Artificial plant bidirectional constitutive strong promoter and application thereof

Abstract

The invention provides an artificial plant bidirectional constitutive strong promoter, and particularly relates to a high-activity short transcription enhancer fragment which is formed by connecting three plant endogenous fragments and a plant virus in series and serves as a transcription activation basic element. A minimum core promoter element Mini 35S from a cauliflower mosaic virus and a minimum core promoter element CsVMV mini-pro functional module from a cassava vein mosaic virus are respectively integrated at two ends, so that the plant constitutive strong promoter with bidirectional starting capability is constructed. According to the invention, a synthetic quadruple STE transcription enhancer, a Mini 35S promoter and a CsVMV mini-pro promoter are functionally coupled to form an expression regulation tool with broad-spectrum adaptability to plant species, and two operably connected target gene expression cassettes can be simultaneously driven by a short regulation sequence to realize synergistic efficient expression in plant cells.

Description

Technical Field

[0001] The present invention belongs to the field of plant genetic engineering, and particularly relates to an artificial plant bidirectional constitutive strong promoter and its application. Background Art

[0002] In the field of plant genetic improvement, the precise regulation of quantitative traits depends on the complex metabolic network formed by the coordinated action of multiple genes. Therefore, the demand for the coordinated expression of multiple genes is increasing. However, traditional multi-gene transformation techniques such as multiple transformations, co-transformation by particle bombardment, and multi-gene fusion generally have systematic defects such as a long operation cycle (usually 18 - 24 months), low transformation efficiency (the average efficiency of particle bombardment < 15%), gene expression imbalance (the proportion imbalance rate of metabolic pathway products > 70%), and gene silencing caused by sequence homology (the incidence rate is as high as 42 - 67%), which severely restricts the process of directed improvement of complex agronomic traits. The functional limitations of the core element of gene expression regulation - the promoter further exacerbate the above problems: Although constitutive strong promoters (such as CaMV 35S) have broad-spectrum expression characteristics, their single functional module is difficult to adapt to the requirements of multi-gene co-regulation, and repeated use easily leads to co-suppression phenomena. In addition, the viral-derived sequences have biosafety risks, resulting in long-term limitation of the synthesis flux of target products in metabolic engineering (for example, the typical expression level in tobacco BY-2 cells is only 0.9 g / L).

[0003] Therefore, synthetic biology strategies have developed an asymmetric promoter system by reconstructing promoter elements (such as integrating the T7 phage RNA polymerase - TATA box complex regulatory unit, synthetic enhancer arrays, and insulators). Experimental verification shows that it can increase the expression intensity of foreign genes in monocotyledonous plants by 8.3 ± 0.7 times, and combined with the CRISPR / Cas12i-mediated multi-gene targeted integration technology (transformation efficiency 78.6%, co-expression stability of T1 generation 92.4%), precise dosage control of key enzyme genes in metabolic pathways has been achieved. It is worth noting that bidirectional promoters (BDPs), as emerging regulatory tools, can avoid the epigenetic silencing effect caused by repeated promoters in traditional tandem vectors by driving the synchronous expression of reverse transcription gene pairs, and their endogenous sequence design can significantly reduce the risk of homologous recombination.

[0004] Currently, the construction of artificial bidirectional promoters mainly adopts the method of reverse connection of two unidirectional promoters. However, the activity and stability of existing artificial bidirectional promoters are difficult to meet the requirements of gene expression for complex trait improvement, and further exploration is still needed; the construction of recombinant vectors also needs to be further optimized. Therefore, using plant endogenous promoters to construct plant expression vectors that can carry multiple functional genes and reduce sequence homology is crucial for improving the expression level and long-term stability of foreign genes. Summary of the Invention

[0005] The present invention uses the tandem of plant endogenous enhancers as the transcriptional activation basic element to construct an artificial plant bidirectional constitutive strong promoter, so as to overcome the problems of low gene expression level, poor stability and easy gene silencing in the existing transgenic technology.

[0006] The present invention adopts the following technical solutions: A synthetic quadruple STE transcriptional enhancer (4×STE), which includes STEos007, STEos008 and STEos006 screened from the rice genome and STEvs011 from the cauliflower mosaic virus 35S promoter.

[0007] Preferably, it is composed of STEos007, STEos008 and STEos006 screened from the rice genome and STEvs011 from the cauliflower mosaic virus 35S promoter in series in sequence.

[0008] Among them, the nucleotide sequence of the STEos007 is as shown in SEQ ID No.4, the nucleotide sequence of the STEos008 is as shown in SEQ ID No.5; the nucleotide sequence of the STEos006 is as shown in SEQ ID No.6; the nucleotide sequence of the STEvs011 is as shown in SEQ ID No.7.

[0009] Among them, the nucleic acid sequence of the synthetic quadruple STE transcriptional enhancer (4×STE) is as shown in SEQ ID No.3.

[0010] An artificial plant bidirectional constitutive strong promoter containing the above synthetic quadruple STE transcriptional enhancer.

[0011] Furthermore, the artificial plant bidirectional constitutive strong promoter contains a minimal core promoter at both ends of the synthetic quadruple STE transcriptional enhancer.

[0012] Furthermore, the minimal core promoters are the minimal core promoter CsVMVmini-pro of the cassava vein mosaic virus and Mini 35S based on the cauliflower mosaic virus (CaMV) 35S promoter.

[0013] Furthermore, the nucleotide sequence of the CsVMV mini-pro is as shown in SEQ ID No.2; the nucleotide sequence of the Mini 35S is as shown in SEQ ID No.8.

[0014] Preferably, the artificial plant bidirectional constitutive strong promoter comprises Mini 35S, synthetic quadruple STE transcriptional enhancer 4×STE, CsVMV mini-pro, and At Psak UTR; the CsVMV mini-pro is connected to the 5' end of 4×STE in a reverse complementary head-to-head manner, the Mini 35S is connected in the forward direction to the 3' end of 4×STE, and 4×STE is located in the shared sequence region between CsVMV mini-pro and Mini 35S; the At Psak UTR is connected to the 3' end of CsVMV mini-pro.

[0015] More preferably, the nucleotide sequence of the artificial plant bidirectional constitutive strong promoter is as shown in SEQ ID No.10.

[0016] On the other hand, a target gene expression cassette is connected upstream and / or downstream of the artificial plant bidirectional constitutive strong promoter.

[0017] Wherein, the target gene expression cassette is multiple genes concatenated by P2A.

[0018] As a specific application, the target gene expression cassette is RUBY or GUS .

[0019] Preferably, the nucleotide sequence of the GUS reporter gene RUBY is as shown in SEQ ID No.11, and the nucleotide sequence of the

[0020] reporter gene GUS is as shown in SEQ ID No.12.

[0021] Preferably, the nucleic acid sequence of the artificial plant bidirectional constitutive strong promoter carrying the RUBY reporter gene is as shown in SEQ ID No.13.

[0022] Preferably, the nucleic acid sequence of the artificial plant bidirectional constitutive strong promoter carrying the GUS and RUBY double reporter genes is as shown in SEQ ID No.15.

[0023] A plant transformation construct comprising the above artificial plant bidirectional constitutive strong promoter.

[0024] An application of the above artificial plant bidirectional constitutive strong promoter in constructing a plant multi-gene co-expression system.

[0025] The beneficial effects of the present invention are as follows: Based on the transcriptional activation basic element formed by tandem of plant endogenous enhancers, the present invention constructs a plant bidirectional promoter capable of simultaneously initiating the expression of two genes. Compared with the conventional multi-gene transformation system, the present invention can overcome the drawbacks of traditional transgenic technologies and significantly improve the level and stability of gene expression.

[0026] In the research of multi-gene genetic transformation, there are problems such as cumbersome and time-consuming operations, low expression efficiency and stability, potential biosafety of virus-derived sequences, and prone to gene silencing phenomenon based on sequence homology, which urgently require further optimization of gene expression vectors. The unique structure of the bidirectional promoter greatly reduces the workload of constructing gene expression vectors, avoids the problem of gene silencing, and significantly improves the expression level and stability of foreign genes; at the same time, the transcriptional activation element formed by tandem of rice endogenous enhancers significantly enhances the compatibility with the plant host genome, reduces the plant's own immune response, thereby achieving precise regulation and efficient and stable expression of foreign genes. It has made great contributions to the continuous improvement of transgenic technologies and given inspiration and reference to relevant researchers in the field of crop improvement.

[0027] The bidirectional promoter of the present invention can be combined with technologies such as CRISPR-Cas9, RNAi, and artificial intelligence to lay a technical and theoretical foundation in many fields such as molecular design breeding, production of important biomass, research on gene function and molecular mechanism. Brief Description of the Drawings

[0028] Figure 1 It is a schematic diagram of the structure of a plant endogenous bidirectional promoter with dual reporter genes.

[0029] Figure 1 The driven reporter genes are marked RUBY and GUS The relative positions and transcription directions are shown, and the arrows indicate the transcription direction.

[0030] Figure 2 It is a Sanger sequencing verification diagram of the bidirectional promoter sequence region in the bidirectional promoter expression vector.

[0031] Figure 3 It is a schematic diagram of the structure of the CaMV35S promoter (control group).

[0032] Figure 3 In the control group in GUS 、 RUBY The relative positions and transcription directions of the two CaMV 35 promoters in the control group driving the expression of the reporter genes respectively are shown, and the arrows indicate the transcription direction.

[0033] Figure 4 It is in the same tobacco leaf RUBYExpression comparison diagram of reporter genes.

[0034] Figure 4 In it, on the left is driven by a bidirectional promoter RUBY ; on the right is driven by the CaMV35S promoter RUBY .

[0035] Figure 5 It is the expression comparison diagram of reporter genes in the same tobacco leaf GUS Expression comparison diagram of reporter genes.

[0036] Figure 5 In it, on the left is driven by a bidirectional promoter GUS ; on the right is driven by the CaMV35S promoter GUS . Specific implementation manners

[0037] The method of the present invention will be described in detail below in conjunction with examples, but they cannot be construed as limiting the protection scope of the present invention.

[0038] Example 1 Cassava vein mosaic virus minimal core promoter (CsVMV mini-pro) element The cassava vein mosaic virus promoter (CsVMV, as shown in SEQ ID No. 1) is a polynucleotide sequence with a full length of 517 bp. This sequence can be divided into three major parts, and the second part contains a 123-bp minimal core promoter (CsVMV mini-pro, as shown in SEQ ID No. 2).

[0039] CsVMV mini-pro can bind to RNA polymerase II and other transcription initiation-related factors, provide a basic action site for transcription initiation, and initiate gene transcription; at the same time, the TATA-box in the sequence determines the transcription direction.

[0040] Example 2 Synthetic quadruple STE transcriptional enhancer element The synthetic quadruple STE transcriptional enhancer (4×STE, as shown in SEQ ID No. 3) is composed of STEos007 (100 bp, SEQ ID No. 4), STEos008 (100 bp, SEQ ID No. 5), and STEos006 (100 bp, SEQ ID No. 6) screened from the endogenous rice genome of plants, and STEvs011 (123 bp, SEQ ID No. 7) from the cauliflower mosaic virus 35S promoter, which are concatenated in series to further enhance the transcriptional level and the stability of gene expression. The mechanism of enhancing transcription is mainly by binding to transcription factors, changing the chromatin structure, making the transcription start site more easily recognized and bound by RNA polymerase and related transcription factors, thereby promoting the initiation of transcription and improving the transcriptional efficiency of genes.

[0041] Example 3 Design of bidirectional promoter The bidirectional promoter consists of four major parts: Mini 35S (46 bp, SEQ ID No. 8), 4×STE, CsVMV mini-pro, and AtPsak UTR (41 bp, SEQ ID No. 9), and its nucleotide sequence is as shown in SEQ ID No. 10. Among them, CsVMV mini-pro is connected upstream of the 5' end of 4×STE in a head-to-head manner with reverse complementarity. The Mini 35S is connected downstream of the 3' end of 4×STE in the forward direction. 4×STE is located in the shared sequence region in the middle of this gene pair (CsVMV mini-pro and Mini 35S).

[0042] Mini 35S (SEQ ID No. 8) connected to the 3' end of the synthetic quadruple STE transcriptional enhancer is constructed based on the 35S promoter of cauliflower mosaic virus (CaMV). The core region of the 35S promoter is selected, part of the enhancer sequence is removed, the key elements for the promoter to function are retained, and the TATA-box in the sequence determines the transcription direction.

[0043] At Psak UTR (SEQ ID No. 9) is the untranslated region sequence of the K subunit gene in Arabidopsis thaliana photosystem I. As a translation enhancer, it can effectively improve the protein production level and is not gene-specific. Its length and sequence have a significant impact on the expression effect. Shorter AtPsaKUTR usually shows higher expression efficiency than longer sequences.

[0044] Example 4 Bidirectional promoter with GUS reporter gene GUS (SEQ ID No. 11, source: E .Coli The reporter gene is fused with the upstream of the bidirectional promoter to form a bidirectional promoter with GUS the reporter gene. Among them, the upstream of the bidirectional reporter promoter is connected to the 3'-end of CsVMV mini-pro and At Psak UTR to form a bidirectional promoter with GUS the reporter gene, and its nucleic acid sequence is as shown in SEQ ID No.13.

[0045] Example 5 Bidirectional promoter with RUBY the reporter gene RUBY (SEQ ID No.12, source: Beta vulgaris The reporter gene is fused with the downstream of the bidirectional promoter to form a bidirectional promoter with RUBY the reporter gene. Among them, the downstream of the bidirectional reporter promoter is connected to RUBY the reporter gene to form a bidirectional promoter with RUBY the reporter gene, and its nucleic acid sequence is as shown in SEQ ID No.14.

[0046] Example 6 Bidirectional promoter with dual reporter genes GUS (SEQ ID No.11, source: E . Coli ), and RUBY (SEQ ID No.12, source: Beta vulgaris The dual reporter genes are respectively fused with both ends of the bidirectional promoter to form a bidirectional promoter with dual reporter genes, and its nucleotide sequence is as shown in SEQ ID No.15. Among them, GUS it is connected to the 3'-end of At Psak through CsVMV mini-pro in a reverse complementary orientation, RUBY and is connected to the 5'-end of Mini 35S in a forward orientation.

[0047] Example 7 Plant transformation construct The plant genetic transformation construct is specifically a recombinant plasmid containing the bidirectional promoter element with dual reporter genes in Example 6.

[0048] The construct characteristically contains the following combination of elements: (a) Assembling the linearized pRUBY vector backbone (Genesand) by In-Fusion ® directed cloning technology.

[0049] (b) By Phanta ®A high-fidelity DNA polymerase (Vazyme) was used to amplify DNA fragments with 20 - 25 bp homologous sequences designed at their ends to ensure precise assembly.

[0050] The specific construction process included: using the In-Fusion ® cloning system (Genesand) for multi-fragment recombination. After transformation, positive clones were screened in LB medium containing 50 μg / ml streptomycin sulfate. Plasmid extraction was carried out using the EndoFree Plasmid Midi Kit (Cwbio) and the FastPure Gel DNA Extraction Mini Kit (Vazyme) for product purification, and the integrity of the vector structure was confirmed by first-generation sequencing. Figure 2 It is a Sanger sequencing verification diagram of the bidirectional promoter sequence region in the bidirectional promoter expression vector. Through sequencing analysis, the obtained sequence was exactly the same as the theoretical length of the target fragment (639 bp), its nucleotide sequence was completely matched with the expected design, and no base deletions, insertions or mismatches were detected. The results showed that the recombinant vector containing the target bidirectional promoter was correctly constructed.

[0051] As Figure 1 shown in Table 1, the bidirectional promoter construct achieved bidirectional expression regulation through a specific combination of promoter elements. The first expression cassette was configured with this bidirectional promoter and 5'-end regulatory elements to drive the upstream GUS gene, and the second expression cassette was configured with this bidirectional promoter and 3'-end regulatory elements to drive the RUBY gene. The two achieved co-expression regulation through the said bidirectional promoter. This technical solution effectively solved the technical limitations of the traditional single-promoter vector system in co-expression regulation and provided a new tool platform for plant gene co-expression research.

[0052] Table 1 Bidirectional promoter construct .

[0053] Example 8 Transient tobacco infection mediated by Rhizobium rhizogenes (1) Preparation of Rhizobium rhizogenes cell bodies for transient infection of tobacco leaves The constructed recombinant vector of the artificial plant bidirectional promoter was transformed into Escherichia coli competent cells DH5α (ATCC 53868) by heat shock method to obtain Escherichia coli cells containing the recombinant vector of the bidirectional promoter. The transformed Escherichia coli was inoculated into LB medium containing the corresponding vector resistance and cultured with shaking at 37 °C for 8-10 h to obtain an Escherichia coli bacterial solution. The recombinant plasmid was extracted from the Escherichia coli cells and transformed into Rhizobium rhizogenes competent cells K599 (NCPPB2659) by chemical transformation method to obtain Rhizobium rhizogenes containing the artificial plant bidirectional constitutive strong promoter. The transformed Rhizobium rhizogenes was inoculated into TY medium containing the corresponding vector resistance and cultured with shaking at 28 °C for 12-16 h to obtain a Rhizobium rhizogenes bacterial solution.

[0054] Among them, each liter of TY medium contains 75 mg of spectinomycin, 5 g of tryptone, 3 g of yeast extract and 1 M calcium chloride. Each liter of resistant LB medium contains 50 mg of streptomycin sulfate, 10 g of tryptone, 5 g of yeast extract and 1 M calcium chloride.

[0055] (2)Transient tobacco infection The tobacco leaves were infected by injection method. First, they were dark-treated for 8 h, and then placed in the light condition for culture for 24-40 h. The growth temperature was 19-23 °C, the humidity was 65%-75%, and the light intensity during growth was 150 μmol·m -2 ·s -1 . Observe the color of the area injected with the bacterial solution on the plant leaves to judge RUBY whether the reporter gene is expressed. The leaves of positive plants show pink in the area injected with the bacterial solution, which is the phenomenon of betacyanin accumulation; judge whether the reporter gene is expressed by GUS histochemical staining method GUS and blue spots appear near the leaf veins and in some mesophyll cells of the leaves of positive plants.

[0056] Recombinant vectors driven by two CaMV35S promoters respectively RUBY and GUS the reporter gene (such as Figure 3 and Table 2) were used to infect tobacco leaves as blank controls.

[0057] Table 2 Control constructs .

[0058] (3) RUBY Positive detection Detection was carried out by observation method. The RUBY reporter system used in the present invention can visualize the positive transformed tobacco leaves under naked eye conditions, that is, the positive transformed leaves with betacyanin accumulation show pink marks. Such as Figure 4As shown, on the right is the expression driven by the CaMV35S promoter (control group), and on the left is the expression driven by the bidirectional promoter of the present invention (experimental group). From the results, it can be seen that significant pink markings appear on both sides of the leaf, indicating that the bidirectional promoter has the transcriptional activity to drive RUBY the expression. RUBY Through the results, it can be seen that significant pink markings appear on both sides of the leaf, indicating that the bidirectional promoter has the transcriptional activity to drive RUBY the transcription.

[0059] (4) GUS Positive detection Detected by histochemical staining. The GUS reporting system used in the present invention can visualize the positive transformed tobacco leaves under histochemical staining, that is, β-glucuronidase hydrolyzes the substrate X-Gluc (5-bromo-4-chloro-3-indolyl-β-D-glucoside), and blue spots appear near the tobacco leaf veins and in the mesophyll cells. As Figure 5 shown, on the right is the expression driven by the CaMV35S promoter (control group), and on the left is the expression driven by the bidirectional promoter of the present invention (experimental group). Through histochemical staining, it can be seen that significant blue markings appear on both sides of the leaf, indicating that the bidirectional promoter has the transcriptional activity to drive GUS the expression. GUS Through histochemical staining, it can be seen that significant blue markings appear on both sides of the leaf, indicating that the bidirectional promoter has the transcriptional activity to drive GUS the transcription.

[0060] (5) The significant advantages of the bidirectional promoter construct In the control group recombinant vector, two repeated CaMV35S promoters are used, which are likely to cause gene silencing due to sequence homology; while the artificial plant bidirectional constitutive strong promoter of the present invention can use one promoter to simultaneously initiate two genes, avoiding repeated promoter sequences and reducing the risk of gene silencing; at the same time, the plant endogenous enhancer significantly improves the compatibility with the plant genome, reduces the immune response, and realizes the precise regulation and efficient and stable expression of foreign genes.

Claims

1. A synthetic quadruple STE transcriptional enhancer, characterized in that, It includes three endogenous highly efficient STEs screened from the rice genome and one highly efficient STE screened from the cauliflower mosaic virus; the nucleotide sequences of the three endogenous highly efficient STEs screened from the rice genome are respectively shown as SEQ ID No. 4 - 6, and the nucleotide sequence of the one highly efficient STE screened from the cauliflower mosaic virus is shown as SEQ ID No.

7.

2. The synthetic quadruple STE transcriptional enhancer according to claim 1, wherein It is composed of four such STEs in series.

3. An artificial plant bidirectional constitutive strong promoter comprising the synthetic tetrameric STE transcriptional enhancer as claimed in claim 1 or 2.

4. The artificial plant bidirectional constitutive strong promoter according to claim 3, characterized in that, It respectively includes a minimal core promoter at both ends of the synthetic tetrameric STE transcriptional enhancer.

5. The artificial plant bidirectional constitutive strong promoter according to claim 4, characterized in that, It includes Mini35S, the synthetic tetrameric STE transcriptional enhancer 4×STE, and CsVMV mini - pro; the CsVMV mini - pro is connected to the 5' end of 4×STE in a reverse complementary head - to - head manner, the Mini 35S is connected forward to the 3' end of 4×STE, and the 4×STE is located in the shared sequence region between CsVMV mini - pro and Mini 35S.

6. The artificial plant bidirectional constitutive strong promoter according to claim 3, wherein The target gene expression cassette can be connected to the upstream and / or downstream of the artificial plant bidirectional constitutive strong promoter, and the target gene expression cassette is multiple genes concatenated through P2A.

7. A plant transformation construct comprising the artificial plant bidirectional constitutive strong promoter as claimed in any one of claims 3 - 6.

8. Use of the artificial plant bidirectional constitutive strong promoter as claimed in any one of claims 3 - 6 in constructing a plant multi - gene co - expression system.

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