CN117844803A - sgRNA of targeted OsSHMT1 gene, sgRNA expression vector and application thereof - Google Patents
sgRNA of targeted OsSHMT1 gene, sgRNA expression vector and application thereof Download PDFInfo
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- CN117844803A CN117844803A CN202311361620.3A CN202311361620A CN117844803A CN 117844803 A CN117844803 A CN 117844803A CN 202311361620 A CN202311361620 A CN 202311361620A CN 117844803 A CN117844803 A CN 117844803A
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- 239000013604 expression vector Substances 0.000 title claims abstract description 20
- FUZZWVXGSFPDMH-UHFFFAOYSA-N hexanoic acid Chemical compound CCCCCC(O)=O FUZZWVXGSFPDMH-UHFFFAOYSA-N 0.000 claims abstract description 22
- 108091028043 Nucleic acid sequence Proteins 0.000 claims abstract description 8
- 239000002773 nucleotide Substances 0.000 claims abstract description 8
- 125000003729 nucleotide group Chemical group 0.000 claims abstract description 8
- 241000209094 Oryza Species 0.000 claims description 16
- 235000007164 Oryza sativa Nutrition 0.000 claims description 16
- 235000009566 rice Nutrition 0.000 claims description 16
- 238000000034 method Methods 0.000 claims description 12
- 206010020649 Hyperkeratosis Diseases 0.000 claims description 7
- 241001494479 Pecora Species 0.000 claims description 7
- 235000014113 dietary fatty acids Nutrition 0.000 claims description 6
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- 150000004665 fatty acids Chemical class 0.000 claims description 6
- 125000003275 alpha amino acid group Chemical group 0.000 claims description 5
- 230000001131 transforming effect Effects 0.000 claims description 5
- 241000589158 Agrobacterium Species 0.000 claims description 4
- 238000012408 PCR amplification Methods 0.000 claims description 4
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- IAJOBQBIJHVGMQ-UHFFFAOYSA-N 2-amino-4-[hydroxy(methyl)phosphoryl]butanoic acid Chemical compound CP(O)(=O)CCC(N)C(O)=O IAJOBQBIJHVGMQ-UHFFFAOYSA-N 0.000 description 1
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Abstract
The invention belongs to the technical field of genetic engineering, and particularly relates to sgRNA of a targeted OsSHMT1 gene, an sgRNA expression vector and application thereof. The invention designs the sgRNA in the 7 th exon region of the OsSHMT1 gene, the nucleotide sequence of the OsSHMT1 gene is shown as SEQ ID NO.1, the sgRNA comprises at least one of the nucleotide sequences shown as SEQ ID NO.2-4, and the gene editing is carried out on the OsSHMT1 gene, so that the tolerance of plants to the caproic acid can be obviously improved.
Description
Technical Field
The invention belongs to the technical field of genetic engineering, and particularly relates to sgRNA of a targeted OsSHMT1 gene, an sgRNA expression vector and application thereof.
Background
Chemical weed control is an essential measure and an important sign of modern agriculture, the evolution of drug resistance of weeds under the selection pressure of herbicides is a necessary rule and a realistic hazard, 1581 cases of weed organisms generate drug resistance to 167 herbicides worldwide and are increasingly stronger, and the development of the weeds faces the dilemma of 'no drug is available' and the weed damage is rampant.
Phytotoxins have been increasingly recognized as a novel herbicide for different processes in plants (maci as et al, 2019). Among several medium-chain fatty acids, such as pelargonic acid (CH) 3 (CH 2 ) 7 CO 2 H) Identified as highly potent, broad spectrum, low risk of developing herbicide resistant phytotoxins (Coleman and Penner,2008; real et al 2021). The recently studied caproic acid (C8H 16O2, CAP) can exhibit high herbicidal activity by disrupting chloroplasts and mitochondria in leaf cells, thus becoming an effective weed herbicide (Li et al 2018; li et al 2019 b). In field trials, CAP showed much faster herbicidal activity than the commercial herbicides glufosinate-ammonium and glyphosate. The sheep fatty acid can be used as a substitute of a main herbicide and has the advantages of simple production, safety, high efficiency and the like. We have recently found that Salicomia Herbacea hydroxymethyltransferase (CcSHMT 1) is a potential CAP herbicide target. The efficient low-risk herbicide and herbicide-resistant crop is the most direct and effective way for solving the harm of weeds, and particularly the targeted herbicide creation and gene editing selective breeding of herbicide-resistantAnd (3) crops. The plant source herbicide has little influence on farmland environment, and is a preferable scheme for controlling weeds. CRISPR/Cas gene editing technology is an emerging genetic engineering technology in recent years, which is a DNA cleavage technology mediated by guide rna, and various editing systems have been developed for Cas differences, including Cas9, cpf1, cms1, C2, etc. The CRISPR/Cas editing technique can implement three kinds of fixed point editing: the first is a site-directed knockout of a gene. The second is to make homologous substitutions to the target to replace the target sequence or site-directed insertion. The third is single base editing. Single base editing is a gene editing method that uses the CRISPR/Cas system to target deaminase to a specific site in the genome to modify a specific base. This method has been successfully used in rice. The invention applies a third method to carry out base editing on the 7 th exon sequence of the OsSHMT1 gene to obtain CAP-resistant crops.
Disclosure of Invention
In order to obtain the anti-caproic acid crop, the invention provides the following technical scheme:
an sgRNA of a targeted OsSHMT1 gene, wherein the nucleotide sequence of the OsSHMT1 gene is shown as SEQ ID NO.1, and the sgRNA comprises at least one of the nucleotide sequences shown as SEQ ID NO. 2-4.
Preferably, the amino acid sequence of the OsSHMT1 gene is shown as SEQ ID NO. 5.
An sgRNA expression vector comprising an sgRNA comprising at least one of the nucleotide sequences set forth in SEQ ID nos. 2-4.
A method of making an sgRNA expression vector, the method comprising the steps of:
(1) Designing sgRNA in the 7 th exon region of an OsSHMT1 gene, wherein the nucleotide sequence of the OsSHMT1 gene is shown as SEQ ID NO. 1;
(2) Inserting the obtained sgRNA into an expression vector to obtain the sgRNA expression vector.
Preferably, in step (2) the expression vectors are evoapobe 1-SpRYCas9n, evoCDA1-SpRYCas9n and ABE8e-SpRYCas9n.
A method of breeding a rice variety resistant to caproic acid, the method comprising the steps of:
(1) Designing sgRNA in the 7 th exon region of the OsSHMT1 gene;
(2) Inserting the obtained sgRNA into an expression vector to obtain the sgRNA expression vector;
(3) Transforming rice callus by using an sgRNA expression vector through agrobacterium, screening by using a culture medium containing sheep fatty acid to obtain rice positive seedlings, and identifying 194 th, 207 th or 209 th sites of an OsSHMT1 gene by using PCR amplification or molecular sequencing; the primer group adopted by the PCR amplification is any one of the following groups:
first primer set:
F:TGTGTGCTGATACTAAGAAGATTTC SEQ ID NO.6，
R:AAACGAAATCTTCTTAGTATCAGCA SEQ ID NO.7；
second primer set:
F:TGTGTGCTGAAATCTTCTTAGTATC SEQ ID NO.8，
R:AAACGATACTAAGAAGATTTCAGCA SEQ ID NO.9；
third primer set:
F:TGTGTGATACTAAGAAGATTTCAGC SEQ ID NO.10，
R:AAACGCTGAAATCTTCTTAGTATCASEQ ID NO.11。
preferably, in step (1) the sgRNA comprises at least one of the nucleotide sequences as shown in SEQ ID NO. 2-4.
The application of the OsSHMT1 gene in cultivation of anti-caproic acid crops is disclosed, and the nucleotide sequence of the OsSHMT1 gene is shown as SEQ ID NO. 1.
Preferably, the amino acid sequence of the OsSHMT1 gene is shown as SEQ ID NO. 5.
The invention has the beneficial effects that:
the invention designs the sgRNA in the 7 th exon region of the OsSHMT1 gene, the nucleotide sequence of the OsSHMT1 gene is shown as SEQ ID NO.1, the sgRNA comprises at least one of the nucleotide sequences shown as SEQ ID NO.2-4, and the gene editing is carried out on the OsSHMT1 gene, so that the tolerance of plants to the caproic acid can be obviously improved.
Drawings
FIG. 1 is a schematic diagram of a base editor carrier; evoapobe 1-SpRYCas9n base editor, evoCDA1-SpRYCas9n base editor, ABE8e-SpRYCas9n base editor, respectively;
FIG. 2 shows the sequence structure of the SHMT gene;
FIG. 3 shows the sequencing peaks of the resistance mutation sites of the SHMT gene generated by transforming CBE and ABE base editing libraries of rice;
FIG. 4 shows the statistics of OsSHMT1 gene resistance mutation sites generated by transforming CBE base editing library of rice;
FIG. 5 shows the phenotype of the resistance mutation site of the OsSHMT1 gene generated by transforming a CBE base editing library of rice;
FIG. 6 is T-DNA and genotyping of T1 generation plants;
FIG. 7 shows 5 strain mutants (BJ-8, BJ-9, BJ-52, BJ-69, BJ-299 and BJ-120) and wild type in caproic acid; (0.69, 1.39, 2.78, 4.17, 8.34 and 11.12 mM) for 3 d;
FIG. 8 is a graph showing the results of 5 strain mutants (BJ-8, BJ-9, BJ-52, BJ-69 and BJ 120) and wild type after 7 days of 2.78mM sheep fatty acid treatment;
FIG. 9 inhibition of mutant and wild type seedlings by caproic acid treatment.
Detailed Description
In order to make the objects, technical solutions and advantages of the present invention more apparent, the technical solutions in the embodiments of the present invention will be clearly and completely described in the following in conjunction with the embodiments of the present invention, and it is apparent that the described embodiments are some embodiments of the present invention, but not all embodiments. All other embodiments, which can be made by those skilled in the art based on the embodiments of the invention without making any inventive effort, are intended to be within the scope of the invention.
EXAMPLE 1 library construction and screening of herbicide-resistant mutation sites
1. Constructing a base editing library targeting a specific structural domain of the endogenous OsSHMT1 gene of rice.
The rice evoCDA1-SpRYCas9n (CBE) and evoAPOBEC1-SpRYCas9n (CBE) with high construction efficiency and wide editing range are utilized, and the ABE8e-SpRYCas9n (ABE) base editor is utilized. The base editor can realize the base conversion of C/G- > T/A (CBE) or A/T- > G/C (ABE) within a certain sequence window (Komor et al, 2016), and evoCDA1 and evoAPOBEC1 are optimized on the basis of the base editor studied previously, and the efficiency of targeted base editing is further improved (Thuronyi et al, 2019; wang et al, 2022). SpRYSAS 9 was modified from SpCas9, spCas9 recognizing mainly the PAM sequence of NGG, while SpRYSAS 9 greatly reduced the requirement for the PAM motif, almost any three bases could be used as the PAM sequence, but with higher recognition efficiency for NRN than NYN (R=G/A, Y=C/T) (Walton et al 2020). In order to further increase the efficiency of rice base editing and expand the range of base editing, a new deaminase and Cas9 protein were designed to BE fused together, and based on the previously developed Anc689BE4 max-nmas 9 and ABEmax-nmas 9 base editors (Wang et al, 2019), new evoapobe 1-SpRYCas9n (CBE), evoCDA1-SpRYCas9n (CBE) and ABE8e-SpRYCas9n (ABE) base editors were formed by replacing the deaminase and Cas9 protein (fig. 1, 2).
The rice endogenous OsSHMT1 gene (loc_os03g 52840) consists of 15 exons, encoding 514 amino acids, where we determined exon 7 of OsSHMT1 as the mutation target region (fig. 2). We set specific segments in the CT domain as base-edited target regions, introduce DNA sequences into the CRISPR-GE website (Xie et al, 2017), design sg targets with NRN (r=r=g/a) as PAM motif, and co-generate 29 sg. The sg sequences are sent to a biological engineering limited company (Shanghai) for synthesis and then cloned to evoAPOBEC1-SpRYCas9n, evoCDA1-SpRYCas9n and ABE8e-SpRYCas9n vectors respectively to form a base editing library targeting the endogenous OsSHMT1 gene of rice.
The nucleotide sequence of the OsSHMT1 gene is shown as SEQ ID NO.1, and is specifically as follows:
GTGTGGTGGGTGCTGGGGCTGATCACTGCAGATAAAAACGCCACGCGAATCCACTACCTCCCAAACCCCGAGACCCCCCCGCCTCCACCACCACCCGCCGCTCGCCGCTCGCCCACCATGGCCATGGCGACGGCGCTCCGCAAGCTCTCCTCCGACGCCCTCCGCCGCCAGCCGCTCTCCCGCATCACCCCGCTCTACTACATGGTTCCGCTCTTCTTCCTCCATTTTTTCTTTAATTTTTTTTTGTTTATATCGTTGCCCTCTTGAGAAATGGTGCTTAATCCGTTCCATGGATTGGTTTGGGTTCGTGCGCTCTCGCAGGCGTCCCTGCCGGCGACGGAGGAGAGATCCGGAGTCACCGTACGTGCTCTCTCTCTCTCTCTCTCTCTTGACTATGTGTTCCGTTGCTCGTTGGTGTCTGTGGTTTGTTCATTCGTTTTTTTTTTATTATTATTTTGTTTGTGTGTTGGATGCAGTGGCCGAAGCAGCTGAACGCGCCGCTGGAGGAGGTGGATCCCGAGATCGCCGACATCATCGAGCACGAGAAGGCCCGCCAATGGAAGGTAACCAGCAACCACCAAAAAAAAAGTGCGTGGATTGTTCAGTGTTTGGTCCACCGACGCATTGCTGAATTGTGGCAATCTGTTTGGTGCAGGGTCTGGAGCTCATCCCGTCGGAGAACTTCACCTCGGTGTCAGTGATGCAGGCGGTGGGATCCGTCATGACCAACAAGTACAGCGAGGGGTACCCCGGCGCGAGATACTACGGTGGAAACGAGTACGGTTGATTGCGTTCTATCGGTTGCCTTTGTTCAGATTATTTGGGTGTTAAAAATGTTTAATCTGAAAGAGAAATCAGTGCCTGATGGGAGTGAATGATGTGAATTTCTGGTGGCTCTGGGTTTTCCTTGCGTGCAGATACATTGATATGGCCGAGTCATTGTGCCAGAAACGTGCTTTGGAGGCCTTCCGCTTGGACCCAGCGAAATGGGGAGGTAAATCATTTTCACTCTTTATCTTGCCTTGTCATGTTTTTATATGAACTCCAGCTTTCATGCGCATCAATAGCCTTTAGTCCTCTTTTGTTTCCTGCTTTGGGCGATGTAAATGAAAAAAAAAAAGGATAGGGGCAAAAAGGTTCCTTTGGATAAGACACAGTTTTTATGGTTCCATAGTTCACAAAAAAAAAAAAAGGGATTTTGATCAGAAAATTTCTTCTAGGTTCTTAATTTACTTAGCTTCCTCACTAGTACAATGAAAAGATTACATCGTTTATAAACCACTCGGTTCTTCAGTAACCTGAATACAAATGGTACACGAGTGGTCCTATTCATTTATATTTTTCGAATTGTTTGCTTTCATTATTAACCGCTTTGTACTTAAATTTATCTAGAATACTTATTCTCAAAATTTTAGTTTTCTTTCTCCAAAAAGGTGGCATTCTAACTTGACAACCAATTTCTACCTCTGCAGTGAATGTGCAACCTCTATCAGGGTCACCTGCCAACTTCCATGTTTACACTGCCCTATTGAAACCACATGAGAGAATCATGGCTTTGGATCTTCCTCATGGTGGACATCTTTCTCACGGCTACCAGGTACATGGGTCTGGACTTCTTCATTGCCTCCTTACTACTAGTTGTTTCAGTTGGCATGCAAGCATAATGCTTCCTTCCTTGTTGTCTTTCTTTGTCCTCACAAGTTTTTCAATTCTCTCATCGTTACTATATACCAATGAGTTTCTTCCCCCTCAATTCTTAGTTATGTTCAGATATAATGCTCTTGGTACTCTCATCCTCCCATGGCTTAGTTGTTCAACTTCTTGATGGTTGCAGACTGATACTAAGAAGATTTCAGCAGTTTCGATATTCTTTGAGACAATGCCCTACAGATTGGATGAAAGCACTGGCTTGATTGATTATGATCAGGTGCAATGTCCTCCTTATCTGTTTTGTTTTAATGCCATTTAACTTATTGAATTCTCTATCATTCTTTTATAGGTATGAGTTTTCATTGGACAAAACGTGTTCTTTTTGCATCTAGCAGATTCCACAGCTGAAAATATGGTTTCAAGTAGCATTATCTAGGATATTTTTCATTGCCTCATATCTCTTTCAATTGTGAAATTATTTCAAGATGAATTTCTGCTGACATGCTTGATTTGCTACTTCTGCACAGATGGAGAAAAGTGCCGTTCTTTTTAGGCCAAAGTTGATCGTTGCGGGTGCAAGTGCATATGCGCGTCTTTATGACTATGACCGCATGCGGAAGGTGAATTTTACTGCTCAATAGTGCATGCTGAATCTTCACCTTCTGTAATCAGTCAAATAATTCACTTGTTATGCAATCTGCAGGTTTGTGACAAGCAGAAGGCAATACTTCTAGCAGATATGGCACATATCAGTGGGCTTGTCGCAGCTGGTGTTGTTCCATCTCCTTTTGATTATGCAGATGTAGTGACTACCACTACTCACAAGTCACTCCGTGGACCACGTGGAGCCATGATCTTTTACAGGAAGGGGGTGAAAGGAGTAAACAAGCAAGGCAAAGAGGTATATACTTGAGCAGAACAATAGCAAGATAGTAGCGTAGGGTGTTGGTACTGTTTCTTCTCCATCCTGTGACTCTGTGAGCTTCAAAAAAATAAAAATGCCTAATGACCCTCTACCGTCTAGGTTATGTATGACTTTGAGGACAAGATCAATGCTGCTGTCTTCCCAGGTCTGCAAGGTGGACCACATAATCATACCATTACTGGCTTAGCTGTTGCGCTTAAGCAGGTCTGTAGCACTTCTGATCCCTGATGTTTTATATTTTATAGATCACATTGTTTGCCTTTGTCTTGGACTCTAAAAGGACTTTCTAACTTCCAGGCAACTACTCCGGAGTACAGAGCTTATCAAGAGCAAGTTATGAGTAACTGTGCAAAATTTGCACAGGTATGTGTTCAAATTTTGCTAGGTTTTTTTTTTTATCTACAATATGGCTTGATCTTTCTTTATTTCACAACACTTAATAATGATTTTGTTCTTCACTTAGTTCACCTCAGCATGTTACGAATTGCAAATTATTGGTTTTTCAACATGATGAATTGTGCTTTAACATTTGAACCACTTCCTTATCTGGTGGCAATTTATCACTTCCAGAGCTTGACAGCAAAAGGCTACGAACTTGTCTCTGGTGGGACTGACAACCATTTAGTGTTGGTAAATCTCAAGAGCAAGGTAAGACAAACATGTGGGACGCTTTGGGTGTTTTGAGATATTTTAGTTCTATCTTTCAGTTTCTCTTATGAGTGCTCACCAATAAACTTTCAGCTGCTTCCTTTTAAAAAATATCGATTGATGTCTATTTAACCTCAAGCTTGCTGTTTCTGTATATAAATGATAGCTAAATGTGCTTTGCAGATAAACTTGTTTTTTTTTTTCAAATTTTTATGCCATATGGGGACAATTATCATCCTCTTCAAATTTCATGTTTAGGGCATAGATGGTTCAAGAGTGGAGAAGGTTTTAGAAAACGTGCACATTGCAGCAAACAAGAACACAGTTCCTGGTGATGTTTCAGCTATGGTACCAGGAGGCATCAGGATGGGTAAAATCTGCCCCTATGTGGTTTTTTTTTTAATTTACTTAAAATAGATTTGCAAAATCTCAATATCAGCTCCATGATCATGCAGGAACCCCAGCACTGACCTCAAGAGGATTTGTTGAGGAGGACTTTGCTAAGGTTGCTGATTTCTTCGATGCAGCAGTGAACTTGGCTTTGAAGGTTAAGGCTGCAGCAGGTCTGTCAAATTCCTAGTTTTTTTTTTGTTTGAAAATGTTGAGAATACAAAACAATTGTTGCATGCATTGAGGTGCCCTCATATGATACTTCACCCTAGGATCATCGTATTCATCATTTTGTTTTTCCCCTTGCAATTTAGGTGGAACAAAACTGAAGGACTTTGTTGCCACTTTGCAATCTGATAGCAACATTCAATCCGAGATTGCAAAACTTCGCCATGATGTGGAGGAATATGCAAAACAGTTCCCCACAATTGGGTTTGAGAAAGAAACCATGAAGTACAAGAACTAAGAAACTTTGAATGGAACAGCAAGGTATTGTCCTGACAGATAAATTGGCTCCATTTTTTTTTAGCATGCTGCATGCAGTATATTAGTTGCCTAGGCCTTGGCTCCGATCATGAACCATGTTTACTCTTTCATTGTTGCCTCAGCTTGCACCCCTTCCAGTTCTTCCACCACCGCTCCTTCCTGCATGCCGTTTTCCAGAAGCAAATTAACTGTGCATTTCTGCTATCAGTCACCGAATTCTTCTGTCGCATTTATTTCAGGGTAAAAGAAAAGGCATCAAGCTGAATTCCTGAGGTGACTGTTGGAATTCTTGCAAGAACAAGTCGGTGTAAACATATATCCATGGAGTGCCATCTTATGTAAAAGGGACCCCTGGCATTTTACAGCGTGTGGAAACTTTGTCAATAGTTCTTATCGTAGACACCTACTGTAAGATGTTATGCTAATGCTATATTAACCTTCACTATCTTCTTGGACAAGCAGTTACACATACTTTGGTGTATTCTGTGAATAATTCGCATGATTGCGGAATTTTTCGTGTTTATAAATCGTAACTTGTAATCTTTTGGCCCTGCACGCAATTTGAAAGCCCTCGATCGGATTGTCGTTTACTGCACACAA。
the amino acid sequence of the OsSHMT1 gene is shown in SEQ ID NO. 5:
>MAMATALRKLSSDALRRQPLSRITPLYYMASLPATEERSGVTWPKQLNAPLEEVDPEIADIIEHEKARQWKGLELIPSENFTSVSVMQAVGSVMTNKYSEGYPGARYYGGNEYIDMAESLCQKRALEAFRLDPAKWGVNVQPLSGSPANFHVYTALLKPHERIMALDLPHGGHLSHGYQTDTKKISAVSIFFETMPYRLDESTGLIDYDQMEKSAVLFRPKLIVAGASAYARLYDYDRMRKVCDKQKAILLADMAHISGLVAAGVVPSPFDYADVVTTTTHKSLRGPRGAMIFYRKGVKGVNKQGKEVMYDFEDKINAAVFPGLQGGPHNHTITGLAVALKQATTPEYRAYQEQVMSNCAKFAQSLTAKGYELVSGGTDNHLVLVNLKSKGIDGSRVEKVLENVHIAANKNTVPGDVSAMVPGGIRMGTPALTSRGFVEEDFAKVADFFDAAVNLALKVKAAAGGTKLKDFVATLQSDSNIQSEIAKLRHDVEEYAKQFPTIGFEKETMKYKN*。
29 sgRNAs and detection primers and Tm temperatures thereof are as follows:
sgRNA1：
CTGATACTAAGAAGATTTC SEQ ID NO.2
f TGTGTGCTGATACTAAGAAGATTTC SEQ ID No.6 Tm temperature=61.1 ℃,
r AAACGAAATCTTCTTAGTATCAGCA SEQ ID No.7 Tm temperature = 57.0 ℃;
sgRNA2
CTGAAATCTTCTTAGTATC SEQ ID NO.3
f TGTGTGCTGAAATCTTCTTAGTATC SEQ ID.8tm temperature=62.7deg.C
R AAACGATACTAAGAAGATTTCAGCA SEQ ID No.9 Tm temperature=58.7°c
sgRNA3：
ATACTAAGAAGATTTCAGC SEQ ID NO.4
F TGTGTGATACTAAGAAGATTTCAGC SEQ ID No.10 Tm temperature=62.7℃
R AAACGCTGAAATCTTCTTAGTATCA SEQ ID No.11 Tm temperature=58.7°c
sgRNA4：
GAAACTGCTGAAATCTTCTT
F TGTGTGGAAACTGCTGAAATCTTCTT Tm temperature=62.7℃
R AAACAAGAAGATTTCAGCAGTTTC Tm temperature=58.7 DEG C
sgRNA5：
TAAGAAGATTTCAGCAGTTT
F TGTGTGTAAGAAGATTTCAGCAGTTT Tm temperature=61.1℃
R AAACAAACTGCTGAAATCTTCTTA Tm temperature=57.0℃
sgRNA6：
TCAGCAGTTTCGATATTCTT
F TGTGTGTCAGCAGTTTCGATATTCTT Tm temperature=62.7℃
R AAACAAGAATATCGAAACTGCTGA Tm temperature=58.7 DEG C
sgRNA7：
TCAAAGAATATCGAAACTGC
F TGTGTGTCAAAGAATATCGAAACTGC Tm temperature=62.7 ℃ R:
AAACGCAGTTTCGATATTCTTTGA Tm temperature=58.7℃
sgRNA8：
AGCAGTTTCGATATTCTTTG
F TGTGTGAGCAGTTTCGATATTCTTTG Tm temperature=62.7 ℃ R:
AAACCAAAGAATATCGAAACTGCT Tm temperature = 58.7 ℃ sgRNA9:
GTCTCAAAGAATATCGAAAC
f TGTGTGGTCTCAAAGAATATCGAAAC Tm temperature=62.7 ℃ R:
AAACGTTTCGATATTCTTTGAGAC Tm temperature=58.7℃
sgRNA10：
TTCGATATTCTTTGAGACAA
F TGTGTGTTCGATATTCTTTGAGACAA Tm temperature=61.1℃
R AAACTTGTCTCAAAGAATATCGAA Tm temperature=57.0℃
sgRNA11：
GGCATTGTCTCAAAGAATAT
F TGTGTGGGCATTGTCTCAAAGAATAT Tm temperature=62.7℃
R AAACATATTCTTTGAGACAATGCC Tm temperature=58.7 DEG C
sgRNA12：
TCTTTGAGACAATGCCCTAC
F TGTGTGTCTTTGAGACAATGCCCTAC Tm temperature=65.8 ℃ R:
AAACGTAGGGCATTGTCTCAAAGA Tm temperature = 62.1 ℃ sgRNA13:
TCTGTAGGGCATTGTCTCAA
f TGTGTGTCTGTAGGGCATTGTCTCAA Tm temperature=65.8 ℃ R:
AAACTTGAGACAATGCCCTACAGA Tm temperature=62.1℃
sgRNA14：
TGAGACAATGCCCTACAGAT
F TGTGTGTGAGACAATGCCCTACAGAT Tm temperature=65.8 ℃ R:
AAACATCTGTAGGGCATTGTCTCA Tm temperature=62.1℃
sgRNA15：
GAGACAATGCCCTACAGATT
F TGTGTGGAGACAATGCCCTACAGATT Tm temperature=65.8 ℃ R:
AAACAATCTGTAGGGCATTGTCTC Tm temperature=62.1℃
sgRNA16：
ACAATGCCCTACAGATTGGA
F TGTGTGACAATGCCCTACAGATTGGA Tm temperature=65.8 ℃ R:
AAACTCCAATCTGTAGGGCATTGT Tm temperature=62.1℃
sgRNA17：
TCATCCAATCTGTAGGGCAT
F TGTGTGTCATCCAATCTGTAGGGCAT Tm temperature=65.8 ℃ R:
AAACATGCCCTACAGATTGGATGA Tm temperature=62.1℃
sgRNA18：
TGCCCTACAGATTGGATGAA
F TGTGTGTGCCCTACAGATTGGATGAA Tm temperature=65.8℃
AAACTTCATCCAATCTGTAGGGCA Tm temperature=62.1 DEG C
sgRNA19：
TGCTTTCATCCAATCTGTAG
F TGTGTGTGCTTTCATCCAATCTGTAG Tm temperature=64.3℃
R AAACCTACAGATTGGATGAAAGCA Tm temperature=60.4℃
sgRNA20：
GTGCTTTCATCCAATCTGTA
F TGTGTGGTGCTTTCATCCAATCTGTA Tm temperature=64.3℃
R AAACTACAGATTGGATGAAAGCAC Tm temperature=60.4℃
sgRNA21：
AGTGCTTTCATCCAATCTGT
F TGTGTGAGTGCTTTCATCCAATCTGT Tm temperature=64.3℃
R AAACACAGATTGGATGAAAGCACT Tm temperature=60.4℃
sgRNA22
TACAGATTGGATGAAAGCAC
F TGTGTGTACAGATTGGATGAAAGCAC Tm temperature=64.3℃
R AAACGTGCTTTCATCCAATCTGTA Tm temperature=60.4℃
sgRNA23：
ACAGATTGGATGAAAGCACT
F TGTGTGACAGATTGGATGAAAGCACT Tm temperature=64.3℃
R AAACAGTGCTTTCATCCAATCTGT Tm temperature=60.4℃
sgRNA24：
GCCAGTGCTTTCATCCAATC
F TGTGTGGCCAGTGCTTTCATCCAATC Tm temperature=67.4℃
R AAACGATTGGATGAAAGCACTGGC Tm temperature=63.8
sgRNA25
ATTGGATGAAAGCACTGGCT
F TGTGTGATTGGATGAAAGCACTGGCT Tm temperature=65.8℃
AAACAGCCAGTGCTTTCATCCAAT Tm temperature=62.1 DEG C
sgRNA26：
GATGAAAGCACTGGCTTGAT
F TGTGTGGATGAAAGCACTGGCTTGAT Tm temperature=65.8℃
AAACATCAAGCCAGTGCTTTCATC Tm temperature=62.1 DEG C
sgRNA27：
AGCACTGGCTTGATTGATTA
F TGTGTGAGCACTGGCTTGATTGATTA Tm temperature=64.3℃
R AAACTAATCAATCAAGCCAGTGCT Tm temperature=60.4
sgRNA28：
ATCATAATCAATCAAGCCAG
F TGTGTGATCATAATCAATCAAGCCAG Tm temperature=62.7℃
R AAACCTGGCTTGATTGATTATGAT Tm temperature=58.7 DEG C
sgRNA29：
TGATCATAATCAATCAAGCC
F TGTGTGTGATCATAATCAATCAAGCC Tm temperature=62.7℃
R AAACGGCTTGATTGATTATGATCA Tm temperature=58.7 DEG C
2. Selection of rice genetic transformation and caproic acid resistance mutants
The constructed base editing library is transformed into callus of rice japonica rice variety Xiushui 134 through agrobacterium, and transgenic positive callus is screened out through hygromycin. In the positive callus differentiation stage, we added 20uM of oriented OsSHMT1 caproic acid on the medium: . On the culture medium, callus transferred into an empty vector cannot differentiate to form seedlings, while callus transferred into a CBE or ABE base editing library can differentiate normally to form seedlings when the OsSHMT1 gene realizes base editing to cause amino acid mutation and the mutation site can generate resistance to the caproic acid.
3. Genotyping of resistant mutants
The CBE base editing library is transformed by agrobacterium to obtain tens of T0 generation positive seedlings, leaf DNA of the screened resistant plants is extracted, and the target region of the OsSHMT1 gene is identified by PCR and Sanger sequencing (figure 3), so that the plants have one or more base substitutions in the target region relative to a wild type sequence, one or more amino acid mutations are caused, and 25 different genotypes are obtained (figure 4). T0 generation plants were planted in soil after acclimatization and some genotypes failed to grow properly and die (fig. 5). In the T1 generation, T-DNA isolation was identified by PCR reaction, and Sanger sequencing was used to genotype the target region of the SHMT gene (FIG. 6), and further experiments were performed to find T-DNA free and homozygous mutants.
4. Tolerance of resistant mutants to sheep fatty acids
The homozygous mutant of T-DNA isolation of the T2 generation was subjected to the experiment of sheep acid resistance (FIG. 7, FIG. 8). Most genotypes were not significantly different from wild type. Only 3 genotype (T194A, D207N and D209N) mutations showed tolerance to capric acid, whereas wild type plants died 3 days after 2.78mM CAP treatment (fig. 8). For the T194A, D207N and D209N mutants, the EC50 of the sheep fatty acids were 2.791mM, 3.378mM and 2.514mM, respectively, whereas the EC50 of the wild type plants was 1.675mM (FIG. 9). This data indicates that the tolerance of the OsSHMT1 gene to the mutated caproic acid is heritable.
Claims (9)
1. The sgRNA targeting the OsSHMT1 gene is characterized in that the nucleotide sequence of the OsSHMT1 gene is shown as SEQ ID NO.1, and the sgRNA comprises at least one of the nucleotide sequences shown as SEQ ID NO. 2-4.
2. The sgRNA of claim 1, wherein the amino acid sequence of the OsSHMT1 gene is shown in SEQ ID No. 5.
3. An sgRNA expression vector comprising the sgRNA of claim 1.
4. A method of making the sgRNA expression vector of claim 3, comprising the steps of:
(1) Designing sgrnas in the 7 th exon region of the OsSHMT1 gene of claim 1;
(2) Inserting the obtained sgRNA into an expression vector to obtain the sgRNA expression vector.
5. The method of claim 4, wherein in step (2) the expression vectors are evoapobic 1-SpRYCas9n, evoCDA1-SpRYCas9n, and ABE8e-SpRYCas9n.
6. A method of breeding a rice variety resistant to caproic acid, said method comprising the steps of:
(1) Designing sgrnas in the 7 th exon region of the OsSHMT1 gene of claim 1;
(2) Inserting the obtained sgRNA into an expression vector to obtain the sgRNA expression vector;
(3) Transforming rice callus by using an sgRNA expression vector through agrobacterium, screening by using a culture medium containing sheep fatty acid to obtain rice positive seedlings, and identifying 194 th, 207 th or 209 th sites of an OsSHMT1 gene by using PCR amplification or molecular sequencing; the primer group adopted by the PCR amplification is any one of the following groups:
first primer set:
F:TGTGTGCTGATACTAAGAAGATTTC SEQ ID NO.6
R:AAACGAAATCTTCTTAGTATCAGCA SEQ ID NO.7；
second primer set:
F:TGTGTGCTGAAATCTTCTTAGTATC SEQ ID NO.8
R:AAACGATACTAAGAAGATTTCAGCA SEQ ID NO.9；
third primer set:
F:TGTGTGATACTAAGAAGATTTCAGC SEQ ID NO.10
R:AAACGCTGAAATCTTCTTAGTATCA SEQ ID NO.11。
7. the method of claim 6, wherein in step (1) the sgRNA comprises at least one of the nucleotide sequences set forth in SEQ ID nos. 2-4.
The application of the OsSHMT1 gene in cultivation of anti-caproic acid crops is characterized in that the nucleotide sequence of the OsSHMT1 gene is shown as SEQ ID NO. 1.
9. The use according to claim 8, wherein the amino acid sequence of the OsSHMT1 gene is shown in SEQ ID No. 5.
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