CN116606821A - Plant salt-alkali-resistant protein GsSIE3, and coding gene and application thereof - Google Patents

Abstract

A plant salt-alkali resistant protein GsSIE3, a coding gene and application thereof belong to the technical field of genetic engineering. In order to improve salt and alkalinity resistance of soybean, the invention provides a GsSIE3 protein with an amino acid sequence shown as SEQ ID NO.2 and a nucleotide sequence which codes the protein and is shown as SEQ ID NO.1, experiments prove that the GsSIE3 gene can respond to salt and alkaline stress reaction, and the GsSIE3 and the GsSnRK1 are physically related, and the GsSnRK1 can phosphorylate the GsSIE3 at a T514 site. Co-expression of GsSnRK1 and GsSIE3 in soybean hairy roots shows that the combination of GsSnRK1 (wt) and GsSIE31 (wt) can significantly increase the resistance of soybeans to saline-alkali stress, and reveals the molecular mechanism that GsSnRK1 and GsSIE3 are cooperatively involved in regulating the salt-alkali tolerance of plants.

Description

A kind of plant salt-alkali resistance protein GsSIE3 and its coding gene and application

technical field

The invention relates to a plant salt-alkali resistance protein GsSIE3 and its coding gene and application, belonging to the field of biotechnology.

Background technique

Salt stress is one of the environmental stress factors affecting crop yields in many parts of the world. Soybean (Glycinemax), as an important crop widely planted in my country, has lost many salt-resistant genes during the long-term artificial breeding process, making soybean more sensitive to salt stress in the soil, and wild soybean (Glycine soja) as a cultivated soybean Relative species have strong environmental adaptability and are important germplasm resources.

In recent years, it has become possible to excavate the key regulatory genes of salt tolerance through genetic engineering, and to improve crop salt tolerance through molecular breeding of genetic engineering technology, thereby increasing crop yield. However, the important prerequisite for its realization is to mine the key regulatory genes of salt tolerance.

Contents of the invention

In order to solve the problem of how to improve the salt-alkaline tolerance of plants, the present invention provides a plant salt-alkali resistance protein GsSIE3, the amino acid sequence of which is shown in SEQ ID NO.2.

Further defined, the plant salt-alkali resistance protein GsSIE3 also includes a fusion protein obtained by linking a tag to the N-terminal and/or C-terminal of the protein whose amino acid sequence is shown in SEQ ID NO.2.

To further define, in order to facilitate the purification of the protein whose amino acid sequence is shown in SEQ ID NO.2, an HA tag or a Myc tag can be attached to the amino-terminal or carboxy-terminal of the protein whose amino acid sequence is shown in SEQ ID NO.2.

The above-mentioned plant salt-alkali-resistant protein GsSIE3 is a protein having the following amino acid residue sequence: the 477th to 522th amino acid residue sequence from the carboxyl end of the amino acid residue sequence SEQ ID NO.2 is the ubiquitin ligase active region RING-Ubox domain , has a highly conserved C ₃ HC ₄ RING motif, and its RING motif belongs to the HC subgroup, which belongs to RING-Ubox E3 ubiquitin ligase.

T514 in the amino acid residue sequence from the 477th to the 522nd amino acid residue sequence at the carboxyl end of the amino acid residue sequence SEQID NO.2 in the ubiquitin ligase active domain of the above-mentioned plant salt-alkali-resistant protein GsSIE3 is a site regulated by phosphorylation.

The present invention also provides the coding sequence of the above-mentioned plant salt-alkali resistance protein GsSIE3, which is shown in SEQ ID NO.1.

Further defined, the coding sequence also includes any of the following:

1) cDNA molecule or DNA molecule as shown in SEQ ID NO.1;

2) A cDNA molecule or a genomic DNA molecule that has more than 75% identity with the coding sequence shown in SEQ ID NO.1 and encodes the protein GsSIE3.

Further defined, the coding sequence also includes a cDNA molecule or a genomic DNA molecule that hybridizes with the coding sequence defined in 1) or 2) above and encodes the protein GsSIE3.

To further define, the coding sequence may be DNA, such as cDNA, genomic DNA or recombinant DNA, or RNA, such as mRNA or hnRNA.

Those skilled in the art can easily use known methods, such as directed evolution and point mutation methods, to mutate the nucleotide sequence encoding the GsSIE3 protein of the present invention. Those artificially modified nucleotides that have 75% or more identity to the nucleotide sequence encoding the GsSIE3 protein, as long as they encode the GsSIE3 protein and have the same function, are derived from the nucleotide sequence of the present invention and are equivalent to Sequences of the invention.

A pair of primers for amplifying the full-length coding sequence of the above-mentioned GsSIE3 protein or a fragment thereof also belongs to the protection scope of the present invention.

The present invention also provides a biological material related to the above coding sequence, the biological material is any one of the following materials:

A1) an expression cassette containing said coding sequence;

A2) a recombinant vector containing the coding sequence;

A3) A recombinant microorganism containing the coding sequence.

To further define, the above-mentioned biological material can also be any one of the following materials:

A4) a recombinant vector containing the expression cassette described in A1);

A5) a recombinant microorganism containing the expression cassette described in A1);

A6) A recombinant microorganism containing the recombinant vector described in A2).

Further defined, the biological material also includes recombinant microorganisms containing the recombinant vector described in A4).

In the above biological materials, the vector can be a plasmid, a cosmid, a phage or a viral vector.

In the above biological materials, the microorganisms can be yeast, bacteria, algae or fungi, such as Agrobacterium.

Among the above-mentioned biological materials, the expression cassette (GsSIE3 gene expression cassette) described in A1) containing the nucleic acid molecule encoding the GsSIE3 protein refers to the DNA capable of expressing the GsSIE3 protein in the host cell. A terminator that terminates transcription of GsSIE3 may also be included. Further, the expression cassette may also include an enhancer sequence. Promoters that can be used in the present invention include, but are not limited to, constitutive promoters, tissue-, organ- and development-specific promoters, and inducible promoters. Examples of promoters include, but are not limited to: the constitutive promoter 35S of cauliflower mosaic virus, the wound-inducible promoter from tomato, leucine aminopeptidase ("LAP", Chao et al. (1999) Plant Physiol 120: 979-992), a chemically inducible promoter from tobacco, pathogenesis-related 1 (PR1) (inducible by salicylic acid and BTH (benzothiadiazole-7-thiohydroxyacid S-methyl ester), tomato protease Inhibitor II promoter (PIN2) or LAP promoter (both can be induced by methyl jasmonate), heat shock promoter (US Patent 5187267), tetracycline-inducible promoter (US Patent 5057422), seed-specific promoter ( Such as millet seed-specific promoter pF128 (Chinese patent 200710099169.7)), seed storage protein-specific promoters (for example, the promoters of phaseolin, napin, oleosin and soybean beta conglycin (Beachy et al. (1985) EMBO J.4 :3047-3053)). They can be used alone or in combination with other plant promoters. All references cited herein are cited in full. Suitable transcription terminators include, but are not limited to: Agrobacterium nopaline synthase terminator (NOS terminator), cauliflower mosaic virus CaMV 35S terminator, tml terminator, pea rbcS E9 terminator and nopaline and octopine synthase terminators (see, e.g.: Odell et al. ( ¹⁹⁸⁵ ) Nature 313 Rosenberg et al. (1987) Gene, 56:125; Guerineau et al. (1991) Mol.Gen.Genet, 262:141; Proudfoot (1991) Cell, 64:671; Sanfacon et al. Genes Dev., 5: 141; Mogen et al. (1990) Plant Cell, 2:1261; Munroe et al. (1990) Gene, 91:151; Ballad et al. (1989) Nucleic Acids Res. 17:7891; Joshi et al. (1987) Nucleic Acids Res. .,15:9627).

An existing expression vector can be used to construct a recombinant vector containing the expression cassette of the GsSIE3 gene. The plant expression vectors include binary Agrobacterium vectors and vectors that can be used for plant microprojectile bombardment and the like. Such as pAHC25, pBin438, pCAMBIA1302, pCAMBIA2301, pCAMBIA1301, pCAMBIA1300, pBI121, pCAMBIA1391-Xa or pCAMBIA1391-Xb (CAMBIA Company), etc. The plant expression vector may also include the 3' untranslated region of the foreign gene, that is, the polyadenylation signal and any other DNA fragments involved in mRNA processing or gene expression. The polyadenylic acid signal can guide polyadenylic acid to be added to the 3' end of the mRNA precursor, such as Agrobacterium crown gall tumor induction (Ti) plasmid gene (such as nopaline synthase gene Nos), plant gene (such as soybean The untranslated region transcribed at the 3' end of the storage protein gene) has similar functions. When using the gene of the present invention to construct plant expression vectors, enhancers can also be used, including translation enhancers or transcription enhancers, and these enhancer regions can be ATG initiation codons or adjacent region initiation codons, etc. The reading frames of the sequences are identical 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 order to facilitate the identification and screening of transgenic plant cells or plants, the plant expression vectors used can be processed, such as adding genes (GUS gene, luciferase gene, etc.) genes, etc.), antibiotic marker genes (such as the nptll gene that confers resistance to kanamycin and related antibiotics, the bar gene that confers resistance to the herbicide phosphinothricin, and the hph gene that confers resistance to the antibiotic hygromycin , and the dhfr gene that confers resistance to methotrexate, the EPSPS gene that confers resistance to glyphosate) or the chemical resistance marker gene (such as the herbicide resistance gene), the mannose-6- that provides the ability to metabolize mannose Phosphate isomerase gene. Considering the safety of the transgenic plants, the transformed plants can be screened directly by adversity without adding any selectable marker gene.

The present invention also provides the above-mentioned plant salt-alkali resistance protein GsSIE3, the application of the above-mentioned coding sequence or the above-mentioned biological material in improving the salt tolerance of soybean.

Further defined, the application is to overexpress protein GsSIE3 or co-express protein GsSIE3 and GsSnRK1 protein in plants.

The present invention also provides the above-mentioned plant salt-alkali resistance protein GsSIE3, the application of the above-mentioned coding sequence or the above-mentioned biological material in improving soybean alkali resistance.

Further defined, the application is to overexpress protein GsSIE3 or co-express protein GsSIE3 and GsSnRK1 protein in plants.

The present invention also provides a method for cultivating transgenic soybean hairy roots with salt tolerance, the method is to introduce the coding gene of protein GsSIE3 into soybean hairy roots, or introduce the coding gene of protein GsSIE3 and the coding gene of GsSnRK1 protein Introduced into soybean hairy roots; the nucleotide sequence of the gene encoding the protein GsSIE3 is shown in SEQ ID NO.1.

Further defined, the soybean hairy root is the soybean hairy root induced by Agrobacterium rhizogenes K599.

The present invention also provides a method for cultivating transgenic soybean hairy roots with alkali resistance, the method is to introduce the coding gene of protein GsSIE3 into soybean hairy roots, or introduce the coding gene of protein GsSIE3 and the coding gene of GsSnRK1 protein Introduced into soybean hairy roots; the nucleotide sequence of the gene encoding the protein GsSIE3 is shown in SEQ ID NO.1.

Further defined, the soybean hairy root is the soybean hairy root induced by Agrobacterium rhizogenes K599.

In one embodiment of the present invention, the gene encoding the GsSIE3 protein is the nucleotide sequence shown in SEQ ID NO.1 through the recombinant vector pPBEL-BiFC-GsSnRK1-GsSIE3 containing the expression cassette of the gene encoding the GsSIE3 protein pPBEL-BiFC-GsSnRK1(K49M)-GsSIE3 was introduced into Agrobacterium rhizogenes K599. The recombinant vectors pPBEL-BiFC-GsSnRK1-GsSIE3 and pPBEL-BiFC-GsSnRK1(K49M)-GsSIE3 are inserted between the EcoRI sites of the pPBEL-BiFC vector with the nucleotide sequence shown in SEQ ID NO.1, and The vector obtained by keeping other sequences of the pPBEL-BiFC vector unchanged. The recombinant vector pPBEL-BiFC-GsSnRK1-GsSIE3 expresses GsSnRK1 protein and GsSIE3 protein, and PBEL-BiFC-GsSnRK1(K49M)-GsSIE3 expresses GsSnRK1(K49M) protein and GsSIE3 protein.

In one embodiment of the present invention, the transgenic soybean hairy root can be understood as the transgenic hairy root obtained by transforming the cotyledon of the target plant with the GsSIE3 gene. The gene can also be transferred into other varieties of the same species, including in particular commercial varieties, using conventional breeding techniques. The transgenic plants include seeds, callus, whole plants and cells.

Beneficial effects of the present invention:

The present invention has discovered an E3 ubiquitin ligase GsSIE3 related to plant salinity stress, which is sensitive to NaCl and NaHCO ₃ , and analysis by qRT-PCR and RT-PCR shows that the GsSIE3 gene is dominantly expressed in wild soybean roots, Moreover, the expression of GsSIE3 was significantly increased after being induced by NaCl and NaHCO ₃ stress, and it could respond to salt stress. The physical association of GsSIE3 and GsSnRK1 was confirmed by yeast binary hybridization validation, GST-pulldown validation, and co-immunoprecipitation assays. Furthermore, it was determined that GsSnRK1 can phosphorylate GsSIE3 at T514. The ubiquitin ligase activity of GsSIE3 was detected by transient transformation technology and HA-Ub antibody biochemically. Salt-alkali stress can activate GsSnRK1, and it was found that the phosphorylation of GsSIE3 by GsSnRK1 was necessary for its ubiquitin ligase activity. GsSnRK1 and GsSIE3 were co-expressed in soybean hairy roots, and the combination of GsSnRK1(wt) and GsSIE3(wt) was found to significantly enhance the resistance of soybean to saline-alkali stress, revealing the new function of GsSIE3 and its regulation mechanism on plant saline-alkali tolerance . Then, we analyzed the phenotype and physiological indicators of various transgenic soybean plants under saline-alkali stress, and the results showed that under saline-alkali stress, the transgenic chimeric soybean plants that overexpressed GsSnRK1 and GsSIE3 alone grew well, and co-expressed GsSnRK1(wt) and GsSIE3(wt) transgenic chimera soybean plants grew better than transgenic chimeric soybean plants overexpressing GsSnRK1 and GsSIE3 alone. This provides new clues for the new function of GsSIE3 and its regulation mechanism on plant tolerance to saline-alkali stress.

Description of drawings

Fig. 1 is the result figure of analyzing the expression situation of GsSIE3 gene in different tissue parts by qRT-PCR and RT-PCR after saline-alkali treatment; Wherein, A in Fig. 1 is each part of GsSIE3 gene in 3-week-old wild soybean plants B in Figure 1 is the expression level of GsSIE3 gene after being treated with different stress solutions, and C in Figure 1 is the expression level of GsSIE3 gene in different tissue parts of 3-week-old wild soybean plants detected by RT-PCR, Figure 1 D in the figure is the expression level of GsSIE3 gene in 3-week-old wild soybean plants after RT-PCR detection of different stress solutions;

Figure 2 is a graph of the multiple alignment results of the amino acid sequences of GsSIE3 family proteins;

Figure 3 is a diagram of the functional domain and RING domain of GsSIE3 E3 ubiquitin ligase; wherein, A in Figure 3 is a diagram of the functional domain of GsSIE3 E3 ubiquitin ligase, and B in Figure 2 is the diagram of GsSIE3 E3 ubiquitin ligase RING domain map;

Figure 4 is a diagram of the physical association results of GsSIE3 and GsSnRK1 confirmed by yeast binary hybridization;

Figure 5 is a map of the subcellular localization of GsSIE3;

Figure 6 is the results of the interaction between GsSIE3 and GsSnRK1 confirmed by GST-Pulldown and Co-IP; among them, A in Figure 6 is the in vitro protein interaction diagram, and B in Figure 6 is the Co-IP analysis of GsSnRK1 and GsSIE3 Interaction results in plant cells;

Figure 7 is the analysis results of ubiquitin ligase activity of GsSIE3 protein; wherein, A in Figure 7 is the self-ubiquitination map of GsSIE3; B in Figure 7 is the self-ubiquitination site map of GsSIE3, and C in Figure 7 GsSIE3 polyubiquitination reaction map;

Figure 8 is the results of Phos-tag ^TM and Western blot detection of GsSnRK1 phosphorylation of GsSIE3, wherein, A in Figure 8 is the results of in vitro phosphorylation analysis using specific phosphorylated antibodies, and B in Figure 8 is Phos- The results of in vitro phosphorylation analysis of GsSIE3 by tag technology; C in Figure 8 is the result of in vitro phosphorylation site analysis of GsSIE3 by Phos-tag technology, T514A is GsSIE3 (T514A), K49M is GsSnRK1 (K49M), Figure 8 D in Figure 8 is the result of in vitro phosphorylation site analysis of GsSIE3 re-validated by Phos-tag technology, and E in Figure 8 is the result of in vitro phosphorylation site analysis again using specific phospho-antibodies;

Figure 9 is a graph showing the effect of phosphorylation sites on ubiquitin ligase activity; A in Figure 9 is a graph showing the effect of GsSnRK1 mutation sites on ubiquitin ligase activity; B in Figure 9 is a mutation in GsSIE3 phosphorylation sites Effect on ubiquitin ligase activity result graph;

Figure 10 is a graph showing the stability of GsSnRK1 affecting GsSIE3;

Figure 11 is a graph showing the analysis results of GsSnRK1 phosphorylated GsSIE3 in soybean hairy roots;

Fig. 12 is 200mM NaCl and _50mMNaHCO The phenotype diagram of transgenic chimera soybean after treatment;

Fig. 13 is 200mM NaCl and _50mMNaHCO Physiological index analysis result figure of transgenic chimera soybean after treatment; Wherein, A and B in Fig. 13 are biomass analysis, C and D in Fig. 13 are root length analysis;

Fig. 14 is 200mM NaCl and _50mMNaHCO Physiological index analysis results figure of transgenic chimeric soybean after treatment; Wherein, A and D in Fig. 14 are chlorophyll content analysis, B and E in Fig. 14 are malondialdehyde content analysis, Fig. C and F in 14 are proline content analysis;

Figure 15 is a graph of the physiological index analysis results of transgenic chimeric soybeans after 200mM NaCl treatment; wherein, A in Figure 15 is a graph of trypan blue staining analysis results, B in Figure 15 is a DAB staining analysis result graph, and in Figure 15 C is the result of NBT staining analysis;

Figure 16 is a graph of the physiological index analysis results of transgenic chimeric soybeans after treatment with 50mM _NaHCO3 ; wherein, A in Figure 16 is a graph of trypan blue staining analysis results, B in Figure 16 is a graph of DAB staining analysis results, and in Figure 16 C is the result of NBT staining analysis.

Detailed ways

The experimental methods used in the following examples, if no special instructions, are conventional methods; materials, reagents, etc. used in the following examples, if no special instructions, can be obtained from commercial sources; Quantitative experiments were performed three times, and the results were averaged.

Wild soybean G07256 seeds in the following examples, Agrobacterium rhizogenes K599, Saccharomyces cerevisiae (Saccharomyces cerevisiae) AH109, pET-32b, pGADT7 and pGBKT7 vectors, pPBEL-BiFC vector, fusion protein prokaryotic expression recombinant vector pGEX-4T-1 -GsSnRK1.1, pET32b-Myc-GsSnRK1.1, pET32b-Flag-GsGRIK1 and recombinant proteins are all disclosed in the patent application number CN202210037002.2, which can be obtained from Northeast Agricultural University.

The Escherichia coli competent Trans1-T1 Phage Resistant Chemically Competent Cell in the following examples is a product of Quanshijin Company; the Saccharomyces cerevisiae competent Y2HGold Chemically Competent Cell in the following examples is a product of Shanghai Weidi Biotechnology Co., Ltd.

See Table 1 for the nucleotide sequences corresponding to the primers involved in the following examples.

The corresponding nucleotide sequence of the primer involved in the embodiment of table 1

Example 1: Cloning of soybean E3 ubiquitin ligase GsSIE3 gene and analysis of its expression pattern

1. Processing of plant material

The plump wild soybean (Glycine soja) G07256 seeds were selected, treated with concentrated HgSO ₄ for 10 min, rinsed with sterilized water for 3-4 times, placed on wet filter paper, and treated in the dark at 4°C for vernalization for 3 days. In an artificial climate chamber, wild soybean seedlings were cultivated with 1/4 Hoagland nutrient solution for 3 weeks to obtain 3-week-old seedlings. The growth conditions are: 24°C, 60% relative humidity, and a photoperiod of 16 hours of light and 8 hours of darkness.

2. RNA extraction

Refer to the PlantRNAKit manual to extract total RNA from wild soybean seedlings, and immediately reverse-transcribe the extracted total RNA or store it at -80°C.

3. Acquisition of cDNA

Using the total RNA obtained in the above step 2 as a template, cDNA was obtained by reverse transcription using the TransScript One-Step gDNARemovalandcDNASynthesis SuperMix kit.

4. PCR amplification

Using the cDNA obtained in step 3 as a template, use GsSIE3-Clone-F (SEQ ID NO.3) and GsSIE3-Clone-R (SEQ ID NO.4) primers and PrimeSTARMax DNAPolymerase kit for PCR amplification to obtain PCR amplification product.

The PCR amplified product was detected by 1% agarose gel electrophoresis to obtain a band with a molecular weight slightly greater than 1kb, and the agarose gel recovery kit was used to recover the PCR amplified product, which was combined with the pEASY-Blunt Simple CloningKit vector (TRANSGEN BIOTECH ) connection to obtain a recombinant plasmid, which was named pEASY-Blunt Simple-GsSIE3, and transformed into Escherichia coli Trans1-T1 competent cells and then sent for sequencing.

Sequencing results showed that: PCR amplification obtained an amplified product with a size of 1569bp, and its nucleotide sequence was as shown in SEQ ID NO.1, which was named GsSIE3 gene, and the ORF was the 1-1569th position of SEQ ID NO.1. The amino acid sequence of the protein encoded by the GsSIE3 gene is shown in SEQ ID NO.2.

SEQ ID NO.1:

ATGCTTGCATATGGGGTACACTTGAACGAGTTGAATCTAAAATGTGTTCTTATGATCATATTGATGATGATATTACCCATCCTAGGGTTGTTTTTCTGGCTAGAGAACAAGTTGTCCCATAATTCCAGTGAGGCCAATCATTACTCCAAATGGAAGGAACATTTGCAGATCAGTGATGAAATAAATACCCTATTTTGTGAACAAGATGAGAACAGTACTAGT GCACACTGTGCTTGCTCCTTCTGTGGAAGATTAAGCAACATAGTCACGAGATGCTCACGCTGCAAAGCTGCTATATATTGCTCGAATGCTTGCCATGTTAAGCATTGGAGGATTTGCCATAAATATGAATGCGTTGAGAAAGAAGGGTCACAAGATCAGCAGGAATCACCGTTTCATGGAACCCATTGCCTTATCATGGAGCCTGAAAATGGTAAGTTCTCATTCAGT GAAGTTATTTGAGCAGAGATCATATAAGGGAGATGTTTACTATGTCGAAGGAGGAGAAAACAGTGCTGAGGTCAGTGATGAAACAGCTCTAAAGTGTAACGATGGCTGTGCAGTGTGTGGCAATCCAAGCTCTAAAGTATGCTCAAGGTGCAAAGCCATAAAATATTGCTCACAAACATGCCAACATTTCGATTGGAGATCTGGGCATAAGTTTCAATGTCTT GTTGAGAAGGCAAATACAACTGAAAAAGCAATTGTCAAATCAAGGAAGACCTGCAAACGGAAATGTTGTAAATCTCACGAATTCGGATGAGGTAGAAGATAATGCTCATTCATCTAGTCCTCTTCGCTTGGAATTTACTCAGGAAACACCAGTTCCAAAGGCCCTGACTCGAAGTTCTTTGTCTCTGGAAGCAACCAATAATGCTCAGAAGGAGATTCAAGATCAATTGA CAAGCCTAGAAGAAGAATTGGCAAAGATAAAAGAGGAGAACATGTCATTACTATCAGAGCGCGACGCATGGGAAGTGCGAGCAAGGAATTCCATAGATAGACTTTATAGCTTCAGGAAAGAAAATGAGCACCAGCTGTTTATTTTGAAGCATGAAAATGAATTGATGTCAAATGCTGAGAAGCAATCACGTCAAATGGTTAATAGTTTATCTCAGAGGCTACACTGC TTGCAGATTGCAGTGGAAAGTGGAGTTGAAGAGAGGAAAAAACAAGAAGAATATATACATATGTTGCAGAATGAATGTGCTAAGGTTAAGATAGAGCTACAGAACAGAACAAGTGCGTCGAAAGGCTTACAGTAGAGCTGGATAAGAACACTCAATTTCCTAGGAGAATAACTGAAGAAACAGGACAAATATTAGTCAATGCTTTAAGTGAAATTGCAGCTGTTGAAT CCAATGCTAACTGTGCTGAGGTGTCCCCTGCCAATTAGTTTGAGCAGAAATCCAACCTTTACAACACAGGGTTGTTCAATTTGCCTAGCCAATGAGAAGAACATGGCCTTTGGTTGTGGACACATGACTTGTTTAGAGTGTGGATCAAAAATTCGCAAGTGTCATATGCCGAAGGAAGATCACCATTCGTATCAGATTGTTTCCTGATTAA

SEQ ID NO.2:

MLAYGVHLNELNLKCVLMIILMMILPILGLFFWLENKLSHNSSEANHYSKWKEHLQISDEINTLFCEQDENSTSAHCACSFCGRLSNIVTRCSRCKAAIYCSNACHVKHWRICHKYECVEKEGSQDQQESPFHGTHCLIMEPENGKFSFSEVIEQRSYKGDVYYVEGGENSAEVSDETALKCND GCAVCGNPSSKVCSRCKAIKYCSQTCQHFDWRSGHKFQCLVEKANTTEKAIVNQGRPANGNVVNLTNSDEVEDNAHSSSPLRLEFYSGNTSSKALTRSSLSLEATNNAQKEIQDQLTSLEEELAKIKEENMSLLSERDAWEVRARNSIDRLYSFRKENEHQLFILKHENELMSNAEKQSRQMVNS LSQRLHCLQIAVESGVEERKKQEEYIHMLQNECAKVKIELQEQNKCVERLTVELDKNTQFPRRITEETGQILVNALSEIAAVESNANCAEVSLPISLSRNPTFTTQGCSICLANEKNMAFGCGHMTCLECGSKIRKCHICRRKITIRIRLFPD

5. Real-time fluorescent quantitative PCR analysis of the expression pattern of GsSIE3

Using primers GsSIE3-qPCR-F (SEQ ID NO.5) and GsSIE3-qPCR-R (SEQ ID NO.6) on the cDNA of each organ of 3-week-old wild soybean seedlings and after 200mM NaCl or 50mMNaHCO ₃ or 15% PEG Or qRT-PCR amplification of cDNA from 3-week-old wild soybean seedlings treated with 10μMABA for 1h.

According to the qRT-PCR results, it can be seen that the GsSIE3 gene is expressed in various organs of wild soybean plants, and the expression level in the roots is relatively high, indicating that the GsSIE3 gene may be involved in the response to salt stress (see A and C in Figure 1). The results after treating wild soybean plants with NaCl and NaHCO ₃ for 1 h showed that the expression of GsSIE3 could be induced by NaCl and NaHCO ₃ (see B and D in Figure 1 ).

6. RT-PCR analysis of the expression pattern of GsSIE3 under NaCl and NaHCO ₃ stress

3-week-old wild soybean seedlings were treated with 100mM NaCl and 50mM NaHCO ₃ for 1 hour, respectively, and samples were taken at designated times. The results showed that the transcript level of GsSIE3 was up-regulated by NaCl and NaHCO ₃ , and the expression of GsSIE3 increased by about 2-fold and 3-fold.

Using GeneDOC to compare the amino acid sequences of the GsSIE3 protein with the SIE3 family proteins in different plants, it was found that the similarity between the GsSIE3 protein and the GmSIE3 in soybean was 100%, and the C-terminus of the SIE3 family protein was highly conserved, while the N-terminus was diverse. (See Figure 2).

According to the SMART database, the functional domain and domain characteristics of GsSIE3 were searched. The results are shown in Figure 3. The GsSIE3 protein contains four functional domains, the most important of which is the conserved RING-Ubox domain at the C-terminal. The amino acid sequence comparison analysis shows that, GsSIE3 contains a complete ORF of 1566bp, encoding 522 amino acids, and the protein molecular weight is about 58KDa. GsSIE3 has a highly conserved C3HC4 RING motif with other homologous proteins, and its RING motif belongs to the HC subgroup, and the RING domain is located at positions 477 to 522, near the C-terminus, and E3 ubiquitin ligase activity requires RING and Zn ²⁺ Conserved Cys and His residues.

Example 2: Interaction between GsSnRK1 and GsSIE3

1. Verification of the interaction between GsSnRK1 and GsSIE3 by yeast binary hybridization

(1) Construction of pGBKT7-GsSnRK1 and pGADT7-GsSIE3 expression vectors

1. Acquisition of GsSnRK1 gene

Using the total cDNA of wild soybean as a template, use BD-GsSnRK1-SmaIF (SEQ ID NO.7) and BD-GsSnRK1-SalIR (SEQ ID NO.8) primers and PrimeSTAR Max DNAPolymerase kit for PCR amplification to obtain PCR amplification The product, the GsSnRK1 gene.

2. Construction of recombinant vector pGBKT7-GsSnRK1

The pGBKT7 vector and the above-mentioned PCR amplification product were digested and ligated with restriction endonucleases SmaI and SalI respectively to obtain the pGBKT7-GsSnRK1 recombinant vector, and the pGBKT7-GsSnRK1 recombinant vector was sequenced and verified.

The sequencing results showed that the pGBKT7-GsSnRK1 recombinant vector was obtained by replacing the DNA fragment between the SmaI and SalI restriction sites of the pGBKT7 vector with the GsSnRK1 gene and keeping other sequences of the pGBKT7 vector unchanged. The pGBKT7-GsSnRK1 recombinant vector expresses GsSnRK1 protein.

3. Construction of recombinant vector pGADT7-GsSIE3

Using the pEASY-Blunt Simple-GsSIE3 plasmid as a template, AD-GsSIE3-SmaIF (SEQ ID NO.9) and AD-GsSIE3-SmaIR (SEQ ID NO.10) primers and PrimeSTARMax DNAPolymerase kit were used for PCR amplification to obtain PCR amplification increase product. The pGADT7 vector was digested with the restriction endonuclease SmaI, and the SmaI restriction site and the homology arm with the vector part were added to the upstream and downstream of the GsSIE3 gene by PCR. The pGADT7 vector was single-digested with SmaI, and the digested product was recovered and purified by gel, and then ligated with the above-mentioned PCR product by using homologous recombination enzyme. After the correct identification, the obtained vector was named pGADT7-GsSIE3.

The sequencing results showed that the pGADT7-GsSIE3 recombinant vector was obtained by inserting the SmaI restriction site of the pGADT7 vector into the GsSIE3 gene through homologous recombination and keeping other sequences of the pGADT7 vector unchanged. The pGADT7-GsSIE3 recombinant vector expresses GsSIE3 protein.

(2) Transform yeast Y2HGold

The two vectors pGBKT7-GsSnRK1 and pGADT7-GsSIE3, the two vectors pGBKT7 empty vector and pGADT7-GsSIE3, the two vectors pGBKT7-GsSnRK1 and pGADT7, and the two vectors pGBKT7 and pGADT7 were transformed into yeast Y2HGold to obtain the plasmid pGBKT7- GsSnRK1 and pGADT7-GsSIE3, pGBKT7 and pGADT7-GsSIE3, pGBKT7-GsSnRK1 and pGADT7, pGBKT7 and pGADT7 yeast Y2HGold, for the specific steps of transforming yeast competent cells, please refer to the specific operation of the Y2HGold Chemically Competent Cell transformation of Vid Biotech.

The results are shown in Figure 4, on SD/-Trp/-Leu/-His (containing 20mM 3-AT) medium, the yeast strains containing pGBKT7-GsSnRK1 and pGADT7-GsSIE3 recombinant vectors in the test group can grow normally, while the blank The control group pGBKT7 and pGADT7 and the yeast strains of the negative control group pGBKT7 and pGADT7-GsSIE3, pGBKT7-GsSnRK1 and pGADT7 could not grow normally. protein interactions.

2. Arabidopsis protoplasts verify the interaction and localization of GsSIE3

(1) Construction of pCAM3301-EGFP-GsSIE3 vector

1. Acquisition of GsSIE3 gene

Using the above pEASY-Blunt Simple-GsSIE3 plasmid as a template, use pCAM3301-EGFP-GsSIE3 F (SEQ ID NO.11) and pCAM3301-EGFP-GsSIE3 R (SEQ ID NO.12) primers and PrimeSTARMaxDNAPolymerase (TaKaRa) kit for PCR Amplify to obtain the PCR amplification product, that is, the GsSIE3 gene.

2. Construction of pCAM3301-EGFP-GsSIE3 vector

The pCAM3301-EGFP vector was digested with restriction endonuclease BamHI (New England Biolabs), ligated to obtain the pCAM3301-EGFP-GsSIE3 recombinant vector, and the pCAM3301-EGFP-GsSIE3 recombinant vector was sequenced and verified.

The sequencing results showed that the pCAM3301-EGFP-GsSIE3 recombinant vector was obtained by inserting the BamHI restriction site of the pCAM3301-EGFP vector into the GsSIE3 gene and keeping other sequences of the pCAM3301-EGFP vector unchanged. The recombinant vector pCAM3301-EGFP-GsSIE3 expressed GsSIE3 protein.

(2) Arabidopsis protoplast transformation

The above-mentioned pCAM3301-EGFP-GsSIE3 vector was transformed into Arabidopsis protoplasts by the polyethylene glycol method (for specific methods, please refer to the instructions of Zhongkeruitai Plant Protoplast Preparation and Transformation Kit), and the transformed pCAM3301-EGFP-GsSIE3 vector and The Arabidopsis protoplasts of pCAM3301-EGFP empty vector were loaded into slices and observed by laser confocal microscope. The results are shown in Figure 5: GsSIE3 protein localized in the cytoplasm.

(3) Extraction of Arabidopsis protoplast protein and detection by Western blot

The related plasmids were transiently expressed in Arabidopsis protoplasts and the total protein was extracted, and the interaction between GsSnRK1 and GsSIE3 was analyzed by Co-IP technology. HA-GsSIE3 was immunoprecipitated (i.e. IP) from all lysates using anti-HA, and Myc-GsSnRK1 protein was detected by Western blot with anti-Myc antibody; and Myc-GsSnRK1 was immunoprecipitated from all lysates using anti-Myc GsSnRK1, and HA-GsSIE3 protein was detected by Western blot with anti-HA antibody. The results shown in Figure 6 (B) show that there is an interaction between GsSnRK1 and GsSIE3, and protein complexes can be formed.

3. In vitro interaction between GsSnRK1 and GsSIE3

(1) Construction of protein expression vector

1. Construction of recombinant vector pET32b-GsSnRK1

1) Acquisition of GsSnRK1 gene

Using the above pGBKT7-GsSnRK1 plasmid as a template, use pET-HA-GsSnRK1-SalIF (SEQ ID NO.13) and pET-HA-GsSnRK1-XhoIR (SEQ ID NO.14) primers and PrimeSTARMax DNAPolymerase (TaKaRa) kit for PCR Amplify to obtain a PCR amplification product, namely the GsSnRK1 gene.

2) Construction of recombinant vector pET32b-GsSnRK1

Use restriction endonucleases SalI (New England Biolabs) and XhoI (New England Biolabs) to carry out double digestion and ligation of the pET32b vector and the above PCR amplification product respectively to obtain the pET32b-GsSnRK1 recombinant vector, and perform sequencing verification on the pET32b-GsSnRK1 recombinant vector .

Sequencing results showed that the pET32b-GsSnRK1 recombinant vector was obtained by replacing the DNA fragment between the SalI and XhoI restriction sites of the pET32b vector with the GsSnRK1 gene and keeping other sequences of the pET32b vector unchanged. The pET32b-GsSnRK1 recombinant vector expresses GsSnRK1 protein.

2. Construction of recombinant vector pET32b-GsSIE3

1) Acquisition of GsSIE3 gene

Using the above pEASY-Blunt Simple-GsSIE3 plasmid as a template, use pET-HA-GsSIE3-EcoRVF (SEQ ID NO.17) and pET-HA-GsSIE3-EcoRVR (SEQ ID NO.18) primers and PrimeSTAR MaxDNAPolymerase (TaKaRa) reagent The cassette is amplified by PCR to obtain a PCR amplified product, namely the GsSIE3 gene.

2) Construction of recombinant vector pET32b-GsSIE3

The pET32b vector was digested with a restriction endonuclease EcoRV (New England Biolabs), and then GsSIE3 was ligated with the digested and purified vector with a homologous recombinase to obtain the pET32b-HA-GsSIE3 recombinant vector.

Sequencing results showed that the pET32b-GsSIE3 recombinant vector was obtained by inserting the GsSIE3 gene after single enzyme digestion of EcoRV of the pET32b vector, and keeping other sequences of the pET32b vector unchanged. The pET32b-GsSIE3 recombinant vector expresses GsSIE3 protein.

(2) GST-Pull down

50 μl of GST-SnRK1 protein purified after prokaryotic expression was incubated with GST filler in PBS solution at 4°C for 2 hours, then incubated with His-GsSIE3 at 4°C for 2 hours, then washed with PBS solution, and detected by Western blot , the results shown in Figure 6 A, GsSIE3 can be combined with GsSnRK1 in vitro.

(3) GsSIE3 ubiquitin ligase activity analysis

1. Functional analysis of GsSIE3 ligase ligation mode

In order to determine the connection mode of polyubiquitin chains, we constructed Flag-Ub(WT), Flag-Ub(K48) and Flag-Ub(K63) vectors, and purified them for in vitro ubiquitination assay, the results are shown in Figure 7 As shown in A in A and C in 7, the results using the K48 protein were basically the same as those using the WT protein, while the K63 ubiquitin ligase activity almost disappeared, and the same results were obtained in the in vivo ubiquitination assay.

2. Autoubiquitination of GsSIE3

The RING-Ubox domain and its four mutants (K9R, K26R, K29R, K36R) were constructed. As a result, as shown in B in Fig. 7, the addition of the wild-type RING-Ubox domain and its mutants resulted in the accumulation of E3-Ub, and the formation of high-molecular-weight bands was clearly observed. K29R and K36R mutants have similar activity of the RING-Ubox domain. K9R and K26R mutants are less ubiquitinated than wild-type RING-Ubox domains. These data suggest that K9 and K26 of the RING-Ubox domain may be the autoubiquitination sites of GsSIE3.

(4) Phosphorylation analysis of GsSnRK and GsSIE3

GsSnRK1 protein performs phosphorylation function and prediction of GsSnRK1 phosphorylation site on GsSIE3

The phosphorylation function of GsSnRK1 protein and the phosphorylation site of GsSnRK1 on GsSIE3 were predicted by online tools (http://ppsp.biocuckoo.org).

The results showed that the 49th amino acid lysine (K) of GsSnRK1 protein was an important amino acid for GsSnRK1 to perform the phosphorylation function, and the threonine (T) at the 514th position of GsSIE3 protein was the putative phosphorylation site of GsSnRK1.

1. Construction of recombinant vector pET32b-GsSnRK1(K49M)

1) Acquisition of GsSnRK1(K49M) gene

We replaced the base AAG encoding the 49th amino acid on the GsSnRK1 gene sequence with ATG, so that the 49th amino acid of the GsSnRK1 protein was mutated from lysine (K) to methionine (M), and we re-synthesized the mutation The modified GsSnRK1 gene was named GsSnRK1(K49M), and the GsSnRK1(K49M) protein encoded by the GsSnRK1(K49M) gene has no phosphorylation function.

Using the GsSnRK1(K49M) gene as a template, using pET-HA-GsSnRK1(K49M)-SalIF(SEQ ID NO.15) and pET-HA-GsSnRK1(K49M)-XhoIR(SEQ ID NO.16) primers and PrimeSTARMaxDNAPolymerase(TaKaRa) The kit performs PCR amplification to obtain a PCR amplification product, that is, the GsSnRK1 (K49M) gene with a restriction site.

2) Construction of recombinant vector pET32b-GsSnRK1(K49M)

Use restriction endonuclease SalI (New England Biolabs) and XhoI (New England Biolabs) respectively to pET32b vector and above-mentioned PCR amplification product to carry out double digestion, connect, obtain pET32b-GsSnRK1 (K49M) recombinant vector, pET32b-GsSnRK1 (K49M) ) recombinant vector for sequencing verification.

The sequencing results showed that the pET32b-GsSnRK1(K49M) recombinant vector was obtained by replacing the DNA fragment between the SalI and XhoI restriction sites of the pET32b vector with the GsSnRK1(K49M) gene and keeping other sequences of the pET32b vector unchanged. The pET32b-GsSnRK1(K49M) recombinant vector expresses the GsSnRK1(K49M) protein.

2. Construction of recombinant vector pET32b-GsSIE3(T514A)

1) Acquisition of GsSIE3(T514A) gene

We replaced the base ACC encoding the 514th amino acid on the GsSIE3 gene sequence with GCC, so that the 514th amino acid of the GsSIE3 protein was mutated from threonine (T) to alanine (A), using a nucleotide sequence such as SEQ The primer pair described in ID NO.23 and SEQID NO.24 amplified the T514 site mutation gene by PCR and named it GsSIE3 (T514A). The GsSIE3 (T514A) protein encoded by the GsSIE3 (T514A) gene does not have phosphorylation by the GsSnRK1 protein Ability.

Using the GsSIE3(T514A) gene as a template, using pET-HA-GsSIE3(T514A)-EcoRV(SEQ ID NO.19) and pET-HA-GsSIE3(T514A)-EcoRVR(SEQ ID NO.20) primers and PrimeSTARMaxDNAPolymerase(TaKaRa) The kit performs PCR amplification to obtain a PCR amplification product, that is, the GsSIE3 (T514A) gene with a restriction site.

2) Construction of recombinant vector pET32b-GsSIE3(T514A)

The pET32b vector was single-digested with restriction endonuclease EcoRV, and then GsSIE3(T514A) was ligated with the digested and purified vector with homologous recombinase to obtain the pET32b-HA-GsSIE3(T514A) recombinant vector.

Sequencing results showed that the pET32b-GsSIE3(T514A) recombinant vector was obtained by inserting the GsSIE3(T514A) gene after EcoRV single enzyme digestion of the pET32b vector, and keeping other sequences of the pET32b vector unchanged. The pET32b-GsSIE3(T514A) recombinant vector expresses GsSIE3(T514A) protein.

(5) Protein expression and purification

The protein expression vectors pET32b-GsSnRK1, pET32b-GsSnRK1(K49M), pET32b-GsSIE3 and pET32b-GsSIE3(T514A) were transformed into competent Escherichia coli BL21 respectively. BL21 Escherichia coli containing pET32b-GsSnRK1, pET32b-GsSnRK1(K49M), pET32b-GsSIE3 and pET32b-GsSIE3(T514A) protein expression vectors were respectively obtained and induced for protein expression.

The expressed GsSnRK1, GsSnRK1(K49M), GsSIE3 and GsSIE3(T514A) proteins were purified respectively, and the purification of GsSnRK1 and GsSnRK1(K49M) proteins was carried out using the Myc fusion protein purification kit provided by Shanghai Guyan Industrial Co., Ltd., the specific steps See the kit instruction for details; GsSIE3 and GsSIE3(T514A) proteins were purified using the Kangwei Century His-Tagged Protein Purification Kit kit, and the specific steps were detailed in the kit instruction.

(6) Phos-tag ^TM detection of phosphorylation of GsSIE3 by GsSnRK1

Phos-tag ^TM kits were used to detect the phosphorylation levels of GsSnRK1 to GsSIE3 and GsSnRK1 to GsSIE3 (T514A), respectively. For details, see the Phos-tag ^TM kit instructions.

The results are shown in Figure 8 B, Figure 8 C and Figure 8 D: GsSnRK1 has a phosphorylation effect on GsSIE3, and GsSnRK1 has no phosphorylation effect on GsSIE3 (T514A). It is proved that GsSnRK1 protein has a phosphorylation effect on GsSIE3 protein, and the 514th amino acid T of GsSIE3 is the key phosphorylation site of GsSnRK1.

(7) Western blot detection of phosphorylation of GsSIE3 by GsSnRK1

The phosphorylation levels of GsSnRK1 to GsSIE3, GsSnRK1 to GsSIE3 (T514A), and GsSnRK1 (K49M) to GsSIE3 (T514A) were detected by Western blot. The pPKDsub antibody was used to detect the presence of phosphorylation, the HA antibody was used to detect the content of GsSIE3 and GsSIE3 (T514A), and the Myc antibody was used to detect the content of GsSnRK1 and GsSnRK1 (K49M).

The results are shown in A in Figure 8 and E in Figure 8: the pPKDsub antibody was used to detect that GsSnRK1 had a phosphorylation effect on GsSIE3, while GsSnRK1 had no phosphorylation effect on GsSIE3 (T514A) and GsSnRK1 (K49M) on GsSIE3. It was once again proved that the GsSnRK1 protein has a phosphorylation effect on the GsSIE3 protein, and the 49th amino acid K of GsSnRK1 is an important amino acid for GsSnRK1 to perform the phosphorylation function, and the 514th amino acid T of GsSIE3 is the key phosphorylation site of GsSnRK1.

(8) GsSnRK1 regulates the function of GsSIE3 ubiquitin ligase

Phosphorylated GsSIE3 was incubated with GsSnRK1 and its GsSnRK1(K49M) separately for ubiquitination experiments.

Results As shown in A in Fig. 9, the phosphorylated form of GsSIE3 enhanced its ligase activity. Then, the ability of the phosphorylation state of GsSIE3 to affect its E3 ligase activity was also examined. GsSIE3 and its GsSIE3(T514A), GsSIE3(T495A) and Flag-Ub were purified for ubiquitination analysis. As a result, as shown in B in FIG. 9 , the autoubiquitination activity of GsSIE3(T514A) was reduced, and similar results were observed by anti-Flag western blot analysis.

(9) GsSnRK1 regulates the stability of GsSIE3

Different transgenic hairy root crude protein extracts (WT, GsSnRK1, GsSnRK (K49M)) were incubated with purified GsSIE3 and GsSIE3 (T514A).

The results are shown in Figure 10, in the presence of ATP, the degradation rate of GsSIE3 in wild type was much faster than that of GsSnRK1(K49M) and GsSIE3(T514A), but slower than that of GsSnRK1 overexpressing plants. These results suggest that GsSnRK1 may regulate the stability of GsSIE3 and that GsSnRK1-mediated phosphorylation of GsSIE3 is required for its degradation. To further confirm the results, we generated GsSnRK1-overexpressing and GsSnRK1(K49M)-overexpressing transgenic hairy roots in a GsSIE3-overexpressing background and treated them with cycloheximide (CHX) to inhibit subsequent protein synthesis. Results As shown in Fig. 10, overexpression of GsSnRK1 resulted in decreased GsSIE3 protein level. In contrast, overexpression of GsSnRK1(K49M) was more stable. Furthermore, in the transgenic hairy roots on the background of GsSIE3(T514A), the degradation of GsSIE3 was much slower than that of wild-type GsSIE3. The addition of MG132 can effectively reduce protein degradation. These results suggest that phosphorylation of GsSIE3 by GsSnRK1 negatively regulates its stability and its degradation by the 26S proteasome.

Example 3: Genetic transformation of GsSIE3 and expression analysis in transgenic soybean

1. Genetic transformation of transgenic soybean hairy roots mediated by Agrobacterium rhizogenes K599 and phenotype analysis of plants under saline-alkali stress

1. Construction of pPBEL-BiFC-GsSnRK1(K49M)-GsSIE3 expression vector

1) Obtaining the GsSnRK1(K49M) gene Using the above-mentioned GsSnRK1(K49M) gene as a template, BiFC-GsSnRK1(K49M)-SmaIF(SEQ ID NO.21) and BiFC-GsSnRK1(K49M)-SmaIR(SEQ ID NO.22 ) primers and PrimeSTARMax DNA Polymerase (TaKaRa) kit for PCR amplification to obtain the PCR amplification product, namely the GsSnRK1 (K49M) gene with restriction sites.

2) Construction of pBEL-BiFC-GsSnRK1(K49M)-GsSIE3 vector

Use restriction endonucleases SmaI and SalI to perform double enzyme digestion on the above-mentioned PBEL-BiFC-GsSnRK1-GsSIE3 vector and the PCR amplification product of the above-mentioned GsSnRK1(K49M) gene, and connect them to obtain PBEL-BiFC-GsSnRK1(K49M)-GsSIE3 For the recombinant vector, the PBEL-BiFC-GsSnRK1(K49M)-GsSIE3 recombinant vector was sequenced and verified.

The sequencing results showed that the pBEL-BiFC-GsSnRK1(K49M)-GsSIE3 recombinant vector was replaced by the GsSnRK1(K49M) gene between the SmaI and SalI restriction sites of the pBEL-BiFC-GsSnRK1-GsSIE3 vector, and the pBEL-BiFC - Other sequences of the GsSnRK1-GsSIE3 vector remain unchanged. The obtained vector pPBEL-BiFC-GsSnRK1(K49M)-GsSIE3 recombinant vector expresses GsSnRK1(K49M) and GsSIE3 proteins.

2. Construction of pPBEL-BiFC-GsSnRK1-GsSIE3(T514A) expression vector

1) Obtaining the GsSIE3(T514A) gene Using the above-mentioned GsSIE3(T514A) gene as a template, BiFC-GsSIE3(T514A)-PmlIF(SEQ ID NO.27) and BiFC-GsSIE3(T514A)-PmlIR(SEQ ID NO.28 ) primers and PrimeSTARMax DNA Polymerase (TaKaRa) kit for PCR amplification to obtain the PCR amplification product, namely the GsSIE3 (T514A) gene with restriction sites.

2) Construction of PBEL-BiFC-GsSnRK1-GsSIE3(T514A) vector

The above-mentioned PBEL-BiFC-GsSnRK1-GsSIE3 vector was single-digested with the restriction endonuclease PmlI, and connected by homologous recombinase to obtain the PBEL-BiFC-GsSnRK1-GsSIE3 (T514A) recombinant vector, and the PBEL-BiFC-GsSnRK1- The GsSIE3(T514A) recombinant vector was sequenced and verified.

The sequencing results showed that the pBEL-BiFC-GsSnRK1-GsSIE3(T514A) recombinant vector replaced the GsSIE3 fragment between the PmlI restriction sites of the pBEL-BiFC-GsSnRK1-GsSIE3 vector with the GsSIE3(T514A) gene, and maintained the pBEL-BiFC- The other sequences of the GsSnRK1-GsSIE3 vector were unchanged. The obtained vector pPBEL-BiFC-GsSnRK1-GsSIE3(T514A) recombinant vector expresses GsSnRK1 and GsSIE3(T514A) proteins.

3. Construction of pPBEL-BiFC-GsSnRK1-GsSIE3 expression vector

1) Acquisition of GsSnRK1 and GsSIE3 genes

BiFC-GsSnRK1-SmaIF and BiFC-GsSnRK1-SmaIR primers, BiFC-GsSIE3-PmlIF and GsSIE3-PmlIR primers, and PrimeSTARMax DNA Polymerase (TaKaRa) kit were used to obtain GsSnRK1 and GsSIE3 PCR products according to the above method.

2) Construction of pPBEL-BiFC-GsSnRK1-GsSIE3 expression vector

The PBEL-BiFC-GsSnRK1(K49M)-GsSIE3 vector was single-digested with the restriction endonuclease PmlI, and connected by homologous recombinase to obtain the PBEL-BiFC-GsSnRK1(K49M)-GsSIE3(T514A) recombinant vector. The PBEL-BiFC-GsSnRK1-GsSIE3 recombinant vector was verified by sequencing.

Sequencing results showed that the pBEL-BiFC-GsSnRK1-GsSIE3 recombinant vector was a replacement of the GsSIE3 fragment between the PmlI restriction sites of the pBEL-BiFC-GsSnRK1-GsSIE3 vector with the GsSIE3 gene and maintained the pBEL-BiFC-GsSnRK1-GsSIE3 vector. The sequence is unchanged. The obtained vector pPBEL-BiFC-GsSnRK1-GsSIE3 recombinant vector expresses GsSnRK1 and GsSIE3 proteins.

2. Obtaining hairy roots of transgenic soybean

Sow soybean Williams82 seeds in the soil (soil: vermiculite = 1:1) about 1-2 cm deep. Place them in a constant temperature climate box at 28°C during the day/20°C at night, water daily, and take 6d-old seedlings whose cotyledons have not yet unfolded for K599 infection. K599 containing recombinant vectors pPBEL-BiFC-GsSnRK1, PBEL-BiFC-GsSnRK1(K49M)-GsSIE3, PBEL-BiFC-GsSIE3, PBEL-BiFC-GsSnRK1-GsSIE3(T514A), pBEL-BiFC-GsSnRK1-GsSIE3 The Agrobacterium rhizogenes bacteria liquid and the K599 Agrobacterium rhizogenes bacteria liquid without any carrier were injected into the cotyledon nodes of soybean, and covered with film after infection. After the hairy root grows, bury the infected site and the lower part with vermiculite to keep it moist, 28°C, 14h light/10h dark, 28°C during the day/20°C at night, culture for 30d, and keep in a humid environment . After 30 days of hairy root growth, when the hairy root grows to about 10 cm, subtract the main root, bury the composite plant in the mixed soil (nutrient soil: vermiculite = 1:1), and water once every 3 days . After 45 days of hairy root growth, it was used for identification and subsequent phenotype analysis.

3. Identification of transgenic soybean hairy roots

1) Identification of pPBEL-BiFC-GsSnRK1, PBEL-BiFC-GsSnRK1(K49M)-GsSIE3, PBEL-BiFC-GsSIE3, PBEL-BiFC-GsSnRK1-GsSIE3(T514A), pBEL-BiFC-GsSnRK1-GsSIE3 transgenic soybean hairy roots

Use the PCR method to take soybean hairy roots with a length of 5mm, put them into a centrifuge tube and add 35μl LysisBufferA, heat at 95°C for 10min, and take 1μl of the supernatant after standing still as a template for the PCR reaction system. BiFC-FW (SEQ ID NO.25) and BIFC-RW (SEQ ID NO.26) primers and PrimeSTARMax DNAPolymerase (TaKaRa) kit were used for PCR amplification, and pPBEL-BiFC-GsSnRK1, PBEL-BiFC- The specific gene fragments carried by GsSnRK1(K49M)-GsSIE3, PBEL-BiFC-GsSIE3, PBEL-BiFC-GsSnRK1-GsSIE3(T514A), pBEL-BiFC-GsSnRK1-GsSIE3 vectors were detected to obtain PCR amplification products. The specific fragment was cloned, indicating that the relevant target gene had been expressed in the transgenic soybean hairy root.

2. Analysis of GsSnRK1 phosphorylation of GsSIE3 in transgenic soybean hairy roots

1. Analysis of phosphorylated GsSIE3 in transgenic soybean hairy root GsSnRK1 in vivo

Using the corresponding plasmids pPBEL-BiFC, pPBEL-BiFC-GsSnRK1, pPBEL-BiFC-GsSnRK1-GsSIE3, pPBEL-BiFC-GsSnRK1(K49M)-GsSIE3, pPBEL-BiFC-GsSnRK1-GsSIE3(T514A), pPBEL-BiFC-GsSnR K1 (K49M)-GsSIE3(T514A) Agrobacterium rhizogenes K599 rooted Williams82 soybeans, and after 30 days of hairy roots, the main root was subtracted, and the hairy roots were genotyped by the above PCR method to confirm the purpose The gene integrates into the plant chromosome. After 45 days of hairy root growth, the transgenic chimera soybean plants were treated with 200mM NaCl for 2h, and then the total protein of hairy root was extracted. The expression of Myc-GsSnRK1 and HA-GsSIE3 in total protein was confirmed using anti-Myc and anti-HA antibodies, respectively.

The results are shown in FIG. 11 , Myc-GsSnRK1 and its mutants and HA-GsSIE3 and its mutants were all expressed in the corresponding hairy roots. Transgenic soybean composite plants were treated with 200 mM NaCl or 50 mM NaHCO ₃ , and proteins were extracted from transgenic hairy roots (+MG132). It is known that the activity of GsSnRK1 is usually activated by phosphorylation. To determine whether GsSnRK1 is activated by salt stress, the phosphorylation level of GsSnRK1 in soybean was determined using AMPKα (Thr172) antibody. Western blot showed that GsSnRK1 could be phosphorylated before 200mM NaCl treatment, and the degree of phosphorylation was enhanced after 200mM NaCl treatment. At the same time, in order to verify whether GsSnRK1 can phosphorylate downstream ubiquitin ligases after being activated, a PKD antibody was used to detect the phosphorylation level of GsSIE3. Treatment with 200 mM NaCl could enhance the activity of GsSnRK1, resulting in an increase in the level of phosphorylated GsSIE3, but could not phosphorylate GsSIE3 (T514A), and GsSnRK1 (K49M) could not phosphorylate GsSIE3. These results are consistent with in vitro phosphorylation. These results suggest that GsSnRK1 may act as an upstream kinase of phospho-GsSIE3, which is essential for salt tolerance in soybean.

3. Phenotype and physiological index analysis of transgenic soybean plants under saline-alkali stress

1. Phenotype analysis of transgenic soybean plants

After they were cultured in Hoagland's nutrient solution or Hoagland's nutrient solution containing 200mM NaCl for 10 days, the phenotype and related physiological data were analyzed. By statistically analyzing physiological indicators such as root length, fresh weight, dry weight, chlorophyll content, trypan blue staining, NBT staining, and DAB staining, all experiments were technically repeated and biologically repeated 3 times.

As shown in Figure 11, under normal conditions, the growth status of plants in each group is similar, but after salt treatment, overexpression of empty load plants, co-expression of GsSnRK1(K49M)/GsSIE3(wt), co-expression of GsSnRK1(wt)/GsSIE3 (T514A) and co-expressing GsSnRK1(K49M)/GsSIE3(T514A) transgenic chimera soybean plants showed growth arrest and severe leaf yellowing and wilting. The growth status of the transgenic chimeric soybean plants co-expressing GsSnRK1(wt)/GsSIE3(wt) was the best, and the growth status of the transgenic chimeric soybean plants overexpressing GsSnRK1 and GsSIE3 was between the two.

As shown in Figure 12, under 200 mM NaCl treatment, GsSnRK1/GsSIE3 had healthier leaves, exhibited the highest salt tolerance and the highest root length and biomass, while GsSnRK1(K49M)/GsSIE3 or GsSnRK/GsSIE3 ( T514A) mutants grow slowly, exhibit severe wilting of leaves, and exhibit lower plant resistance, which indicates that GsSnRK1 may play an upstream kinase regulatory role on GsSIE3, sense stress signals, and then phosphorylate GsSIE3, thereby responding to stress react. As shown in Figure 13, compared with 200 mM NaCl treatment, 50 mM NaHCO ₃ treatment significantly reduced the phenotypes exhibited by plants, the increase in biomass and the increase in root length. Notably, GsSIE3 also exhibited some resistance, suggesting that GsSIE3 endows plants with tolerance to salt stress in soybean. Overall, the kinase activity of GsSnRK1 is essential for the function of GsSIE3, and GsSnRK1 negatively regulates the stability of GsSIE3 protein, thereby enhancing the resistance of plants to saline-alkali stress. Chlorophyll can carry out photosynthesis and absorb light energy, and its content affects the strength of photosynthesis, and some adverse environmental conditions will affect the synthesis of chlorophyll. As shown in Figure 14, before the stress treatment, the chlorophyll content of each group was basically the same. After treatment with 200mM NaCl and 50mM NaHCO3, the roots of the complex were injured, resulting in a decrease in the chlorophyll content of the leaves of the complex. Among them, the chlorophyll content of GsSnRK1(K49M)/GsSIE3 and GsSnRK1/GsSIE3(T514A) mutants decreased the fastest, while GsSnRK1/ The chlorophyll of GsSIE3 decreased the least, so it can be seen that GsSnRK1/GsSIE3 is more salt-tolerant, while GsSnRK1(K49M)/GsSIE3 and GsSnRK1/GsSIE3(T514A) mutants are more sensitive to stress. Malondialdehyde (MDA) is produced when the body is aging or damaged under stress, and its tissue or organ membrane lipid peroxidation is produced. Its content is closely related to the aging of the body and stress damage. The higher the peroxide content , the higher the MDA content, the greater the damage to the plant. Before the stress treatment, the content of MDA in each group was basically the same. After treatment with 200 mM NaCl and 50 mM NaHCO3, GsSnRK1(K49M)/GsSIE3 and GsSnRK1/GsSIE3(T514A) mutants had the highest MDA content, while GsSnRK1/GsSIE3 had the lowest MDA content, which indicated that GsSnRK1/GsSIE3 was more abundant than GsSnRK1(K49M)/GsSIE E3 and GsSnRK1 The /GsSIE3(T514A) mutant is more salt-tolerant. Proline (Pro) also reflects a certain stress resistance to a certain extent, the higher the Pro content, the stronger the plant resistance. As shown in Figure 14, before the stress treatment, the Pro content of each group was basically the same. After treatment with 200 mM NaCl and 50 mM NaHCO3, GsSnRK1/GsSIE3 had the highest Pro content, while GsSnRK1(K49M)/GsSIE3 and GsSnRK1/GsSIE3(T514A) mutants had the lowest Pro content, which also indicated that the co-expression complex was more salt-tolerant than the mutants .

Under salt stress, in order to maintain the internal environment, cells will die actively and orderly, and produce H ₂ O ₂ and O ₂ ^- to regulate various physiological and biochemical processes. As shown in Figure 15 and Figure 16, after 200mM NaCl treatment, trypan blue, DAB and NBT staining showed that compared with the empty vector, the leaves of GsSIE3 and GsSnRK1 were partially dead and oxidized by O ₂ ^- to produce a small amount of H ₂ O ₂ , However, most leaves of GsSnRK1(K49M)/GsSIE3 and GsSnRK1/GsSIE3(T514A) mutants died and were oxidized by O2- to produce a large amount of H ₂ O ₂ . 50mM NaHCO3 treatment produced a similar phenomenon, which suggested that the transgenic hairy roots might have an effect on the aerial part. Normally, GsSIE3(T514A) can inhibit the normal function of endogenous GsSIE3, and the phosphorylation status of Thr514 in GsSIE3 is critical for the function of this protein in response to NaCl and NaHCO3 stress.

Although the present invention has been disclosed above with preferred embodiments, it is not intended to limit the present invention. Any person familiar with this technology can make various changes and modifications without departing from the spirit and scope of the present invention. Therefore The scope of protection of the present invention should be defined by the claims.

Claims (10)

1. A plant salt-alkali resistance protein GsSIE3, characterized in that the amino acid sequence of the plant salt-alkali resistance protein GsSIE3 is shown in SEQ ID NO.2. 2. The coding sequence of the plant salt-alkali resistance protein GsSIE3 according to claim 1, characterized in that, the coding sequence is as shown in SEQ ID NO.1. 3. A biological material related to the coding sequence of claim 2, wherein the biological material is any one of the following materials: A1) an expression cassette containing said coding sequence; A2) a recombinant vector containing the coding sequence; A3) A recombinant microorganism containing the coding sequence. 4. The biomaterial according to claim 3, wherein the biomaterial is any one of the following materials: A4) a recombinant vector containing the expression cassette described in A1); A5) a recombinant microorganism containing the expression cassette described in A1); A6) A recombinant microorganism containing the recombinant vector described in A2). 5. The application of the plant salt-alkali-resistant protein GsSIE3 according to claim 1, the coding sequence according to claim 2, or the biological material according to any one of claims 3 or 4 in improving soybean salt tolerance. 6. The application according to claim 5, characterized in that, the application is overexpressing protein GsSIE3 or co-expressing protein GsSIE3 and GsSnRK1 protein in plants. 7. The application of the plant salt-alkali resistance protein GsSIE3 according to claim 1, the coding sequence according to claim 2, or the biological material according to any one of claims 3 or 4 in improving soybean alkali tolerance. 8. The application according to claim 7, characterized in that, the application is to overexpress protein GsSIE3 or co-express protein GsSIE3 and GsSnRK1 protein in plants. 9. A method for cultivating a salt-tolerant transgenic soybean hairy root, characterized in that, the method is to introduce the coding gene of protein GsSIE3 into soybean hairy root, or the coding gene of protein GsSIE3 and the coding of GsSnRK1 protein The gene is introduced into soybean hairy roots; the nucleotide sequence of the gene encoding the protein GsSIE3 is shown in SEQ ID NO.1. 10. A method for cultivating a transgenic soybean hairy root with alkali resistance, characterized in that, the method is to import the coding gene of protein GsSIE3 into soybean hairy root, or the coding gene of protein GsSIE3 and the coding of GsSnRK1 protein The gene is introduced into soybean hairy roots; the nucleotide sequence of the gene encoding the protein GsSIE3 is shown in SEQ ID NO.1.