CN109385408B

CN109385408B - Application of SmSIP1 protein and related biological materials thereof in promoting degradation of squalene synthase

Info

Publication number: CN109385408B
Application number: CN201710684337.2A
Authority: CN
Inventors: 黄璐琦; 荣齐仙; 姜丹; 林慧馨; 陈宜均
Original assignee: Institute of Materia Medica of CAMS
Current assignee: Institute of Materia Medica of CAMS
Priority date: 2017-08-11
Filing date: 2017-08-11
Publication date: 2021-07-06
Anticipated expiration: 2037-08-11
Also published as: CN109385408A

Abstract

The invention discloses an application of SmSIP1 protein and a coding gene thereof in promoting squalene synthase degradation. Experiments prove that the SmSIP1 protein has the activity of ubiquitin ligase, and the SmSIP1 protein can promote the degradation of squalene synthase and regulate the content of squalene synthase. The invention lays a foundation for deeply researching the action mechanism of squalene synthase in sterol and triterpene metabolic pathways, and provides a theoretical basis for further research and industrial production of sterol and triterpene biosynthesis.

Description

Application of SmSIP1 protein and related biological materials thereof in promoting degradation of squalene synthase

Technical Field

The invention belongs to the technical field of biology, and particularly relates to application of SmSIP1 protein and related biological materials thereof in regulation of squalene synthase content, in particular to application of SmSIP1 protein and related biological materials thereof in promotion of squalene synthase degradation and regulation of squalene synthase content in a sterol synthesis pathway in salvia miltiorrhiza.

Background

With the rapid development of life science research, people find that the sequence information of the genome can not completely explain various life processes and phenomena, and the protein as the embodiment of cell activity and function has irreplaceable effect in the life process. However, proteins, as direct executives of vital activities, also require different degrees of processing and modification to function. Post-translational modifications (PTMs) of proteins can change the spatial conformation, activity, stability and their properties in interaction with other molecules, thereby participating in the regulation of diverse vital activities of the body. More than 400 posttranslational modifications have been identified, and common modifications are phosphorylation, ubiquitination, methylation, acetylation, glycosylation, SUMO, nitrosylation, and oxidation, among others. It is estimated that 50-90% of proteins in humans undergo post-translational modifications, which is the effect of such post-translational modifications, such that one gene does not correspond to only one protein, thereby imparting more complexity to the life process.

The ubiquitination process has attracted increasing attention in various forms of post-translational modification of proteins, and nobel chemical awarded three scientists who made pioneer contributions in the study of "ubiquitin-mediated protein degradation processes". Ubiquitination, similar to protein phosphorylation signal transduction, primarily acts to regulate the levels of those proteins that are both environmentally stimulated and constitutively regulated. Ubiquitination modification of proteins is therefore involved in many physiological processes of vital activity, such as: cell proliferation and differentiation regulation, cell cycle progression, protein transport, DNA repair, organelle development, quality control of endoplasmic reticulum proteins, apoptosis, and cell response to stress, among others.

Ubiquitination is an important post-translational protein modification process in eukaryotic cells, and plays a role in regulating and controlling the growth and development of organisms and the adaptability of the organisms to the surrounding environment, wherein ubiquitin molecules are polypeptides consisting of 76 highly conserved amino acids. The process is that target protein is marked by ubiquitin molecule and then degraded after being recognized by proteasome. The process of labeling target protein is completed by the co-mediated catalysis of E1 ubiquitin activating enzyme, E2 ubiquitin conjugated enzyme and E3 ubiquitin ligase. The El ubiquitin activating enzyme relies on ATP to provide energy to catalyze the ubiquitin molecules to be combined with substrate protein, simultaneously activates the ubiquitin molecules to be transferred to E2 ubiquitin conjugating enzyme, and finally labels target protein by combining E3 ubiquitin ligase with lysine residue. E3 ubiquitin ligase plays a key role in target protein specific recognition, and ubiquitin ligase E3 can be classified into single subunit types such as HECT, RING/U-box and multi-subunit types such as SCF complex, late-stage promoting complex (APC), CUL3-BTB, CUL4-DDB, etc. according to its subunit composition and action mechanism. Studies have shown that more than 5% of the predicted proteins (more than 1400 genes) in the arabidopsis proteome are involved in the ubiquitination/26S proteasome pathway. Of these proteins, only a few encoded the E1 enzyme (with two isoforms), the 37 predicted E2, 26S proteasome components, and other factors (such as deubiquitinating enzymes). Whereas more than 1400 genes encode E3 ubiquitin ligases involved in ubiquitination-dependent proteasome degradation pathways. The diverse variety of E3 ubiquitin ligases makes it involved in specific proteasome degradation pathways. The specificity of E3 for recognizing the target protein can also be increased through the combination of different E2 and E3. Therefore, the diversity of E3, and the diversity of E3 in combination with E2, not only can regulate different types of ubiquitination modifications, but also can specifically regulate target proteins.

Squalene synthase (SS or SQS) is a key enzyme in the biosynthesis of terpenes such as triterpenes, sterols and cholesterol, and its content and activity determine the yield of the subsequent product. Few reports are currently reported on the presence or absence of post-translational modifications of squalene synthase. Timothy p. et al found that rapid degradation of the TSS protein occurred between 45-50h after treatment of tobacco suspension cells with fungal elicitors, and that there was a PEST sequence associated with the regulation of protein degradation between amino acids 75-89 of the TSS protein, thus presumably post-translational modification of the TSS protein.

Salvia miltiorrhiza is a traditional Chinese medicine in China, is a salvia plant in Labiatae, has the effects of removing blood stasis, relieving pain, activating blood, stimulating menstrual flow, cooling blood, eliminating carbuncle, clearing away heart fire and relieving restlessness, and is commonly used for treating diseases of cardiovascular systems, blood systems and the like. The strain is considered to be an ideal model organism for traditional Chinese medicine research due to the characteristics of wide distribution region, strong vitality, short generation period, mature tissue culture and transgenic technology system, small genome, small chromosome number and the like.

Disclosure of Invention

An object of the present invention is to provide a novel use of the SmSIP1 protein.

The invention provides application of SmSIP1 protein serving as E3 ubiquitin ligase.

The invention also provides application of the SmSIP1 protein in regulating the content of squalene synthase.

The invention also provides application of the SmSIP1 protein in preparing a product for regulating and controlling the content of squalene synthase.

In the application, the control of the squalene synthase content is to promote the degradation of squalene synthase.

In the application, the SmSIP1 protein is the protein of A1) or A2) or A3) or A4) as follows:

A1) the amino acid sequence is a protein shown in a sequence 2;

A2) a fusion protein obtained by connecting a label to the N end and/or the C end of the protein shown in the sequence 2;

A3) the protein with the same function is obtained by substituting and/or deleting and/or adding one or more amino acid residues in the amino acid sequence shown in the sequence 2;

A4) and (b) a protein having a homology of 75% or more than 75% with the amino acid sequence shown in the sequence 2 and having the same function.

Wherein, the sequence 2 consists of 274 amino acid residues.

In order to facilitate the purification of the protein in A1), the amino terminal or the carboxyl terminal of the protein shown in the sequence 2 in the sequence table can be connected with the label shown in the table 1.

TABLE 1 sequence of tags

Label (R)	Residue of	Sequence of
			Poly-Arg	5-6 (typically 5)	RRRRR
Poly-His	2-10 (generally 6)	HHHHHH
			FLAG	8	DYKDDDDK
Strep-tag II	8	WSHPQFEK
			c-myc	10	EQKLISEEDL

In a specific embodiment of the invention, the fusion protein described in a2) is a protein obtained by connecting an MBP tag to the N-terminus of the SmSIP1 protein shown in sequence 2.

The protein of A2) above may be artificially synthesized, or may be obtained by synthesizing the coding gene and then performing biological expression.

The gene encoding the protein of A2) above can be obtained by deleting one or several amino acid residues from the DNA sequence shown in SEQ ID No. 1, and/or by carrying out missense mutation of one or several base pairs, and/or by attaching a coding sequence of the tag shown in Table 1 or MBP tag to the 5 'end and/or 3' end thereof.

The protein according to A3), wherein the substitution and/or deletion and/or addition of one or more amino acid residues is a substitution and/or deletion and/or addition of not more than 10 amino acid residues.

The above-mentioned "homology" in A4) includes an amino acid sequence having 75% or more, or 80% or more, or 85% or more, or 90% or more, or 95% or more homology to the amino acid sequence represented by the sequence 2 of the present invention.

Another object of the present invention is to provide a novel use of biological material related to the SmSIP1 protein.

The invention provides application of biological materials related to SmSIP1 protein in preparation of E3 ubiquitin ligase.

The biological material related to the SmSIP1 protein is any one of the following B1) to B16):

B1) a nucleic acid molecule encoding a SmSIP1 protein;

B2) an expression cassette comprising the nucleic acid molecule of B1);

B3) a recombinant vector comprising the nucleic acid molecule of B1);

B4) a recombinant vector comprising the expression cassette of B2);

B5) a recombinant microorganism comprising the nucleic acid molecule of B1);

B6) a recombinant microorganism comprising the expression cassette of B2);

B7) a recombinant microorganism containing the recombinant vector of B3);

B8) a recombinant microorganism containing the recombinant vector of B4);

B9) a transgenic animal cell line comprising the nucleic acid molecule of B1);

B10) a transgenic animal cell line comprising the expression cassette of B2);

B11) a transgenic animal cell line containing the recombinant vector of B3);

B12) a transgenic animal cell line containing the recombinant vector of B4);

B13) a transgenic plant cell line comprising the nucleic acid molecule of B1);

B14) a transgenic plant cell line comprising the expression cassette of B2);

B15) a transgenic plant cell line comprising the recombinant vector of B3);

B16) a transgenic plant cell line comprising the recombinant vector of B4).

In the above application, the nucleic acid molecule of B1) is a gene as shown in 1) or 2) or 3) as follows:

1) the coding sequence is a cDNA molecule or a genome DNA molecule shown in a sequence 1;

2) a cDNA molecule or a genome DNA molecule which has 75 percent or more than 75 percent of identity with the nucleotide sequence defined by 1) and codes SmSIP1 protein;

3) a cDNA molecule or a genome DNA molecule which is hybridized with the nucleotide sequence limited by 1) or 2) under strict conditions and codes SmSIP1 protein.

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

Wherein, the sequence 1 consists of 825 nucleotides, and the coding sequence 2 shows the amino acid sequence.

The nucleotide sequence encoding the SmSIP1 protein of the present invention can be readily mutated by one of ordinary skill in the art using known methods, such as directed evolution and point mutation. Those nucleotides which have been artificially modified to have 75% or more identity to the nucleotide sequence encoding the SmSIP1 protein are derived from and identical to the nucleotide sequence of the present invention as long as they encode the SmSIP1 protein and have the same function.

The term "identity" as used herein refers to sequence similarity to a native nucleic acid sequence. "identity" includes nucleotide sequences that are 75% or more, or 85% or more, or 90% or more, or 95% or more identical to the nucleotide sequence of a protein consisting of the amino acid sequence shown in coding sequence 2 of the present invention. Identity can be assessed visually or by computer software. Using computer software, the identity between two or more sequences can be expressed in percent (%), which can be used to assess the identity between related sequences.

The above-mentioned identity of 75% or more may be 80%, 85%, 90% or 95% or more.

In the above application, the stringent conditions are hybridization and membrane washing 2 times at 68 ℃ for 5min in a solution of 2 XSSC, 0.1% SDS, and hybridization and membrane washing 2 times at 68 ℃ for 15min in a solution of 0.5 XSSC, 0.1% SDS; alternatively, hybridization was carried out at 65 ℃ in a solution of 0.1 XSSPE (or 0.1 XSSC), 0.1% SDS, and the membrane was washed.

In the above application, the vector may be a plasmid, a cosmid, a phage, or a viral vector.

In the above application, the microorganism may be yeast, bacteria, algae or fungi, such as Agrobacterium.

In the above applications, the transgenic plant cell line does not comprise propagation material.

The invention also provides application of the biological material in regulating the content of squalene synthase.

The invention also provides application of the biological material in preparing a product for regulating and controlling the content of squalene synthase.

The invention selects the salvia miltiorrhiza squalene synthase SmSQS1 as a research target, constructs a cDNA library through a yeast two-hybrid system, and screens a candidate protein SmSIP1 which has interaction with SmSQS1, wherein the length of the candidate protein SmSIP 1is 274 amino acids. The interaction between SmSQS1 and SmSIP 1is found by yeast gyration verification and in vivo LUC experiments. The SmSIP1 protein is prepared by a prokaryotic expression method, and the SmSIP1 protein is further proved to have the activity of in vitro ubiquitin ligase and can promote the degradation of SmSQS 1. The invention lays a foundation for deeply researching the action mechanism of SmSQS1 in the metabolic pathways of sterol and triterpenes, and provides a theoretical basis for further research and industrial production of biosynthesis of sterol and triterpenes.

Drawings

Fig. 1is a SmSIP1 bioinformatics analysis.

FIG. 2is a phylogenetic tree constructed based on SmSIP1 and other 43 protein sequences.

FIG. 3 shows the results of prokaryotic expression detection of SmSIP1 protein.

Figure 4 is a study of SmSIP1 promoting SmSQS1 degradation.

Detailed Description

The experimental procedures used in the following examples are all conventional procedures unless otherwise specified.

Materials, reagents and the like used in the following examples are commercially available unless otherwise specified.

In the quantitative tests in the following examples, three replicates were set up and the results averaged.

Coli BL21, E.coli DH5 α, pGADT7 and PGBKT7 vectors in the following examples were purchased from Takara Bio Inc.; gateway expression vectorPurchased from Invitrogen corporation; the RNA extraction reagent Trizol (200 mL standard) was purchased from Invitrogen; RevertAId H Minus Reverse Transcriptase (# EP0451), RiboLock RNase Inhibitor (# E00381), Oligod (T)₁₈Primer (# S0131) and Taq DNA polymerase were both purchased from Fermentas; the gel recovery kit was purchased from petaike corporation; t4DNA ligase, restriction enzymes Kpn I, EcoR I, Nde I, BamH I and SalI were all available from Beijing, Inc. (NEB), N.J.Biotechnology.

The pMAL-c2 vector of the following examples is described in the literature "Zhang, Y., C.Yang, Y.Li, N.Zheng, H.Chen, Q.ZHao, T.Gao, H.Guo and Q.Xie (2007)' SDIR1is a RING finger E3 ligand gene regulation strain-responsive antigenic signaling in Arabidopsis.plant Cell 19(6): 1912-1929", publicly available from the Applicant, the biomaterial being only used for the repetition of the relevant experiments of the present invention and not being useful for other purposes.

The pCAMBIA1307-6myc vector in the following examples is described in the literature "Lin, H., Y.Yang, R.Quan, I.Mendoza, Y.Wu, W.Du, S.ZHao, K.S.Schumaker, J.M.pardo and Y.Guo (2009), Phosphorylation of SOS3-LIKE CALCIUM BINDING PROTEIN8by SOS2PROTEIN kinase stabilization of the PROTEIN complex and regulation of salt and tolerance in Arabidopsis plant Cell 21(5): 1607-1619", publicly available from the Applicant, and the biomaterial is only used for repeating the experiments related to the present invention and is not used for other purposes.

pENTR vectors in the following examples are described in the documents "Guo, J., X.Ma, Y.Cai, Y.Ma, Z.Zhan, Y.J.ZHou, W.Liu, M.Guan, J.Yang, G.Cui, L.kang, L.Yang, Y.Shen, J.Tang, H.Lin, X.Ma, B.jin, Z.Liu, R.J.Peters, Z.K.ZHao and L.Huangang (2016). Cytochrome P450 pro-sensitivity leads to a biofunctional biochemical pathway for supporting shishines. New Phytol 210(2): 525-" available from the Applicant, which biomaterial is only useful for repeating the experiments relevant to the present invention and is not useful for other public uses.

The pKK7FWG2.0 vectors of the following examples are described in the literature "Yu, L., X.Tan, B.Jiang, X.Sun, S.Gu, T.Han and W.Hou (2014.). A peroxiral long-chain-CoA synthesis from Glycine max absorbed in lipid degradation.PLoS One 9(7): e100144", publicly available from the Applicant, the biomaterials being used only for the repetition of the experiments relating to the invention and not for other uses.

The pCAMBIA-NLuc vectors and pCAMBIA-CLuc vectors in the following examples are described in the documents "Chen, H., Y.Zou, Y.Shang, H.Lin, Y.Wang, R.Cai, X.Tang and J.M.Zhou (2008); recovery luminescence compensation analysis for protein-protein interactions in plants. plant Physiol 146(2): 368-376", publicly available from the Applicant ", and the biomaterials are used only for the repetition of the experiments relating to the present invention and are not used for other purposes.

The following examples of Agrobacterium tumefaciens EHA105 are described in the references "Cui, G., L.Duan, B.jin, J.Qian, Z.Xue, G.Shen, J.H.Snyder, J.Song, S.Chen, L.Huang, R.J.Peters and X.Qi (2015). Functional transformation of variant Synthesis in the Medicinal Plant Salvia militaria. Plant Physiol 169(3):1607 &1618", publicly available from the Applicant, and the biomaterials are used only for the repetition of the experiments relating to the present invention and are not useful for other purposes.

The Yeast AH109 in the following examples is described in the document "Moeini-Naghani, I., and Navartanam, D.S. (2016. Yeast Two-Hybrid Screening to Test for Protein-Protein Interactions in the Audio System and Audio and vehicular Research: Methods and Protocols, 95-107." publicly available from the Applicant, and this biomaterial was used only for the repetition of the experiments relating to the present invention and was not used for other purposes.

Crude extracts of Arabidopsis thaliana E2UBC32 protein expression in the following examples are described in the literature "Cui, f., Liu, l., Zhao, Q., Zhang, z., Li, Q., Lin, b., Wu, y., Tang, s.and Xie, Q. (2012. Arabidopsis ubiquitin coupling enzyme UBC32is an ERAD component which is in a branched-dimensional saline strain. the Plant Cell,24(1), 233-.

MYC-GFP and p19 bacteria are described in the publications "Liu, L., Y.Zhang, S.Tang, Q.Zhao, Z.Zhang, H.Zhang, L.Dong, H.Guo and Q.Xie (2010) and An effective system to detect protein solubilization by imaging filtration in Nicotiana benthamiana. plant J61 (5): 893-.

MBP protein, wheat El protein expression crude extracts and ubiquitin proteins (His-Ub proteins) in the following examples are described in the literature "ZHao, Q., M.Tian, Q.Li, F.Cui, L.Liu, B.yin and Q.Xie (2013). A plant-specific in vision inactivation analysis system. plant J74 (3): 524-533"), and are publicly available from the applicant, and the biomaterials are used only for repeating the relevant experiments of the present invention and are not used for other purposes.

Example 1 obtaining and validation of SmSIP1 protein interacting with Salvia miltiorrhiza squalene synthase

Construction and evaluation of first-and second-hybrid cDNA library of red-rooted salvia yeast

1. Selection of test materials

The invention selects 2-year-old Salvia miltiorrhiza plants as research materials, samples are collected from 2008 month 4, and are collected regularly at about 10, 20 and 30 days per month until the end of 7 months, and the samples are stored at-80 ℃ for later use, and are identified as labiatae Salvia miltiorrhiza Salvia Miltrorrhiza by a chrysolon researcher of Chinese academy of sciences.

2. construction and evaluation of cDNA library

Respectively extracting RNA of the salvia miltiorrhiza material collected in each month by using a Trizol kit, equivalently mixing, separating mRNA, performing reverse transcription by using total salvia miltiorrhiza RNA as a template and oligo (dT) as a primer to synthesize single-stranded cDNA, synthesizing double-stranded cDNA, connecting Aadptor, removing short-piece short cDNA, connecting the double-stranded cDNA with a pGADT7 vector, and transforming escherichia coli to obtain a salvia miltiorrhiza yeast double-hybrid cDNA library. The quality of the library was evaluated by calculating the library capacity of the library, recombination rate and the size of the insert. The colony count of the transformed plate was calculated to obtain a library capacity of 1.50X 10 for the original library⁶cfu, recombination rate 95.3%, length of insert rangedThe fragment length is 0.5-5.0 Kpb, and the average length of the fragment is more than 1.0Kpb, which indicates that the constructed cDNA library has higher quality.

Two, construction of 'bait' vector and self-activation detection of SmSQS1

1. Construction of "bait" vectors

(1) PCR amplification is carried out by taking a plasmid SmSQS1(GeneBank, accession number: FJ768961.1) as a template and adopting SmSQS1-F and SmSQS1-R primers to obtain a SmSQS1 target fragment; the SmSQS1 target fragment was purified by the GeneJET PCR Purification Kit to obtain purified SmSQS 1. The primer sequences are as follows:

SmSQS 1-F: 5'-TTTTTTCATATGATGGGGAGTTTACGTGCGATTTTGAG-3', containing Nde I enzyme cutting sites;

SmSQS 1-R: 5'-TTTTGGATCCGTTAGACGGCTCTCTTTGGTGCAAAG-3', containing BamH I cleavage sites.

(2) The purified SmSQS1 and PGBKT7 vectors are subjected to double enzyme digestion and connection by using restriction enzymes Nde I and BamH I respectively to obtain recombinant vectors pGBKT7-SmSQS1, and sequencing verification is carried out on the recombinant vectors.

The sequencing result shows that: the recombinant vector pGBKT7-SmSQS 1is a vector obtained by inserting a DNA fragment (GeneBank, accession number: FJ768961.1) of a SmSQS1 coding region into Nde I and BamH I enzyme cutting sites of a pGBKT7 vector and keeping other sequences of the pGBKT7 vector unchanged.

3. Self-activation detection of SmSQS1

(1) Transferring the recombinant vector pGBKT7-SmSQS1 into an AH109 yeast competent cell to obtain yeast containing pGBKT7-SmSQS 1;

transferring the pGBKT7 vector into an AH109 yeast competent cell to obtain a yeast containing pGBKT 7;

(2) pGADT7 vector was transformed into a yeast containing pGBKT7-SmSQS1 and a yeast containing pGBKT7, respectively, and then inoculated on SD/-Trp/-Leu and SD-Trp/-Leu/-His/-Ade plates, respectively, and cultured upside down at 30 ℃ for 5-7 days.

The results show that: SmSQS 1is unable to activate reporter gene expression, is not self-activating and can be used as bait protein for screening yeast two-hybrid library.

Screening of SmSIP1 protein interacting with SmSQS1

1. Screening for proteins interacting with SmSQS1

The recombinant vector pGBKT7-SmSQS 1is used as a bait protein, and a cDNA library of salvia miltiorrhiza yeast double-hybrid is screened by a yeast sequential transformation method to obtain a positive clone. The method comprises the following specific steps:

(1) yeast colonies (2-3 mm in diameter) containing pGBKT7-SmSQS1 were picked on SD/-Trp plates and inoculated into 50ml SD/-Trp liquid medium, cultured at 30 ℃ and 200rpm for 18-24h to the yeast OD₆₀₀About 1.5;

(2) OD in step (1)₆₀₀1.5 Yeast containing pGBKT7-SmSQS1 transferred 1:40 to 300ml SD/-Trp liquid medium, 30 deg.C, 250rpm continued shaking for 12-16h to OD₆₀₀About 0.6;

(3) OD in step (2)₆₀₀Putting 0.6 yeast containing pGBKT7-SmSQS1 into a 50mL centrifuge tube, centrifuging at room temperature for 5min at 1000g, and collecting cells;

(4) adding 50mL of sterile ddH to the cells collected in step (3)₂O, suspend the pellet with vortex;

(5) centrifuging at room temperature at 1000g for 5min, removing supernatant, and collecting precipitate;

(6) repeating (4) and (5);

(7) suspending the precipitate in step (6) with 3ml of sterile 100mmoL/L LiAc per tube, performing water bath at 30 ℃ for 15min, and collecting yeast cells;

(8) preparing a PEG/LiAc solution, wherein the formula is shown in a table 1;

TABLE 1 Yeast transformation reaction System (360. mu.L)

(9) Dividing 100 mu L of yeast cells in the step (7) into 8 centrifugal tubes of 1.5mL on average, and centrifuging at room temperature for 30s at 13,000 rpm;

(10) removing supernatant, and collecting cell precipitate; adding precooled PEG/LiAc solution to the cell pellet (360 uL per tube), suspending the pellet by vortex and mixing uniformly;

(11) water bath at 30 deg.C for 30min, and mixing the cells once every 5 min;

(12) heat-shocking in 42 deg.C water bath for 30min, mixing the cells once every 5 min;

(13) centrifuging at 13,000rpm for 30 s;

(14) removing supernatant, collecting precipitate, adding 600 μ L ddH to the precipitate₂O suspended cells, coated on 50 SD/-Trp/-Leu/-His/-Ade plates 150mm in diameter; culturing in 30 deg.C incubator for 7-10 days, and observing the result;

(15) mix well with 1 tube before plate coating, aspirate 1 μ L ddH dissolved in 199 μ L with pipette₂In O, mixing uniformly;

(16) then 10. mu.L of ddH dissolved in 190. mu.L were aspirated from the tube₂In O, uniformly mixing, repeating the steps in the same manner, and repeatedly diluting for 2 times;

(17) there were 3 different concentrations after dilution, respectively: 1: 200. 1: 2000. 1: 20, 000;

(18) 3 tubes of cell suspensions with different concentrations are respectively smeared on 3 SD/-Trp/-Leu plates with the diameter of 90mm, the plates are placed in an incubator at 30 ℃ for 3-4 days, the results are observed, and colonies are counted for calculating the transformation efficiency.

Finally, SmSQS 1is used as bait protein, and a yeast two-hybrid system is used for screening proteins interacting with pGBKT7-SmSQS1 from a salvia miltiorrhiza cDNA library to obtain 529 positive clones.

2. Acquisition and sequence analysis of SmSIP1 protein interacting with SmSQS1

Selecting all single colony clones, culturing for 5-7 days in SD/-Trp-Leu-His-Ade four-deficient culture medium at 250rpm, extracting plasmids of all positive clone yeast strains, converting escherichia coli, amplifying, extracting plasmids, sequencing, and performing Blast analysis on the obtained sequences and a GenBank database. Through sequencing analysis, 26 proteins which can possibly interact with SmSQS1 are obtained preliminarily. The protein with the complete sequence of the coding region is selected and named SmSIP1, the amino acid sequence of the protein is shown as the sequence 2, and the coding gene sequence of the SmSIP1 protein is shown as the sequence 1.

3. Sequence analysis of SmSIP1 protein

(1) Bioinformatic analysis of SmSIP1 protein

Homology analysis was carried out using the SmSIP1 protein sequence (GenBank, accession number: KY270500) shown in sequence No. 2 using the website of the national center for Biotechnology information (http:// www.ncbi.nlm.nih.gov). The transmembrane domain was predicted using TMHMMServer v.2.0(http:// www.cbs.dtu.dk/services/TMHMM-2.0 /); the functional domains were predicted using SMART (http:// SMART. embl-heidelberg. de /).

The SmSIP1 protein was predicted by SMART protein sequence analysis program (http:// SMART. embl-heidelberg. de /) to have a possible transmembrane region at C-terminus (amino acids 266 in SEQ ID NO: 2), a conserved RING finger domain at N-terminus (amino acids 41-89 in SEQ ID NO: 3HC 4) and an IBR domain between amino acids 108-170 in SEQ ID NO: 2, as shown in FIG. 1.

(2) Phylogenetic analysis of the SmSIP1 sequence

Phylogenetic trees were constructed based on the SmSIP1 sequence and the other 43 protein sequences using MEGA7.0 software. The constructed phylogenetic tree is shown in fig. 2. The results show that SmSIP1 belongs to the Plant II subfamily branch.

Verification analysis of SmSIP1 and SmSQS1 interaction

(one) Torulopsis gyratory yeast validation of the interaction of SmSIP1 with SmSQS1

SmSIP1 and SmSQS1 Torulopsis were used to verify the interaction of SmSIP1 with SmSQS 1. The method comprises the following specific steps: transforming the pGADT7-SmSIP1 plasmid obtained by screening in the third step into AH109 yeast containing pGBKT7-SmSQS1, respectively coating the recombinant yeast on SD/-Trp/-Leu and SD-Trp/-Leu/-His/-Ade plates after transforming the AH109 yeast, and then putting the plates in an incubator at 30 ℃ for culturing for 5-7 days. The empty vector pGADT7 was also transformed with AH109 yeast containing pGBKT7-SmSQS1 as a control.

The results show that: all yeast co-transformed combinations grew normally on the SD-Trp-Leu plates, indicating that all combinations were successfully transformed. However, the recombinant yeast containing pGBKT7-SmSQS and pGADT7-SmSIP1 on the SD-Trp-Leu-His-Ade screening plate grew normally, whereas the recombinant yeast containing pGBKT7-SmSQS alone (control group) failed to grow normally. The SmSIP1 protein and the SmSQS1 protein can be specifically combined with each other to activate the expression of a reporter gene.

(II) luciferase complementary imaging technology verifies the interaction of SmSIP1 and SmSQS1

Luciferase complementation imaging technology is a method of rapidly analyzing interaction between a protein and a protein, the protein interacting with a small molecule compound, which divides Luciferase (LUC) into two parts of an N-terminal part (Nluc) and a C-terminal part (Cluc), which cannot function by spontaneous recombination, but when they are fused with two proteins capable of interacting with each other, respectively, recombinant luciferase activity is initiated. The invention utilizes the technology to verify the interaction of SmSIP1 protein and SmSQS1 protein. The method comprises the following specific steps:

1. gene cloning and vector construction

(1) Cloning of genes

1) Design of primers

Corresponding primers are designed according to the restriction enzyme cutting site information of the vectors pCAMBIA-CLuc and pCAMBIA-NLuc and the sequences of SmSQS1 and SmSIP 1. The primer sequences are as follows:

pCAMBIA-CLuc-SIP 1-KpnI-F: 5'-TTTTTTGGTACCATGGGAAACACTCTGCAAAAGC-3', containing KpnI enzyme cutting site;

pCAMBIA-CLuc-SIP 1-SalI-R: 5'-TTTTTTGTCGACTTATGTAGGAGTATTATTCCTAA-3', containing SalI cleavage sites;

pCAMBIA-NLuc-SQS 1-KpnI-F: 5'-TTTTTTGGTACCATGGGGAGTTTACGTGCGAT-3', containing KpnI enzyme cutting site;

pCAMBIA-NLuc-SQS 1-SalI-R: 5'-TTTTTTGTCGACGACGGCTCTCTTTGGTGCAA-3', containing SalI cleavage sites.

2) Cloning of genes

Carrying out PCR amplification by using a plasmid SmSIP1 as a template and primers pCAMBIA-CLuc-SIP1-KpnI-F and pCAMBIA-CLuc-SIP1-SalI-R to obtain a SmSIP1 target fragment;

the plasmid SmSQS 1is used as a template, and primers pCAMBIA-NLuc-SQS1-KpnI-F and pCAMBIA-NLuc-SQS1-SalI-R are adopted for PCR amplification to obtain a SmSQS1 target fragment.

(2) Construction of recombinant vectors

Carrying out double enzyme digestion on the SmSIP1 target fragment and the vector pCAMBIA-CLuc by using restriction enzymes Kpn I and Sal I respectively, and connecting to obtain a recombinant vector pCAMBIA-CLuc-SmSIP 1; carrying out double enzyme digestion on a SmSQS1 target fragment and the vector pCAMBIA-NLuc by using restriction enzymes Kpn I and Sal I respectively, and connecting to obtain a recombinant vector pCAMBIA-NLuc-SmSQS 1;

the recombinant vector pCAMBIA-CLuc-SmSIP1 and the recombinant vector pCAMBIA-NLuc-SmSQS1 were sequenced, respectively. The sequencing result shows that: the recombinant vector pCAMBIA-CLuc-SmSIP 1is obtained by inserting a SmSIP1 coding region DNA fragment shown in a sequence 1 between Kpn I and Sal I enzyme cutting sites of the pCAMBIA-CLuc vector and keeping other sequences of the vector pCAMBIA-CLuc unchanged. The recombinant vector pCAMBIA-NLuc-SmSQS 1is obtained by inserting a DNA fragment (GeneBank, accession number: FJ768961.1) of a coding region of SmSQS1 into a cleavage site between Kpn I and Sal I of the vector pCAMBIA-NLuc and keeping other sequences of the vector pCAMBIA-NLuc unchanged.

2. Preparation of bacterial liquid

(1) Preparation of recombinant bacterium

The recombinant vectors pCAMBIA-NLuc-SmSQS1 and pCAMBIA-CLuc-SmSIP1 are respectively transferred into EHA105 Agrobacterium tumefaciens competent cells to obtain recombinant bacteria pCAMBIA-NLuc-SmSQS1/EHA105 and pCAMBIA-CLuc-SmSIP1/EHA 105.

Respectively transferring the empty vectors pCAMBIA-NLuc and pCAMBIA-CLuc into the EHA105 Agrobacterium tumefaciens competent cell to obtain recombinant bacteria pCAMBIA-NLuc/EHA105 and pCAMBIA-CLuc/EHA 105.

(2) Cultivation of recombinant bacteria

Recombinant bacteria pCAMBIA-NLuc-SmSQS1/EHA105 and pCAMBIA-CLuc-SmSIP1/EHA105, recombinant bacteria pCAMBIA-NLuc/EHA105 and pCAMBIA-CLuc/EHA105 and p19 were shake-cultured overnight at 28 ℃ in a liquid LB medium containing kanamycin (Kana) and rifampicin (Rif) until OD₆₀₀The value is about 0.6-1.0. Detection of each bacterium by ultraviolet spectrophotometerOD of (1)₆₀₀。

(3) Preparation of the suspension

1) Calculating the volume number of required various bacteria: 0.5OD₆₀₀unit/ml×0.5ml＝0.25OD₆₀₀unit＝OD₆₀₀Absorbance x number of volumes of bacterial suspension (ml).

2) Taking various bacteria with corresponding volumes obtained by calculation, and mixing the bacteria as follows:

control group 1: pCAMBIA-NLuc bacterial liquid, pCAMBIA-CLuc-SmSIP1 bacterial liquid and p19 bacterial liquid;

control group 2: pCAMBIA-CLuc bacterial liquid, pCAMBIA-NLuc-SmSQS1 bacterial liquid and p19 bacterial liquid;

experimental groups: pCAMBIA-CLuc-SmSIP1 bacterial liquid, pCAMBIA-NLuc-SmSQS1 bacterial liquid and p19 bacterial liquid.

3) The mixed bacterial solution of each group was centrifuged at 13000rpm for 1 minute and then resuspended in 500ul infection suspension (10mM MgCl. RTM.)₂10mM MES and 200. mu.M acetosyringone), incubating at 28 ℃ for 3-5 hours, and standing to obtain a control group 1 suspension, a control group 2 suspension and an experimental group suspension respectively.

3. Genetic transformation of tobacco

(1) Sowing the seeds of the native tobacco in a flowerpot, culturing in a greenhouse for sprouting, transplanting when two young leaves grow, culturing under the conditions of 25 ℃ of temperature, 54 percent of relative humidity and 16 h/8 h of illumination for 6 weeks to obtain the tobacco to be infected for later use.

(2) And (3) injecting the control group 1 suspension prepared in the step (2), the control group 2 suspension and the experimental group suspension into the back of the tobacco to be infected by using a 1ml injector respectively, injecting completely without leaving a gap as far as possible, marking the injected tobacco leaves by using a line hanging label, and culturing for 2-3 days. Spraying fluorescein (luciferin) with concentration of 100mM on the surface of tobacco leaf, and standing in dark for 5-10 min. The CCD imaging device was cooled with light to take a photograph, obtaining LUC images and calculating the luminous intensity.

The experimental results show that: only the experimental group (transformed with both the recombinant vector pCAMBIA-CLuc-SmSIP1 and the recombinant vector pCAMBIA-NLuc-SmSQS1) could detect a stronger fluorescence signal, while the control group (transformed with only the recombinant vector pCAMBIA-CLuc-SmSIP1 or pCAMBIA-NLuc-SmSQS1) could not detect a fluorescence signal. The SmSQS1 protein and the SmSIP1 protein are also shown to have interaction in plants.

Example 2 prokaryotic expression of SmSIP1 protein

Design of primers

Corresponding primers are designed according to the enzyme cutting site information of the pMAL-c2 vector and the SmSIP1 sequence. The primer sequences are as follows:

pMAL-c2-SmSIP 1-BamHI-F: 5'-TTTTTTGGATCCATGGGAAACACTCTGCAAAAGCT-3', containing BamHI enzyme cutting site;

pMAL-c2-SmSIP 1-PstI-R: 5'-TTTTTTCTGCAGTCATCTGAAGCACCATTCGGAAC-3', containing a PstI cleavage site.

Second, construction of expression vector

The SmSIP1 sequence with the transmembrane region removed and shown in the 1 st to 729 th positions of the sequence 1is inserted between BamHI and PstI enzyme cutting sites of a pMAL-c2 vector, and other sequences of the pMAL-c2 vector are kept unchanged to obtain a recombinant vector pMAL-c2-SmSIP1 DT.

Expression of mbp-SmSIP1DT fusion protein

Transferring the recombinant vector pMAL-c2-SmSIP1DT into Escherichia coli E.coli BL21 by a heat shock method, culturing for 12-16h (overnight) in an incubator at 37 ℃, picking out a monoclonal colony, and culturing overnight in 2mL LB liquid medium (Amp200 mug/mL); after the culture, PCR identification is carried out, 20 mu L of positive bacteria liquid is taken and transferred into 2mL LB liquid culture medium (Amp200 mu g/mL), and the mixture is subjected to shaking culture on a shaking table at 220rpm at 37 ℃ until OD is reached₆₀₀About 0.6 to about 1.0; take 1mL of OD₆₀₀Centrifuging the bacterial solution of about 0.6-1.0 at 4 ℃ for 3min at 5000g, and collecting thalli; then, resuspending the thalli by using sterile water, centrifuging again to collect the thalli, and transferring the thalli into a 5OmL LB liquid culture medium (Amp400 mu g/mL); shaking and culturing at 37 deg.C and 220rpm until cell density OD of BL21 thallus₆₀₀When the value reaches 0.6-1.0, adding IPTG inducer with final concentration of 0.4mM for induction culture; centrifuging at 4 deg.C and 3000g for 5min, collecting thallus, washing with precooled PBS buffer solution for 2 times, and collecting BL21 thallus; resuspending BL21 thallus with PBS buffer solution, placing in ice bath for ultrasonic bacteria breaking (ultrasonic l0s, interval 10s, total 6 times) until the bacteria liquid becomes clear and transparent to obtain Escherichia coli BL21 broken liquid (containing mbp-SmSIP1DT fusion)Synalbumin); the broken liquid of the Escherichia coli BL21 is centrifuged at 15000g for 30min at 4 ℃, and the supernatant (containing mbp-SmSIP1DT fusion protein) is collected to be detected by SDS-PAGE (sodium dodecyl sulfate-polyacrylamide) gel electrophoresis. The results of the electrophoresis are shown in FIG. 3,

lanes

1 and 5 represent uninduced pMAL-c 2;

lanes

2 and 6 are Induction pMAL-c 2;

lanes

3 and 7 are uninduced pMAL-c2-SmSIP1 DT;

lanes

4 and 8 are Induction pMAL-c2-SmSIP1 DT. The results show that: the mbp-SmSIP1DT fusion protein mainly exists in thallus lysis supernatant, the molecular weight is about 68.73kDa, and the mbp-SmSIP1DT fusion protein is SmSIP1 protein with mbp tag at the N terminal.

Purification of fusion protein mbp-SmSIP1DT

And (3) purifying the supernatant (containing mbp-SmSIP1DT fusion protein) obtained in the step three to obtain the purified mbp-SmSIP1 fusion protein.

1. Taking 20-40uL MBP agarose beads (GE healthcare) to a 1.5mL Eppendorf tube, washing the agarose beads with column buffer solution, centrifuging at 2000rpm for 2min, discarding the supernatant, and repeating the washing twice;

2. adding the supernatant (containing mbp-SmSIP1DT fusion protein) of step three, supplementing the total volume to 1ml with column buffer solution, and slowly rotating at room temperature for 1 h;

3. agar spheres were washed by adding 1ml of 50mM Tris (pH 8.0), centrifuged at 2000rpm for 2min at room temperature, and the supernatant carefully removed with a pipette tip;

4. repeating the step 3 five times, and washing off non-specifically bound protein to obtain purified mbp-SmSIP1 fusion protein.

Example 3 ubiquitination Activity analysis of SmSIP1 protein

In vitro ubiquitination reaction was performed in the presence of ATP-supplied energy, wheat E1 (having E1 ubiquitin activating enzyme activity), arabidopsis thaliana E2 (having E2 ubiquitin binding enzyme activity), and ubiquitin protein (His-Ub purified protein), and ubiquitination activity of SmSIP1 protein was analyzed. The method comprises the following specific steps:

1. preparation of ubiquitin system

Preparing a ubiquitin system, and dividing the ubiquitin system into a control group, -E1 group, -E2 group, -ub group, -E3 group and an experimental group according to different components in the ubiquitin system. The components are as follows (table 2):

(1) control group: 1uL MBP protein, 1.5uL 20 × reaction buffer (1M Tris pH 7.5, 40mM ATP (sigma), 100mM MgCl₂40mM DTT), 3uL wheat El (wheat El protein expression crude extract, about 50ng), 3uL Arabidopsis E2 (Arabidopsis E2UBC32 protein expression crude extract, about l00ng), 2uL ubiquitin protein (about 4ug), sterile water to total volume of 30 uL;

(2) -group E1: 4uL of the purified mbp-SmSIP1 fusion protein of example 2, 1.5uL of 20 × reaction buffer (1M Tris pH 7.5, 40mM ATP (sigma), 100mM MgCl₂40mM DTT), 3uL Arabidopsis E2 (Arabidopsis E2UBC32 protein expression crude extract, about l00ng), 2uL ubiquitin protein (His-Ub purified protein, about 4ug), sterile water was added to a total volume of 30 uL;

(3) -group E2: 4uL of the purified mbp-SmSIP1 fusion protein of example 2, 1.5uL of 20 × reaction buffer (1M Tris pH 7.5, 40mM ATP (sigma), 100mM MgCl₂40mM DTT), 3uL wheat El (wheat El protein expression crude extract, about 50ng), 2uL ubiquitin protein (His-Ub purified protein, about 4ug), sterile water to a total volume of 30 uL;

(4) -group ub: 4uL of the purified mbp-SmSIP1 fusion protein of example 2, 1.5uL of 20 × reaction buffer (1M Tris pH 7.5, 40mM ATP (sigma), 100mM MgCl₂40mM DTT), 3uL wheat El (wheat El protein expression crude extract, about 50ng), 3uL Arabidopsis E2 (Arabidopsis E2UBC32 protein expression crude extract, about l00ng), sterile water to a total volume of 30 uL;

(5) -group E3: 4uL of the purified mbp-SmSIP1 fusion protein of example 2, 3uL of wheat El (wheat El protein expression crude extract, about 50ng), 3uL of Arabidopsis E2 (Arabidopsis E2UBC32 protein expression crude extract, about l00ng), 2uL of ubiquitin protein (His-Ub purified protein, about 4ug), sterile water was added to a total volume of 30 uL;

(6) experimental groups: 4uL of the purified mbp-SmSIP1 fusion protein of example 2, 1.5uL of 20 × reaction buffer (1M Tris pH 7.5, 40mM ATP (sigma), 100mM MgCl₂40mM DTT), 3uL wheat El (wheat El protein expression crude extract, about 50ng), 3uL Arabidopsis E2 (Arabidopsis E2UBC32 protein expression crude extract, aboutl00ng), 2uL ubiquitin protein (His-Ub purified protein, about 4ug), sterile water was added to make a total volume of 30 uL.

TABLE 2 formulation of ubiquitin System

2. Respectively placing the ubiquitin systems prepared in the step 1 on an Eppendorf Thermomixer comfort instrument, and reacting for 90min at 30 ℃ and 900 rpm;

3. adding 10uL 4 xSDS-PAGE protein loading buffer solution into the tube to terminate the reaction, and heating with boiling water for 5 min;

4. centrifuging at room temperature at 13,000rpm for 3min, and performing SDS-PAGE (sodium dodecyl sulfate-polyacrylamide gel electrophoresis) on a 15uL sample;

5. and (5) after the electrophoresis is finished, membrane switching is carried out, and Western hybridization detection is carried out by using Nickel-HRP.

The results show that: SmSIP1 can add more and more ubiquitin proteins to itself to form a large molecule MBP-SmSIP1-Ubn complex, and at the same time, no polyubiquitination modification signal was detected after 90min of reaction in the control group (MBP) and the reaction group lacking wheat E1(-E1 group) or arabidopsis E2(-E2 group). Thus indicating that the prokaryotic expression SmSIP1 has specific E3 ubiquitin ligase activity.

Example 4 SmSIP1 protein promotes SmSQS1 degradation

First, construction of recombinant vector

1. Design of primers

Corresponding primers are designed according to the restriction site information of the vectors pCAMBIA1307-6myc and pENTR and the sequences SmSQS1 and SmSIP 1. The primer sequences are as follows: pCAMBIA1307-6myc-SmSQS 1-SalI-F: 5'-TTTTTTGTCGACATGGGGAGTTTACGTGCGAT-3', respectively; pCAMBIA1307-6myc-SmSQS 1-KpnI-R: 5'-TTTTTTGGTACCGACGGCTCTCTTTGGTGCAA-3', respectively; pENTR-SmSIP 1-F: 5'-CACCATGGGAAACACTCTGCAAAAGCTCC-3', respectively; pENTR-SmSIP 1-R: 5'-GTCGACTTATGTAGGAGTATTATTCCTAA-3' are provided.

2. Cloning of the Gene of interest

(1) PCR amplification is carried out by taking SmSQS1 as a template and adopting primers pCM1307-SmSQS1-F and pCM1307-SmSQS1-R to obtain a SmSQS1 target fragment;

(2) SmSIP 1is used as a template, and pENTR-SmSIP1-F and pENTR-SmSIP1-R primers are adopted for PCR amplification to obtain a fragment of the complete coding region of SmSIP 1.

3. Construction of recombinant vectors

(1) Cloning the whole coding region fragment of SmSIP1 into a pENTR vector by using a directive TOPO cloning kit (Invitrogen), and then subcloning the whole coding region fragment of SmSIP1 into a pKK7FWG2.0 vector by using a Gateway LR Enzyme Mix (Invitrogen) to obtain a recombinant vector SmSIP 1-pKK7FWG2.0;

(2) the SmSQS1 target fragment and the vector pCAMBIA1307-6MYC are subjected to double enzyme digestion and connection by using restriction enzymes SalI and KpnI respectively to obtain a recombinant vector 35S, namely MYC-SmSQS 1.

Second, construction of recombinant bacteria

1. Preparation of Agrobacterium competence

Agrobacterium EHA105 competent cells were prepared as follows:

1) taking the preserved agrobacterium EHA105, streaking and activating on a YEB solid culture medium containing corresponding antibiotic resistance, and carrying out 24-36 h in a constant temperature incubator at 28 ℃ until a single colony grows out;

2) a single colony of Agrobacterium was picked and inoculated into 5mL of YEB broth containing the corresponding antibiotic. Shake culturing at 28 deg.C and 250rpm for 24-36 h until the Agrobacterium liquid OD₆₀₀About 0.5 to about 0.6;

3) adding the bacterial liquid into YEB liquid culture medium containing corresponding antibiotics at a ratio of 2% for dilution, and performing shake culture at 28 deg.C and 250rpm to OD₆₀₀0.5 to 0.6;

4) centrifuging at 4 deg.C and 5000rpm for 10min, and collecting thallus;

5) adding non-resistant YEB solution with the same volume as the culture solution in the enlarged culture, and re-suspending;

6) centrifuging at 4 deg.C and 5000rpm for 10min, and collecting thallus;

7) adding sterilized 70mmol/L CaCl for enlarging the volume of culture solution 1/10 during culture₂Solution (pre-cooled), resuspension;

8) subpackaging the resuspended bacterial liquid into 100 mu L of small portions, and placing the small portions on ice for later use;

9) if the product needs to be preserved, 15-20% of glycerin with the final concentration can be added for freezing and preserving at the temperature of minus 80 ℃.

2. Transformation of Agrobacterium

Transforming the recombinant vector 35S MYC-SmSQS1 into EHA105 Agrobacterium tumefaciens competent cells, culturing on a YEB culture medium plate, and culturing in an incubator at 28 ℃ for 48h to obtain recombinant Agrobacterium containing 35S MYC-SmSQS 1;

transforming the recombinant vector SmSIP1-pK7FWG2,0 into EHA105 Agrobacterium tumefaciens competent cells, culturing on a YEB culture medium plate, and culturing in an incubator at 28 ℃ for 48h to obtain recombinant Agrobacterium containing SmSIP1-pK7FWG2, 0;

culturing 35S-MYC-GFP transformed EHA105 Agrobacterium tumefaciens competent cells on a YEB culture medium plate in an incubator at 28 ℃ for 48h to obtain the recombinant Agrobacterium containing 35S-MYC-GFP.

Third, tobacco genetic transformation

1. Agrobacterium injection

Tobacco NB for genetic transformation was planted in a greenhouse (16h light/8 h dark, 70% relative humidity, temperature 22 ℃) and 5-7 weeks old tobacco leaves were used for agrobacterium injection. The method comprises the following specific steps:

injecting the recombinant agrobacterium containing 35S MYC-SmSQS1 into tobacco leaf, and sampling after injecting for 3 days to obtain MYC-SmSQS1 sample;

injecting the recombinant agrobacterium tumefaciens containing SmSIP1-pK7FWG2,0 into tobacco leaves, and sampling after injecting for 3 days to obtain a SmSIP1-GFP sample;

injecting the recombinant agrobacterium containing 35S MYC-GFP into the tobacco leaf, and sampling after injecting for 3 days to obtain a GFP sample.

2. Protein extraction

Extracting proteins in the MYC-SmSQS1 sample, the SmSIP1-GFP sample and the GFP sample respectively to obtain a MYC-SmSQS1 protein extracting solution, a SmSIP1-GFP protein extracting solution and a GFP protein extracting solution respectively. The method comprises the following specific steps: collecting the permeated part of each tobacco leaf, grinding the leaf tissue in liquid nitrogen to obtain ground tissue, and placing the ground tissue in extraction buffer NB1(50mM T) on iceRIS-MES(pH 8.0)，0.5M sucrose，1mM MgCl₂10mM EDTA, 5mM DTT, protease inhibitor cocktail complementMini tables (Roche, http:// www.roche.com /) to obtain a protein extract.

SmSQS1 degradation promotion by SmSIP1

1. Adding protein synthesis inhibitor CHX (sigma company) with final concentration of 50 μ M into MYC-SmSQS1 protein extract, SmSIP1-GFP protein extract and GFP protein extract in step three, respectively, suspending leaf tissue, and centrifuging at 4 deg.C and 16000g for 30min to obtain MYC-SmSQS1 protein extract, SmSIP1-GFP protein extract and GFP protein extract, respectively.

2. Mixing MYC-SmSQS1 protein extract and SmSIP1-GFP protein extract at a ratio of 1:2 to obtain experimental group sample (SmSIP 1-GFP); meanwhile, MYC-SmSQS1 protein extract and GFP protein extract were mixed at a ratio of 1:2 to serve as a control sample (GFP).

3. The experimental group sample and the control group sample are respectively placed in an Eppendorf Thermomixer for 30 ℃ and are cultured by gentle shaking, and then the samples are respectively taken out when the samples are cultured for 0h, 1h, 2h and 4 h. SDS buffer was added to the sample, the reaction was stopped after boiling for 10 minutes for western detection, and MYC-SmSQS1 protein was detected using anti-Myc antibody (available from sigma), and SmSIP1-GFP and GFP proteins were detected using anti-GFP antibody (available from sigma).

The results are shown in FIG. 4 (Ponceau S means loading control). As can be seen from the figure: the amount of SmSQS1 protein gradually decreased in both groups of samples with time, and the degradation rate of SmSQS1 protein was significantly higher in the experimental group of samples (SmSIP1-GFP) than in the control group of samples (GFP). The amount of SmSQS1 protein in the experimental group sample (SmSIP1-GFP) at reaction 2h is reduced, the amount of SmSQS1 protein is reduced along with the increase of time after reaction 4h, and the SmSQS1 protein is proved to be capable of promoting the degradation of SmSQS1 protein and is used for regulating the content of SmSQS1 protein.

Claims

The application of SmSIP1 protein as E3 ubiquitin ligase;

the SmSIP1 protein is the protein of A1) or A2) as follows:

A1) the amino acid sequence is a protein shown in a sequence 2;

A2) and (b) a fusion protein obtained by connecting a tag to the N-terminal and/or the C-terminal of the protein shown in the sequence 2.
The application of SmSIP1 protein in regulating and controlling the content of squalene synthase;

the SmSIP1 protein is the protein of A1) or A2) as follows:

A1) the amino acid sequence is a protein shown in a sequence 2;

A2) and (b) a fusion protein obtained by connecting a tag to the N-terminal and/or the C-terminal of the protein shown in the sequence 2.
The application of SmSIP1 protein in preparing a product for regulating and controlling the content of squalene synthase;

the SmSIP1 protein is the protein of A1) or A2) as follows:

A1) the amino acid sequence is a protein shown in a sequence 2;

A2) and (b) a fusion protein obtained by connecting a tag to the N-terminal and/or the C-terminal of the protein shown in the sequence 2.
4. Use according to claim 2 or 3, characterized in that: the squalene synthase content is regulated to promote the degradation of squalene synthase.
5. Application of biological materials related to SmSIP1 protein in preparation of E3 ubiquitin ligase;

the biological material related to the SmSIP1 protein is any one of the following B1) to B12):

B1) a nucleic acid molecule encoding a SmSIP1 protein;

B2) an expression cassette comprising the nucleic acid molecule of B1);

B3) a recombinant vector comprising the nucleic acid molecule of B1);

B4) a recombinant vector comprising the expression cassette of B2);

B5) a recombinant microorganism comprising the nucleic acid molecule of B1);

B6) a recombinant microorganism comprising the expression cassette of B2);

B7) a recombinant microorganism containing the recombinant vector of B3);

B8) a recombinant microorganism containing the recombinant vector of B4);

B9) a transgenic plant cell line comprising the nucleic acid molecule of B1);

B10) a transgenic plant cell line comprising the expression cassette of B2);

B11) a transgenic plant cell line comprising the recombinant vector of B3);

B12) a transgenic plant cell line comprising the recombinant vector of B4);

the SmSIP1 protein is the protein of A1) or A2) as follows:

A1) the amino acid sequence is a protein shown in a sequence 2;

A2) and (b) a fusion protein obtained by connecting a tag to the N-terminal and/or the C-terminal of the protein shown in the sequence 2.
6. Use of the biological material as claimed in claim 5 for modulating squalene synthase content.
7. Use of the biological material as claimed in claim 5 for the preparation of a product for modulating squalene synthase content.
8. Use according to claim 6 or 7, characterized in that: the squalene synthase content is regulated to promote the degradation of squalene synthase.
9. Use according to any one of claims 5 to 7, characterized in that: B1) the nucleic acid molecule is a cDNA molecule or a genome DNA molecule shown in a sequence 1.