Oat endogenous Ubiquitin promoter AsUbi10 with high transcriptional activity

Abstract

The invention discloses a Ubiquitin promoter AsUbi10 with high endogenous transcriptional activity of oats and a preparation method of the Ubiquitin promoter AsUbi10. The invention provides a DNA (deoxyribonucleic acid) molecule which is any one of the following: A1) a DNA molecule containing a nucleotide sequence as shown in a sequence 1; a2) a DNA molecule which is obtained by substitution, deletion or addition of one or more nucleotides in A1) and has the same function. According to the present invention, the candidate gene AsUbi10 of the oat Ubiquitin promoter is screened from the genes of the oat Ubiquitin family; according to the chemiluminescence average fluorescence intensity and relative luciferase activity judgment, AsUbi10p shows efficient promoter transcriptional activity, which indicates that AsUbi10p can be used as an ideal promoter for driving a Cas gene in an oat CRISPR-Cas system.

Description

Technical Field

[0001] This invention belongs to the field of biotechnology and relates to endogenous high transcriptional activity in oats. Ubiquitin The promoter is AsUbi10. Background Technology

[0002] Oats, one of the world's top eight grain crops, are hailed as the "King of Functional Grains." From a functional perspective, oats' extremely high nutritional value and health benefits stem from a variety of components in the grain, such as protein, dietary fiber, vitamins, minerals, and unsaturated fatty acids. The abundant dietary fiber and high-quality protein in oat grains offer health benefits in lowering blood pressure, blood lipids, and cholesterol. Oat breeding has broad prospects, with future breeding directions focusing on increasing yield, improving quality, and enhancing stress resistance. In recent years, gene editing technology has provided a new path to break down the genetic barriers to improving oat stress resistance and quality. Optimizing oat gene editing systems and screening for highly active key components of gene editing systems can lay the technical foundation for the subsequent application of gene editing technology in oat breeding.

[0003] Plant ubiquitin gene ( Ubiquitin Promoters have been widely used in monocotyledonous plants and serve as key components of gene editing systems. Multiple researchers have successfully demonstrated their advantages, including high initiation efficiency, low methylation levels, and stable genetic traits (Christensen et al., 1992). From the perspective of promoter structure, Ubiquitin The promoter's structure conforms to the characteristics of a Pol II promoter. Pol II promoters are typically used to drive the expression of protein-coding genes. In the CRISPR-Cas9 gene editing system, Cas9 is a protein-coding gene, therefore the Pol II promoter is used to drive the expression of the Cas9 protein.

[0004] Unlocking the endogenous high efficiency of oats Ubiquitin Promoters to improve gene editing efficiency and stability. Summary of the Invention

[0005] The technical problem solved by this invention is to provide the oat endogenous Ubiquitin promoter AsUbi10 with high transcriptional activity.

[0006] To address the aforementioned technical problems, the first aspect of the present invention provides a DNA molecule, which is any of the following: A1) A DNA molecule containing the nucleotide sequence shown in Sequence 1; A2) A DNA molecule that has undergone substitution, deletion, or addition of one or more nucleotides in A1) and has the same function.

[0007] In some embodiments, A1) is the DNA molecule shown in sequence 1; In a second aspect, the present invention provides biological materials related to the DNA molecule described in the first aspect, including any one of B1) to B7) below: B1) An expression cassette containing the DNA molecule described in the first aspect; B2) A recombinant vector containing the DNA molecule described in the first aspect; B3) A recombinant vector containing the expression cassette described in B1); B4) Recombinant microorganisms containing the DNA molecules described in the first aspect; B5) Recombinant microorganisms containing the expression cassette described in B1); B6) Recombinant microorganisms containing the recombinant vector described in B2); B7) Recombinant microorganisms containing the recombinant vector described in B3); B8) A nucleic acid construct containing the DNA molecule described in the first aspect.

[0008] In the biological materials described above, the nucleic acid construct comprises the DNA molecule described in the first aspect and a nucleic acid molecule operably linked to the DNA molecule.

[0009] The nucleic acid molecule is a gene or sgRNA.

[0010] In the aforementioned biological materials, B1) the expression cassette of the DNA molecule (promoter) described in the first aspect refers to DNA capable of driving the expression of a target gene in a host cell. This DNA may include not only the target gene but also a terminator to terminate the transcription of the target gene. Furthermore, the expression cassette may also include an enhancer sequence.

[0011] Recombinant vectors containing promoters can be constructed using existing expression vectors.

[0012] In the aforementioned biological materials, the carrier may be a plasmid, a granule, a bacteriophage, or a viral vector.

[0013] In the aforementioned biological materials, the microorganisms may be yeast, bacteria, algae, or fungi. Among them, bacteria may be Agrobacterium tumefaciens or Corynebacterium glutamicum (…). Corynebacterium glutamicum ), lactic acid fermenting short bacilli, yellow short bacilli ( brevibacterium flavum Corynebacterium pingeri () Corynebacterium pekinense ), ammonia-eating short bacilli, blunt-toothed rod-shaped bacilli or pantothecin ( Pantoea ).

[0014] Thirdly, the present invention provides the use of the DNA molecule described in the first aspect as a promoter.

[0015] Fourthly, the present invention provides the application of the DNA molecule described in the first aspect as a promoter in initiating the expression of plant nucleic acid molecules.

[0016] In the application described above, the plant is oat.

[0017] In the applications described above, the nucleic acid molecule is a gene or sgRNA encoding nucleic acid.

[0018] Fifthly, the present invention provides a method for driving the expression of nucleic acid molecules in plants, comprising the following steps: using the DNA molecule described in the first aspect as a promoter to drive the expression of nucleic acid molecules in plants.

[0019] In the method described above, the plant is oats.

[0020] In the method described above, the nucleic acid molecule is a gene or sgRNA encoding nucleic acid.

[0021] In some embodiments, the gene is a luciferase reporter gene. Luciferase.

[0022] This invention is derived from oats Ubiquitin oats were selected from family genes Ubiquitin promoter candidate genes AsUbi10 Based on the average fluorescence intensity of chemiluminescence and the relative luciferase activity, AsUbi10p It exhibits highly efficient promoter transcriptional activity, suggesting that it can serve as an ideal promoter for driving sgRNA in the oat CRISPR-Cas system. Attached Figure Description

[0023] Figure 1 For containing different AsUbi Schematic diagram of promoter dual-luciferase series vectors.

[0024] Figure 2 For oats AsUbi A schematic diagram of the electrophoretic bands of a promoter PCR clone.

[0025] Figure 3 For containing different AsUbi Chemiluminescence image of the dual-luciferase vector of the promoter.

[0026] Figure 4 For different AsUbi Average fluorescence intensity of the promoter.

[0027] Figure 5 For different AsUbi Transient expression activity of the promoter. Detailed Implementation

[0028] The present invention will now be described in further detail with reference to specific embodiments. The given embodiments are merely illustrative of the invention and not intended to limit its scope. The embodiments provided below can serve as a guide for further improvements by those skilled in the art and do not constitute a limitation on the invention in any way.

[0029] Unless otherwise specified, the experimental methods used in the following examples are conventional methods, performed according to the techniques or conditions described in the literature in this field or according to the product instructions. Unless otherwise specified, the materials and reagents used in the following examples are commercially available.

[0030] Unless otherwise specified, the quantitative experiments in the following examples are all repeated three times, and the results are averaged.

[0031] The materials used in the following examples are seeds of the oat variety 'Yumai L.3', which were obtained from the Crop Germplasm Resources Center of the Institute of Crop Science, Chinese Academy of Agricultural Sciences.

[0032] Example 1: Oats AsUbi promoter cloning 1. Oats AsUbi Cloning of promoter truncated forms This experiment will use corn Ubiquitin The CDS sequence of the gene was analyzed using BLAST based on a laboratory oat transcriptome database to mine endogenous oat genes. Ubiquitin Promoters. The obtained candidate genes were selected from oats based on homology and transcriptional activity. Ubiquitin Selected from gene families Pepsico1_Contig7823 , Pepsico1_Contig2555 As the final candidate genes, they were named respectively. AsUbi10 , AsUbi11 .

[0033] Using oat genomic DNA as a template, AsU10FW-Rec and AsU11FW-Rec were amplified with AsU10_11RV-Rec, respectively, to obtain the promoters AsUbi10p and AsUbi11p. The primers are shown in Table 1. The AsUbi10p promoter band size was 2348 bp, and the AsUbi11p promoter band size was 1958 bp. Based on the marker band size, the PCR products were of the expected size.

[0034] Table 1 shows... AsUbi10 , AsUbi11 List of primers for gene promoters

[0035] Note: Lowercase letters represent sequences that overlap with the vector, and uppercase letters represent gene-specific primers.

[0036] Electrophoretic detection such as Figure 2 As shown, it can be seen that the expected size of the segment is obtained.

[0037] The PCR products were sequenced, and the results are as follows: AsUbi10p The nucleotide sequence is sequence 1; AsUbi11p The nucleotide sequence is sequence 2.

[0038] 2. Construction of a dual-luciferase expression vector Take the primer pair pG3GBFw-SHS (5'-GTG AGC TCT GAA AGC TTA TAC TAG TTT TCT CCATAA TAA TGT GTG AGT AG-3') / pG3GBRv-SKA (5'-CAT GAG CTC ATG GTA CCA TGG CGCGCC GGC ACT GGC CGT CGT TTT AC -3') Amplification of pG3GB411-BWM vector (Literature: Qiang Zhang, YuZhang, Min-Hui Lu, Yi-Ping Chai, Yuan-Yuan Jiang, Yun Zhou, Xue-Chen Wang, Qi-Jun Chen. A novel ternary vector system united with morphogenic genesenhances CRISPR / Cas delivery in maize. Plant Physiol. 2019, 181(4):1441-1448.doi: The backbone fragment (3.7 kb) in (10.1104 / pp.19.00767.) was self-ligated by SacI restriction enzyme to obtain the pG3box vector.The CmYLC fragment (NCBI GenBank ID AF364175) was amplified using primers CmFW-HindIII (5'-AGA AGC TTG GCA GAC ATA CTG TCC CAC A-3') / Rluc-Cm-RV (5'-TGG ATC ATA AAC TTT CGA AGT CAT TCT AGA TGC TTA GCT CTT ACC TGT-3'). The vector pGreenII was amplified using primers Cm-Rluc-FW (5'-ACA GGT AAG AGC TAAGCA TCT AGA ATG ACT TCG AAA GTT TAT GAT CCA-3') / RlucRV-Spe (5'-AAA ACT AGTTTA TTG TTC ATT TTT GAG AAC TCG CTC A-3'). The Rluc fragment (940bp) on 0800-LUC (Shanghai Newp Biotechnology Co., Ltd., plasmid number: V010545) was amplified with primers CmFW-HindIII / RlucRV-Spe to obtain the fusion fragment CmYLC+Rluc obtained by mixing the two PCR products. The CmYLC:Rluc fragment was recovered and digested with HindIII / SpeI to obtain the vector pG3box, which was then ligated to obtain the vector pG3CmRluc.The Luc fragment (1.6 kb) on the vector pGreenII 0800-LUC (Shanghai Newpu Biotechnology Co., Ltd., plasmid number: V010545) was amplified using primers Luc-FW-Kpn (5'-AAA GGT ACC AGC TCG AGA TGG AAG ACG CCA AAA ACATAA AGA-3') / Luc-RV-Sac (5'-ATT GAG CTC TTA CAC GGC GAT CTT TCC GCC-3'). HSP18.2 in the vector pG3GB411-BWM was amplified using primer pairs HSPter-FW-Sac (5'-AAG AGC TCA TAT GAA GAT GAA GAT GAA ATA TTTGGT-3') / HSPter-RV-Hind (5'- GTA AAG CTT CTT ATC TTT AAT CAT ATT CCA TAG TCCATA CCA-3'). The termination sequence was used to digest the Luc fragment with KpnI / SacI and the HSP18.2 termination sequence with SacI / HindIII, respectively, and then ligated into the vector pG3CmRluc. The PCR fragment (Luc fragment) and the vector pG3CmRluc were then digested with KpnI / SacI and ligated to obtain pG3Cmdualluc.

[0039] Dual-luciferase expression vector pG3Cmdualluc- AsUbi10p To obtain the above 1 AsUbi10p The vector was obtained by replacing the fragment between the KpnI and SacI restriction sites linked to the vector pG3Cmdualluc. AsUbi10p Located in the luciferase reporter gene Luciferase Upstream of the signal, to detect its promoter transcriptional activity.

[0040] Dual-luciferase expression vector pG3Cmdualluc- AsUbi11p To obtain the above 1 AsUbi11p The vector was obtained by replacing the fragment between the KpnI and SacI restriction sites in the vector pG3Cmdualluc. AsUbi11p Located in the luciferase reporter gene Luciferase Upstream of the signal, to detect its promoter transcriptional activity.

[0041] Dual luciferase expression vector pG3Cmdualluc-AsUbi-pro (vector names for different promoters are pG3Cmdualluc-...) AsUbi10p or pG3Cmdualluc- AsUbi11p The graph of ) is as follows Figure 1 As shown.

[0042] 3. Chemiluminescence analysis AsU3 promoter transcriptional activity 1) Chemiluminescence diagram The above dual-luciferase expression vectors were transformed into Agrobacterium EHA105 competent cells, and the recombinant Agrobacterium was obtained after PCR positive identification, which was then used for infection.

[0043] Each recombinant Agrobacterium was cultured overnight in LB liquid medium. After collecting the cells, they were resuspended in WPBS-C solution (formula from the literature: Wang N, Ryan L, Sardesai N, Wu E, Lenderts B, Lowe K, Che P, Anand A, Worden A, van Dyk D, Barone P, Svitashev S, Jones T, Gordon-Kamm W. Leaf transformation for efficient random integration and targeted genomemodification in maize and sorghum. Nat Plants. 2023 Feb;9(2):255-270. doi:10.1038 / s41477-022-01338-0. Epub 2023 Feb 9. PMID: 36759580; PMCID:PMC9946824.). The resuspended solution with an OD value between 0.4 and 0.8 was used for infection. The infection material was callus induced by mature embryos of 'Yama L.3'. After 20 minutes of inoculation with the resuspension, Agrobacterium was removed with filter paper and transferred to WPBS-CAS solid medium containing acetylsuccinone (AS) (AS and WPBS-C solution were mixed and the concentration of AS was 100 uM). After three days of co-culture, the co-cultured oat callus material was obtained.

[0044] The vector used in this experiment contains a luciferase reporter gene. LuciferaseIn vitro chemiluminescence can be detected using a dual-color infrared laser imaging system to determine the expression of the luciferase gene. The analytical instrument used in this experiment was a dual-color infrared laser imaging system (ODYSSEYClx) from the Beijing Regional Center for Large-Scale Instruments for Life Sciences, Institute of Botany, Chinese Academy of Sciences. The substrate used was D-luciferin potassium salt. The preparation method of the D-luciferin potassium salt solution was as follows: D-luciferin potassium salt (CAS Registry No. 115144-35-9) was dissolved in distilled water and mixed thoroughly to achieve a final concentration of 30 mg / mL in the stock solution. The D-luciferin potassium salt stock solution was then aliquoted into brown Eppendorf tubes and stored at -20°C in the dark.

[0045] A small amount of co-cultured oat callus material was immersed in a 0.3 mg / mL fluorescein potassium salt solution after filtration and sterilization, and allowed to stand for 30 s. Then it was placed directly on a WPBS subculture medium solid plate (formulation from the literature: Wang N, Ryan L, Sardesai N, Wu E, Lenderts B, Lowe K, Che P, Anand A, Worden A, vanDyk D, Barone P, Svitashev S, Jones T, Gordon-Kamm W. Leaf transformation for efficient random integration and targeted genome modification in maize and sorghum. Nat Plants. 2023 Feb;9(2):255-270. doi: 10.1038 / s41477-022-01338-0.Epub 2023 Feb 9. PMID: 36759580; PMCID: PMC9946824.); dark treatment 5 The image is a chemiluminescence image obtained from a dual-color infrared laser imaging system.

[0046] Chemiluminescence imaging results as follows Figure 3 As shown, both AsUbi promoters can drive the expression of fluorescent genes in the dual-luciferase expression vector, exhibiting a certain fluorescence intensity. The AsUbi10p-driven fluorescent expression showed stronger activity, with high-brightness fluorescence detected, while the AsUbi11p-driven fluorescent expression showed medium-brightness fluorescence.

[0047] 2) Average fluorescence intensity of different AsUbi promoters ImageJ can help assess key parameters such as staining intensity and calculate the positivity index; therefore, this software is used to analyze chemiluminescence intensity. ImageJ transforms the results of chemiluminescence imaging into visualized data, converting the fluorescence image into a single-channel (monochrome) fluorescence image. The grayscale value of each pixel represents the fluorescence intensity at that point. The formula for the fluorescence intensity of a specific region is: Fluorescence Intensity (Mean) = Sum of Fluorescence Intensities in that Region (IntDen) / Area of ​​that Region (Area). The chemiluminescence images of the two dual-luciferase expression vectors mentioned above are imported into ImageJ for analysis.

[0048] The results are as follows Figure 4 As shown, different AsUbi Average fluorescence intensity of promoter chemiluminescence. AsUbi10p (134.339) fluorescence intensity was significantly higher than AsUbi11p (98.619). The results show that, AsUbi10p The activity of the driving fluorescent gene is stronger.

[0049] 4. Analysis of Dual-Luciferase Experiment AsU3 promoter transcriptional activity A portion of co-cultured oat callus material obtained from promoters AsUbi10p and AsUbi11p was used, and the activities of firefly luciferase and reniform luciferase were detected sequentially using a Dual-Luciferase® Reporter Assay System. The reniform luciferase activity (Rluc value) and firefly luciferase activity (Fluc value) were measured. After standardization (using reniform luciferase activity as an internal control), the fluorescence characteristics of different promoters were evaluated using relative fluorescence intensity (RLA) values ​​according to the following formula.

[0050] RLA = Fluc / Rluc The results are as follows Figure 5 As shown, it can be seen that AsUbi10p The relative luciferase activity was the strongest, reaching 0.53, significantly higher than that of other enzymes. AsUbi11p The trend is consistent with the average fluorescence intensity of chemiluminescence.

[0051] therefore, AsUbi10p It is a highly transcriptionally active endogenous promoter from oats that drives the expression of target genes. It can serve as an ideal promoter for driving sgRNA in oat CRISPR-Cas systems.

[0052] The present invention has been described in detail above. For those skilled in the art, the invention can be practiced in a wide range of ways with equivalent parameters, concentrations, and conditions without departing from its spirit and scope, and without requiring unnecessary experiments. Although specific embodiments have been given, it should be understood that further modifications can be made to the invention. In summary, according to the principles of the invention, this application is intended to include any changes, uses, or improvements to the invention, including changes made using conventional techniques known in the art that depart from the scope disclosed herein. Some of the essential features can be applied within the scope of the following appended claims.

Claims

1. A DNA molecule, which is any of the following: A1) A DNA molecule containing the nucleotide sequence shown in Sequence 1; A2) A DNA molecule that has undergone substitution, deletion, or addition of one or more nucleotides in A1) and has the same function.

2. A biological material related to the DNA molecule of claim 1, comprising any one of B1) to B7) below: B1) An expression cassette containing the DNA molecule of claim 1; B2) A recombinant vector containing the DNA molecule of claim 1; B3) A recombinant vector containing the expression cassette described in B1); B4) Recombinant microorganisms containing the DNA molecule of claim 1; B5) Recombinant microorganisms containing the expression cassette described in B1); B6) Recombinant microorganisms containing the recombinant vector described in B2); B7) Recombinant microorganisms containing the recombinant vector described in B3); B8) A nucleic acid construct containing the DNA molecule of claim 1.

3. The biomaterial according to claim 2, characterized in that: The nucleic acid construct comprises the DNA molecule of claim 1 and a nucleic acid molecule operably linked to the DNA molecule.

4. The use of the DNA molecule of claim 1 as a promoter.

5. The use of the DNA molecule according to claim 1 as a promoter in initiating the expression of plant nucleic acid molecules.

6. The application according to claim 5, characterized in that: The plant in question is oats.

7. The application according to claim 5 or 6, characterized in that: The nucleic acid molecule is a gene or sgRNA encoding nucleic acid.

8. A method for driving the expression of nucleic acid molecules in plants, comprising the following steps: using the DNA molecule of claim 1 as a promoter to drive the expression of nucleic acid molecules in plants.

9. The method according to claim 8, characterized in that: The plant in question is oats.

10. The method according to claim 8 or 9, characterized in that: The nucleic acid molecule is a gene or sgRNA encoding nucleic acid.

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