CN107099544A - The PL LbCpf1 RVR genes of identification specific site and its application in paddy gene target practice - Google Patents

Abstract

The invention provides a PL‑LbCpf1‑RVR gene for identifying specific sites in rice gene targeting and its application. During the rice gene targeting experiment, the present invention unexpectedly obtained a new PL‑LbCpf1‑RVR gene, and found that the use of the PL‑LbCpf1‑RVR gene for rice shearing can identify specific sites and identify more genomes site. And the present invention provides an expression cassette and an expression vector constructed based on the gene, and the application of the expression cassette and the expression vector in rice gene editing. The present invention uses the obtained PL‑LbCpf1‑RVR to construct a plant expression vector, construct a rice targeting vector, and introduce it into rice cells to cause DNA double-strand shearing of rice specific gene loci, thereby realizing rice gene targeting, and obtaining Transgenic rice plants.

Description

The PL-LbCpf1-RVR gene and its specific loci identified in rice gene targeting application

technical field

The invention relates to the technical fields of biotechnology and plant genetic engineering. Specifically, the present invention relates to a PL-LbCpf1-RVR gene for identifying specific sites in gene targeting and its application in rice gene targeting.

Background technique

The CRISPR/Cas gene editing system has become an important means for plant functional gene research and molecular breeding. Traditional CRISPR/Cas systems mostly use Cas9 protein as an endonuclease to cause site-specific cleavage at target sites in the plant genome, thereby introducing site-specific mutations. Recently, another Cas protein, Cpf1, was found from bacteria to also have endonuclease activity. Compared with Cas9 protein, Cpf1 has some characteristics in cutting principle and editing mode. For example: Cas9 recognizes a cytosine-rich PAM (Protospacer adjacent motif) sequence at the 3' end, while Cpf1 recognizes a thymine-rich PAM sequence at the 5' end; Cas9 usually creates blunt-ended DNA near the 5' upstream of PAM Cpf1 cuts sticky ends at the downstream distal end of PAM; Cas9 only has DNA shearing activity, while Cpf1 can both cut DNA and process RNA; Cas9 requires a long artificial RNA sequence for targeted editing, Cpf1 only needs a shorter crRNA sequence to complete the targeting.

The most commonly used Cpf1 in plants is derived from LbCpf1 of Lachnospiraceae bacterium ND2006. LbCpf1 can effectively recognize the TTTV (V=A/C/G) sequence in the genome as a PAM, thereby targeting its downstream sequence. Compared with the NGG PAM recognized by Cas9, the number of editable genes of LbCpf1 is limited.

However, there is currently no general and feasible method to increase the number of LbCpf1 editable sites and ensure the mutation efficiency of LbCpf1 gene in crop gene targeting, and the number of existing LbCpf1 genes with high mutation efficiency is limited. Therefore, it is urgently desired to provide more LbCpf1 genes that provide more editing sites in crop gene targeting, but such genes are often unavailable, and there is no established theory or method for people to find them. Such genes provide a theoretical basis.

Contents of the invention

In view of the above problems, the present invention hopes to provide a LbCpf1-RVR gene that recognizes a specific site in crop gene targeting.

Specifically, in the first aspect, the present invention provides a new PL-LbCpf1-RVR gene recognized by PAM, named PL-LbCpf1-RVR, and the PL-LbCpf1-RVR gene at least comprises SEQ in the sequence listing The nucleotide sequence shown in ID NO.1. The PL-LbCpf1-RVR gene of the present invention can recognize more editable sites during the transgenic cultivation of rice.

Preferably, the gene consists of the nucleotide sequence shown in SEQ ID NO:1. Compared with the existing LbCpf1 gene, the editing scope of the PL-LbCpf1-RVR gene obtained in the present invention is wider.

In the second aspect, the present invention provides a plant expression vector containing the PL-LbCpf1-RVR gene. The construction method of the plant expression vector is to use the NotI/SacI enzyme cutting site to cut the pHUN600 vector with NotI/SacI and recover it. Since the synthetic PL-LbCpf1-RVR sequence has NotI/SacI enzyme cutting sites at both ends, it can T4 ligase was used to connect PL-LbCpf1-RVR to the pHUN600 vector to obtain the plant expression vector pHUN-RVRPL-LbCpf1-RVR (pHUN 6b11).

On the other hand, on the basis of the expression vector, the present invention constructs a corresponding gene targeting vector according to the actual needs of the experiment.

In another aspect, the present invention provides an expression cassette, which is characterized in that the expression cassette contains the above-mentioned PL-LbCpf1-RVR gene.

On the other hand, the present invention provides a kind of application of above-mentioned gene, expression cassette or carrier, it is characterized in that, described application comprises using described PL-LbCpf1-RVR gene to complete the shearing of DNA double strand in rice body, and in self-repair Under the action of the system, transgenic plants or plant parts with mutation sites are obtained.

In another aspect, the present invention provides a pHUN-PL-LbCpf1-RVR (pHUN 6b11) expression vector (which contains the PL-LbCpf1-RVR gene), on the basis of the expression vector, only simple annealing, The targeting vector (pHUN 6b11-DL) of the specific gene can be obtained by enzyme digestion and ligation, and the method for introducing the targeting vector into rice cells comprises the following steps:

(1) dehulling and sterilizing the rice seeds, separating the embryos, and placing them on the callus induction medium to produce secondary callus;

(2) Secondary callus is transferred to new callus induction medium for pre-cultivation;

(3) The callus obtained in step (2) was contacted with the Agrobacterium carrying the targeting vector (pHUN 6b11-DL) of PL-LbCpf1-RVR for 15 minutes;

(4) Transfer the callus of step (3) to a petri dish with three sterile filter papers (adding 2.5-3.5mL Agrobacterium suspension medium) on the upper pad, and cultivate for 48 hours at 21-23°C;

(5) the callus of step (4) is placed on the pre-selection medium and cultivated for 5-7 days;

(6) transfer the callus of step (5) on the selection medium to obtain resistant callus;

(7) transfer the resistant callus to the differentiation and regeneration medium to differentiate into shoots; and

(8) The shoots of step (7) are transferred to the rooting medium to take root.

Wherein the seed in the step (1) is a mature seed; the induction medium in the step (1), (2) is the induction medium listed in Table 1 of the instructions; The Agrobacterium contact is that the callus is soaked in the Agrobacterium suspension; the Agrobacterium suspension medium in the step (4) is the suspension medium listed in Table 1 of the instructions; in the step (5) The pre-selection medium is the pre-screening medium listed in Table 1 of the instructions; the screening medium in the step (6) is the screening medium listed in Table 1 of the instructions; the differentiation in the step (7) The regeneration medium is the differentiation regeneration medium listed in Table 1 of the instructions; the rooting medium in the step (8) is the rooting medium listed in Table 1 of the instructions.

In a preferred embodiment, the rice is japonica rice, more preferably, the rice is japonica Nipponbare.

Exemplary formulation of medium in Table 1

"N6majors" mentioned in the table means that [NO3-]/[NH4+]=40mM/10mM in the N6majors.

In a preferred embodiment, the nucleotide sequence of the PL-LbCpf1--RVR marker gene is the nucleotide sequence shown in SEQ ID NO:1, specifically as:

ATGTCCAAGCTGGAGAAGTTTACAAACTGTTACAGCCTCTCCAAAACCCTCAGGTTTAAAGCGATCCCGGTGGGCAAGACCCAGGAGAACATCGACAACAAGAGGCTCCTGGTGGAAGACGAGAAGCGCGCCGAAGACTACAAGGGCGTGA AGAAGCTGCTCGAT AGGTACTACCTCAGCTTTATTAACGACGTGCTGCACAGCATCAAACTCAAGAATCTCAACAACTACATCTCCCTCTTCCGCAA AAAGACCCGCACCGAGAAGGAGAACAAGGAGCTGGAGAACCTGGAGATCAACCTCCGCAAGGAAATCGCCAAAGCGTTCAAGG GCAATGAAGGGTACAAGAGCCTCTTCAAGAAAGACATCATCGAAACTATCCTCCCAGAGTTTCTCGATGACAAGGACGAGATC GCGCTGGTGAACTCCTTTAACGGGTTCACAACCGCGTTTACCGGCTTCTTTGATAACAGGGAAAATATGTTCTCCGAGGAGGC CAAGTCCACCAGCATCGCCTTCAGGTGTATCAACGAGAACCTCACCCGCTACATTTCCAATATGGACATTTTCGAGAAGGTGG ATGCGATCTTCGATAAGCACGAGGTGCAGGAGATCAAAGAGAAGATTCTCAATTCCGATTATGACGTCGAGGATTTCTTCGAA GGGGAGTTCTTTAATTTTGTGCTCACACAAGAGGGCATTGACGTGTACAACGCGATTATCGGGGGCTTCGTCACAGAGTCCGG GGAGAAGATTAAGGGGCTGAATGAGTACATCAATCTGTACAATCAGAAGACCAAGCAGAAACTGCCGAAATTCAAGCCGCTCTACAAGCAAGTCCTGTCCGATAGGGAAAGCCTCTCCTTCTACGGCGAGGGCTATACCAGCGACGAGGAGGTGCTGGAAGTCTTC CGCAACACACTGAATAAGAATAGCGAGATTTTCTCCTCCATCAAGAAGCTCGAGAAGCTCTTTAAGAACTTTGACGAG TACAG CTCCGCCGGGATTTTCGTGAAGAACGGGCCGGCGATCAGCACCATCTCCAAGGACATCTTTGGCGAGTGGAACGTCATCAGGG ACAAGTGGAACGCCGAGTACGACGACATCCACCTGAAGAAGAAGGCGGTGGTGACCGAGAAGTATGAGGACGATCGCAGGAAG TCCTTCAAAAAAATCGGCTCCTTCAGCCTCGAACAGCTCCAGGAGTATGCCGATGCGGATCTGTCCGTCGTCGAGAAGCTGAA GGAAATCATCATTCAGAAGGTCGACGAGATCTATAAAGTGTACGGGTCCAGCGAGAAGCTGTTCGACGCCGACTTTGTGCTCG AGAAGTCCCTCAAAAAGAATGACGCCGTGGTGGCCATTATGAAAGACCTGCTCGACTCCGTGAAGTCCTTCGAAAATTACATT AAAGCGTTCTTTGGGGAGGGGAAGGAAACTAACAGGGATGAGTCCTTCTATGGCGACTTTGTCCTCGCGTACGACATCCTGCT GAAGGTCGACCACATTTACGACGCGATCCGCAACTACGTGACACAGAAGCCGTACTCCAAAGACAAGTTCAAGCTGTACTTCC AGAACCCGCAATTTATGCGCGGCTGGGACAAGGATGTCGAGACAGACAGGCGCGCGACAATTCTCCGCTATGGCTCCAAATACTATCTGGCCATCATGGACAAGAAGTACGCGAAGTGCCTGCAGAAGATCGACAAAGACGACGTCAATGGCAACTATGAAAAGAT CAACTACAAGCTGCTGCCGGGCCCGAACAAGATGCTCCCGAAGGTGTTCTTCAGCAAGAAGTGGATGGCCTACTACAATCCAA GCGAGGATATTCAGAAAATCTATAAAAACGGGACCTTCAAGAAGGGGGACATGTTTAACCTCAACGACTGCCACAAGCTCATT GATTTCTTCAAGGATAGCATTTCCCGCTACCCGAAATGGTCCAATGCGTACGATTTTAACTTCTCCGAGAC AGAAAAGTACAA AGACATCGCGGGCTTTTACAGGGAGGTGGAGGAGCAAGGGTATAAAGTTTCTTTTGAATCCGCGAGCAAGAAGGAAGTCGACA AGCTCGTCGAGGAGGGCAAGCTCTACATGTTCCAAATTTATAACAAGGACTTTTCCGACAAGAGCCATGGGACCCCAAACCTC CACACCATGTACTTCAAACTGCTCTTTGACGAGAACAACCACGGGCAAATCAGGCTGAGCGGCGGCGCCGAATTATTCATGCG CAGGGCCTCCCTCAAGAAGGAAGAGCTGGTCGTCCATCCAGCCAATTCCCCGATCGCGAACAAGAACCCGGACAATCCGAAAA AGACCACCACCCTGTCCTACGACGTCTACAAGGACAAACGCTTCAGCGAAGACCAGTACGAATTACACATCCCAATTGCGATT AATAAGTGCCCAAAGAATATCTTCAAAATTAATACAGAGGTCAGGGTGCTGCTCAAACACGACGACAATCCGTATGTCATCGGCATTGACAGGGGCGAGCGCAATCTGCTCTATATCGTGGTCGTGGATGGGAAGGGCAATATTGTGGAGCAGTACTCCCTGAACG AGATTATCAACAACTTCAATGGGATTAGGATTAAGACCGACTATCACAGCCTGCTCGACAAGAAAGAAAAAGAGAGGTTTGAG GCCCGCCAAAACTGGACCTCCATTGAGAATATCAAAGAATTAAAGGCCGGCTATATTTCCCAAGTCGTCCACAAGATCTGCGA GCTGGTGGAGAAATATGACGCCGTGATTGCGCTCGAAGACTTAAATTCTGGGTTCAAGAACTCCCGCGTGAAGGTGGAAAAAC AGGTGTATCAGAAATTCGAGAAAATGCTGATCGACAAACTCAATTATATGGTGGATAAGAAGTCCAACCCGTGTGCCACAGGG GGCGCGCTGAAGGGCTATCAGATCACCAACAAGTTCGAGAGCTTCAAGAGCATGAGCACCCAGA ACGGGTTTATTTTCTACAT CCCGGCGTGGCTCACCTCCAAGATTGACCCGAGCACCGGCTTCGTGAACCTCCTGAAGACAAAGTATACCTCCATTGCCGACA GCAAGAAGTTTATCTCCTCCTTCGACCGCATTATGTATGTGCCGGAGGAGGACCTCTTCGAGTTCGCCCTCGACTACAAAAAC TTCAGCCGCACAGATGCGGATTACATCAAGAAGTGGAAGCTGTACTCCTACGGGAACAGGATCCGCATCTTCAGGAATCCAAA AAAAAATAACGTCTTTGACTGGGAGGAAGTGTGCCTGACATCCGCCTACAAGGAACTGTTCAATAAATACGGCATCAATTACCAGCAGGGCGACATTCGCGCCCTCCTCTGTGAGCAGTCCGACAAAGCGTTTTACTCCAGCTTCATGGCCCTCATGTCCCTGATG CTCCAAATGAGGAATAGCATCACAGGGCGCACCGACGTCGACTTCCTCATCAGCCCGGTGAAGAACTCCGACGGGATCTTTTA CGACTCCCGCAACTATGAGGCGCAAGAGAATGCGATCCTCCCGAAGAACGCCGATGCGAACGGGGCCTATAATATCGCCAGGA AAGTGCTCTGGGCCATCGGGCAGTTCAAAAAGGCGGAGGATGAGAAGCTCGACAAGGTGAAAATTGCCATTTCCAACAAGGAG TGGCTGGAGTACGCGCAGACCTCCGTGAAGCACAAAAGGCCGGCGGCCACGAAAAAGGCCGGCCAGGCAAAAAAGAAAAAGGG ATCCTACCCATACGATGTTCCAGATTACGCTTATCCCTACGACGTGCCTGATTATGCATACCCATATGATGTCCCCGACTATG CCTAA

Description of drawings

Fig. 1 to Fig. 3 are PL-LbCpf1-RVR gene obtained in the present invention and original LbCpf1-RVR nucleotide sequence alignment. Wherein Optimized is the nucleotide sequence of the PL-LbCpf1-RVR obtained in the present invention, and Original is the nucleotide sequence of the original LbCpf1-RVR.

Fig. 4 is the amino acid sequence alignment of the protein encoded by the PL-LbCpf1-RVR obtained in the present invention and the original LbCpf1-RVR gene. Optimized is the amino acid sequence encoded by the PL-LbCpf1-RVR gene obtained in the present invention, and Original is the amino acid sequence encoded by the original LbCpf1-RVR.

Fig. 5 is a schematic diagram of the PHUN6b11 (LbCpf1-RVR) vector plasmid.

Figure 6 is an example of the mutant form of the DL gene locus in the T0 generation of transgenic plants.

detailed description

Embodiments of the present invention are described below in conjunction with the accompanying drawings. It should be noted that the following embodiments are only used to illustrate exemplary implementations of the present invention, but not to limit the present invention. Certain equivalent changes and obvious improvements can be made to the present invention by those skilled in the art.

Unless otherwise specified, the operations in the following specific embodiments are performed by common conventional operations in the art. Those skilled in the art can easily obtain teachings about such routine operations from the prior art, for example, with reference to the textbooks Sambrook and David Russell, Molecular Cloning: A Laboratory Manual, 3rd ed., Vols 1, 2; Charles Neal Stewart, Alisher Touraev, Vitaly Citovsky and Tzvi Tzfira, Plant Transformation Technologies, etc. The medicinal raw materials, reagents, materials, etc. used in the following examples are all commercially available products unless otherwise specified.

Embodiment 1——The acquisition of PL-LbCpf1-RVR gene

The inventors of the present application tried to transform the LbCpf1-RVR gene from Escherichia coli in various ways, unexpectedly obtained a new DNA sequence, and added a rice-preferred stop codon TGA to the end of the DNA sequence to form A new gene, the gene is named PL-LbCpf1-RVR, its sequence is shown in SEQ ID NO: 1, and its sequence comparison with LbCpf1-RVR is shown in Figure 1.

Further analysis of its base composition, the results are shown in Table 2. It is known from Table 2 that the GC content of PL-LbCpf1 is as high as 53.06%, significantly higher than that of LbCpf1 which is 50.09%. In this way, the gene structure is more stable, because 3 hydrogen bonds can be formed between GC and 2 between AT.

Table 2 Gene base composition analysis of PL-LbCpf1-RVR and LbCpf1-RVR.

Analysis-RVR The protein amino acid sequences encoded by LbCpf1-RVR and LbCpf1-RVR genes, the comparison results are shown in FIG. 4 . It can be seen from Figure 4 that the amino acid sequences of the two are completely consistent.

After the designed PL-LbCpf1-RVR gene was sent to Suzhou Jinweizhi Biotechnology Co., Ltd. for synthesis, it was connected to the PUC57-AMP vector to form a PUC57-AMP-PL-LbCpf1-RVR vector, and loaded into E. coli XL-blue strain.

Example 2—Construction of Plant Targeting Vector Containing PL-LbCpf1-RVR Gene

From Escherichia coli XL-blue containing the PUC57-AMP-PL-LbCpf1-RVR vector, use the Axygen plasmid extraction kit to extract the plasmid, digest with NotI/SacI, and recover the PL-LbCpf1-RVR fragment. At the same time, NotI/SacI enzyme was used to linearize pHUN600, recover pHUN600, and connect the above-mentioned PL-LbCpf1-RVR fragment and pHUN600 fragment with T4 ligase (purchased from TaKaRa Company) to obtain the plant expression vector pHUN600-PL-LbCpf1 - RVR (Figure 3), named pHUN 6b11.

The nucleotide sequence TATC AAAGCTGCCAAGCCAGATATCCCTCACAG at position 6370-6398 in the rice DL gene (LOCOs03g0215200) was selected as the targeting site. The target site sequence was fused to pHUN6b11 to form pHUN6b11-DL. The plant expression vector was transformed into Agrobacterium tumefaciens EHA105 strain (preserved by Rice Research Institute, Anhui Academy of Agricultural Sciences) by freeze-thaw method for genetic transformation.

Example 3—the genetic transformation of rice using pHUN6b11-DL as a targeting vector and the acquisition of mutants.

1. Induction and pre-culture of mature embryo callus

The mature seeds of Nipponbare (the Paddy Rice Research Institute of Anhui Academy of Agricultural Sciences are preserved) are shelled, and the seeds with normal appearance and cleanness without mildew are selected, shaken for 90 sec with 70% alcohol, and pour off the alcohol; then use 50% sodium hypochlorite ( The concentration of available chlorine in the stock solution is greater than 4%. Add 1 drop of Tween20) solution per 100 milliliters to clean the seeds, and shake for 45 minutes (180 r/min) on a shaker. Pour off the sodium hypochlorite, wash with sterile water 5-10 times until there is no smell of sodium hypochlorite, finally add sterile water, soak overnight at 30°C. Use a scalpel to separate the embryos along the aleurone layer, put the scutellum up on the induction medium (see Table 1 for ingredients), 12 embryos/dish, and culture in the dark at 30°C to induce callus.

Two weeks later, spherical, rough, light yellow secondary callus appeared, and pre-cultivation operation could be performed, that is, the secondary callus was transferred to a new callus induction medium, and pre-cultivated in the dark at 30°C for 5 days. After pre-cultivation, small particles in good condition and vigorous division were collected with a spoon into a 50 mL sterile centrifuge tube for Agrobacterium infection.

2. Cultivation of Agrobacterium strains and preparation of suspension

Streak the Agrobacterium strain EHA105 containing the pHUN6b11-DL carrier on an LB plate containing 50mg/L kanamycin (see Table 1 for ingredients), culture in the dark at 28°C, and inoculate the activated Agrobacterium with a sterile loop after 24 hours. Inoculate to fresh 50mg/L kanamycin LB plates for second activation, and culture overnight at 28°C in the dark. Add 20-30 mL of Agrobacterium suspension medium (see Table 1 for ingredients) into a 50 mL sterile centrifuge tube, scrape off the twice activated Agrobacterium with an inoculation loop, and adjust OD660 (Optical density660nm, 660nm absorbance value) to about 0.10 -0.25, stand at room temperature for more than 30 minutes.

3. Infection and co-cultivation

Add the Agrobacterium suspension to the prepared callus (see step 1), soak for 15 minutes, and shake gently from time to time. After soaking, pour off the liquid (drip the liquid as far as possible), use sterile filter paper to absorb excess Agrobacterium bacterium liquid on the surface of the callus, and dry it with sterile wind in an ultra-clean bench. Put three pieces of sterile filter paper on a 100×25mm disposable sterile Petri dish, add 2.5mL of Agrobacterium suspension medium, disperse the blotted callus evenly on the filter paper, and incubate in the dark at 23°C for 48h.

4. Pre-screening and screening culture

After the co-cultivation, the co-cultured calli were uniformly spread in the pre-selection medium (see Table 1 for composition), and cultured in the dark at 30° C. for 5 days. After the pre-screening culture, transfer the callus to the screening medium (see Table 1 for ingredients), pick up 25 callus in each dish, and culture in the dark at 30°C. After 2-3 weeks, the resistant callus The growth is obvious, and the differentiation and regeneration operation can be carried out.

5. Differentiation and regeneration

For each independent transformant, 2-3 fresh small particles with good growth status were selected and transferred to the differentiation regeneration medium (see Table 1 for the composition). Five independent transformants were received per culture dish. Light culture was carried out at 28°C, the light cycle was 16h light and 8h dark, and the light intensity was 3000-6000lx.

6. Rooting and transplanting

When the shoots differentiated from the resistant callus grew to about 2 cm, each independent transformant only took one well-grown shoot, moved it to the rooting medium (see Table 1 for the composition), and cultivated it under light at 28° C. 16h light and 8h dark, the light intensity is 3000-6000lx. Two weeks later, select seedlings with well-developed root systems, wash off the medium with water, and transplant them into the soil.

7. Molecular identification

Before transplanting, rice leaf samples were taken, and DNA was extracted by CTAB method. The resulting genomic DNA samples were used for PCR analysis. The PCR primers used to amplify PL-LbCpf1-RVR were 5'-TTCACAACCGCGTTTACC-3' and 5'-TCACCACCGCCTTCTTCT-3', resulting in a 492bp fragment. The PCR components were first held at 95°C for 5 minutes, followed by 32 cycles of 94°C for 45 seconds, 56°C for 45 seconds, 72°C for 45 seconds, and a final extension at 72°C for 10 minutes. Eight transgenic plants were randomly selected, all of them were positive after identification, and the positive rate reached 100%.

Leaf DNA was extracted from all the 48 transgenic plants, and the resulting genomic DNA samples were used for PCR analysis. The PCR primers used to amplify the PDS gene fragment were 5'-CATGGATATTTAGCGGATCA-3' and 5'-GGAGATATTGGGGATGAGTAGA-3', resulting in a 155bp fragment. The PCR components were first held at 95°C for 5 minutes, followed by 32 cycles of 94°C for 30 seconds, 60°C for 30 seconds, 72°C for 30 seconds, and a final extension at 72°C for 10 minutes. The PCR products were sequenced. The measured results were compared with the wild-type sequence. Point mutation occurred in 8 of the 48 transgenic plants tested; the mutation efficiency was 16.7%. It shows that the modified Lbcpf1-RVR can cut the rice genome, and some mutant forms are shown in FIG. 6 .

The PL-LbCpf1-RVR gene of the present invention can not only be effectively applied in the transgenic cultivation of rice to realize cutting, but also compared with the existing LbCpf1 gene, in the transgenic cultivation of rice, the PL-LbCpf1- The editing range of the RVR gene is wider. It has been confirmed by theoretical demonstration and experimental verification of some of them. Compared with the existing LbCpf1, it has an editing range of at least 1.5 parts when rice is cut, which is the ratio of editable sites. Lbcpf1 is nearly twice as large. Lbcpf1 can recognize 1.5 million loci on the rice genome, and PL-Lbcpf1-RVR can recognize nearly 2.3 million loci. The gene of the present invention can recognize both TTTV site and TATV site.

sequence listing

<110> Rice Research Institute of Anhui Academy of Agricultural Sciences

<120> Identification of PL-LbCpf1-RVR gene RVR that specifically provides more editing sites in rice gene targeting and its application

<130> HCI20170170

<160> 5

<170> PatentIn version 3.3

<210> 1

<211> 3822

<212>DNA

<213> PL-LbCpf1--RVR

<400> 1:

atgtccaagc tggagaagtt tacaaactgt tacagcctct ccaaaaccct caggtttaaa 60

gcgatcccgg tgggcaagac ccaggagaac atcgacaaca agaggctcct ggtggaagac 120

gagaagcgcg ccgaagacta caagggcgtg aagaagctgc tcgataggta ctacctcagc 180

tttattaacg acgtgctgca cagcatcaaa ctcaagaatc tcaacaacta catctccctc 240

ttccgcaaaa agacccgcac cgagaaggag aacaaggagc tggagaacct ggagatcaac 300

ctccgcaagg aaatcgccaa agcgttcaag ggcaatgaag ggtacaagag cctcttcaag 360

aaagacatca tcgaaactat cctcccagag tttctcgatg acaaggacga gatcgcgctg 420

gtgaactcct ttaacgggtt cacaaccgcg tttaccggct tctttgataa cagggaaaat 480

atgttctccg aggaggccaa gtccaccagc atcgccttca ggtgtatcaa cgagaacctc 540

acccgctaca tttccaatat ggacattttc gagaaggtgg atgcgatctt cgataagcac 600

gaggtgcagg agatcaaaga gaagattctc aattccgatt atgacgtcga ggatttcttc 660

gaagggggagt tctttaattt tgtgctcaca caagaggggca ttgacgtgta caacgcgatt 720

atcgggggct tcgtcacaga gtccggggag aagattaagg ggctgaatga gtacatcaat 780

ctgtacaatc agaagaccaa gcagaaactg ccgaaattca agccgctcta caagcaagtc 840

ctgtccgata gggaaagcct ctccttctac ggcgagggct ataccagcga cgaggaggtg 900

ctggaagtct tccgcaacac actgaataag aatagcgaga ttttctcctc catcaagaag 960

ctcgagaagc tctttaagaa ctttgacgag tacagctccg ccggggatttt cgtgaagaac 1020

gggccggcga tcagcaccat ctccaaggac atctttggcg agtggaacgt catcagggac 1080

aagtggaacg ccgagtacga cgacatccac ctgaagaaga aggcggtggt gaccgagaag 1140

tatgaggacg atcgcaggaa gtccttcaaa aaaatcggct ccttcagcct cgaacagctc 1200

caggagtatg ccgatgcgga tctgtccgtc gtcgagaagc tgaaggaaat catcattcag 1260

aaggtcgacg agatctataa agtgtacggg tccagcgaga agctgttcga cgccgacttt 1320

gtgctcgaga agtccctcaa aaagaatgac gccgtggtgg ccattatgaa agacctgctc 1380

gactccgtga agtccttcga aaattacatt aaagcgttct ttggggaggg gaaggaaact 1440

aacagggatg agtccttcta tggcgacttt gtcctcgcgt acgacatcct gctgaaggtc 1500

gaccacattt acgacgcgat ccgcaactac gtgacacaga agccgtactc caaagacaag 1560

ttcaagctgt acttccagaa cccgcaattt atgcgcggct gggacaagga tgtcgagaca 1620

gacaggcgcg cgacaattct ccgctatggc tccaaatact atctggccat catggacaag 1680

aagtacgcga agtgcctgca gaagatcgac aaagacgacg tcaatggcaa ctatgaaaag 1740

atcaactaca agctgctgcc gggcccgaac aagatgctcc cgaaggtgtt cttcagcaag 1800

aagtggatgg cctactacaa tccaagcgag gatattcaga aaatctataa aaacgggacc 1860

ttcaagaagg gggacatgtt taacctcaac gactgccaca agctcattga tttcttcaag 1920

gatagcattt cccgctaccc gaaatggtcc aatgcgtacg attttaactt ctccgagaca 1980

gaaaagtaca aagacatcgc gggcttttac aggggaggtgg aggagcaagg gtataaagtt 2040

tcttttgaat ccgcgagcaa gaaggaagtc gacaagctcg tcgaggaggg caagctctac 2100

atgttccaaa tttataacaa ggacttttcc gacaagagcc atgggacccc aaacctccac 2160

accatgtact tcaaactgct ctttgacgag aacaaccacg ggcaaatcag gctgagcggc 2220

ggcgccgaat tattcatgcg cagggcctcc ctcaagaagg aagagctggt cgtccatcca 2280

gccaattccc cgatcgcgaa caagaacccg gacaatccga aaaagaccac caccctgtcc 2340

tacgacgtct acaaggacaa acgcttcagc gaagaccagt acgaattaca catcccaatt 2400

gcgattaata agtgcccaaa gaatatcttc aaaattaata cagaggtcag ggtgctgctc 2460

aaacacgacg acaatccgta tgtcatcggc attgacaggg gcgagcgcaa tctgctctat 2520

atcgtggtcg tggatgggaa gggcaatatt gtggagcagt actccctgaa cgagattatc 2580

aacaacttca atgggattag gattaagacc gactatcaca gcctgctcga caagaaagaa 2640

aaagagaggt ttgaggcccg ccaaaactgg acctccattg agaatatcaa agaattaaag 2700

gccggctata tttcccaagt cgtccacaag atctgcgagc tggtggagaa atatgacgcc 2760

gtgattgcgc tcgaagactt aaattctggg ttcaagaact cccgcgtgaa ggtggaaaaa 2820

caggtgtatc agaaattcga gaaaatgctg atcgacaaac tcaattatat ggtggataag 2880

aagtccaacc cgtgtgccac aggggggcgcg ctgaagggct atcagatcac caacaagttc 2940

gagagcttca agagcatgag cacccagaac gggtttattt tctacatccc ggcgtggctc 3000

acctccaaga ttgacccgag caccggcttc gtgaacctcc tgaagacaaa gtatacctcc 3060

attgccgaca gcaagaagtt tatctcctcc ttcgaccgca ttatgtatgt gccggaggag 3120

gacctcttcg agttcgccct cgactacaaa aacttcagcc gcacagatgc ggattacatc 3180

aagaagtgga agctgtactc ctacgggaac aggatccgca tcttcaggaa tccaaaaaaa 3240

aataacgtct ttgactggga ggaagtgtgc ctgacatccg cctacaagga actgttcaat 3300

aaatacggca tcaattacca gcagggcgac attcgcgccc tcctctgtga gcagtccgac 3360

aaagcgtttt actccagctt catggccctc atgtccctga tgctccaaat gaggaatagc 3420

atcacagggc gcaccgacgt cgacttcctc atcagcccgg tgaagaactc cgacgggatc 3480

ttttacgact cccgcaacta tgaggcgcaa gagaatgcga tcctcccgaa gaacgccgat 3540

gcgaacgggg cctataatat cgccaggaaa gtgctctggg ccatcgggca gttcaaaaag 3600

gcggaggatg agaagctcga caaggtgaaa attgccattt ccaacaagga gtggctggag 3660

tacgcgcaga cctccgtgaa gcacaaaagg ccggcggcca cgaaaaaggc cggccaggca 3720

aaaaagaaaa agggatccta cccatacgat gttccagatt acgcttatcc ctacgacgtg 3780

cctgattatg catacccata tgatgtcccc gactatgcct aa 3822

<210> 2

<211> 18

<212>DNA

<213> Primer

<400> 2:

ttcacaaccg cgtttacc 18

<210> 3

<211> 22

<212>DNA

<213> Primer

<400> 3:

tcaccaccgc cttcttct 18

<210> 4

<211> 21

<212>DNA

<213> Primer

<400> 4:

catggatatt ttagcggatc a 21

<210> 5

<211> 22

<212>DNA

<213> Primer

<400> 5:

ggagatattg gggatgagta ga 22

Claims (9)

1. a kind of PL-LbCpf1-RVR genes that specific site is recognized in paddy gene target practice, it is characterised in that the PL- LbCpf1-RVR genes comprise at least the nucleotide sequence as shown in SEQ ID NO.1 in sequence table.

2. a kind of RVR according to claim 1 can realize the PL-LbCpf1-RVR bases of target practice in paddy gene target practice Cause, it is characterised in that the PL-LbCpf1-RVR genes include the nucleotide sequence shown in SEQ ID NO.1 in sequence table Variant.

3. a kind of expression cassette, it is characterised in that the PL-LbCpf1-RVR genes described in claim 1 are included in the expression cassette.

4. a kind of expression vector, it is characterised in that the expression vector includes the PL-LbCpf1-RVR bases described in claim 1 Expression cassette described in cause or claim 3.

5. the expression cassette described in gene, claim 3 described in a kind of claim 1 or the carrier described in claim 4 should With, it is characterised in that the shearing to rice genome is realized in the application using the PL-LbCpf1-RVR genes, is obtained Obtain the genetically modified plants containing mutational site or plant part.

6. a kind of beaten using the expression vector establishment specific gene containing the PL-LbCpf1-RVR genes described in claim 1 Targeting vector, the method for targeting vector Introduced into Rice cell comprises the steps：

(1) rice paddy seed is shelled, sterilize after embryo is separated, be placed on callus inducing medium and be cured with producing secondary Injured tissue；

(2) the secondary callus is transferred to new callus inducing medium and carries out preculture, obtain callus；

(3) callus obtained in step (2) is contacted 15 minutes with Agrobacterium, wherein, institute is introduced in the Agrobacterium State and the PL-LbCpf1- that the RVR recognizes specific site in paddy gene target practice is carried in targeting vector, the targeting vector RVR genes；

(4) callus after step (3) processing is transferred to and be lined with thereon in the culture dish of aseptic filter paper, 21-23 DEG C of culture 48 hours；

(5) cultivated 5-7 days on screening and culturing medium before the callus after step (4) processing is placed in；

(6) callus after step (5) processing is shifted on screening and culturing medium, to obtain resistant calli；

(7) resistant calli is transferred to seedling differentiation in differentiation and regeneration culture medium；With

(8) seedling obtained in step (7) is transferred in root media and taken root.

7. the expression vector establishment specific gene targeting vector of PL-LbCpf1-RVR genes according to claim 6, will beat The method of targeting vector Introduced into Rice cell, it is characterised in that the paddy rice is japonica rice.

8. the expression vector establishment specific gene targeting vector of PL-LbCpf1-RVR genes according to claim 6, will beat The method of targeting vector Introduced into Rice cell, it is characterised in that methods described also includes carrying out molecule mirror to obtained paddy rice sample It is fixed.

9. the expression vector establishment specific gene targeting vector of PL-LbCpf1-RVR genes according to claim 8, will beat The method of targeting vector Introduced into Rice cell, it is characterised in that the PCR primer that the Molecular Identification is used for 5 '- TTCACAACCGCGTTTACC-3 ' and 5 '-TCACCACCGCCTTCTTCT-3 '.