecules, or an expression cassette, a recombinant vector, a recombinant microorganism or a transgenic cell line containing any of the above-mentioned nucleic acid molecules, which is at least one of the following b1)-b2):

[0024] b1) regulating plant resistance to drought stress;

[0025] b2) regulating plant resistance to salt stress;

[0026] b3) Regulate the seed germination ability of plants under ABA treatment conditions.

[0027] In addition, the present invention also protects the use of any of the above-mentioned proteins SlRD1, or any of the above-mentioned nucleic acid molecules, or an expression cassette, a recombinant vector, a recombinant microorganism or a transgenic cell line containing any of the above-mentioned nucleic acid molecules in cultivating drought-resistant and / or salt-tolerant transgenic plants.

[0028] In the above-mentioned protein S1RD1 or the above-mentioned use, the plant is any one of the following c1) to c3): c1) dicotyledonous plants; c2) tomato; c3) tomato 'Micro-Tom'; c4) Arabidopsis thaliana; c5) wild-type Arabidopsis thaliana 'col'.

[0029] In addition, the present invention also protects a method for cultivating transgenic plants, comprising the following steps: increasing the expression level of any of the above-mentioned proteins SlRD1 in the starting plant to obtain a transgenic plant; under drought stress and salt stress conditions, the main root growth and / or seed germination rate of the transgenic plant is increased compared with the control group plants.

[0030] The "increasing the expression level of any of the above-mentioned proteins SlRD1 in the starting plant" can be achieved by methods well known in the art such as multiple copies, changing promoters, regulatory factors, and transgenics to increase the expression level and / or activity of protein SlRD1.

[0031] In the above method, the "increasing the expression level and / or activity of the protein S1RD1 in the starting plant" can be achieved by introducing a nucleic acid molecule encoding any of the above-mentioned proteins S1RD1 into the starting plant.

[0032] The "introduction of a nucleic acid molecule encoding any of the above-mentioned proteins SlRD1 into the starting plant" can be achieved by introducing a recombinant vector containing a nucleic acid molecule encoding any of the above-mentioned proteins SlRD1 into the starting plant. The "recombinant vector containing a nucleic acid molecule encoding any of the above-mentioned proteins SlRD1" can be the recombinant plasmid pBI121-SlRD1.

[0033] Finally, the present invention also protects a plant breeding method, comprising the following steps: increasing the content of any of the above-mentioned proteins SlRD1 in the plant, thereby improving the resistance of the transgenic plant to drought and salt stress; and at the same time, improving the sensitivity of the transgenic plant seeds to ABA.

[0034] Any of the plants described above is any one of the following c1) to c3): c1) a dicotyledonous plant; c2) tomato; c3) tomato 'Micro-Tom'; c4) Arabidopsis thaliana; c5) wild type Arabidopsis thaliana 'col'.

[0035] Beneficial effects of the present invention:

[0036] The present invention finds that overexpression of SlRD1 promotes the elongation of the main root of transgenic Arabidopsis thaliana through drought stress, salt stress treatment and ABA treatment, improves the resistance of transgenic Arabidopsis thaliana to drought and salt stress, and at the same time, improves the sensitivity of transgenic plant seeds to ABA. The results of transgenic Arabidopsis thaliana show that SlRD1 has a regulatory function in regulating the drought resistance, salt tolerance and main root development of Arabidopsis thaliana, and it is speculated that the gene may also have similar regulatory functions in the drought resistance, salt tolerance and main root development of tomatoes. The present invention has important research value and application value. BRIEF DESCRIPTION OF THE DRAWINGS

[0037] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings required for use in the embodiments or the description of the prior art will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying creative work.

[0038] Figure 1 Agarose gel electrophoresis was used to detect the amplified fragment of SlRD1;

[0039] Figure 2 The expression of SlRD1 gene in different tissues (A), drought (B), salt stress (C) and ABA (D) treatments;

[0040] Figure 3 SlRD1 expression in Arabidopsis lines overexpressing SlRD1 and WT;

[0041] Figure 4 Phenotypes of Arabidopsis lines overexpressing SlRD1 and WT seedlings (A) and statistical analysis (B);

[0042] Figure 5 Statistical analysis of the phenotypes (A), taproot length (B), and survival rate (C) of Arabidopsis lines overexpressing SlRD1 and WT seedlings under drought and salt treatments;

[0043] Figure 6 The phenotypes of Arabidopsis thaliana lines overexpressing SlRD1 and WT seeds germinated on 1 / 2MS and 1 / 2MS+10μM ABA medium for 5 days (A) and the seed germination rate on 1-7 days of germination (B). DETAILED DESCRIPTION

[0044] To make the purpose, technical solution and advantages of the embodiments of the present invention more clear, the technical solution in the embodiments of the present invention will be clearly and completely described below in conjunction with the accompanying drawings in the embodiments of the present invention.

[0045] Unless otherwise specified, the test materials used in the following examples were purchased from conventional biochemical reagent stores.

[0046] The quantitative tests in the following examples were all repeated three times, and the results were averaged.

[0047] 1 Experimental materials and methods

[0048] 1.1 Plant materials

[0049] Experimental materials used: Micro-Tom tomato and Columbia wild-type Arabidopsis thaliana stored in our laboratory. Tomato seedlings and transplants were carried out in a growth room with a light condition of 26℃ / 16 hours and a dark condition of 18℃ / 8 hours. Samples of roots, stems, leaves, flowers, green-ripe fruits, broken-color fruits and red-ripe fruits were taken respectively. Six-leaf seedlings with basically the same growth were selected, and leaves were treated with PEG6000 (20%), NaCl (200mM and 400mM) and ABA (100μM). Leaf samples were taken at different treatment time points. Seedlings that were not treated under the same environment were used as controls. Five seedlings were selected at each time point, and three biological replicates were set. The sample tissues were quickly frozen in liquid nitrogen and stored at -80℃ for future use. Arabidopsis thaliana was planted in a light incubator with a light condition of 22℃ / 16 hours and a dark condition of 22℃ / 8 hours.

[0050] 1.2 Reagents and consumables

[0051] (1) Restriction endonucleases, PCR reaction system, gel recovery kit, cloning kit, plasmid extraction kit (Shenggong), DNA extraction kit (Tiangen), polysaccharide and polyphenol plant RNA extraction kit (Tiangen), reverse transcription and fluorescence quantification kit (GenStar), etc.

[0052] (2) Other drugs: Agarose was a Spanish product. Peptone, yeast extract, chloroform, isoamyl alcohol, ethanol, isopropanol, sodium chloride, etc. were domestic analytical grade. Kanamycin and ampicillin were purchased from Bao Biotechnology Dalian Co., Ltd. Escherichia coli competent cells DH5α and Agrobacterium competent cells Gv3101 were purchased from Bao Biotechnology Dalian Co., Ltd.

[0053] (3) Culture medium: LB liquid culture medium: 10 g / L tryptone, 5 g / L yeast extract, 10 g / L sodium chloride (NaCl); LB solid culture medium: 10 g / L tryptone, 5 g / L yeast extract, 10 g / L sodium chloride (NaCl), 15 g / L agar powder, fixed to 1 L; LB selective culture medium: before LB plating, add antibiotics of corresponding concentration when the culture medium is autoclaved and cooled to 55 degrees, shake well and then spread on the plate.

[0054] (4) Main instruments: PCR amplifier (BIO-RAD), high-speed centrifuge (Hettich MIKRO 200R), electrophoresis equipment (BIO-RAD), gel imaging system (BIO-RAD), fluorescence quantitative PCR instrument (Applied Biosystems StepOnePlus), etc.

[0055] 2 Test methods and results

[0056] 2.1 Cloning of CDS of Tomato SlRD1 (Cloning of the Gene Encoding Protein SlRD1 (SlRD1 Gene))

[0057] The RNA of different tissues and different treatment samples of tomato was extracted using the Plant Polysaccharide and Polyphenol Total RNA Extraction Kit (Tiangen, Cat No. DP441). The above RNA samples were reverse transcribed and synthesized into cDNA using the StarScript II First-strand cDNA Synthesis Mix WithgDNARemover Kit (GenStar, Cat No. A230). The cDNA of tomato leaves was used as a template and amplified using the specific primers SlRD1-F: ATGGAGTTGAAGTTTCTTCACAT and SlRD1-R:

[0058] CTAGTTCTTAGGGACCCAAACA, PCR amplification was performed using 2×SuperNovaPCR Mix (GenStar, CatNo.A064), reaction system: cDNA template 5μl; forward primer (SlRD1-F): 2.5μl; reverse primer (SlRD1-R): 2.5μl; 2×SuperNovaPCRMix: 25μl; sterile water: 15μl. Reaction conditions: ①98℃, 3min; ②98℃, 15s; ③55℃, 15s; ④72℃, 40s; (②-④ steps 30 cycles); ⑤72℃, 10min. The expansion product was recovered by gel, the purified product was connected to the cloning vector (pMD19-T), transformed into DH5α Escherichia coli cells, single colonies were picked for PCR detection, and sent to Sangon Biotech Co., Ltd. (Xi'an) for sequencing.

[0059] The results showed that the CDS fragment of SRD1 was amplified from Micro-Tom and the fragment of the gene was about 1000 bp. Figure 1 The CDS length of S1RD1 obtained by sequencing was 1098 bp, and the sequence of SEQ ID NO.2 was as follows:

[0060] ATGGAGTTGAAGTTTCTTCACATCCTTACATATCTTTCATTGGCACTAGTGGC

[0061] AAGTCATGCAGCTCTTCCTGTTACCTATTGGAATACTAAGCTGCCTAATACTC

[0062] CAATACCTAAGGCAATCAAAGAATCTCTCCAGCCTTCTGGACTTACGGAAG

[0063] ACAAAAGCACTTCAGTAGATGTAGGCAAAGGTGGAGTAAACGTCGGAGTC

[0064] GACAAGGGCCGCCACCACTCCGGTGGAACAAACGTGAACGTTGGAGGCAA

[0065] TAAAGGCGGCGCGTTAACGTGGACACTCCAGGTGGGACCCACGTAGGCG

[0066] TTGGTAAAGGAGGAGTTGGTGTTACAACCCCCGGCCACCATGGAAAACCA

[0067] CCAGTCTCCGTGGGTGTTCACCCTGGACCCTCACCGTTTGTTTACAATTACG

[0068] CGGCGAAAGACGATCAACTTAACGATAACCCTAACGTGGCGTTATTTTTCCT

[0069] GGAAAAAGATTTGCATGAGGGAAGTAACATGAAGTTGAAGTTTGTGGAAA

[0070] ATGGCAATGGAGCTTCGTTTTTGCCCCGTCAAGAGGCGGATTCGATTCCGTT

[0071] TAGCTCAGAGAAGATGCGGAGATACTTCCACAAGTTTTCAGTGGATGAGGA

[0072] TAGTGAAGAAGGTCAGATTATGAAGAAACAGTAAGAGAATGGAAGAGC

[0073] CAGGTATTAAAGGAGAGGAGAAGTATTGTGCAACTTCCATTAGAATCCATGAT

[0074] TGATTTCACCACATCAAAGTTAGGAACAAAGTGCAACCATTATCAACAGA

[0075] GACACAAAAAGAAAACACAGAAATGCAAAAATACACAATCCTAGGAGCCA

[0076] AGAAAATGGGAAACAACAATGACAAATCAGTAGTTTGTCACAAAACAAAAC

[0077] TATGCCTATGCAGTATTTTATTGTCACAAAACAGAAACAACAGAGTCATACA

[0078] TGGTATCATTAGTTGGTGTTGATGGAACAAAGTTAAGGCTGTGGCTGTTTG

[0079] TCACAAAGACACATCACAATGGAACCCAAAACATTTGGCTTTTAAAGTTCT

[0080] TAAGGTTACACCTGGATCCGTCCCTGTTTGTCATTTCCTTCCACAAGATCAC

[0081] ATTGTTTGGGTCCCTAAGAACTAG

[0082] Bioinformatics analysis showed that the SlRD1 gene encoded 365 amino acids, the relative molecular weight of the protein was 39.55 kD, and the isoelectric point was 7.26.

[0083] The amino acid sequence encoded by the SlRD1 gene is:

[0084] MELKFLHILTYLSLALVASHAALPVTYWNTKLPNTPIPKAIKESLQPSGLTEDKSTSVDVGKGGVNVGVDKGRHHSGGTNVNVGGNKGGGVNVDTPGGTHVGVGKGGVGVTTPGHHGKPPVSVGVHPGPSPFVYNYAAKDDQLNDNPNVALFFLEKDLHEGSNMKLKFVENGNGASFLPRQE ADSIPFSSEKMPEILHKFSVDEDSEEGQIMKKTVRECEEPGIKGEEKYCATSLESMIDFTTSKLGNKVQPLSTETQKENTEMQKYTILGAKKMGNNNDKSVVCHKQNYAYAVFYCHKTETTESYMVSLVGVDGTKVKAVAVCHKDTSQWNPKHLAFKVLKVTPGSVPVCHFLPQDHIVWVPKN

[0085] 2.2 Quantitative analysis of tomato SlRD1 expression

[0086] Real-time fluorescence quantitative PCR (qRT-PCR) was performed using the RealStar Green FastMixture with ROX (2×) kit (GenStar, Cat No. A303) and Applied Biosystems StepOnePlus instrument; quantitative specific primers: SlRD1-QF: GCAATCAAAGAATCTCTCCAGCC and SlRD1-QR: GTTCACGTTTGTTCCACCGG. Reaction system (20 μL): Buffer 2 (10 μL), upstream and downstream primers (10 μM, 0.4 μL), cDNA template (2 μL) and ddH2O (7.2 μL). The reaction program was set as follows: 95°C, 2 min; 95°C, 5 s; 60°C, 30 s (40 cycles); melting curve process: 95°C, 15 s; 60°C, 20 s; 95°C, 15 s. SlEF1α (Solyc06g005060.3) was used as the internal reference gene, and each reaction was repeated 3 times. Calculate the relative expression of genes.

[0087] The results showed that the expression characteristics of SlRD1 in different tissues were analyzed by qRT-PCR, and it was found that the expression level of SlRD1 was the highest in red-ripe fruit, followed by leaves; in addition, SlRD1 was expressed in other tissues to a certain extent ( Figure 2 -A). Under drought treatment, the expression of SlRD1 was significantly upregulated, especially at 1h( Figure 2 -B). Under different salt concentration treatments, the expression of SlRD1 was significantly upregulated. When the salt concentration was 200mM, the expression of SlRD1 was significantly upregulated at 3d, with an increase of 4.3 times; when the salt concentration was 400mM, the expression of SlRD1 was significantly upregulated at 12h, 24h and 3d, especially at 24h, with an increase of 5.7 times ( Figure 2 -C). Under ABA treatment, the expression of SlRD1 was significantly upregulated at different time points of treatment and showed a gradual upward trend ( Figure 2 -D). The above results indicate that SlRD1 may be involved in the response process of drought and salt stress, and may also be involved in the ABA signaling pathway.

[0088] 2.3 Construction of SlRD1 overexpressing vector and Arabidopsis transformation

[0089] Using pBI121 as an overexpression vector, the CDS sequence of SlRD1 was constructed into the pBI121 vector by homologous recombination using specific amplification primers SlRD1-OE-F: CACGGGGGACTCTAGAATGGAGTTGAAGTTTCTTCACAT and SlRD1-OE-R: GACGGCCAGTGAGCTCCTAGTTCTTAGGGACCCAAACA to obtain the recombinant plasmid pBI121-SlRD1 and transform Agrobacterium GV3101.

[0090] Arabidopsis thaliana 'col' was transformed by Agrobacterium-mediated inflorescence infection. Genomic DNA of wild type and positive seedlings was extracted using a plant genomic DNA extraction kit (Tiangen) to check positive transgenic tomato plants with primers 35S: CTATCCTTCGCAAGACCCTTC and SlRD1-YR: CTGACCTTCTTCACTATCCTC. The expression level of SlRD1 was identified by qRT-PCR.

[0091] The results showed that the SlRD1 overexpression vector was constructed by homologous recombination, and the recombinant plasmid (pBI121-SlRD1) was transformed into Arabidopsis thaliana by Agrobacterium-mediated inflorescence infection. RNA of T3 transgenic tomatoes was extracted, reverse transcribed, and qRT-PCR was performed using SlRD1-specific fluorescent quantitative primers. The results showed that the expression level of SlRD1 in transgenic lines (OE#3, OE#6, and OE#8) was significantly increased compared with the control (WT) ( Figure 3 ).

[0092] (4) Phenotypic observation and statistics of transgenic Arabidopsis thaliana overexpressing SlRD1 under drought stress

[0093] Three positive homozygous T3 lines and the wild type were selected and cultured on plates under the same environment, and the taproot length of the Arabidopsis seedlings was observed and counted. The wild type and transgenic Arabidopsis seedlings that had germinated for 3 days were transplanted into 1 / 2MS, 1 / 2MS+100mM mannitol and 1 / 2MS+100mM NaCl medium, respectively, and the growth of the seedlings and the taproot length were observed and counted. The sterilized wild type and transgenic Arabidopsis seeds were respectively placed in 1 / 2MS and 1 / 2MS+10μM ABA medium for germination and growth, and the germination of the seeds was observed and counted at different times.

[0094] Phenotypes and statistics of transgenic Arabidopsis under drought, salt and ABA treatments:

[0095] (1) The sterilized SlRD1-overexpressing transgenic Arabidopsis thaliana (OE#3, OE#6, and OE#8) and wild-type (WT) seeds were spotted in 1 / 2MS medium. The phenotypes of the seedlings after 5 days of growth were observed and statistically analyzed. It was found that the taproot of the transgenic Arabidopsis thaliana was significantly elongated ( Figure 4 ).

[0096] (2) The sterilized SlRD1-overexpressing transgenic Arabidopsis thaliana (OE#3, OE#6, and OE#8) and wild-type (WT) seeds were plated in 1 / 2MS medium. The 3-day-old seedlings were transplanted into 1 / 2MS, 1 / 2MS+100mM mannitol, and 1 / 2MS+100mM NaCl medium, respectively. The phenotypes of the 5-day-old seedlings were observed and statistically analyzed. It was found that the drought resistance and salt tolerance of the transgenic Arabidopsis thaliana were enhanced ( Figure 5 -A), especially under salt treatment, the survival rate of transgenic Arabidopsis was significantly higher than that of wild type ( Figure 5 -C). In addition, under mannitol and salt treatment, the taproot of transgenic Arabidopsis thaliana increased significantly ( Figure 5 -B).

[0097] (3) The sterilized SlRD1-overexpressing transgenic Arabidopsis (OE#3, OE#6, and OE#8) and wild-type (WT) seeds were spotted in 1 / 2MS and 1 / 2MS+10μM ABA medium, respectively, and the seed germination rates from 1d to 7d were observed and counted. It was found that the seed germination rate of transgenic Arabidopsis was significantly reduced under ABA treatment, indicating that overexpression of SlRD1 increases the sensitivity of Arabidopsis seeds to ABA ( Figure 6 -A, B).

[0098] The embodiments described above are only preferred specific implementation modes of the present invention, and the protection scope of the present invention is not limited thereto. Any simple changes or equivalent replacements of the technical solutions that can be obviously obtained by any technician familiar with the field within the technical scope disclosed in the present invention belong to the protection scope of the present invention.

Claims

1. The amino acid sequence of protein S1RD1 is shown in SEQ ID NO.

1.

2. A nucleic acid molecule encoding the protein S1RD1 according to claim 1.

3. The nucleic acid molecule according to claim 2, characterized in that The nucleic acid molecule is any one or more of the following sequences: a1) the coding region is a DNA molecule as shown in SEQ ID NO.2; a2) the nucleotide sequence is the DNA molecule shown in SEQ ID NO.2; a3) a DNA molecule having 75% or more identity with the nucleotide sequence defined in a1) or a2), derived from tomato and encoding the protein S1RD1 according to claim 1; a4) A DNA molecule which hybridizes with the nucleotide sequence defined in a1) or a2) under stringent conditions, is derived from tomato and encodes the protein S1RD1 according to claim 1.

4. An expression cassette, recombinant vector, recombinant microorganism or transgenic cell line containing the nucleic acid molecule according to claim 2 or 3.

5. Use of the protein S1RD1 according to claim 1, or the nucleic acid molecule according to claim 2 or 3, or the expression cassette, recombinant vector, recombinant microorganism or transgenic cell line containing the nucleic acid molecule according to claim 2 or 3, is at least one of the following b1)-b2): b1) regulating plant resistance to drought stress; b2) regulating plant resistance to salt stress; b3) Regulate the seed germination ability of plants under ABA treatment conditions.

6. Use of the protein S1RD1 according to claim 1, or the nucleic acid molecule according to claim 2 or 3, or an expression cassette, a recombinant vector, a recombinant microorganism or a transgenic cell line containing the nucleic acid molecule according to claim 2 or 3 in cultivating drought-resistant and / or salt-tolerant transgenic plants.

7. The protein S1RD1 according to claim 1, or the use according to claim 5 or 6, characterized in that: The plant is any one of the following c1) to c3): c1) a dicot; c2) tomato; c3) tomato 'Micro-Tom'; c4) Arabidopsis thaliana; c5) wild type Arabidopsis thaliana 'col'.

8. A method for cultivating transgenic plants, characterized in that: The method comprises the following steps: increasing the expression level of the protein SlRD1 according to claim 1 in the starting plant to obtain a transgenic plant; under drought stress and salt stress conditions, compared with the control group plants, the main root of the transgenic plant grows, the drought and salt resistance is enhanced, and / or under ABA treatment conditions, the seed germination rate of the transgenic plant is reduced.

9. A plant breeding method, characterized in that: The method comprises the following steps: increasing the content of the protein SlRD1 according to claim 1 in the plant, thereby improving the resistance of the transgenic plant to drought and salt stress; and at the same time, improving the sensitivity of the transgenic plant seeds to ABA.

10. The method according to claim 8 or 9, characterized in that: The plant is any one of the following c1) to c3): c1) a dicotyledon; c2) tomato; c3) tomato 'Micro-Tom'; c4) Arabidopsis thaliana; c5) wild type Arabidopsis thaliana 'col'.

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