Wheat endogenous high-activity TaUbi promoter and application thereof

Abstract

The invention discloses a wheat endogenous high-activity TaUbi promoter and an application of the wheat endogenous high-activity TaUbi promoter. The nucleotide sequence of the TaUbi promoter is as shown in SEQ ID No. 1. Experiments prove that the TaUbi promoter has obviously improved transcriptional activity and endogenous stability, and can greatly improve the expression level of a target gene; the TaUbi promoter drives the expression of Cas9 protein capable of recognizing a specific DNA sequence and cutting, so that the protein yield can be remarkably increased, and the gene editing efficiency is improved. The method has the outstanding advantages of high expression activity, good species specificity, strong stability and the like, is suitable for cultivation of conventional transgenic crops, can be compatible with various gene editing systems such as CRISPR / Cas9 and the like, provides an efficient and reliable multifunctional tool platform for wheat genetic improvement, and can powerfully promote the development of wheat molecular breeding. The method has an important application value.

Description

TECHNICAL FIELD

[0001] The application belongs to the field of plant genetic engineering and genome editing technology, and particularly relates to a wheat endogenous high-activity TaUbi promoter and application thereof. BACKGROUND

[0002] Wheat is an important food crop in the world, and its genetic improvement is of great significance to food security. Traditional breeding methods are time-consuming and inefficient, which cannot meet the needs of modern agricultural production. Transgenic technology and genome editing technology provide efficient means for wheat trait improvement. Promoter is a key element for regulating gene expression, and its activity directly affects the expression level of transgene and the efficiency of gene editing. The ZmUbi promoter from corn, which is widely used in wheat genetic transformation, has obvious limitations. As an exogenous promoter, its expression activity in wheat is insufficient and it is prone to generation silencing, resulting in unstable transgenic expression. Meanwhile, when used to drive the CRISPR / Cas9 system SpCas9 gene, the editing efficiency is restricted due to insufficient expression. In addition, wheat itself lacks known efficient endogenous promoter resources, which seriously limits the application of wheat genetic transformation system. SUMMARY

[0003] The purpose of the present application is to provide a promoter derived from wheat and capable of improving the efficiency of gene editing.

[0004] The present application first protects a specific DNA molecule (i.e. TaUbi promoter). The specific DNA molecule can be a DNA molecule as shown in a1) or a2) or a3): a1) a DNA molecule with a nucleotide sequence as shown in SEQ ID No. 1; a2) a DNA molecule with 75% or more identity to the nucleotide sequence defined in a1) and having a promoter function; a3) a DNA molecule hybridizing to the nucleotide sequence defined in a1) or a2) under stringent conditions and having a promoter function.

[0005] Those skilled in the art can easily mutate the nucleotide sequence of the specific DNA molecule of the present application by using known methods, such as directed evolution and point mutation. Those artificially modified nucleotides with 75% or more identity to the nucleotide sequence of the specific DNA molecule provided by the present application, as long as they have a promoter function, are derived from the nucleotide sequence of the present application and equivalent to the sequence of the present application.

[0006] The term "identity" as used herein refers to sequence similarity with a native nucleic acid sequence. "Identity" includes nucleotide sequences having 75% or more, 80% or more, or 85% or more, or 90% or more, or 95% or more identity with the nucleotide sequence of a specific DNA molecule of the application. Identity can be assessed by eye or by computer software. Using computer software, identity between two or more sequences can be expressed as a percentage (%) which can be used to assess identity between related sequences.

[0007] Expression cassettes containing any of the specific DNA molecules described above are also within the scope of the present application.

[0008] The expression cassette (from 5' to 3') can include a promoter region (comprised of the specific DNA molecule), a transcription initiation region, a gene of interest region, a transcription termination region, and optionally a translational termination region. The promoter region and the gene of interest region can be native / analogous to the host cell, or, the promoter region and the gene of interest region can be native / analogous to each other, or, the promoter region and / or the gene of interest region can be heterologous to the host or to each other. "Heterologous" means a sequence that is foreign to the species from which it is derived, or, if from the same species, has been substantially modified from the native form by deliberate human intervention in either component and / or genomic location. The optionally included transcription termination region can be homologous to the transcription initiation region, to the operably linked gene of interest region, to the host; or; the gene of interest region, the host are foreign or heterologous.

[0009] The expression cassette can also include a 5' leader sequence. The 5' leader sequence can enhance translation.

[0010] In preparing the expression cassette, adapters or linkers can be used to join DNA fragments, or, other manipulations can be involved to provide appropriate restriction sites, removal of excess DNA, removal of restriction sites, etc. To this end, in vitro mutagenesis, primer repair, restriction enzyme cleavage, annealing, re-substitutions, such as transitions and transversions, can be performed.

[0011] The expression cassette can also include a selectable marker gene for screening transformed cells. Selectable marker genes can be used to screen transformed cells or tissues. Marker genes include genes that encode antibiotic resistance. Other selectable markers include phenotypic markers such as fluorescent proteins. The above listed selectable markers are not limiting. Any selectable marker gene can be used with the present application.

[0012] Recombinant plasmids containing any of the specific DNA molecules described above are also within the scope of the present application.

[0013] The recombinant plasmid can be a recombinant plasmid obtained by inserting any of the specific DNA molecules described above into a starting plasmid. Specifically, the recombinant plasmid can be a recombinant plasmid obtained by inserting any of the specific DNA molecules described above into the multiple cloning site of a starting plasmid.

[0014] The starting plasmid can be an expression vector, a cloning vector, or a gene editing vector.

[0015] The recombinant plasmid can include any of the expression cassettes described above containing the specific DNA molecule.

[0016] Specifically, the recombinant plasmid can be the recombinant plasmid pGreenII-4600-Luc, the vector pMWB110-Cas9-TaCCD1-TaUbi, or the single-base editing vector ABE-SpCas9-NGG-TaUbi mentioned in the examples.

[0017] The recombinant plasmid pGreenII-4600-Luc is a recombinant plasmid obtained by replacing the small DNA fragment between the restriction enzymes KpnI and XhoI of the pGreenII 0800-LUC dual luciferase reporter vector with a DNA molecule having the nucleotide sequence shown in SEQ ID No. 1.

[0018] The vector pMWB110-Cas9-TaCCD1-TaUbi is a recombinant plasmid obtained by replacing the maize ubiquitin promoter ZmUbi in the vector pMWB110-Cas9-TaCCD1-ZmUbi with a DNA molecule having the nucleotide sequence shown in SEQ ID No. 1.

[0019] The single-base editing vector ABE-SpCas9-NGG-TaUbi is a recombinant plasmid obtained by replacing the ZmUbi promoter in the single-base editing vector ABE-SpCas9-NGG-ZmUbi with a DNA molecule having the nucleotide sequence shown in SEQ ID No. 1.

[0020] The recombinant microorganism containing the specific DNA molecule also falls within the protection scope of the present application.

[0021] The recombinant microorganism can be obtained by introducing the recombinant plasmid into a starting microorganism.

[0022] The starting microorganism can be a yeast, a bacterium, an alga, or a fungus. The bacterium can be a gram-positive bacterium or a gram-negative bacterium. The gram-negative bacterium can be Agrobacterium tumefaciens (ATCC 33916). Agrobacterium tumefaciens Specifically, the Agrobacterium tumefaciens (ATCC 33916) can be Agrobacterium tumefaciens GV3101. Agrobacterium tumefaciens

[0023] ​The transgenic cell line containing the specific DNA molecule also belongs to the protection scope of the present application.

[0024] The transgenic cell line can be a transgenic plant cell line or a transgenic animal cell line.

[0025] The transgenic cell line containing the specific DNA molecule does not include propagation material.

[0026] The transgenic plant is understood to include not only the first generation transgenic plant obtained by transforming the specific DNA molecule into a receptor plant, but also its offspring. For the transgenic plant, the gene can be propagated in the species, or the gene can be transferred into other varieties of the same species by conventional breeding techniques, especially including commercial varieties. The transgenic plant includes seeds, callus, whole plants and cells.

[0027] The application of any of the above-mentioned specific DNA molecules as a promoter (i.e. TaUbi promoter) also belongs to the protection scope of the present application.

[0028] The application of any of the above-mentioned specific DNA molecules, any of the above-mentioned expression cassettes or any of the above-mentioned recombinant plasmids in promoting the expression of a target gene also belongs to the protection scope of the present application.

[0029] The application of any of the above-mentioned specific DNA molecules, any of the above-mentioned expression cassettes or any of the above-mentioned recombinant plasmids in increasing the expression amount of a target gene also belongs to the protection scope of the present application.

[0030] The application of any of the above-mentioned specific DNA molecules, any of the above-mentioned expression cassettes or any of the above-mentioned recombinant plasmids in improving the gene editing efficiency also belongs to the protection scope of the present application.

[0031] In the above-mentioned applications, the improvement of the gene editing efficiency can be achieved by expressing the coding gene of a nuclease capable of recognizing a specific DNA sequence and cutting in the gene editing system through any of the above-mentioned specific DNA molecules.

[0032] The present application also protects a method for expressing a target gene.

[0033] The method for expressing a target gene protected by the present application can be method A, which can be the expression of a target gene using any of the above-mentioned specific DNA molecules as a promoter.

[0034] The method for expressing a target gene protected by the present application can be method B, which can include the following steps: inserting any of the above-mentioned specific DNA molecules upstream of any target gene or enhancer to initiate the expression of the target gene.

[0035] The method for expressing a target gene according to the present application, specifically method C, can comprise the following steps: inserting the target gene downstream of the specific DNA molecule in any of the above-mentioned expression cassettes, and starting the expression of the target gene by the specific DNA molecule.

[0036] The method for expressing a target gene according to the present application, specifically method D, can comprise the following steps: inserting the target gene downstream of the specific DNA molecule in any of the above-mentioned recombinant plasmids, and starting the expression of the target gene by the specific DNA molecule.

[0037] In any of the above-mentioned methods, the expression amount of the target gene is increased (i.e. overexpression of the target gene).

[0038] The expression of the target gene according to any of the above-mentioned embodiments can be overexpression of the target gene.

[0039] The target gene according to any of the above-mentioned embodiments can be the SpCas9 gene mentioned in the embodiments, The specific DNA molecule according to any of the above-mentioned embodiments can be used as a promoter (high-activity promoter) to express genes (such as exogenous genes, endogenous genes) in plants. The plants can be any of wheat, corn, rice, sorghum, etc. Since the specific DNA molecule is derived from the wheat genome, the problem of epigenetic silencing caused by exogenous sequences in wheat can be avoided.

[0040] The target gene according to any of the above-mentioned embodiments can be an exogenous gene, or an endogenous gene (i.e. derived from the wheat genome).

[0041] The present application also protects a method for improving the efficiency of gene editing, which can be achieved by using the above-mentioned specific DNA molecule to start the expression of the coding gene of a nuclease capable of recognizing and cutting a specific DNA sequence in a gene editing system.

[0042] The gene editing according to any of the above-mentioned embodiments can be single-base editing or prime editing.

[0043] The gene editing system according to any of the above-mentioned embodiments can be a CRISPR / Cas9 gene editing system.

[0044] The nuclease capable of recognizing and cutting a specific DNA sequence according to any of the above-mentioned embodiments can be a Cas9 protein. The Cas9 protein can be specifically an SpCas9 protein.

[0045] This invention, through integrated bioinformatics analysis and experimental verification, identifies for the first time a novel endogenous, highly active TaUbi promoter from the wheat genome. The TaUbi promoter not only exhibits higher transcriptional activity than the ZmUbi promoter, significantly increasing exogenous gene expression, but also avoids epigenetic silencing issues caused by exogenous sequences due to its origin in the wheat genome. More importantly, when used to drive the CRISPR / Cas9 system, the TaUbi promoter can significantly enhance… SpCas9 The TaUbi promoter significantly improves gene expression levels, thereby enhancing gene editing efficiency. It is evident that the TaUbi promoter can replace the traditional ZmUbi promoter, leveraging its significantly enhanced transcriptional activity and endogenous stability to effectively overcome epigenetic silencing caused by exogenous promoters and substantially increase the expression level of target genes. The TaUbi promoter-driven expression of nucleases capable of recognizing and cleaving specific DNA sequences (such as the SpCas9 protein, a core component of the CRISPR / Cas9 system) significantly increases protein yield, thereby improving gene editing efficiency. This invention possesses outstanding advantages such as high expression activity, good species specificity, and strong stability. It is suitable for the cultivation of conventional transgenic crops and compatible with various gene editing systems such as CRISPR / Cas9, Base Editor, and Prime Editor, providing an efficient, reliable, and multifunctional tool platform for wheat genetic improvement. In practical applications, the combination of the TaUbi promoter with different expression vectors can be flexibly selected according to specific needs, which will strongly promote the development of wheat molecular breeding. This invention has significant application value. Attached Figure Description

[0046] Figure 1 Data analysis of candidate promoter expression in different tissues (IWGSC, 2014).

[0047] Figure 2 LUC results for detecting promoter activity.

[0048] Figure 3 shows the Luc / Ren ratios for different promoters.

[0049] Figure 4 The spectrum of the vector pWMB110-Cas9-ZmUbi.

[0050] Figure 5 This is a partial structural diagram of the carriers pMWB110-Cas9-TaCCD1-ZmUbi and pMWB110-Cas9-TaCCD1-TaUbi.

[0051] Figure 6 For some T0 generation transgenic wheat plants bar Protein test results.

[0052] Figure 7 The relative expression amount of the gene in the T0 generation transgenic wheat positive plant SpCas9 The relative expression amount of the gene in the T0 generation transgenic wheat positive plant

[0053] Figure 8 The single base editing vector ABE-SpCas9-NGG-ZmUbi map.

[0054] Figure 9 The single base editing vector ABE-SpCas9-NGG-TaUbi map.

[0055] Figure 10 The gene editing situation of part of the T0 generation transgenic wheat plants in Example 4. DETAILED DESCRIPTION

[0056] The application will be further described in detail below with specific embodiments, and the examples given are only for illustrating 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 of ordinary skill in the art, and do not in any way constitute a limitation on the application.

[0057] Unless otherwise specified, the experimental methods used in the examples are conventional methods in the art, and can be referred to relevant technical literature (such as Sambrook et al. Molecular Cloning: A Laboratory Manual (New York: Cold Spring Harbor Laboratory Press, 1989)) or the instructions for use of relevant reagent products. The materials, reagents, etc. used in the examples, unless otherwise specified, can be obtained through commercial channels.

[0058] In the quantitative test in the following examples, three repeated experiments were set, and the average value was taken.

[0059] Example 1, screening and activity detection of high-activity promoters I. Screening and vector construction of endogenous high-activity promoters in wheat 1. Experimental materials pGreenII 0800-LUC dual luciferase reporter vector is a product of Shanghai Mokang Biotechnology, with product catalog number MF3732-5UG. The pGreenII 0800-LUC dual luciferase reporter vector contains Luciferase a reporter gene and Renilla a luciferase internal reference gene.

[0060] KOD Plus Neo high-fidelity DNA polymerase is a product of TOYOBO LIFE SCIENCE, with product catalog number KOD-401.

[0061] In-Fusion HD Cloning System is a product of TaKaRa, product catalog number is 638910.

[0062] Restriction enzymes AscI, SnabI, PstI, KpnI and XhoI are all products of Thermo Scientific, product catalog numbers are FD1894, FD0404, FD0614, FD0524 and FD0694, respectively.

[0063] DNA purification kit is a product of Beijing Jinge Biological Technology Co., Ltd., product catalog number is TD413.

[0064] 2. Screening and cloning of endogenous high-activity promoters in wheat Based on the data of gene expression database (http: / / 202.194.139.32 / expression / wheat.html) and wheat genome database, bioinformatics analysis was performed to screen 11 candidate promoters highly expressed in wheat, which were TraesCS6B02G293300 promoter, TraesCS7D02G442700 promoter, TraesCS5A02G062600 promoter, TraesCS6D02G248600 promoter, TraesCS6A02G266000 promoter, TraesCS6B02G164600 promoter, TraesCS6A02G198100 promoter, TraesCS2B02G585600 promoter, TraesCS7A02G453300 promoter, TraesCS6B02G093100 promoter and TraesCS5B02G479100 promoter (part of which is shown in Figure 1 ). Then KODPlus Neo high-fidelity DNA polymerase was used to amplify the 11 promoters (reaction conditions: 95℃ pre-denaturation for 5 min; 95℃ denaturation for 30 s, 60℃ annealing for 30 s, 72℃ extension for 30 s, 35 cycles; 72℃ final extension for 5 min), and the amplification products were detected by agarose gel electrophoresis and then purified and recovered using a DNA purification kit (Beijing Jinge Biological Technology Co., Ltd.).

[0065] 3. Construction of reporter vector The pGreenII 0800-LUC dual luciferase reporter vector was digested with restriction enzymes Kpnl and Xhol, and the vector backbone was recovered. Then the TraesCS6B02G293300 promoter obtained in step 2 was connected to the vector backbone by using In-Fusion HD Cloning System, and the connection product was transformed into E. coli Top10 competent cells (a product of Beijing Chengke Biological Technology Co., Ltd., product catalog number DLC112), plated, cultured, and a number of single colonies were obtained. Then colony PCR was performed on the positive colonies by using a primer pair consisting of primer M13F-47: 5'-CGCCAGGGTTTTCCCAGTCACGAC-3' and primer GLP2: 5'-CTTTATGTTTTTGGCGTCTTCC-3'. The positive colonies contained the recombinant plasmid pGreenII-3300-Luc. The sequencing results showed that the recombinant plasmid pGreenII-3300-Luc was obtained by replacing the small piece of DNA between the restriction enzymes Kpnl and Xhol of the pGreenII 0800-LUC dual luciferase reporter vector with the TraesCS6B02G293300 promoter.

[0066] According to the above steps, the TraesCS6B02G293300 promoter is replaced with the TraesCS7D02G442700 promoter, the TraesCS5A02G062600 promoter, the TraesCS6D02G248600 promoter, the TraesCS6A02G266000 promoter, the TraesCS6B02G164600 promoter, the TraesCS6A02G198100 promoter, the TraesCS2B02G585600 promoter, the TraesCS7A02G453300 promoter, the TraesCS6B02G093100 promoter, the TraesCS5B02G479100 promoter and the corn ZmUbi promoter (nucleotide sequence as shown in SEQ ID No. 2) respectively, and the other steps are unchanged, and the recombinant plasmids pGreenII-2700-Luc, pGreenII-2600-Luc, pGreenII-8600-Luc, pGreenII-6000-Luc, pGreenII-4600-Luc, pGreenII-8100-Luc, pGreenII-5600-Luc, pGreenII-3300-Luc, pGreenII-3100-Luc, pGreenII-9100-Luc and pGreenII-ZmUbi-Luc are obtained in turn. The recombinant plasmid pGreenII-ZmUbi-Luc is used as a control.

[0067] II. In vivo imaging fluorescence detection 1. Obtaining of the recombinant Agrobacterium The recombinant plasmid (the recombinant plasmid pGreenII-3300-Luc, the recombinant plasmid pGreenII-2700-Luc, the recombinant plasmid pGreenII-2600-Luc, the recombinant plasmid pGreenII-8600-Luc, the recombinant plasmid pGreenII-6000-Luc, the recombinant plasmid pGreenII-4600-Luc, the recombinant plasmid pGreenII-8100-Luc, the recombinant plasmid pGreenII-5600-Luc, the recombinant plasmid pGreenII-3300-Luc, the recombinant plasmid pGreenII-3100-Luc, the recombinant plasmid pGreenII-9100-Luc or the recombinant plasmid pGreenII-ZmUbi-Luc) is introduced into the Agrobacterium tumefaciens GV3101 competent cell (product of Beijing Chengke Biological Technology Co., Ltd., product catalog number DLC301) by chemical transformation to obtain the recombinant Agrobacterium.

[0068] 2. Tobacco transient transformation and in vivo imaging fluorescent detection (1) The recombinant Agrobacterium single colony obtained in step 1 was inoculated and cultured to OD 600nm 0.6-0.8, and then centrifuged to collect the bacterial cells, which were resuspended in an infection buffer (solutes and their concentrations are 1 mM MgCl2, 0.2 mM MES and 200 μM acetosyringone, solvent is water) to obtain an infection solution.

[0069] (2) After step (1) was completed, 4-6 week old N. benthamiana was used. A needle-free syringe was used to inject the infection solution into the back of the leaf, and 3-5 sites per tobacco plant were injected.

[0070] (3) After step (2) was completed, the injected tobacco plants were incubated at 22°C under 16 h light / 8 h dark conditions for 48-72 hours before fluorescent detection. First, D-luciferin potassium salt substrate solution (1 mM) was sprayed, and after 5 minutes of dark adaptation, a CCD imaging system was used to collect the fluorescent signal, and the relative activity of each promoter was analyzed by software.

[0071] Three biological replicates were set up for each experiment, and at least 5 independent transformation sites were detected for each replicate to ensure the reliability of the data.

[0072] Some of the test results are shown in Figure 2 (A and B are recombinant plasmid pGreenII-ZmUbi-Luc, C is TraesCS6B02G164600 promoter, D is TraesCS6B02G093100 promoter, and E is TraesCS5B02G479100 promoter). The results of the dual luciferase reporter system detection showed that there were significant differences in the expression of the reporter genes driven by different promoters. Among them, the TraesCS6B02G164600 promoter showed the strongest fluorescent signal intensity, and its activity was significantly higher than that of other promoters; the TraesCS6B02G093100 promoter was second, while the fluorescent signal intensity of the TraesCS5B02G479100 promoter was relatively weak, similar to the expression level of the ZmUbi promoter.

[0073] Three, luciferase activity detection 1. The recombinant Agrobacterium single colony obtained in step 2 was inoculated and cultured to OD 600nm 0.6-0.8, and then centrifuged to collect the bacterial cells, which were resuspended in an infection buffer (solutes and their concentrations are 1 mM MgCl2, 0.2 mM MES and 200 μM acetosyringone, solvent is water) to obtain an infection solution.

[0074] 2、After step 1, take 4-6 weeks old N. benthamiana, use a needle-free syringe to inject the infection solution on the back of the leaf, 3-5 sites per plant.

[0075] 3、After step 2, first dark culture for 24 hours, then culture at 22℃, 16 h light / 8 h dark for 48 hours.

[0076] 4、After step 3, cut the injected part of the tobacco and add 100 μL of cell lysis buffer lysis solution, vortex for 1 min, ice bath for 10 min, then centrifuge at 4℃, 12000 rpm for 2 min, collect the supernatant. Take 10 μL of the supernatant to the enzyme-labeled plate, then add 40 μL of Reaction buffer, mix immediately after sucking and beating, and then use the fluorescence detector to detect the luciferase reporter gene activity; add 40 μL of Stop buffer to the enzyme-labeled plate, mix immediately after sucking and beating, and then use the fluorescence detector to detect the renilla luciferase reporter gene activity. By calculating the ratio of firefly luciferase to renilla luciferase, the Luc / Ren ratio (standardized promoter activity) is calculated to quantitatively analyze the expression level.

[0077] Some of the test results are shown in Figure 3 . The results show that the expression efficiency of TraesCS6B02G164600 promoter is the most prominent, and the expression amount driven by the gene reaches more than 3 times of that of ZmUbi promoter (P<0.01), which provides strong evidence for its use as a high-efficiency plant expression vector promoter element. P

[0078] The TraesCS6B02G164600 promoter is named as TaUbi promoter. The nucleotide sequence of the TaUbi promoter is shown in SEQ ID No. 1.

[0079] Example 2, comparison of the starting effect of TaUbi promoter and ZmUbi promoter in wheat I. Construction of recombinant vector The map of vector pWMB110-Cas9-ZmUbi is shown in Figure 4 ​The vector pWMB110-Cas9-ZmUbi is described in the following document: Liu HY, Wang K, Jia ZM, Gong Q, Lin ZS, Lipu Du LP, Pei XW, Ye XG. Editing TaMTL gene induces haploid plants efficiently by optimized Agrobacterium-mediated CRISPR system in wheat. Journal of Experimental Botany, 2020, 71: 1337-1349. 1. The CDS sequence of TaCCD1 (TraesCS5A02G001500) was found from NCBI website (https: / / www.ncbi.nlm.nih.gov / ) by bioinformatics, and the specific SgRNA target sequence of the gene was designed and obtained: 5'-CCGCCTTGAGAACCCAGATCTGG-3'. TaCCD1 TaCCD1 After that, the SgRNA was connected to the vector pWMB110-Cas9-ZmUbi according to the method in the above document, and the vector pMWB110-Cas9-TaCCD1-ZmUbi was obtained. The partial structure diagram of the vector pMWB110-Cas9-TaCCD1-ZmUbi is shown in (a) of Figure 1. Figure 5

[0080] 2. The ZmUbi promoter in the vector pMWB110-Cas9-TaCCD1-ZmUbi was replaced with TaUbi promoter by molecular cloning technology, and the vector pMWB110-Cas9-TaCCD1-TaUbi was obtained. Specifically: the vector pMWB110-Cas9-TaCCD1-ZmUbi was digested with restriction enzyme PstI to specifically remove the corn ubiquitin promoter ZmUbi; the DNA fragment of TaUbi promoter was inserted into the vector backbone by In-Fusion HD Cloning System, and the ligation product was transformed into E. coli competent cells, and positive clones were screened and verified by Sanger sequencing to ensure that the promoter replacement was accurate and no unintended mutations were introduced.

[0081] The partial structure diagram of the vector pMWB110-Cas9-TaCCD1-TaUbi is shown in (b) of Figure 1. Figure 5

[0082] II. Agrobacterium-mediated genetic transformation of wheat ​​​The vector pMWB110-Cas9-TaCCD1-ZmUbi and the vector pMWB110-Cas9-TaCCD1-TaUbi were introduced into wheat Fielder by Agrobacterium-mediated genetic transformation method (referring to Wang et al., The gene TaWOX5 overcomes genotype dependency in wheat genetic transformation. Nature Plants. 2022. 8: 110-117) to obtain T0 generation transgenic wheat plants. The main steps are as follows: 1. The vector (vector pMWB110-Cas9-TaCCD1-ZmUbi or vector pMWB110-Cas9-TaCCD1-TaUbi) was introduced into Agrobacterium tumefaciens GV3101 competent cells by chemical transformation method to obtain recombinant Agrobacterium.

[0083] 2. After step 1 was completed, 4 days before the infection experiment, the recombinant Agrobacterium was inoculated on YEP solid medium containing 50 mg / L Gent (gentamicin), 50 mg / L Kana (kanamycin) and 50 mg / L Rif (rifampicin), and cultured at 28°C in the dark for 3 days. Single colonies were inoculated in 10 mL YEP liquid medium containing 50 mg / L Gent, 50 mg / L Kana and 50 mg / L Rif, and cultured at 28°C, 200 rpm. The bacteria liquid cultured to logarithmic phase was adjusted to OD 600nm value of about 0.6 with MS resuspension liquid (solute and its concentration were 10 g / L glucose and 0.443 g / L MS powder, and solvent was water) to obtain the infection liquid.

[0084] 3. After step 2 was completed, the 14-day-old wheat Fielder embryo was selected and infected with the infection liquid prepared in step 2. The infected embryo was plated on AS co-culture medium (solute and its concentration were 10 g / L glucose, 8 g / L agar, 200 μM acetosyringone and 0.443 g / L MS powder, and solvent was water), and cultured at 25°C in the dark for 3 days.

[0085] 4. After completing step 3, the embryos were transferred to WLS-RES medium (solutes and their concentrations were 2.2 mg / L picloram, 250 mg / L carbenicillin (Cb), 250 mg / L cephalosporin (Cef), and 4.43 g / L MS powder, with water as the solvent) and cultured in the dark for 5 days. Two rounds of selection were then performed sequentially. The first round used WLS-RES medium containing 5 mg / L PPT and cultured in the dark for 14 days. The second round used WLS-RES medium containing 10 mg / L PPT and cultured in the dark for 21 days to obtain callus tissue.

[0086] 5. After completing step 4, the callus tissue was transferred to differentiation medium (solutes and their concentrations were 5 mg / L PPT, 250 mg / L Cb, and 4.43 g / L MS powder, solvent: water) and cultured under light for 2 weeks. The resulting green shoots were isolated and inoculated into rooting medium (solutes and their concentrations were 5 mg / L PPT, 0.5 mg / L IBA, 100 mg / L Cb, and 4.43 g / L MS powder, solvent: water) and cultured for 21 days. After the root system was fully developed, the plants were transplanted into soil to obtain T0 generation transgenic wheat plants.

[0087] III. Obtaining positive T0 generation transgenic wheat plants The PAT / bar rapid test strip (Shanghai Youlong Biotechnology Co., Ltd., product number: AA1032-LS) was used to test T0 generation transgenic wheat plants. bar Rapid detection of proteins.

[0088] Some test results can be found Figure 6 (The two red lines indicate) bar (Protein positive). The results indicate that protein was successfully obtained from T0 generation transgenic wheat plants. bar Protein-positive T0 generation transgenic wheat plants (referred to as T0 generation transgenic wheat positive plants). A total of 26 T0 generation transgenic wheat positive plants were obtained using the vector pMWB110-Cas9-TaCCD1-ZmUbi. A total of 27 T0 generation transgenic wheat positive plants were obtained using the vector pMWB110-Cas9-TaCCD1-TaUbi.

[0089] IV. Detection of positive T0 generation transgenic wheat plants obtained in step three SpCas9 Relative expression level of genes 1. Total RNA was extracted from the leaf tissue of T0 generation transgenic wheat positive plants obtained in step 3 using a plant RNA extraction kit (TransGen, catalog number ER302); the quality and integrity of the RNA were detected by agarose gel electrophoresis and spectrophotometry.

[0090] 2. After completing step 1, use the HiScript All-in-one RT Master Mix for qPCR kit (Kangwei Century, catalog number CW3371M) to reverse transcribe 1 μg of total RNA from T0 generation transgenic wheat into cDNA, thus obtaining the cDNA of T0 generation transgenic wheat.

[0091] The reaction conditions were: 42℃ for 15 minutes, then 85℃ for 5 seconds.

[0092] 3. After completing step 2, using cDNA from T0 generation transgenic wheat as a template, real-time quantitative PCR was performed for detection. SpCas9 Relative gene expression levels (in wheat) TaActin (Genes were used as internal controls). Specifically, the 2×SuperFast UniversalSYBR Master Mix 2.0 kit (Kangwei Century, catalog number CW3360H) was used, and the detection was performed on an Applied Biosystems 7500 Real-Time PCR System.

[0093] The reaction system consisted of 20 μL, including 10 μL of 2×SYBR Master Mix, 1 μL of T0 generation transgenic wheat cDNA, 0.5 μL of forward primer (10 μM), 0.5 μL of reverse primer (10 μM), and 8 μL of nuclease-free water. The reaction program was as follows: 95℃ pre-denaturation for 5 minutes; 95℃ denaturation for 10 seconds, 60℃ annealing, 30 seconds extension, for 45 cycles; finally, melting curve analysis was performed (95℃ for 15 seconds, 60℃ for 1 minute, 95℃ for 15 seconds).

[0094] Each sample was configured with 3 biological replicates, using 23 -△△CT Method Calculation SpCas9 The relative expression level of genes.

[0095] Detection SpCas9 The forward primer for the gene is 5'-TCAAGGCTCTTGTTCGTCAGCA-3', and the reverse primer is 5'-TTGCCGCTCTGCTTATCCCTGA-3'.

[0096] Detection TaActin The forward primer for the gene is 5'-TGACCGTATGAGCAAGGAG-3', and the reverse primer is 5'-CCAGACAACTCGCAACTTAG-3'.

[0097] Some test results can be found Figure 7 (The horizontal axis represents the strain name). The results show that the ZmUbi promoter and the TaUbi promoter... SpCas9The regulation effect of gene expression is significantly different: in the editing strain driven by ZmUbi promoter, only Z23 detects the basic level of SpCas9 gene expression, and Z24, Z25, Z26, Z27 and Z30 have no significant expression; and in the editing strain driven by TaUbi promoter, T1, T2, T3 and T6 SpCas9 significantly express, among which the expression amount of T6 SpCas9 gene is particularly prominent.

[0098] The above results show that the type of promoter is a key factor affecting SpCas9 the expression amount of gene. Compared with ZmUbi promoter, TaUbi promoter can more effectively drive SpCas9 gene to achieve efficient expression in the strain, which provides an important theoretical basis and practical guidance for the selection of promoter in wheat transgenic and gene editing technology.

[0099] Example 3, Effect of TaUbi Promoter on Wheat Gene Editing Efficiency 1. According to the method of reference example 2, the vector (vector pMWB110-Cas9-TaCCD1-ZmUbi or vector pMWB110-Cas9-TaCCD1-TaUbi) is introduced into wheat Fielder to obtain T0 generation transgenic wheat plants.

[0100] 2. The leaf of the T0 generation transgenic wheat plant obtained in step 1 is taken, and the genomic DNA of the T0 generation transgenic wheat plant is extracted by using a DNA extraction kit (Kangwei Century Biotechnology Co., Ltd., product number CW0581M).

[0101] 3. Taking the genomic DNA of the T0 generation transgenic wheat plant as a template, the primer pair for amplifying TaCCD1 the A / B / D genome of the gene is used for PCR amplification, and after agarose gel electrophoresis detection, the correct PCR amplification product is selected and sent to a biological company for sequencing. According to the sequencing results, the editing types are analyzed, the different types of editing plants are statistically arranged, and the mutant materials are reserved.

[0102] The primer pair for amplifying TaCCD1 the A genome of the gene consists of primer F1: 5'-ATCCTGTGCCAATAACAATAAC-3' and primer R1: 5'-TCCAGGAAGAATCTCACTGTAGG-3'.

[0103] The primer pair for amplifying TaCCD1The primer pair for the B genome of the gene consists of primer F2: 5'- ATCCTGTGCCAATAACAATAAC-3' and primer R2: 5'-AACAAGCTCCACAATGAGTTG-3'.

[0104] The primer pair for the D genome of the gene consists of primer F3: 5'- ATCCTGTGCCAATAACAATAAC-3' and primer R3: 5'-GCTCAATGCCTCAGATGAACTA-3'. TaCCD1

[0105] T0 generation transgenic wheat plants carrying different vectors were successfully obtained by Agrobacterium-mediated genetic transformation. Analysis of the gene editing efficiency of T0 generation transgenic wheat plants showed (see Table 1) that the gene editing vector containing the TaUbi promoter showed higher editing efficiency, reaching 81.48%, which was significantly better than the editing efficiency of 76.92% of the traditional ZmUbi promoter. This result confirms that the TaUbi promoter can more effectively promote gene editing to occur. It can be seen that the selection of the promoter is one of the key factors affecting the editing efficiency of the CRISPR / Cas9 system.

[0106]

[0107] Example 4, Effect of TaUbi Promoter on Single Base Editing Efficiency of Wheat The map of the single base editing vector ABE-SpCas9-NGG-ZmUbi is shown in Figure 8 . The single base editing vector ABE-SpCas9-NGG-ZmUbi is described in the following document: Wang et al., Application of Nicotinamide to Culture Medium Improves the Efficiency of Genome Editing in Hexaploid wheat. 2023. International Journal of Molecular Sciences, 24, 4416. (Note: The source needs to be more detailed) 1. The ZmUbi promoter in the single base editing vector ABE-SpCas9-NGG-ZmUbi was replaced with the TaUbi promoter using molecular cloning techniques to obtain the single base editing vector ABE-SpCas9-NGG-TaUbi. Specifically as follows: restriction endonuclease ASC and SnabI ​The single base editing vector ABE-SpCas9-NGG-ZmUbi was digested by restriction enzyme, and the ubiquitin promoter ZmUbi of maize was specifically removed. The DNA fragment of TaUbi promoter was inserted into the vector backbone by In-Fusion HD Cloning System. The ligation product was transformed into E. coli competent cells, and positive clones were screened and verified by Sanger sequencing to ensure accurate promoter replacement and no unintended mutations.

[0108] The map of the single base editing vector ABE-SpCas9-NGG-TaUbi is shown in Figure 9 .

[0109] 2. According to the sequences of 1Ax gene (Genebank number: X61009.1), 1Bx gene (Genebank number: X13927.3) and 1Dx gene (Genebank number: X03346.1), the target sequence (sgRNA1: 5'-ACAATATGAGCAGCAAGTCGTGG-3') for 1Ax gene, the target (sgRNA2: 5'-ACAATACGAGCAGCAACCTGTGG-3') for 1Bx gene and the target (sgRNA3: 5'-ACAATACGAGCAGCAAATCGTGG-3') for 1Dx gene were designed. Then, according to the method in the literature (Liu HY, Wang K, Jia ZM, Gong Q, Lin ZS, Lipu Du LP, Pei XW, Ye XG. Editing TaMTL gene induces haploid plants efficiently by optimized Agrobacterium-mediated CRISPR system in wheat. Journal of Experimental Botany, 2020, 71: 1337-1349.), sgRNA1, sgRNA2 and sgRNA3 were respectively combined with the expression cassette of ABE-SpCas9-NGG-TaUbi to obtain the single base editing vectors ABE-SpCas9-NGG-TaUbi-1Ax, ABE-SpCas9-NGG-TaUbi-1Bx and ABE-SpCas9-NGG-TaUbi-1Dx. Asc The single base editing vector ABE-SpCas9-NGG-TaUbi was digested by restriction enzyme, and the ubiquitin promoter ZmUbi of maize was specifically removed. The DNA fragment of TaUbi promoter was inserted into the vector backbone by In-Fusion HD Cloning System. The ligation product was transformed into E. coli competent cells, and positive clones were screened and verified by Sanger sequencing to ensure accurate promoter replacement and no unintended mutations.

[0110] According to the above steps, the single base editing vector ABE-SpCas9-NGG-TaUbi is replaced by the single base editing vector ABE-SpCas9-NGG-ZmUbi, and other steps are unchanged, and the vectors ABE-SpCas9-NGG-ZmUbi-1Ax, ABE-SpCas9-NGG-ZmUbi-1Bx and ABE-SpCas9-NGG-ZmUbi-1Dx are obtained in turn.

[0111] 3、According to the method of reference example 2, the mixed single base editing vector of two different promoters (i.e. the mixing of the vectors ABE-SpCas9-NGG-TaUbi-1Ax, ABE-SpCas9-NGG-TaUbi-1Bx and ABE-SpCas9-NGG-TaUbi-1Dx; the mixing of the vectors ABE-SpCas9-NGG-ZmUbi-1Ax, ABE-SpCas9-NGG-ZmUbi-1Bx and ABE-SpCas9-NGG-ZmUbi-1Dx) is introduced into wheat Fielder, respectively, to obtain T0 generation transgenic wheat plants.

[0112] 4、Take the leaves of the T0 generation transgenic wheat plants obtained in step 3, extract the genomic DNA using a DNA extraction kit (Kangwei Century Biotechnology Co., Ltd., product number CW0581M) to obtain the genomic DNA of the T0 generation transgenic wheat.

[0113] 5、Using the genomic DNA of the T0 generation transgenic wheat obtained in step 4 as a template, PCR amplification is performed using primer pairs for amplifying the 1Ax gene, the 1Bx gene and the 1Dx gene, respectively, and after agarose gel electrophoresis detection, the correct PCR amplification products are selected and sent to a biological company Sanger sequencing, and the editing types are analyzed according to the sequencing results, the different types of edited plants are counted and sorted, and the mutant materials are reserved.

[0114] The primer pair for amplifying the 1Ax gene consists of primer AxP500F: 5'-CTGAAATGGCCTTTAGGAGTTATC-3' and primer Ax294R: 5'-TATACTTTGTTGGAGTTGCTGT-3'.

[0115] The primer pair for amplifying the 1Bx gene consists of primer BxPF: 5'-CCAGAACTAGGATTAAGCCCATTACG-3' and primer Bx553R: 5'-TTGTCCTGGCTGCTGTGAAGT-3'.

[0116] The primer pair for amplifying the 1Dx gene consists of primer Dx5F: 5'-CTAAGCGGTTAGTCCTCTTTGT-3' and primer Dx386R: 5'-GCTTGGCCTGGATAGTATGAAA-3'.

[0117] The gene editing of part of the T0 generation transgenic wheat plants is shown in Table 1. Figure 10 .

[0118] The experimental results are shown in Table 2. The results show that the average editing efficiency of the single base editing vector system containing the TaUbi promoter (i.e. single base editing vector ABE-SpCas9-NGG-TaUbi) reaches 50.00%, which is increased by 160.06% compared with the editing efficiency (19.23%) of the single base editing vector system containing the ZmUbi promoter (i.e. single base editing vector ABE-SpCas9-NGG-ZmUbi). Compared with the ZmUbi promoter, the gene editing efficiency of the single base editing vector system using the TaUbi promoter is significantly improved. It can be seen that the TaUbi promoter can more effectively promote the occurrence of genome single base editing. This substantial improvement in editing efficiency provides a more efficient vector selection for subsequent plant genome editing research.

[0119]

[0120] The above has been described in detail. For those skilled in the art, without departing from the purpose and scope of the present application, and without unnecessary experiments, the present application can be implemented in a wider range under equivalent parameters, concentrations and conditions. Although the present application gives a special example, it should be understood that further improvements can be made to the present application. In short, according to the principle of the present application, this application intends to include any change, use or improvement of the present application, including changes made by conventional techniques known in the art, which deviates from the scope disclosed in the present application.

Claims

1. Specific DNA molecules, as shown in a1), a2), or a3): a1) The nucleotide sequence is that of the DNA molecule shown in SEQ ID No. 1; a2) DNA molecules that have 75% or more identity with the nucleotide sequence defined by a1) and have promoter function; a3) A DNA molecule that hybridizes to a nucleotide sequence defined by a1) or a2) under strict conditions and has promoter function.

2. An expression cassette containing the specific DNA molecule of claim 1.

3. A recombinant plasmid containing the specific DNA molecule of claim 1.

4. A transgenic cell line containing the specific DNA molecule described in claim 1.

5. The application of the specific DNA molecule of claim 1 as a promoter.

6. The application of the specific DNA molecule of claim 1, the expression cassette of claim 2, or the recombinant plasmid of claim 3, is b1) or b2) or b3). b1) Initiate the expression of the target gene; b2) Increase the expression level of the target gene; b3) Improve gene editing efficiency.

7. The application according to claim 6, characterized in that: The improvement of gene editing efficiency This is achieved through the expression of the gene encoding a nuclease that can recognize and cut a specific DNA sequence in the specific DNA molecule-initiated gene editing system described in claim 1.

8. A method for expressing a target gene, namely method A, method B, method C, or method D; Method A involves using the specific DNA molecule described in claim 1 as a promoter to initiate the expression of the target gene; Method B includes the following steps: inserting the specific DNA molecule of claim 1 upstream of any target gene or enhancer to initiate the expression of the target gene; Method C includes the following steps: inserting the target gene downstream of the specific DNA molecule in the expression cassette of claim 2, and having the expression of the target gene initiated by the specific DNA molecule; Method D includes the following steps: inserting the target gene downstream of the specific DNA molecule in the recombinant plasmid of claim 3, and having the expression of the target gene initiated by the specific DNA molecule.

9. The method according to claim 8, characterized in that: The expression level of the target gene was increased.

10. A method for improving gene editing efficiency, achieved by initiating the expression of a gene encoding a nuclease in a gene editing system capable of recognizing and cutting a specific DNA sequence using the specific DNA molecule of claim 1.

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