Gene combination for enhancing anabolic flow of tomato triterpenoids and application of gene combination

Abstract

The invention discloses a gene combination for enhancing anabolic flux of tomato triterpenoids and application of the gene combination. In particular discloses application of a gene combination (genes for coding proteins OSS1, OSS2 and OSS3) to regulation and control of anabolic flow of the tomato triterpenoids and regulation and control of synthesis of precursor substances of the tomato triterpenoids, the triterpenoids and / or sterol compounds. The key gene combination for enhancing the synthetic route of the triterpene precursor substance in the mature tomato fruit is screened and identified. Experiments prove that the gene combination disclosed by the invention can enhance the synthesis of squalene and squalene oxide in tomato fruits and further promote the synthesis of triterpenoids and sterols. The gene combination disclosed by the invention can be used for creating a chassis material for synthesizing substances such as triterpenoids and phytosterol in tomato fruits, is used for producing metabolites such as triterpenoids and phytosterol in plants, and has a good application prospect.

Description

TECHNICAL FIELD

[0001] The present application belongs to the field of biotechnology, and particularly relates to a gene combination for enhancing the metabolic flow of triterpenoid compounds in tomato and application thereof. BACKGROUND

[0002] Triterpenoids are important components of primary and secondary metabolites in plants. Triterpenoids are an important class of terpenoids, and have diverse biochemical regulatory activities. For example, ginsenosides can promote the biosynthesis of proteins and RNA, and regulate and enhance the metabolism and immunity of the body; mogroside can promote insulin secretion, and adjust the immune imbalance caused by insulin-dependent diabetes, and has the effect of preventing and treating diabetes. Phytosterols belong to plant steroidal compounds, and have the effects of reducing blood cholesterol, preventing prostate hypertrophy, inhibiting tumors, inhibiting breast hyperplasia, and regulating immunity. Triterpenoids and phytosterols have the same synthesis precursor 2,3-oxidosqualene, and share the synthesis pathway upstream of 2,3-oxidosqualene.

[0003] Tomato is an annual or perennial herb of the Solanaceae family, and has a large biomass, a wide planting range, genetic stability, a mature genetic transformation system, and has gradually completed high-quality genome sequencing and annotation optimization. Tomato fruits are rich in carotenoids, which belong to tetraterpenoids, so tomato has a vigorous terpenoid synthesis metabolic flow. By promoting the expression of genes related to 2,3-oxidosqualene synthesis, the metabolic flow of tomato can be reprogrammed, so that the tomato fruits can synthesize triterpenoids and phytosterols with high economic value. SUMMARY

[0004] The technical problem to be solved by the present application is how to regulate the triterpenoid compound synthesis metabolic flow in tomato, and how to regulate the synthesis of triterpenoid compound precursors, triterpenoids and / or sterols. The technical problem to be solved is not limited to the technical subject described, and other technical subjects not mentioned herein can be clearly understood by those skilled in the art through the following description.

[0005] To solve the above technical problem, the present application first provides the application of the gene combination in any one of the following:

[0006] A1) in regulating the triterpenoid compound synthesis metabolic flow in tomato;

[0007] A2) in regulating the synthesis of triterpenoid compound precursors, triterpenoids and / or sterols in tomato;

[0008] A3) in regulating the content of triterpenoid compound precursors in tomato;

[0009] A4) use in modulating the ability of a tomato to produce triterpenoids and / or sterols;

[0010] A5) use in creating a pharmaceutical tomato germplasm;

[0011] The combination of genes includes a gene encoding protein OSS1, a gene encoding protein OSS2, and a gene encoding protein OSS3;

[0012] The protein OSS1 can be a protein with an amino acid sequence of SEQ ID No. 2, or a fusion protein with the same function obtained by connecting a tag to the N-terminus and / or C-terminus of SEQ ID No. 2;

[0013] The protein OSS2 can be a protein with an amino acid sequence of SEQ ID No. 4, or a fusion protein with the same function obtained by connecting a tag to the N-terminus and / or C-terminus of SEQ ID No. 4;

[0014] The protein OSS3 can be a protein with an amino acid sequence of SEQ ID No. 6, or a fusion protein with the same function obtained by connecting a tag to the N-terminus and / or C-terminus of SEQ ID No. 6.

[0015] In the above uses, the proteins OSS1 and OSS2 can be derived from tomato, and the protein OSS3 can be derived from Siraitia grosvenorii.

[0016] In the above uses, the connection can be direct connection by a peptide bond, or connection by a linker.

[0017] In order to facilitate purification or detection of the protein, a tag protein can be connected to the amino-terminal or carboxyl-terminal end of a protein consisting of the amino acid sequence shown in SEQ ID No. 2, SEQ ID No. 4, or SEQ ID No. 6 in the sequence listing.

[0018] The tag includes, but is not limited to, a GST (glutathione S-transferase) tag protein, a His tag protein, a MBP (maltose binding protein) tag protein, a Flag tag protein, a SUMO tag protein, a HA tag protein, a Myc tag protein, a LacZ tag protein, a CBD (cellulose binding domain) tag protein, a GFP (green fluorescent protein), a CFP (cyan fluorescent protein), a YFP (yellow-green fluorescent protein), a mCherry (monomeric red fluorescent protein) or an AviTag tag protein. Those skilled in the art know how to select a suitable tag protein according to the desired purpose (e.g. purification, detection or tracking). The use of the tag does not change the function of the target protein, and the purpose of the target protein is for purification, detection or tracking, so the tag protein suitable for the present application is not limited to a specific kind. The tag can be separated from the target protein by chemical cleavage or enzymatic cleavage known in the art.

[0019] The protein OSS1, OSS2 or OSS3 can be artificially synthesized, or a gene encoding the same can be synthesized first and then expressed biologically.

[0020] In the above application, the triterpenoid precursor can be squalene and / or oxidosqualene.

[0021] In the above application, the nucleotide sequence of the gene encoding the protein OSS1 can be as shown in SEQ ID No. 1; the nucleotide sequence of the gene encoding the protein OSS2 can be as shown in SEQ ID No. 3; and the nucleotide sequence of the gene encoding the protein OSS3 can be as shown in SEQ ID No. 5.

[0022] The present application also provides an application of a biological material in any one of the following:

[0023] B1) an application in regulating the metabolic flow of triterpenoids in tomato;

[0024] B2) an application in regulating the synthesis of triterpenoid precursors, triterpenoids and / or sterols in tomato;

[0025] B3) an application in regulating the content of triterpenoid precursors in tomato;

[0026] B4) an application in regulating the ability of tomato to produce triterpenoids and / or sterols;

[0027] B5) an application in creating medicinal tomato germplasm;

[0028] The biological material can be any one of the following:

[0029] C1) an expression cassette containing any one of the gene combinations described herein;

[0030] C2) a recombinant vector comprising any of the gene combinations described herein;

[0031] C3) a recombinant microorganism comprising any of the gene combinations described herein;

[0032] C4) a recombinant host cell comprising any of the gene combinations described herein;

[0033] C5) a transgenic plant cell line comprising any of the gene combinations described herein;

[0034] C6) a transgenic plant tissue comprising any of the gene combinations described herein;

[0035] C7) a transgenic plant organ comprising any of the gene combinations described herein.

[0036] Further, the application described herein can be overexpressing any of the gene combinations described herein in tomato.

[0037] Further, C1) the expression cassette, C2) the recombinant vector, C3) the recombinant microorganism, C4) the recombinant host cell, C5) the transgenic plant cell line, C6) the transgenic plant tissue, and C7) the transgenic plant organ all express any of the genes or gene combinations described herein.

[0038] The gene combinations described herein can comprise an OSS1 gene, an OSS2 gene, and an OSS3 gene. The coding sequence (CDS) of the OSS1 gene is shown in SEQ ID No. 1; the coding sequence (CDS) of the OSS2 gene is shown in SEQ ID No. 3, and the coding sequence (CDS) of the OSS3 gene is shown in SEQ ID No. 5.

[0039] The genes described herein can be DNA genes, such as cDNA genes, genomic genes, etc.; or RNA genes, such as mRNA genes or hnRNA genes, etc.

[0040] Further, C1) the expression cassette, C2) the recombinant vector, C3) the recombinant microorganism, C4) the recombinant host cell, C5) the transgenic plant cell line, C6) the transgenic plant tissue, or C7) the transgenic plant organ further comprises an E8 promoter, an E4 promoter, and a CYP450 promoter.

[0041] Further, the nucleotide sequence of the E8 promoter can be shown in SEQ ID No. 7; the nucleotide sequence of the E4 promoter can be shown in SEQ ID No. 8; and the nucleotide sequence of the CYP450 promoter can be shown in SEQ ID No. 9.

[0042] Further, the expression cassette C1), the recombinant vector C2), the recombinant microorganism C3), the recombinant host cell C4), the transgenic plant cell line C5), the transgenic plant tissue C6) or the transgenic plant organ C7) further contains the signal peptide AtCor15b, SlDXS2 and SlSSU.

[0043] Further, the nucleotide sequence of the gene of the signal peptide AtCor15b (AtCor15b gene) can be as shown in SEQ ID No. 10; the nucleotide sequence of the gene of the signal peptide SlDXS2 (SlDXS2 gene) can be as shown in SEQ ID No. 11; the nucleotide sequence of the gene of the signal peptide SlSSU (SlSSU gene) can be as shown in SEQ ID No. 12.

[0044] Further, in the expression cassette C1), the recombinant vector C2), the recombinant microorganism C3), the recombinant host cell C4), the transgenic plant cell line C5), the transgenic plant tissue C6) or the transgenic plant organ C7), the N terminus of the OSS1 gene is operably linked to the AtCor15b gene and the E8 promoter, which can be at the N terminus of the AtCor15b gene; the N terminus of the OSS2 gene is operably linked to the SlDXS2 gene and the E4 promoter, which can be at the N terminus of the SlDXS2 gene; the N terminus of the OSS3 gene is operably linked to the SlSSU gene and the CYP450 promoter, which can be at the N terminus of the SlSSU gene.

[0045] Those skilled in the art can easily mutate the nucleotide sequence encoding the protein OSS1, OSS2 and / or OSS3 of the present application by using known methods, such as site-directed mutagenesis (including oligonucleotide primer-mediated site-directed mutagenesis, PCR-mediated site-directed mutagenesis and cassette mutagenesis, etc.) or directed evolution (including error-prone PCR, DNA shuffling and in vitro random priming recombination, etc.). Those artificially modified nucleotide sequences having 75% or more identity with the nucleotide sequence encoding the protein OSS1, OSS2 and / or OSS3 of the present application, as long as they encode the protein OSS1, OSS2 and / or OSS3 and have the same function as the protein OSS1, OSS2 and / or OSS3, are derived from the nucleotide sequence of the present application and are equivalent to the sequence of the present application.

[0046] The gene of the protein OSS1, OSS2 or OSS3 (OSS1 gene, OSS2 gene or OSS3 gene) described in the present application can be any nucleotide sequence capable of encoding the protein OSS1, OSS2 or OSS3. In consideration of the degeneracy of codons and the preference of codons of different species, the skilled person can use codons suitable for expression in a particular species as required.

[0047] A recombinant expression vector containing the gene combination (OSS1 gene, OSS2 gene and OSS3 gene) described in the present application can be constructed using an existing plant expression vector. The plant expression vector includes, but is not limited to, binary Agrobacterium vectors and vectors that can be used for plant microprojectile bombardment, etc. The plant expression vector can also comprise a 3' untranslated region of the foreign gene, i.e. a polyadenylation signal and any other DNA fragment involved in mRNA processing or gene expression. The polyadenylation signal can guide the addition of polyadenylate to the 3' end of the mRNA precursor, such as the 3' untranslated region of the Agrobacterium crown gall inducing (Ti) plasmid gene (such as the nopaline synthase Nos gene), the plant gene (such as the soybean storage protein gene), etc.

[0048] When constructing a recombinant plant expression vector using the gene combination described in the present application, any one of the enhancer promoters or constitutive promoters can be added before the transcription initiation nucleotide, including but not limited to the cauliflower mosaic virus (CAMV) 35S promoter, the ubiquitin promoter of maize, which can be used alone or in combination with other plant promoters; in addition, when constructing a plant expression vector using the gene combination of the present application, enhancers, including translation enhancers or transcription enhancers, can also be used. These enhancer regions can be the ATG start codon or the adjacent region start codon, etc., but must be in the same reading frame as the coding sequence to ensure correct translation of the entire sequence. The source of the translation control signal and the start codon is wide and can be natural or synthetic. The translation initiation region can be from the transcription initiation region or the structural gene.

[0049] In order to facilitate the identification and screening of transgenic plant cells or plants, the plant expression vector used can be processed, such as adding genes that can be expressed in plants to produce color changes or luminescent compounds (GUS gene, luciferase gene, etc.), antibiotic markers with resistance (gentamicin marker, kanamycin marker, etc.) or chemical reagent resistance marker genes (such as herbicide resistance genes), etc. For the safety of transgenic plants, no selective marker gene can also be added and the transformed plants can be directly screened.

[0050] In one or more embodiments of the present application, the recombinant vector is pL2-1-OSS1+OSS2+OSS3. The recombinant vector pL2-1-OSS1+OSS2+OSS3 is a recombinant vector obtained by replacing the fragment between the two Bpil enzyme sites of vector pL2-1 with a nucleotide sequence as shown in the DNA molecule of SEQ ID No. 14, while keeping other nucleotide sequences of vector pL2-1 unchanged. pL2-1-OSS1+OSS2+OSS3 contains promoters E8pro, E4pro and CYP450pro, which respectively drive the transcription of genes oxidosqualene synthase 1 gene (OSS1), oxidosqualene synthase 2 gene (OSS2) and oxidosqualene synthase 3 gene (OSS3). The vector structure is as shown in Figure 1 (B) shown.

[0051] In one or more embodiments of the present application, the expression cassette is E4pro-OSS2, E8pro-OSS1 or CYP450pro-OSS3. The nucleotide sequence of the expression cassette E4pro-OSS2 is shown in SEQ ID No. 14 at positions 11592-15254; the nucleotide sequence of the expression cassette E8pro-OSS1 is shown in SEQ ID No. 14 at positions 7450-11567; and the nucleotide sequence of the expression cassette CYP450pro-OSS3 is shown in SEQ ID No. 14 at positions 1908-7405.

[0052] The present application also provides a method for preparing a transgenic tomato, promoting the synthesis of squalene and / or oxidosqualene in tomato or increasing the content of squalene and / or oxidosqualene in tomato, which comprises increasing the content and / or activity of the proteins OSS1, OSS2 and OSS3 in a recipient tomato, to obtain a transgenic tomato with higher content of squalene and / or oxidosqualene than the recipient tomato.

[0053] In the above method, the increase in the content and / or activity of the proteins OSS1, OSS2 and OSS3 in the recipient tomato is achieved by increasing the expression amount of any of the gene combinations described herein in the recipient tomato.

[0054] In the above method, the increase in the expression amount of any of the gene combinations described herein in the recipient tomato is achieved by at least one of the following ways:

[0055] D1) increasing the copy number of the gene combination;

[0056] D2) placing the gene combination under the drive of a strong promoter for expression;

[0057] D3) increasing a regulatory element of the gene combination to overexpress, the regulatory element including an enhancer element, an element to improve mRNA stability, an element to enhance translation efficiency and / or an element to enhance protein secretion;

[0058] D4) increasing a ribosome binding site of the gene combination;

[0059] D5) codon optimizing the gene combination.

[0060] In the above method, the D1) is achieved by introducing any of the gene combinations described herein into the recipient tomato.

[0061] Further, the gene combination can be composed of an OSS1 gene with a nucleotide sequence as shown in SEQ ID No. 1, an OSS2 gene with a nucleotide sequence as shown in SEQ ID No. 3 and an OSS3 gene with a nucleotide sequence as shown in SEQ ID No. 5.

[0062] Further, the D1) can be introducing a single copy or multiple copies of the gene combination into the recipient tomato.

[0063] Further, the D2) can be achieved by replacing the natural promoter of the gene encoding the protein OSS1, OSS2 and / or OSS3 with a strong promoter, or by operably linking a second strong promoter to the gene encoding the protein OSS1, OSS2 and / or OSS3.

[0064] The strong promoter can be an E8 promoter (E8pro), an E4 promoter (E4pro), a CYP450 promoter (CYP450pro), a T7 promoter, a CaMV promoter, an SV40 promoter, an SFFV promoter, a ubq promoter, an ubi promoter and / or an RBCS promoter.

[0065] The enhancer includes but is not limited to a CMV enhancer, an SV40 enhancer and an RSV enhancer.

[0066] In the above method, in the gene combination, the gene encoding the protein OSS1 can be regulated by the E8 promoter; the gene encoding the protein OSS2 can be regulated by the E4 promoter; the gene encoding the protein OSS3 can be regulated by the CYP450 promoter; the nucleotide sequence of the E8 promoter can be as shown in SEQ ID No. 7; the nucleotide sequence of the E4 promoter can be as shown in SEQ ID No. 8; and the nucleotide sequence of the CYP450 promoter can be as shown in SEQ ID No. 9.

[0067] Further, the method for preparing the transgenic tomato, promoting the synthesis of squalene and / or oxidized squalene in tomato, or increasing the content of squalene and / or oxidized squalene in tomato can comprise the following steps:

[0068] (1) constructing a recombinant vector comprising any of the gene combinations described herein;

[0069] (2) introducing the recombinant vector constructed in step (1) into a recipient tomato;

[0070] (3) obtaining the transgenic tomato through screening and identification.

[0071] Further, the introduction described herein includes but is not limited to: transfecting plant cells or tissues by using Ti plasmid, Ri plasmid, plant virus vector, direct DNA transformation, microinjection, electroporation, Agrobacterium-mediated, etc. conventional biological methods, and cultivating the transfected plant cells or tissues into plants.

[0072] In an embodiment of the present application, the method for constructing a recombinant vector is: first, connecting the unit for expressing OSS2 driven by E4 promoter to vector pL1F-4, the unit for expressing OSS1 driven by E8 promoter to vector pL1F-3, and the unit for expressing OSS3 driven by CYP450 promoter to vector pL1F-2 through the first round of Golden gate reaction, and then connecting the three foreign gene expression cassettes E4pro-OSS2 (positions 11592-15254 of SEQ ID No. 14), E8pro-OSS1 (positions 7450-11567 of SEQ ID No. 14) and CYP450pro-OSS3 (positions 1908-7405 of SEQ ID No. 14) to vector pL2-1 through the second round of Golden gate reaction, to obtain the recombinant vector pL2-1-OSS1+OSS2+OSS3.

[0073] Any of the gene combinations described herein, or the expression cassette, recombinant vector, recombinant microorganism, recombinant host cell containing any of the gene combinations described herein are within the scope of protection of the present application.

[0074] The regulation described herein can be up-regulation or down-regulation.

[0075] The regulation of the synthesis of tomato triterpenoids described herein can be to enhance or weaken the synthesis of tomato triterpenoids.

[0076] The regulation of the synthesis of tomato triterpenoid precursors, triterpenoids and / or sterols described herein can be to promote or inhibit the synthesis of tomato triterpenoid precursors, triterpenoids and / or sterols.

[0077] The content of the triterpenoid precursor substance in the tomato can be increased or decreased by regulating the content of the triterpenoid precursor substance in the tomato as described herein.

[0078] The ability of the tomato to produce triterpenoids and / or sterols can be increased or decreased by regulating the ability of the tomato to produce triterpenoids and / or sterols as described herein.

[0079] In this context, the transgenic tomato is understood to include not only the first generation transgenic tomato obtained by introducing the gene combination described herein into the target tomato, but also its offspring. The transgenic tomato includes seeds, callus, whole plants, and cells.

[0080] Based on the genome data of tomato and momordica grosvenori, the triterpenoid metabolic pathway genes were explored, and through extensive and in-depth research, a key gene combination for enhancing the synthesis pathway of triterpenoid precursors in mature tomato fruits was screened and identified, providing a gene module (gene combination) for regulating the synthesis of triterpenoid precursor substances squalene and oxidosqualene in tomato, namely, oxidosqualene synthase 1 gene (OSS1), oxidosqualene synthase 2 gene (OSS2), and oxidosqualene synthase 3 gene (OSS3). Experiments have proved that the gene module can enhance the synthesis of squalene and oxidosqualene in tomato, and further promote the synthesis of triterpenoids and sterols. The present application first screens a key gene combination for enhancing the synthesis pathway of triterpenoid precursors in mature tomato fruits and uses it to regulate the synthesis and metabolic flow of triterpenoids in tomato, thereby increasing the content of triterpenoid precursors in tomato. The present application can be used in synthetic biology research using tomato fruits as a chassis, overexpressing other triterpenoid or plant sterol synthesis pathway key genes based on the material to produce triterpenoid metabolites and plant sterols with medicinal value, or conducting basic research related to the function of triterpenoid and plant sterol synthesis pathway genes. The gene module of the present application can be used to create a chassis material for synthesizing triterpenoids, plant sterols, and other substances in tomato fruits, to produce triterpenoids and sterols and other metabolites in plants, and has excellent application prospects.

[0081] Definition of terms

[0082] In the present application, unless otherwise specified, the scientific and technical terms used herein have the meanings commonly understood by those skilled in the art. Meanwhile, in order to better understand the present application, the definitions and explanations of related terms are provided as follows.

[0083] The term "expression cassette" generally refers to a nucleic acid construct comprising nucleic acid elements sufficient to express a gene of interest. A typical expression cassette comprises a promoter, a MCS (multiple cloning site), and a terminator. An expression cassette can also include a gene of interest, a marker gene (such as a TK gene, a DHFR gene, a CAT gene, and a NEO gene), a ribosome recognition and binding site (SD), a transcription factor binding site (TFBS), an enhancer, a silencer, a repressor, an intron, a poly(A) addition signal sequence, and / or an mRNA splicing signal sequence, etc. The elements in an expression cassette can be directly connected or indirectly connected via a linker.

[0084] The term "vector" generally refers to a vehicle capable of carrying foreign DNA or a gene of interest into a host cell for amplification and / or expression. The vector can be a cloning vector or an expression vector. The vector can be introduced into a host cell by transformation, transduction, or transfection, so that the genetic material elements carried by the vector are amplified and / or expressed in the host cell. A person skilled in the art can select a suitable vector according to the purpose of genetic engineering and the nature of the recipient cell. The vector includes but is not limited to: a plasmid, a phage (such as lambda phage or M13 phage), a cosmid (i.e., a Coles plasmid), a phagemid, a shuttle vector (such as a yeast expression vector), a Ti plasmid, an artificial chromosome (such as a yeast artificial chromosome (YAC), a bacterial artificial chromosome (BAC), a P1 artificial chromosome (PAC), or a Ti plasmid artificial chromosome (TAC)), a viral vector (such as a baculovirus vector, a retrovirus (including a lentivirus), an adenovirus, an adeno-associated virus, a poxvirus, a papillomavirus, a papovavirus (such as SV40), a herpesvirus (such as a herpes simplex virus)). A vector can contain multiple elements for controlling expression, including but not limited to a promoter sequence, a transcription initiation sequence, an enhancer sequence, a selection element, and a reporter gene. In addition, the vector can also contain a replication initiation site. In one or more embodiments of the present application, the vector is pL1F-1, pL1F-2, pL1F-3, pL1F-4, and / or pL2-1.

[0085] The term "microorganism" generally includes bacteria, viruses, fungi, actinomycetes, rickettsia, mycoplasma, chlamydia, spirochetes, algae, and the like. For example, the bacteria can be from Escherichia sp., Erwinia sp., Agrobacterium sp., Flavobacterium sp., Alcaligenes sp., Pseudomonas sp., and Bacillus sp., and the like. The viruses can include rotavirus, baculovirus, retrovirus (e.g., lentivirus), adenovirus, adeno-associated virus, poxvirus, papillomavirus, influenza virus, papovavirus (e.g., SV40), and herpesvirus (e.g., herpes simplex virus), and the like. The fungi can be from Saccharomyces sp., Fusarium sp., Rhizoctonia sp., Verticillium sp., Penicillium sp., Aspergillus sp., and Cephalosporium sp., and the like. The algae can be from Fucus sp., Achnanthes sp., and Boekelovia sp., and the like. In one or more embodiments of the present application, the microorganism is Escherichia coli and / or Agrobacterium tumefaciens.

[0086] The term "host cell" is also referred to as a recipient cell, and generally refers to any type of cell that can be used to introduce a vector, such as a plant cell and an animal cell. The host cell can be understood to mean not only the specific recipient cell, but also the progeny of such a cell, which can not necessarily be completely identical to the original parent cell due to natural, accidental or deliberate mutation and / or alteration, but which is still included in the scope of the host cell.

[0087] The term "recombinant vector" generally refers to a recombinant DNA molecule constructed in vitro by ligating an exogenous gene of interest with a vector, which can be constructed in any suitable manner, as long as the constructed recombinant vector can carry the exogenous gene of interest into a recipient cell, and provide the exogenous gene of interest with the ability to replicate, integrate, amplify, and / or express in the recipient cell.

[0088] The term "recombinant microorganism" generally refers to a recombinant microorganism obtained by manipulating and modifying the genes of a microorganism of interest, so that the function of the recombinant microorganism is changed. For example, an exogenous gene of interest or a recombinant vector is introduced into a microorganism of interest, or the endogenous genes of a microorganism of interest are directly edited.

[0089] The term "recombinant host cell" generally refers to a host cell whose genome has been manipulated and modified so that the recombinant host cell has a functionally changed genome. For example, a foreign gene of interest or a recombinant vector is introduced into the host cell, or the endogenous gene of the host cell is directly genetically edited.

[0090] The term "linked" generally refers to the association of two or more molecules. The linkage can be covalent or non-covalent. The linkage described herein can be directly linked by a peptide bond, or linked by a linker (linker).

[0091] The term "identity" generally refers to the degree of similarity of two (nucleotide or amino acid) sequences at the same positions in an alignment, and is generally expressed as a percentage. The identity described herein can refer to the identity of an amino acid sequence or a nucleotide sequence. Two copies having exactly the same sequence have 100% identity. Those skilled in the art know that the identity of an amino acid sequence or a nucleotide sequence can be determined using an identity search site on the Internet, such as the BLAST page of the NCBI home page website. For example, the value of identity (%) can be obtained by performing a search in Advanced BLAST 2.1 using blastp as the program, setting the Expect value to 10, setting all Filters to OFF, using BLOSUM62 as the Matrix, setting Gap existence cost, Perresidue gap cost, and Lambda ratio to 11, 1, and 0.85 (default values), respectively, and then calculating the identity of the amino acid sequence using the computer program BLAST, especially BLASTP or TBLASTN, using the default parameters. The identity described herein of 75% or more can be 75%, 80%, 85%, 90%, or 95% or more. The identity of 80% or more can be at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% or more.

[0092] The term "overexpression" generally refers to increasing or up-regulating the level and / or activity of a target protein or gene. Overexpression can be achieved by regulation at the genetic level, such as replication, transcription, translation, post-transcriptional modification, and / or post-translational modification of a gene, or by promoting or increasing the amount, activity, and / or function of a target protein at the protein level. The means of overexpression is not particularly limited, and numerous ways of achieving overexpression are well known to those skilled in the art. For example, a nucleic acid molecule to be overexpressed or a nucleic acid molecule encoding a protein to be overexpressed can be placed under the control of a strong promoter; the copy number of one or more genes encoding the proteins described in the present application can be increased; or the strength of a ribosome binding site or Kozak sequence, the stability of mRNA, codon usage, knock-out of suppressor elements, etc. can be increased.

[0093] The term "promoter" generally refers to a site specifically recognized and bound by RNA polymerase, located upstream of the transcriptional initiation site of a structural gene, having a strict orientation and initiating transcription. Because the strength of a promoter determines the efficiency of transcription, different types of promoters can be used in genetic engineering to regulate the expression of key genes, change the metabolic flow in the cell, and thus efficiently produce relevant metabolites. It is known to those skilled in the art that a constitutive strong promoter can be used to overexpress a target gene. To further enhance the expression of a target gene, multiple promoters can also be used in series. When multiple target genes are expressed at high strength at the same time, different promoters of different strengths are often used to express different genes. Typically, hundreds of different promoter and insertion site combinations are constructed and screened to produce a compound, and this process is long and challenging.

[0094] The term "enhancer" generally refers to a DNA sequence that enhances the transcriptional activity of a gene, located upstream or downstream of a structural gene, or in an intron.

[0095] The term "regulatory element" generally refers to a DNA molecule with gene regulatory activity. Regulatory elements that function in plants include promoters, leader sequences, enhancers, introns, and 3' UTRs, etc.

[0096] The term "operably linked" generally refers to the physical and / or functional linkage of a DNA segment to another DNA segment, which linkage allows the segment to function in its intended manner. DNA encoding a gene product can be operably linked to a regulatory element, which can directly or indirectly modulate the transcription of the DNA. For example, when an enhancer is operably linked to DNA encoding a gene product, the transcription of the DNA can be enhanced, and the enhancer can be located upstream of, downstream of, or embedded in the coding region of the DNA.

[0097] The term "introducing" generally refers to the transfer of an exogenous gene into a recipient cell, such as a eukaryotic recipient cell or a prokaryotic recipient cell. The method of introduction is not particularly limited, and any known transformation method can be used as long as it can transfer the gene of interest (e.g., the gene combination of the present application) into the recipient cell. The introduced DNA molecule can be a single copy or multiple copies. The introduction can be integration of the exogenous gene into the host chromosome or expression of the exogenous gene from a plasmid outside the chromosome. The method of introduction can include any one of the following: (1) introducing the gene of interest or a recombinant vector containing the gene of interest into a host bacterium by a chemical transformation method (e.g., Ca 2+ induced transformation method, polyethylene glycol-mediated transformation method, or metal cation-mediated transformation method) or a physical transformation method (e.g., electroporation transformation method). (2) Transducing the gene of interest into the host bacterium by a bacteriophage transduction method. (3) Directly transferring the gene of interest into a plant recipient cell by a physical or chemical method, such as a chemical stimulation method, an electric shock method, a liposome-mediated method, a microinjection method, a gene gun method, a laser microbeam method, a pollen tube channel method, an ultrasonic wave method, an air gun method, and a vortex method. (4) Transferring the gene of interest into a plant recipient cell using a vector as a medium, such as an Agrobacterium Ti plasmid vector (including Ti plasmid-derived vectors such as co-integration vector systems and binary vector systems). (5) Introducing the gene of interest into an isolated animal cell (transfection) by a calcium phosphate co-precipitation method, a liposome-mediated method, an electroporation method, or a viral vector method. (6) Introducing the gene of interest into an in vivo animal cell by a microinjection method, a retroviral vector method, a somatic cell nuclear transfer method, a sperm vector method, or an embryonic stem cell method, and further preparing a transgenic animal.

[0098] The term "codon optimization" generally refers to a technique for increasing the expression level of a protein in an organism by increasing the translation efficiency of a target gene. Codon optimization generally redesigns a gene by avoiding rare codons, using preferred codons, simplifying the secondary structure of mRNA, optimizing repetitive sequences, eliminating restriction enzyme sites, adjusting GC content, and the like, to improve translation efficiency and thus increase protein expression level. BRIEF DESCRIPTION OF DRAWINGS

[0099] Figure 1 The gene module for synthesizing triterpenoid metabolic flow in tomato fruits can enable the target gene to be efficiently expressed in red-ripe fruits. (A) Schematic diagram of the gene module vector for enhancing triterpenoid metabolic flow. (B) Statistical diagram of the expression levels of each gene in the stably genetically transformed plant that enhances triterpenoid metabolic flow. (C) Pictures of the whole plants and fruits of the triterpenoid-enhancing gene module overexpression lines and wild-type materials.

[0100] Figure 2The gene module for synthesizing triterpenoid metabolic flow in tomato fruit can effectively promote the accumulation of squalene and oxidized squalene in tomato fruit. (A) Metabolite ultra-high performance liquid chromatography ion peak graph of tomato red ripe fruit extract of stable genetically transformed gene module plants. The retention time of squalene and oxidized squalene, which are increased in content in the overexpression plants, in gas chromatography is 13.75 minutes and 15.34 minutes, respectively. (B) Schematic diagram of mass spectrometry mass-to-charge ratio of squalene. (C) Schematic diagram of mass spectrometry mass-to-charge ratio of oxidized squalene. (D) Statistical diagram of the content increase degree of squalene and oxidized squalene in the triterpenoid-enhanced gene module overexpression lines. DETAILED DESCRIPTION

[0101] The application will be further described in conjunction with the specific embodiments. The examples given are only to illustrate the application, and are not intended to limit the scope of the application. The examples provided below can serve as a guide for further improvement by those skilled in the art, and do not constitute any limitation on the application.

[0102] In the following examples, the experimental methods are conventional methods, and are performed according to the techniques or conditions described in the literature in the art or according to the product instructions, unless otherwise specified. The materials, reagents, etc. used in the following examples can be obtained from commercial channels, unless otherwise specified.

[0103] The vectors pL1F-1, pL1F-2, pL1F-3, pL1F-4, pL2-1 and pELE-4 in the following examples are described in the following literature: Weber E, Engler C, Gruetzner R, Werner S, Marillonnet S. A modular cloning system for standardized assembly of multigene constructs. PLoS One. 2011 Feb 18; 6(2): e16765. doi: 10.1371 / journal.pone.0016765. PMID: 21364738; PMCID: PMC3041749.

[0104] Tomato M82 in the following examples is described in the following publication: Soyk S, Muller NA, Park SJ, Schmalenbach I, Jiang K, Hayama R, Zhang L, Van Eck J, Jimenez-Gomez JM, Lippman ZB. Variation in the flowering gene SELF PRUNING 5G promotes day-neutrality and early yield in tomato. Nat Genet. 2017 Jan;49(l):162-168. doi: 10.1038 / ng.3733. Epub 2016 Dec 5. PMID: 27918538.

[0105] "Squalene" in the following examples is also known as "Tetraterpenoid" and has the chemical formula C 30 H 50 , CAS number: 7683-64-9. Squalene belongs to the class of triterpenoids and is a precursor for the synthesis of triterpenoids.

[0106] "Oxidosqualene" in the following examples is also known as 2,3-Oxidosqualene and has the chemical formula C 30 H 50 O, CAS number: 7200-26-2. Oxidosqualene belongs to the class of triterpenoid precursors and is a precursor for the synthesis of triterpenoids.

[0107] Example 1, cloning of OSS1 and OSS2 genes

[0108] 1. Extraction of total RNA

[0109] The pericarp part of M82 tomato green mature fruit was taken, and then was quickly frozen in liquid nitrogen, and was ground into powder under low temperature using a mortar. 400-600 mg of the tissue powder was taken, and RNA was extracted using an EASYspin plant RNA extraction kit (Aidlab, product number RN09). First, 10 volumes (1 mL) of a tissue lysis solution RLT and 1 volume (100 μL) of a PLANTaid for removing polysaccharide and polyphenol components were mixed, and then the tissue powder was added and mixed well, and then was centrifuged at 13000 rpm at room temperature for 8 min. The supernatant was taken, 0.5 volumes of anhydrous ethanol was added, and then the mixture was added to an RNA adsorption column, and was centrifuged at 13000 rpm for 2 min to pass through the column. Then, 700 μL of a deproteinization solution was added to the RNA adsorption column, and was centrifuged at 13000 rpm for 30 s to remove impurities. Then, 500 μL of a rinse solution was added, and was centrifuged at 13000 rpm for 30 s to remove impurities. Then, 500 μL of a rinse solution was added again, and was centrifuged at 13000 rpm for 30 s. Then, the RNA adsorption column was centrifuged at 13000 rpm for 2 min to remove residual liquid in the column. Finally, 30 μL of RNA-free water was added, and was centrifuged at 12000 rpm for 1 min to obtain the tomato green mature fruit RNA.

[0110] 2. Reverse transcription to obtain cDNA

[0111] Subsequently, DNA-freeTM kit (Thermo, product number: AM1906) was used to remove DNA in the RNA. 20 μL of the RNA sample was taken, 1 μL of rDNase, and 2 μL of DNase I buffer were added, and then were mixed and incubated at 37°C for 20-30 min. Then, 2.3 μL of DNase inactivation reagent was added, and then was mixed and incubated at room temperature for 2 min. Then, 10000 g centrifugation was performed for 1.5 min, and then the supernatant was taken, and the RNA concentration was determined. The RNA concentration needs to be higher than 100 ng / μL. After obtaining the RNA, FastKing RT kit (with gDNase) cDNA first strand synthesis kit (Tiangen, product number: KR116) was used to reverse transcribe the RNA whose DNA had been digested, and the tomato green mature fruit cDNA was obtained. The specific experimental steps refer to the instruction manual.

[0112] 3. Cloning of target genes

[0113] Tomato M82 green mature fruit cDNA was used as a template, and KOD plus high-fidelity enzyme was used to amplify the gene fragment. Specific primers were designed for the coding regions of OSS1 and OSS2 genes, and the gene numbers were Solyc12g015860 and Solyc01g110290, respectively. The primer sequences are as follows:

[0114] Forward primer OSS1-F: 5'-tggtctcacgccGCTGATCTGAAGAAGAAATT-3' (lower case letters represent the Bsa I enzyme recognition site and the homologous sequence for ligation with the plastid localization signal fragment, upper case letters represent the partial sequence of the gene);

[0115] Reverse primer OSS1-R: 5'-tggtctcagcagCTACTTCTGCCTCTTATAAA-3' (lower case letters represent the Bsa I enzyme recognition site and the homologous sequence for ligation with the terminator fragment, upper case letters represent the partial sequence of the gene);

[0116] Forward primer OSS2-F: 5'-tggtctcagcttATGGGAACATTGAGGGCA-3' (lower case letters represent the Bsa I enzyme recognition site and the homologous sequence for ligation with the plastid localization signal fragment, upper case letters represent the partial sequence of the gene);

[0117] Reverse primer OSS2-R: 5'-tggtctcataccCTAAGACCGGCTGCCAGAAA-3' (lower case letters represent the Bsa I enzyme recognition site and the homologous sequence for ligation with the terminator fragment, upper case letters represent the partial sequence of the gene).

[0118] The cDNA obtained in step 2 was used as a template, and forward primer OSS1-F and reverse primer OSS1-R, and forward primer OSS2-F and reverse primer OSS2-R were used for PCR amplification, respectively. The PCR reaction system for gene fragment amplification is shown in Table 1:

[0119] Table 1, PCR reaction system

[0120] Reagent Amount (μL) cDNA (200 ng / μL) 1 KOD plus enzyme (1.0 U / μL) 1 10 x buffer for KOD-plus 2.5 2 mM dNTPs 2.5 25 mM MgSO4 1.5 10 μM Primer (forward primer) 1 10 μM Primer (reverse primer) 1 ddH2O 14.5 Total reaction system 25

[0121] The PCR reaction program was as follows: 94°C pre-denaturation for 2 min, 94°C denaturation for 15 s, 55°C annealing for 30 s, 68°C extension for 1 min, 34 cycles of amplification; and 68°C extension for 5 min. The amplified OSS1 gene fragment and OSS2 gene fragment were subjected to gel electrophoresis, respectively. 4 μL of the PCR product was taken and added to 1 μL of loading buffer, and electrophoresis was performed in a 1% (5 μL of gel red, 1 g of agarose / 100 mL of 0.5xTAE buffer) agarose gel. After the electrophoresis was completed, a photograph was taken in the gel imaging system, the band size was determined to be consistent with the target fragment, and the Axygen PCR clean up kit (item number: AP-PCR-250G) was used for PCR product recovery, to obtain the gene DNA fragment.

[0122] Take 20 μL PCR product, add 100 μL buffer A and 100 μL isopropyl alcohol, mix, then pass the liquid through the adsorption column, wash twice with 700 μL wash buffer II, then centrifuge at 12000 rpm for 1 minute, add 20 μL elute buffer to elute the gene DNA fragment, and determine the concentration of the gene DNA fragment. Finally, the tomato oxidosqualene synthase 1 OSS1 gene fragment and the tomato oxidosqualene synthase 2 OSS2 gene fragment are obtained.

[0123] The coding sequence (CDS) of the OSS1 gene is shown in SEQ ID No. 1 and consists of 1029 nucleotides.

[0124] The OSS1 gene encodes a protein OSS1, and the amino acid sequence of the protein OSS1 is shown in SEQ ID No. 2.

[0125] The coding sequence (CDS) of the OSS2 gene is shown in SEQ ID No. 3 and consists of 1236 nucleotides.

[0126] The OSS2 gene encodes a protein OSS2, and the amino acid sequence of the protein OSS2 is shown in SEQ ID No. 4.

[0127] Example 2, cloning of the OSS3 gene

[0128] Using the cDNA of the fruit of Momordica cochinchinensis 15 days after pollination as a template, forward primer OSS3-F and reverse primer OSS3-R were used for PCR amplification to obtain the coding sequence of the oxidosqualene synthase 3 gene. The primer sequences are as follows:

[0129] Forward primer OSS3-F: 5'-tggtctcacgccGCTGATCTGAAGAAGAAATT-3' (lower case letters represent the BsaI enzyme recognition site and the homologous sequence for connecting the plastid localization signal fragment, and upper case letters represent the partial sequence of the gene);

[0130] Reverse primer OSS3-R: 5'-tggtctcagcagCTACTTCTGCCTCTTATAAA-3' (lower case letters represent the BsaI enzyme recognition site and the homologous sequence for connecting the terminator fragment, and upper case letters represent the partial sequence of the gene).

[0131] The PCR amplification reaction system and the reaction procedure are the same as those in Example 1.

[0132] The coding sequence (CDS) of the OSS3 gene is shown in SEQ ID No. 5 and consists of 1587 nucleotides.

[0133] The OSS3 gene encodes a protein OSS3, and the amino acid sequence of the protein OSS3 is shown as SEQ ID No. 6.

[0134] Example 3, amplification of the fragment of the plastid localization signal peptide of the tomato fruit-specific promoter

[0135] 1. Amplification of the promoter

[0136] DNA of tomato M82 leaves was extracted by using CTAB extraction method, and the tomato M82 genomic DNA was used as a template to amplify the promoter by using KODplus high-fidelity enzyme. The specific primers were designed in the 5' non-coding region of the corresponding gene to amplify the E8, E4 and CYP450 gene promoters. The primer sequences are as follows:

[0137] Forward primer E8pro-F: 5'-tttggtctcaagcgAATTCTAGGCGGGTTATCTT-3' (the lowercase letters represent the Bsa I enzyme recognition site and the sequence homologous to the vector, and the uppercase letters represent the promoter partial sequence);

[0138] Reverse primer E8pro-R: 5'-tggtctcaCTTCTTTTGCACTGTGAATG-3' (the lowercase letters represent the Bsa I enzyme recognition site, and the uppercase letters represent the promoter partial sequence);

[0139] Forward primer E4pro-F: 5'-tttggtctcaagcgCGGTTGAGCTCAAGATAA-3' (the lowercase letters represent the Bsa I enzyme recognition site and the sequence homologous to the vector, and the uppercase letters represent the promoter partial sequence);

[0140] Reverse primer E4pro-R: 5'-tttggtctcaGCATTGCTCAATCTCTAATAATAT-3' (the lowercase letters represent the Bsa I enzyme recognition site, and the uppercase letters represent the promoter partial sequence);

[0141] Forward primer CYP450pro-F: 5'-tggtctcaagcgGACTGGTAGATGAGAAACAA-3' (the lowercase letters represent the Bsa I enzyme recognition site and the sequence homologous to the vector, and the uppercase letters represent the promoter partial sequence);

[0142] Reverse primer CYP450pro-R: 5'-tctgcagaattctagagctcGGGGTCGACCTTTGATTTTT-3' (the lowercase letters represent the homologous sequence for connecting the promoter and the plastid localization signal fragment, and the uppercase letters represent the promoter partial sequence).

[0143] The PCR amplification reaction system and the reaction procedure were the same as those in Example 1.

[0144] The nucleotide sequence of the E8 promoter (E8pro) is shown in SEQ ID No. 7; the nucleotide sequence of the E4 promoter (E4pro) is shown in SEQ ID No. 8; and the nucleotide sequence of the CYP450 promoter (CYP450pro) is shown in SEQ ID No. 9.

[0145] 2. Amplifying the plastid localization signal peptide gene fragment

[0146] The DNA of Arabidopsis Col leaves was extracted by using the CTAB extraction method, and the plastid localization signal peptide AtCor15b gene fragment was amplified by using the Arabidopsis Col genomic DNA as a template and KOD plus high-fidelity enzyme, and the primer sequences were as follows:

[0147] Forward primer AtCor15b-F: 5'-tggtctcagaagATGGCGATGTCTTTCTCAGG-3' (the lowercase letters represent the Bsa I enzyme recognition site and the homologous sequence connected with the promoter, and the uppercase letters represent the plastid localization signal part sequence);

[0148] Reverse primer AtCor15b-R: 5'-tggtctcaGGCGTAGATCAACGACTTCTT-3' (the lowercase letters represent the Bsa I enzyme recognition site, and the uppercase letters represent the plastid localization signal part sequence).

[0149] The tomato red-ripe fruit cDNA was used as a template, and the plastid localization signal peptide SlDXS2 and SlSSU gene fragments were amplified by using KOD plus high-fidelity enzyme, and the primer sequences were as follows:

[0150] Forward primer SlDXS2-F: 5'-tggtctcaATGCATGGCCCTCAATTTGCTTTC-3' (the lowercase letters represent the Bsa I enzyme recognition site, and the uppercase letters represent the plastid localization signal part sequence);

[0151] Reverse primer SlDXS2-R: 5'-tggtctcaAAGCTCAGCCCTTCCAGGCCAGGC-3' (the lowercase letters represent the Bsa I enzyme recognition site, and the uppercase letters represent the plastid localization signal part sequence);

[0152] Forward primer SlSSU-F: 5'-cgagctctagaattctgcagATGGCTTCCTCTATAGTTTC-3' (lower case letters indicate the homologous sequence for the ligation of the promoter and plastid localization signal fragment, upper case letters indicate the partial sequence of the plastid localization signal);

[0153] Reverse primer SlSSU-R: 5'-tgatccaccatggtactgcGCAACTGACTCTTCCACCGT-3' (lower case letters indicate the homologous sequence for the ligation of the plastid localization signal fragment and the gene fragment, upper case letters indicate the partial sequence of the plastid localization signal).

[0154] The PCR amplification reaction system and the reaction procedure are the same as those in Example 1.

[0155] The nucleotide sequence of the plastid localization signal peptide AtCor15b gene (AtCor15b SP) is shown in SEQ ID No. 10; the nucleotide sequence of the plastid localization signal peptide SlDXS2 gene (SlDXS2 SP) is shown in SEQ ID No. 11; and the nucleotide sequence of the plastid localization signal peptide SlSSU gene (SlSSU SP) is shown in SEQ ID No. 12.

[0156] Example 4, construction of a gene overexpression vector

[0157] 1. First round of Golden gate reaction: the unit for expressing OSS2 driven by the E4 promoter was connected to the vector pL1F-4, the unit for expressing OSS1 driven by the E8 promoter was connected to the vector pL1F-3, and the unit for expressing OSS3 driven by the CYP450 promoter was connected to the vector pL1F-2. The Golden gate reaction system is shown in Table 2.

[0158] Table 2, first round of Golden gate reaction system

[0159]

[0160]

[0161] Subsequently, the following PCR program was run: 37°C for 3 min, 16°C for 4 min, 20 cycles of the above two steps, 50°C for 5 min, 80°C for 10 min. The reaction system was added to competent cells DH5a, which were heat shocked at 42°C for 50 s, and then 400 μL of LB liquid medium without antibiotics was added. After shaking culture at 37°C and 220 rpm for 1 h, the bacterial solution was spread on LB solid medium containing 50 mg / L ampicillin, and the plate was incubated in a 37°C incubator for 16 h. The bacteria were picked, sequenced, and the plasmid of the correct clone was stored at -20°C.

[0162] The above steps constructed the OSS1 gene, the OSS2 gene, and the OSS3 gene into three different vectors, respectively, to finally obtain three recombinant vectors, which were named pL1F-3-E8pro-OSS1, pL1F-4-E4pro-OSS2, and pL1F-2-CYP450pro-OSS3, respectively.

[0163] 2. The second round of Golden gate reaction: three exogenous gene expression cassettes E4pro-OSS2 (positions 11592-15254 of SEQ ID No. 14), E8pro-OSS1 (positions 7450-11567 of SEQ ID No. 14), and CYP450pro-OSS3 (positions 1908-7405 of SEQ ID No. 14) were connected to the vector pL2-1 to form the final vector pL2-1-OSS1+OSS2+OSS3. The Golden gate reaction system is shown in Table 3.

[0164] Table 3. Second round of Golden gate reaction system

[0165] Reagent Amount (μL) pL1F-4-E4 pro-OSS2 (100 ng / μL) 1 pL1F-3-E8 pro-OSS1 (100 ng / μL) 1 pL1F-2-CYP450 pro-OSS3 (100 ng / μL) 1 pL1F-1-NPT II (100 ng / μL) 1 end-linker (100 ng / μL) 1 pL2-1 (100 ng / μL) 1 T4 ligase 1 Bpil 1 10 x T4 ligase buffer 1.5 ddH2O 5.5 Total reaction system 15

[0166] In the above reaction system, the pL1F-1-NPT II vector is a recombinant vector obtained by replacing the fragment between the two BsaI enzyme cutting sites of the vector pL1F-1 with a DNA molecule having the nucleotide sequence shown in SEQ ID No. 13, while keeping other nucleotide sequences of the vector pL1F-1 unchanged. The end-linker is pELE-4.

[0167] Subsequently, the following PCR program was run: 37°C for 3 min, 16°C for 4 min, 20 cycles of the above two steps, 50°C for 5 min, 80°C for 10 min.

[0168] The reaction system was added to competent cells DH5a, heat shocked at 42°C for 50s, then 400μL of LB liquid medium without antibiotics was added, and the bacterial solution was spread on LB solid medium containing 50mg / L of kanamycin at 37°C for 1h. After the plate was placed in a 37°C incubator for 16 hours, the bacteria were picked and sequenced. The correct clone was extracted and stored at -20°C. The correct plasmid was named: pL2-1-OSS1+OSS2+OSS3.

[0169] The structure of the pL2-1-OSS1+OSS2+OSS3 plasmid is described as follows: the fragment between the two Bpil enzyme sites of the vector pL2-1 was replaced by a DNA molecule with the nucleotide sequence shown in SEQ ID No. 14, and the other nucleotide sequences of the vector pL2-1 were kept unchanged to obtain a recombinant vector. The pL2-1-OSS1+OSS2+OSS3 contains the promoters E8pro, E4pro and CYP450pro, which respectively drive the transcription of the genes oxidosqualene synthase 1 gene (OSS1), oxidosqualene synthase 2 gene (OSS2) and oxidosqualene synthase 3 gene (OSS3). The vector structure is as shown in Figure 1 (B).

[0170] Example 5, stable transformation of tomato with the recombinant vector

[0171] The recombinant plasmid pL2-1-OSS1+OSS2+OSS3 obtained in the above Example 4 was transformed into Agrobacterium tumefaciens AGL1 competent cells to obtain recombinant Agrobacterium AGL1 / pL2-1-OSS1+OSS2+OSS3. Through plant genetic transformation mediated by Agrobacterium AGL1 / pL2-1-OSS1+OSS2+OSS3, plants with stable overexpression of OSS1, OSS2 and OSS3 genes driven by E8pro, E4pro and CYP450pro promoters, respectively, were obtained.

[0172] The Agrobacterium genetic transformation process is as follows: the seeds of AC tomato are sowed in the medium (2.15 g / L MS salt, 100 mg / L myo-inositol, 2 mg / l thiamine, 0.5 mg / L vitamin B6, 0.5 mg / L nicotinic acid, 10 g / l sucrose and 8 g / L agar), and the seedlings are grown to 6-8 days, the cotyledons are cut into approximate squares for pre-culturing for 2 days, then the Agrobacterium AGL1 / pL2-1-OSS1+OSS2+OSS3 is adjusted to the OD value in the range of 0.6-0.8, the cotyledons are infected for 10 min, and the cotyledons are co-cultured for 2 days, and then the cotyledons are transferred to the sprouting medium containing 75 mg / L kanamycin. When the bud length is more than 1.5 cm, the bud is transferred to the rooting medium containing 75 mg / L kanamycin for 2-4 weeks, and the well-rooted materials are used for genotype identification, and the following identification primers are used: NPT-F: 5'-CTATTCGGCTATGACTGGGC-3', NPT-R: 5'-AATATCACGGGTAGCCAACG-3', and the positive seedlings are identified by PCR electrophoresis. The transgenic positive plants are transplanted to the greenhouse. The whole plant and fruit of the obtained plants are as shown in FIG. 10C, the whole plant grows normally, and the fruit color is lighter than that of the wild type plant, which is caused by the transformation of part of the terpenoid upstream metabolites for synthesizing carotenoids into the triterpenoid metabolite precursor, and the decrease of the carotenoid content. Figure 1 (C) shown, the whole plant grows normally, and the fruit color is lighter than that of the wild type plant, which is caused by the transformation of part of the terpenoid upstream metabolites for synthesizing carotenoids into the triterpenoid metabolite precursor, and the decrease of the carotenoid content.

[0173] The composition of the sprouting medium containing 75 mg / L kanamycin is 4.3 g / L MS salt, 100 mg / L myo-inositol, 1 ml / L nichia vitamin (1000x), 20 g / L sucrose, 2 mg / L trans-zeatin, 75 mg / L kanamycin, 300 mg / L timentin, and 5.2 g / L agar gel, 0.05 mg / L IAA.

[0174] The composition of the rooting medium containing 75 mg / L kanamycin is 4.3 g / L MS salt, 1 ml / L nichia vitamin (1000x), 30 g / L sucrose, 8 g / L agar, 75 mg / L kanamycin, 300 mg / L timentin, 1 mg / L IAA.

[0175] After obtaining the stably inherited triterpenoid enhancement module transgenic plants, about 600-800 mg of red ripe fruit pulp tissue is ground into powder at low temperature, and the red ripe fruit RNA of the transgenic material is extracted. The extraction method is shown in step 1 of Example 1. The cDNA obtained after reverse transcription is used as a template for fluorescence quantitative PCR experiment to identify the expression level of the target gene. The fluorescence quantitative PCR experiment system is configured as shown in Table 4:

[0176] Table 4, fluorescence quantitative PCR reaction system

[0177] Reagent Amount (μL) TB Green Premix Ex Taq 5 PCR forward primer (10 μM) 0.4 PCR reverse primer (10 μM) 0.4 cDNA 0.4 (20 ng) ddH2O 3.8 Total volume 10

[0178] The fluorescence quantitative PCR program was run as shown in Table 5:

[0179] Table 5, fluorescence quantitative PCR reaction program

[0180]

[0181] The fluorescence quantitative PCR primer sequences involved are shown in Table 6:

[0182] Table 6, fluorescence quantitative PCR primer sequences

[0183]

[0184]

[0185] The results are shown in Figure 1 (C) The three genes in the gene module that enhance the synthesis of triterpenoid compounds in tomato fruit were all highly expressed in the tomato overexpression lines.

[0186] Example 6, metabolite detection of tomato fruit

[0187] About 100 g of red ripe tomato fruit was quickly frozen in liquid nitrogen, and ground into a uniform powder using a high-throughput tissue grinder at 30 Hz for 70 s. The powder was freeze-dried in a freeze dryer for 24-48 h.

[0188] 1 g of fruit powder was weighed, 10 mL of extraction solution (methanol: acetone = 1:1) was added, and the mixture was shaken and mixed, ultrasonically treated at 40 kHz for 30 min, and repeated once, with ice added to keep the water bath temperature below 30°C.

[0189] Centrifugation was performed at 11000 rpm for 15 min at 4°C, 6 mL of supernatant was aspirated, and vacuum pump centrifugation was performed for 60 min. The composition and content of metabolites in the fruit were detected by gas chromatography-mass spectrometry.

[0190] The results are shown in Figure 2 As shown in

[0191] The application has been described in detail. For those skilled in the art, the application can be implemented in a wider range under the same parameters, concentrations and conditions without departing from the spirit and scope of the application and without unnecessary experiments. Although the application gives a special example, it should be understood that the application can be further improved. In summary, according to the principle of the application, the application intends to include any change, use or improvement of the application, including the change made by the conventional technology known in the art, which is out of the range disclosed in the application.

Claims

1. Use of a gene combination in any one of the following: A1) regulating the metabolic flow of triterpenoids in tomato; A2) regulating the synthesis of triterpenoid precursors, triterpenoids and / or sterols in tomato; A3) regulating the content of triterpenoid precursors in tomato; A4) regulating the ability of tomato to produce triterpenoids and / or sterols; A5) creating medicinal tomato germplasm; the gene combination comprises a gene encoding protein OSS1, a gene encoding protein OSS2 and a gene encoding protein OSS3; the protein OSS1 is a protein with an amino acid sequence of SEQ ID No. 2, or a fusion protein with the same function obtained by connecting a tag to the N-terminus and / or C-terminus of SEQ ID No. 2; the protein OSS2 is a protein with an amino acid sequence of SEQ ID No. 4, or a fusion protein with the same function obtained by connecting a tag to the N-terminus and / or C-terminus of SEQ ID No. 4; the protein OSS3 is a protein with an amino acid sequence of SEQ ID No. 6, or a fusion protein with the same function obtained by connecting a tag to the N-terminus and / or C-terminus of SEQ ID No.

6.

2. Use according to claim 1, characterized in that, the triterpenoid precursor is squalene and / or oxidosqualene.

3. Use according to claim 1 or 2, characterized in that, the nucleotide sequence of the gene encoding protein OSS1 is shown in SEQ ID No. 1; the nucleotide sequence of the gene encoding protein OSS2 is shown in SEQ ID No. 3; and the nucleotide sequence of the gene encoding protein OSS3 is shown in SEQ ID No.

5.

4. Use of a biological material in any one of the following: B1) regulating the metabolic flow of triterpenoids in tomato; B2) regulating the synthesis of triterpenoid precursors, triterpenoids and / or sterols in tomato; B3) regulating the content of triterpenoid precursors in tomato; B4) regulating the ability of tomato to produce triterpenoids and / or sterols; B5) creating medicinal tomato germplasm; the biological material is any one of the following: C1) an expression cassette containing the gene combination of any one of claims 1-3; C2) a recombinant vector containing the gene combination of any one of claims 1-3; C3) a recombinant microorganism containing the gene combination of any one of claims 1-3; C4) a recombinant host cell containing the gene combination of any one of claims 1-3; C5) a transgenic plant cell line containing the gene combination of any one of claims 1-3; C6) a transgenic plant tissue containing the gene combination of any one of claims 1-3; C7) a transgenic plant organ containing the gene combination of any one of claims 1-3.

5. A method for making a transgenic tomato, for promoting the synthesis of or increasing the content of squalene and / or oxidosqualene in a tomato, characterized in that, The method comprises increasing the content and / or activity of the proteins OSS1, OSS2 and OSS3 as claimed in claim 1 in a recipient tomato, to obtain a transgenic tomato with higher content of squalene and / or oxidosqualene than the recipient tomato.

6. The method of claim 5, wherein, The increase of the content and / or activity of the proteins OSS1, OSS2 and OSS3 as claimed in claim 1 in the recipient tomato is achieved by increasing the expression level of the gene combination as claimed in any one of claims 1-3 in the recipient tomato.

7. The method of claim 6, wherein, The increase of the expression level of the gene combination as claimed in any one of claims 1-3 in the recipient tomato is achieved by at least one of the following: D1) increasing the copy number of the gene combination; D2) placing the gene combination under the expression of a strong promoter; D3) increasing the regulatory elements of the gene combination to overexpress, the regulatory elements including enhancer elements, elements to improve mRNA stability, elements to enhance translation efficiency and / or elements to enhance protein secretion; D4) increasing the ribosome binding site of the gene combination; D5) codon optimizing the gene combination.

8. The method according to claim 6 or 7, characterized in that, The D1) is achieved by introducing the gene combination as claimed in any one of claims 1-3 into the recipient tomato.

9. The method of claim 8, wherein, In the gene combination, the gene encoding the protein OSS1 is regulated by the E8 promoter; the gene encoding the protein OSS2 is regulated by the E4 promoter; the gene encoding the protein OSS3 is regulated by the CYP450 promoter; the nucleotide sequence of the E8 promoter is shown in SEQ ID No. 7; the nucleotide sequence of the E4 promoter is shown in SEQ ID No. 8; the nucleotide sequence of the CYP450 promoter is shown in SEQ ID No.

9.

10. The gene combination as claimed in any one of claims 1-3, or an expression cassette, a recombinant vector, a recombinant microorganism, a recombinant host cell containing the gene combination as claimed in any one of claims 1-3.

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