CN119410702B - Application of leymus chinensis U6 gene promoter proLcU a - Google Patents

Abstract

The present invention discloses an application of a Leymus chinensis U6 gene promoter proLcU6a, and belongs to the field of biotechnology. The Leymus chinensis U6 gene promoter proLcU6a having a nucleotide sequence as shown in SEQ ID NO: 18 provided by the present invention has a promoter activity, can drive the transcription of sgRNA targeting the Leymus chinensis gene, thereby constructing a CRISPR-Cas9 gene editing system driven by the promoter proLcU6a to sgRNA transcription, and realizes the editing of the Leymus chinensis target gene, which can be applied in the technical fields of Leymus chinensis genetic transformation, molecular breeding, and the like.

Description

Application of leymus chinensis U6 gene promoter proLcU a

Technical Field

The invention belongs to the technical field of biology, and particularly relates to a novel application of a leymus chinensis U6 gene promoter (belonging to leymus chinensis RNA polymerase III type promoter, abbreviated as proLcU a in the text), in particular to an application in driving sgRNA transcription of targeted leymus chinensis genes, constructing a CRISPR-Cas9 gene editing vector of targeted editing leymus chinensis genes, genetic transformation of leymus chinensis and molecular breeding.

Background

Herba Caprae Seu Ovis (Leymus chinensis (Trin.) Tzvel.) belonging to genus Leymus of Gramineae is a perennial rhizome plant. The leymus chinensis is widely distributed on the European Asia continental, is a main colonisation seed of the European Asia strawberries, has the characteristics of high nutritive value, good palatability, strong penetration and encroachment capability of rhizome, capability of coiling and holding soil, water and soil conservation and the like, has excellent properties of cold resistance, salt and alkali resistance, drought resistance and the like, and is pasture with important economic value and ecological value. However, the problem of low germination rate, low heading rate and low fruiting rate of the leymus chinensis in production generally exists, the heterozygosity is high, the genome is complex, the generation period is long, and the creation of new leymus chinensis germplasm is difficult to quickly realize through traditional hybridization breeding. Recently, along with the rapid development of gene editing technology, the technology can be used for introducing mutation at a specific position of a leymus chinensis genome to quickly and efficiently create new leymus chinensis germplasm so as to realize the improvement of leymus chinensis germplasm.

The CRISPR/Cas9 gene editing system consists of a single-stranded guide RNA (sgRNA) and a Cas9 protein with endonuclease function, and is a gene editing technology with important application potential. The sgRNA can guide the Cas9 enzyme to directionally cut target sites, so that the precise editing of target genes is realized. At present, the CRISPR/Cas9 gene editing system is widely applied to important crops such as rice, corn, wheat, soybean, tomatoes and the like, and plays an important role in improving plant yield, improving plant quality, enhancing plant resistance and the like. Compared with the crops, the research and application of the gene editing technology on pasture are lagging.

To increase the efficiency of gene editing, researchers have engineered the elements of the CRISPR/Cas9 gene editing system. U3/U6 promoter is an important element for driving sgRNA transcription, the efficiency of which affects the efficiency of gene editing, for example Lin Hao subject group achieves 10.35% editing efficiency of T0 generation by using the alfalfa itself MtU6 promoter to drive the transcription of sgRNA targeting PDS gene (Meng Y et al . Targeted mutagenesis by CRISPR/Cas9 system in the model legume Medicago truncatula. Plant Cell Rep. 2017 Feb;36(2):371-374. doi: 10.1007/s00299-016-2069-9. Epub 2016 Nov 11. PMID: 27834007.）., although U3 promoter has been successfully used in gene editing of multiple species, there is a certain difference in U3/U6 promoter between different species, heterologous driving sgRNA transcription activity may be limited, and multiple U3/U6 promoters are often present in the same species, and there is a difference in transcription activity, therefore cloning and using a suitable plant endogenous U3/U6 promoter is of great significance to the establishment and refinement of CRISPR/Cas9 gene editing system of that species.

The leymus chinensis is an important pasture resource in China. The establishment of the CRISPR/Cas9 gene editing system of the leymus chinensis provides reliable technical support for the basic research, variety improvement and germplasm resource creation of the leymus chinensis, and provides for the development and utilization of leymus chinensis resources and the alleviation of the problem of forage shortage in China. However, the research on the leymus chinensis U6 promoter is still lacking so far, and no endogenous promoter with higher transcriptional activity is suitable for leymus chinensis, which limits the application of the CRISPR/Cas9 gene editing system in the aspects of leymus chinensis basic research, germplasm innovation and the like. Therefore, an endogenous U6 promoter with higher transcription activity in the leymus chinensis is found and cloned, a CRISPR/Cas9 gene editing system suitable for the leymus chinensis is constructed, and the method has important research significance and application value for leymus chinensis functional gene research and genetic breeding.

Disclosure of Invention

Aiming at one or more of the problems in the prior art, the invention clones and obtains the RNA polymerase III type promoter proLcU a of the endogenous U6 snRNA gene of the leymus chinensis in the leymus chinensis for the first time, and the proLcU a promoter has higher transcription activity through detection, can drive the U6 snRNA gene at the downstream of the promoter to be efficiently expressed in the leymus chinensis, and can drive the transcription of the sgRNA of the targeted leymus chinensis gene (such as GW2 gene), thereby being capable of being used for constructing a CRISPR/Cas9 gene editing system of the targeted leymus chinensis gene and further providing an efficient tool for genetic transformation and molecular breeding research of the leymus chinensis. The invention is mainly realized by the following technical scheme.

The first aspect of the invention provides an application of a leymus chinensis U6 gene promoter in driving sgRNA transcription of a targeted leymus chinensis gene, wherein the leymus chinensis U6 gene promoter is named proLcU a, and the nucleotide sequence of the leymus chinensis U6 gene promoter is shown as SEQ ID NO. 18.

In some embodiments, the leymus chinensis gene may include, but is not limited to, a GW2 gene. In fact, the leymus chinensis U6 gene promoter proLcU a provided by the invention can be used for driving the sgRNA transcription of any target leymus chinensis genes.

In some embodiments, the nucleotide sequence of the sgRNA targeting the leymus chinensis GW2 gene is shown in SEQ ID NO. 19.

The second aspect of the present invention provides a leymus chinensis gene editing vector, which may comprise a promoter proLcU a having a nucleotide sequence shown as SEQ ID NO. 18.

In some embodiments, the leymus chinensis gene editing vector may further comprise a Cas9 coding sequence and a sgRNA coding sequence targeting the leymus chinensis gene, wherein the promoter proLcU a drives transcription of the sgRNA coding sequence.

In some embodiments, the leymus chinensis gene may be a leymus chinensis GW2 gene, and the nucleotide sequence of the sgRNA coding sequence is shown in SEQ ID NO. 19.

In a third aspect, the invention provides an isolated nucleic acid molecule having a nucleotide sequence as set forth in SEQ ID NO. 18.

The use of the promoter proLcU a mentioned in the first aspect of the present invention, or the leymus chinensis gene editing vector described in the second aspect of the present invention, or the isolated nucleic acid molecule described in the third aspect of the present invention, for targeted editing of leymus chinensis genes, genetic transformation of leymus chinensis, and leymus chinensis molecular breeding is also within the scope of the present invention.

In a fourth aspect, the present invention provides a method for detecting the promoter activity of the leymus chinensis U6 gene promoter proLcU a, which comprises the following operations:

(S1) constructing a leymus chinensis gene editing vector transcribed by a sgRNA driven by a promoter proLcU a, further comprising a Cas9 expression element and a reporter group expression element, and transferring the constructed leymus chinensis gene editing vector into leymus chinensis protoplasts for expression by a PEG 4000-mediated leymus chinensis protoplast transient transformation method;

(S2) observing whether the sheep grass original plastid has fluorescence of the reporter group by a fluorescence microscope, and

(S3) extracting genome DNA from the fluorescent sheep grass original plastid with the reporter group obtained in the step (S2), amplifying the genome DNA to obtain a fragment of the target gene, and carrying out enzyme digestion on the fragment;

If the cleavage result in the step (S3) shows that the fragment of the target gene is only partially and completely cleaved, the leymus chinensis U3 gene promoter proLcU a has a promoter activity.

In some embodiments, the target gene may be a GW2 gene and the sgRNA has a nucleotide sequence as shown in SEQ ID NO. 19.

The fifth aspect of the present invention also provides a leymus chinensis gene editing vector for detection of the promoter activity of leymus chinensis U6 gene promoter proLcU a, comprising an sgRNA expression element, a Cas9 expression element and a reporter group expression element, wherein in the sgRNA expression element, transcription of the sgRNA is driven by promoter proLcU a.

Drawings

FIG. 1 shows the abundance of the expression of the U6 snRNA in leaves, roots and ears of leymus chinensis, wherein the color gradient characterizes the log2 FPKM of the snRNA, the red color represents high expression abundance, and the green color represents low expression abundance.

FIG. 2 is a schematic diagram showing the structure of a plasmid of proLcU a-driven leymus chinensis gene editing vector.

FIG. 3 shows the results of PEG-mediated transient transformation of sheep grass original plastids.

FIG. 4 shows the XbaI cleavage results of the amplified product of the fragment of GW2-sgRNA in the DNA of PEG-mediated sheep grass original plastid.

Detailed Description

The invention is further illustrated below in conjunction with specific examples. It should be understood that the detailed description and specific examples, while indicating the invention, are intended for purposes of illustration only and are not intended to limit the scope of the invention.

Hereinafter, only certain exemplary embodiments are briefly described. As will be recognized by those of skill in the pertinent art, the described embodiments may be modified in various different ways without departing from the spirit or scope of the present invention. Accordingly, the drawings and description are to be regarded as illustrative in nature and not as restrictive.

The methods used in the examples below are conventional methods unless otherwise specified, and the specific procedures can be found in the guidelines for molecular cloning experiments "（"Molecular Cloning: A Laboratory Manual" Sambrook,J.,Russell, David W.,Molecular Cloning: A Laboratory Manual,3rd edition,2001,NY,Cold Spring Harbor）.

The various biomaterials described in the examples were obtained by merely providing an experimental route for achieving the objectives of the specific disclosure and should not be construed as limiting the source of biomaterials of the present invention. In fact, the source of the biological material used is broad, and any biological material that is available without violating law and ethics may be used instead as suggested in the examples. The test materials used in the examples described below, unless otherwise specified, were purchased from conventional biochemical reagent stores.

The nucleotides referred to in the examples below can all be synthesized by the prior art.

Example 1 screening of U6 Gene promoters suitable for use in a CRISPR/Cas9 Gene editing System of leymus chinensis.

(1) According to the conservation of U6 snRNA among different species (such as Arabidopsis thaliana, tomato, rice, corn, wheat, etc.), alignment (BLASA) of U6 snRNA sequence SlU (SEQ ID NO: 5), rice U6 snRNA sequence OsU6-112939753（SEQ ID NO:6）、OsU6-112939464（SEQ ID NO:7）、OsU6-112938844（SEQ ID NO:8）、OsU6-112938467（SEQ ID NO:9）, corn U6 snRNA sequence ZmU-118476585 (SEQ ID NO: 10), zmU6-111591274 (SEQ ID NO: 11), zmU6-111590679 (SEQ ID NO: 12) and wheat U6 snRNA sequence TridiU6-119359853 (SEQ ID NO: 13), tridiU-119351499 (SEQ ID NO: 14), tridiU-119342326 (SEQ ID NO: 15) with small RNAseq sequence of sheep grass measured in the earlier stage of the laboratory was performed using Arabidopsis thaliana U6 snRNA sequence AtU-26 (SEQ ID NO: 1), atU-1 (SEQ ID NO: 2), atU6.3 (SEQ ID NO: 3), atU6.4 (SEQ ID NO: 4), tomato U6 snRNA sequence SlU (SEQ ID NO: 5). The expression level (i.e., the expression abundance) of U6 snrnas encoded by a plurality of different sites in the genome of leymus chinensis in the leymus chinensis leaves (leaf), ears (panicle) and roots (root) is determined by calculating the U6 SNRNA FPKM value in leymus chinensis, and the result is shown in fig. 1, wherein red represents the high expression level of the corresponding U6 snrnas and green represents the low expression level of the corresponding U6 snrnas. As can be seen from the results shown in FIG. 1, the U6 snRNA encoded by a plurality of sites in the leymus chinensis can be highly expressed in different tissues of the leymus chinensis, and the results show that the promoters of the U6 snRNA have higher transcriptional activity in cells of different tissues of the leymus chinensis, and can effectively drive the transcription of downstream U6 snRNA. However, of these U6 snrnas, only the U6 snRNA indicated by the arrow in fig. 1 (designated as LcU a snRNA herein) was able to amplify the promoter region upstream thereof in the following step (2), and thus the following procedure was intended to obtain the upstream promoter region of LcU a snRNA and verify the promoter activity thereof.

(2) The method comprises the steps of amplifying a promoter region 500 bp upstream of LcU a snRNA by designing primers F and R (shown below) by using leymus chinensis genome DNA as a template:

F:CCATGTACTCTACCATATATG(SEQ ID NO:16)

R:CAGTGTGGGTGCAAGCGTGGG(SEQ ID NO:17)。

(3) PCR cloning was performed in a 50. Mu.L system using KOD FX high-fidelity enzyme (TOYOBO code. No.: KFX-101) to obtain PCR amplification products. Wherein the PCR amplification reaction procedure comprises 95 ℃ pre-denaturation for 2 min,98 ℃ denaturation for 10s,58 ℃ annealing for 30s,68 ℃ extension for 30s,35 cycles, and 68 ℃ final extension for 5min. The PCR amplification reaction system consisted of 1. Mu.L of template, 1.5. Mu.L of primer F (10. Mu.M), 1.5. Mu.L of primer R (10. Mu.M), 2x PCR buffer for KOD FX:25. Mu.L of primer R, 10. Mu.L of 2 mM dNTPs, 1. Mu.L of KOD FX and 10. Mu.L of ddH 2O.

(4) Cloning the amplified product obtained in the step (3) onto pEasy Blunt vector (full-scale gold code. No. CB 101-01), transforming escherichia coli DH5 alpha, picking up recombinant monoclonal and sequencing to obtain a leymus chinensis U6 gene promoter with the length of 500 bp, which is named as proLcU a, and the nucleotide sequence of the leymus chinensis U6 gene promoter is shown as SEQ ID NO. 18.

Example 2 Activity verification of the leymus chinensis U6 Gene promoter proLcU a.

(1) Vector construction for verifying proLcU a promoter activity specifically included the following procedures.

(1.1) Primers were designed for ligating proLcU a-GW2 sgRNA (representing GW2 sgRNA driven by proLcU a promoter (nucleotide sequence CCAGGAUGGGGUAUUUCUAG (SEQ ID NO: 19)) with pJIT163-Cas9-GFP vector (see SHAN ET AL NAT Biotechnol. 2013 Aug;31 (8): 686-8. Doi: 10.1038/nbt.2650) by homologous recombination:

F1:GACGGGGATCGCATGCCATGTACTCTACCATATA(SEQ ID NO:20)

R1:CTAGAAATACCCCATCCTGGCAAGCAGTGTGGGTGCAAGCG(SEQ ID NO:21)

F2:CAGGATGGGGTATTTCTAGGTTTTAGAGCTAGAAATAGCA(SEQ ID NO:22)

R2:GATCTCGAGATATCGaaaaaaagcaccgactcggtgc(SEQ ID NO:23)。

(1.2) cloning positive strain proLcU a as template, amplifying with primers F1 and R1 to recover fragment 1, amplifying with primers F2 and R2 to recover fragment 2 as fragment 2, and after full mixing of fragment 1, fragment 2 and SphI digested pJIT163-Cas9-GFP vector fragment with homologous recombinase for 1 hour at 50 ℃, obtaining proLcU a driven leymus chinensis gene editing vector (targeted editing leymus chinensis GW2 gene) named pLcU a-GW2-pJIT163-Cas9-GFP (also named LcU a ¬ _pJIT 163-Cas9-GFP), whose plasmid structure schematic diagram is shown in FIG. 2, and also can be used as proLcU a promoter activity detection vector comprising sgRNA and Cas9 expression element driven by leymus chinensis U6 promoter proLcU a, and simultaneously introducing GFP element as reporter gene.

(2) PEG4000 mediated transient transformation of sheep grass original biomass demonstrated proLcU a activity, and the specific procedures were as follows.

(2.1) Cutting leaves of two week old Chinese wildrye seedlings to 1 mM size, placing in a small beaker, preparing an enzymolysis solution (Cellulase R-10 (cellulase R-10,Yakult Pharmaceutical Industry Co., Ltd. Code.No : L0012） 0.3 g、Macerozyme R-10（Yakult Pharmaceutical Industry Co., Ltd. Code.No : L0021）0.15 g、Mannitol（ mannitol) 2.1844 g, MES (fatty acid sodium methane sulfonate; sigma-aldrich code. No: M2933) 0.04264 g to 20 mL, adjusting pH to 5.8 with KOH, water-bath at 55 ℃ for 10 min, adding CaCl2 0.02g, BSA (bovine serum albumin) 0.02 g), at room temperature, 50-70 rpm, and under dark conditions, hydrolyzing about 3 h, diluting the enzymolysis solution containing protoplasts with 1/2 volume of W5 solution (154 mM NaCl, 125 mM CaCl2, 5mM KCl, and 4mM MES, pH 5.7), rinsing 100 pore mesh filter with W5 solution, filtering undissolved leaves (operation is light), washing leaves in 1/2 volume of W5 solution with the filter, and collecting the enzymolysis filtrate.

(2.2) The filtrate was packed with 50 mL centrifuge tubes, centrifuged 2 min with slow acceleration (acceleration set to 1 or 2) of 100-200 g to precipitate protoplasts, the supernatant was removed as much as possible, the protoplasts were gently resuspended with ice-pre-chilled W5 solution, and left to stand on ice for 30 min.

(2.3) Centrifugation at 3 min was slowly accelerated (acceleration was set to 1 or 2) at room temperature with 100-200 g a to precipitate the protoplasts at the bottom of the tube, and the W5 solution was removed as much as possible without loss of protoplasts, followed by resuspension of the protoplasts with the appropriate amount of MMG solution (1M Mannitol 4 mL, 1M MgCl2 0.15 mL, 200 mM MES 0.2 mL).

(2.4) Taking a new 2.0 mL centrifuge tube, adding 20. Mu.g of proLcU a-driven leymus chinensis gene editing vector (pLcU a-GW2-pJIT163-Cas 9-GFP) successfully constructed thereto, adding 100. Mu.L of protoplast (obtained in step (2.3)), and gently mixing.

(2.5) 100. Mu.L of PEG4000 solution was added and the tube was gently inverted to allow complete mixing and transformation was induced at room temperature in the dark from 20-30 min.

(2.6) Diluting the transformation mixture with 1 mL of W5 solution at room temperature, then gently inverting the shaking centrifuge tube to complete the mixing to terminate the transformation reaction, centrifuging at 100 g at room temperature for 5: 5min, removing the supernatant, and repeating once.

(2.7) Adding 200 mu L W solution to gently resuspend protoplast, inducing protoplast at 25 ℃ under dark condition overnight at about 48: 48 h, observing whether the sheep protoplast has GFP green fluorescence by fluorescence microscope (488 nm), and the observation result is shown in FIG. 3, wherein BF and GFP respectively represent the results obtained by photographing the same visual field under Bright Field (BF) and green fluorescent protein channel (GFP), namely BF is all protoplast under the visual field, GFP is protoplast capable of emitting green fluorescence under the visual field (namely successful transformation of vector), and the result shows that the transformation efficiency of protoplast is higher (> 50%).

(3) Genomic DNA of the sheep grass original protoplast was extracted by CTAB method, the fragment of GW2-sgRNA in the protoplast DNA was amplified using specific primers F3 (GCATGTACTTTGATTGTTTGC (SEQ ID NO: 24)) and R3 (GTTCTACCATGAGCTTCTGC (SEQ ID NO: 25)), and the PCR product was digested by using restriction enzyme XbaI, and the digestion was observed. The results are shown in fig. 4, wherein pLcU a-GW2 represents the experimental group, i.e., the result of amplification and cleavage using DNA extracted from protoplasts transformed with pLcU a-GW2-pJIT163-Cas9-GFP vector, CK represents the control group, i.e., the result of amplification and cleavage using DNA extracted from protoplasts not transformed with vector, it is seen that the fragment of GW2 in the DNA from protoplasts of the experimental group is only partially cleaved completely (see the result of three bands represented by lane 2), and the non-cleaved part is the GW2 fragment where gene editing occurs, thereby proving that proLcU a has promoter activity that can be used to drive transcription of sgrnas targeting a leymus gene (e.g., GW2 gene) in CRISPR-Cas9 gene editing vector, and thus can target edit the gene.

According to the basic theory of molecular biology, a promoter can drive transcription of a downstream gene, the transcriptional activity of which is determined by the sequence of the promoter itself, rather than the sequence of the downstream gene it drives. The U3 or U6 promoter is a class of promoter sequences (Ma, hongming et al . "Pol III promoters to express small RNAs: delineation of transcription initiation." Molecular Therapy-Nucleic Acids 3 (2014).）,, U3/U6 promoters in different species) that are recognized by the PolIII polymerase and transcribed into small RNAs downstream (including SMALL INTERFERING RNA, short HAIRPIN RNA, and guide RNAs, etc.) and are widely used to drive transcription of sgrnas targeting different genes in gene editing (Kor, SAKSHI DHARMENDRA et al . "RNA Pol III promoters—Key players in precisely targeted plant genome editing." Frontiers in Genetics 13 (2023): 989199.）. thus demonstrated proLcU a promoter activity above, and can be used to drive transcription of sgrnas targeting the leymus chinensis GW2 gene in CRISPR-Cas9 gene editing vectors, the leymus chinensis proLcU a promoter can also be used to drive transcription of sgrnas targeting other genes of leymus chinensis.

In summary, the invention obtains the RNA polymerase III type promoter proLcU a of the leymus chinensis U6 snRNA gene, verifies that the leymus chinensis U6 snRNA gene has a starting activity, can be used for driving the transcription of the sgRNA of the targeted leymus chinensis gene, and can construct a CRISPR-Cas9 gene editing system driven by the proLcU a promoter to transcribe the sgRNA. Therefore, the promoter proLcU a provided by the invention can be applied to a CRISPR-Cas9 gene editing system, and can provide a high-efficiency and accurate gene editing tool for genetic transformation, germplasm innovation, germplasm rapid improvement research and the like of leymus chinensis.

It should be noted that the above-mentioned embodiments are merely preferred embodiments of the present invention, and the present invention is not limited to the above-mentioned embodiments, but may be modified or some of the technical features thereof may be replaced by other technical solutions described in the above-mentioned embodiments. Any modification, equivalent replacement, improvement, etc. made within the spirit and principle of the present invention should be included in the protection scope of the present invention.

Claims (8)

1. An application of a leymus chinensis U6 gene promoter in driving sgRNA transcription of a targeted leymus chinensis gene, wherein the leymus chinensis U6 gene promoter is named proLcU a, and the nucleotide sequence of the leymus chinensis U6 gene promoter is shown as SEQ ID NO. 18.

2. The use of claim 1, wherein the leymus chinensis gene comprises a GW2 gene.

3. The use according to claim 2, wherein the nucleotide sequence of sgRNA targeting the leymus chinensis GW2 gene is shown as SEQ ID No. 19.

4. A leymus chinensis gene editing vector comprises a promoter proLcU a with a nucleotide sequence shown as SEQ ID NO. 18.

5. The leymus chinensis gene editing vector of claim 4, further comprising a Cas9 coding sequence and a sgRNA coding sequence targeting a leymus chinensis gene, wherein the promoter proLcU a drives transcription of the sgRNA coding sequence.

6. The leymus chinensis gene editing vector according to claim 5, wherein the leymus chinensis gene is leymus chinensis GW2 gene, and the nucleotide sequence of the sgRNA coding sequence is shown as SEQ ID NO. 19.

7. An isolated nucleic acid molecule having the nucleotide sequence shown in SEQ ID NO. 18.

8. Use of the promoter proLcU a mentioned in any one of claims 1-3, or the leymus chinensis gene editing vector of any one of claims 4-6, or the isolated nucleic acid molecule of claim 7, for targeted editing of leymus chinensis genes, leymus chinensis genetic transformation, and leymus chinensis molecular breeding.