CN106591335A - Codon vegetalization-transformed LbCpf1 gene and application thereof - Google Patents

Abstract

The invention provides a codon-planted LbCpf1 gene and application thereof. The invention synthesizes the LbCpf1 gene by utilizing rice optimized codon design and synthesis. And the present invention provides an expression cassette and an expression vector, as well as the application of the expression cassette and the expression vector in rice gene editing. The invention utilizes the transformed LbCpf1 gene to construct a plant expression vector, and then constructs a rice targeting vector, which is introduced into rice cells to cause DNA double-strand shearing of rice-specific gene sites. The invention realizes rice gene targeting, because the LbCpf1 modified by codon plantization is suitable for genome splicing of rice.

Description

A kind of LbCpf1 gene of codon phytochemical transformation and application thereof

technical field

The invention relates to the technical fields of biotechnology and plant genetic engineering. Specifically, the present invention relates to the application of a codon-modified LbCpf1 gene in rice gene targeting.

Background technique

CRISPR-nuclease technology is a eukaryotic site-specific gene editing technology developed in recent years. There are two main types of nucleases involved in this technology: Cas9 nuclease and Cpf1 nuclease. Since CRISPR-Cas9 technology was discovered in 2012, due to its simple operation and high efficiency, it has been widely used in gene editing of zebrafish, mice, rats and other animals and plants such as Arabidopsis thaliana, tobacco, sweet orange, corn and rice middle. CRISPR-Cpf1 was discovered in the past two years and has been successfully applied to gene editing in rats and other animals. Compared with CRISPR-Cas9, CRISPR-Cpf1 has the following advantages: 1. Cpf1 can not only cleave DNA, but also cleave RNA; 2. Cpf1 can independently process pre-crRNA to form mature crRNA; 3. Cpf1 protein cleaves DNA double The formation of sticky ends after the chain is more conducive to the occurrence of substitutions or the deletion or insertion of large fragments to a certain extent; 4. Cpf1 provides more sites for gene editing.

But the LbCpf1 currently used was isolated from the prokaryote Escherichia coli, and prokaryotes and eukaryotes have different codon preferences and base compositions. For example, monocotyledonous plants represented by rice have high GC content and stronger codon preference than species such as bacteria and dicotyledonous plants. Therefore, direct use of LbCpf1 without artificial optimized design will affect its expression efficiency in eukaryotic cells, thereby affecting its cutting efficiency of DNA double strands. In addition, since LbCpf1 comes from Escherichia coli, it may have adverse effects on the genome of the transformed recipient, and may also cause concerns about its safety.

Contents of the invention

In view of the above problems, the present invention hopes to transform LbCpf1 into phytocodons. The present invention aims to transform the codons of the LbCpf1 gene into an expression vector through phytochemical transformation, construct a corresponding targeting vector on this basis, and then realize rice-specific gene editing through rice genetic transformation.

Specifically, in the first aspect, the present invention provides a codon phytochemically modified LbCpf1 gene named plant LbCpf1, the LbCpf1 gene at least comprising the nucleotides shown in SEQ ID NO.1 in the sequence listing sequence.

Preferably, the gene consists of the nucleotide sequence shown in SEQ ID NO:1. The sequence is obtained based on the codons of the model crop rice, that is, under the premise of maintaining the coding amino acid sequence unchanged, the codons selected by the inventor from the codons related to rice genes are used to replace the original codons. The phytotransformed plant LbCpf1 sequence is obtained, and then chemically synthesized. Using the gene of the invention can obviously improve the cutting efficiency.

In the second aspect, the present invention provides a plant expression vector containing the plant LbCpf1 gene. The construction method of the plant expression vector is to use the NotI/SacI restriction site to digest the pHUN600 vector with NotI/SacI and recover it. Since the synthetic plant LbCpf1 sequence has NotI/SacI restriction sites at both ends, it can be connected by T4 The enzyme ligated plantLbCpf1 to the pHUN600 vector to obtain the plant expression vector pHUN-plant LbCpf1 (pHUN 611).

On the other hand, on the basis of the expression vector, the corresponding gene targeting vector is constructed according to the actual needs of the experiment.

On the other hand, the present invention provides an expression cassette, characterized in that the expression cassette contains the above-mentioned LbCpf1 gene.

On the other hand, the present invention provides an application of the above-mentioned gene, expression cassette or vector, characterized in that the application includes utilizing the LbCpf1 gene of the codon phytochemical transformation to complete the cutting of the DNA double strand in the rice body, and Under the action of the self-repair system, transgenic plants or plant parts with mutation sites are obtained.

In another aspect, the present invention provides a pHUN-plantLbCpf1 (pHUN 611) expression vector (which contains the LbCpf1 gene of the codon phytochemical transformation), on the basis of the expression vector only need to carry out simple annealing, enzyme digestion The targeting vector (pHUN 611-BEL) of the specific gene can be obtained by 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 611-BEL) of plantLbCpf1 for 15 minutes;

(4) Transfer the callus of step (3) to a petri dish with three sterile filter papers (adding 2.5-3.5 mL of 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 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, wherein the rice is japonica, more preferably, the rice is japonica Nipponbare.

Exemplary formulation of medium in Table 1

The "optimized N6 macroelement" mentioned in the table refers to [NO3-]/[NH4+]=40mM/10mM in the N6 macroelement.

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

atgtccaagctggagaagtttacaaactgttacagcctctccaaaaccctcaggtttaaagcgatcccggtgggcaagacccaggagaacatcgacaacaagaggctcctggtggaagacgagaagcgcgccgaagactacaagggcgtgaagaagctgctcgataggtactacctcagctttattaacgacgtgctgcacagcatcaaactcaagaatctcaacaactacatctccctcttccgcaaaaagacccgcaccgagaaggagaacaaggagctggagaacctggagatcaacctccgcaaggaaatcgccaaagcgttcaagggcaatgaagggtacaagagcctcttcaagaaagacatcatcgaaactatcctcccagagtttctcgatgacaaggacgagatcgcgctggtgaactcctttaacgggttcacaaccgcgtttaccggcttctttgataacagggaaaatatgttctccgaggaggccaagtccaccagcatcgccttcaggtgtatcaacgagaacctcacccgctacatttccaatatggacattttcgagaaggtggatgcgatcttcgataagcacgaggtgcaggagatcaaagagaagattctcaattccgattatgacgtcgaggatttcttcgaaggggagttctttaattttgtgctcacacaagagggcattgacgtgtacaacgcgattatcgggggcttcgtcacagagtccggggagaagattaaggggctgaatgagtacatcaatctgtacaatcagaagaccaagcagaaactgccgaaattcaagccgctctacaagcaagtcctgtccgatagggaaagcctctccttctacggcgagggctataccagcgacgaggaggtgctggaagtcttccgcaacacactgaataagaatagcgagattttctcctccatcaagaagctcgagaagctctttaagaactttgacgagtacagctccg ccgggattttcgtgaagaacgggccggcgatcagcaccatctccaaggacatctttggcgagtggaacgtcatcagggacaagtggaacgccgagtacgacgacatccacctgaagaagaaggcggtggtgaccgagaagtatgaggacgatcgcaggaagtccttcaaaaaaatcggctccttcagcctcgaacagctccaggagtatgccgatgcggatctgtccgtcgtcgagaagctgaaggaaatcatcattcagaaggtcgacgagatctataaagtgtacgggtccagcgagaagctgttcgacgccgactttgtgctcgagaagtccctcaaaaagaatgacgccgtggtggccattatgaaagacctgctcgactccgtgaagtccttcgaaaattacattaaagcgttctttggggaggggaaggaaactaacagggatgagtccttctatggcgactttgtcctcgcgtacgacatcctgctgaaggtcgaccacatttacgacgcgatccgcaactacgtgacacagaagccgtactccaaagacaagttcaagctgtacttccagaacccgcaatttatggggggctgggacaaggataaagagacagactaccgcgcgacaattctccgctatggctccaaatactatctggccatcatggacaagaagtacgcgaagtgcctgcagaagatcgacaaagacgacgtcaatggcaactatgaaaagatcaactacaagctgctgccgggcccgaacaagatgctcccgaaggtgttcttcagcaagaagtggatggcctactacaatccaagcgaggatattcagaaaatctataaaaacgggaccttcaagaagggggacatgtttaacctcaacgactgccacaagctcattgatttcttcaaggatagcatttcccgctacccgaaatggtccaatgcgtacgattttaacttctccgagacagaaaagtacaaagacatcgc gggcttttacagggaggtggaggagcaagggtataaagtttcttttgaatccgcgagcaagaaggaagtcgacaagctcgtcgaggagggcaagctctacatgttccaaatttataacaaggacttttccgacaagagccatgggaccccaaacctccacaccatgtacttcaaactgctctttgacgagaacaaccacgggcaaatcaggctgagcggcggcgccgaattattcatgcgcagggcctccctcaagaaggaagagctggtcgtccatccagccaattccccgatcgcgaacaagaacccggacaatccgaaaaagaccaccaccctgtcctacgacgtctacaaggacaaacgcttcagcgaagaccagtacgaattacacatcccaattgcgattaataagtgcccaaagaatatcttcaaaattaatacagaggtcagggtgctgctcaaacacgacgacaatccgtatgtcatcggcattgacaggggcgagcgcaatctgctctatatcgtggtcgtggatgggaagggcaatattgtggagcagtactccctgaacgagattatcaacaacttcaatgggattaggattaagaccgactatcacagcctgctcgacaagaaagaaaaagagaggtttgaggcccgccaaaactggacctccattgagaatatcaaagaattaaaggccggctatatttcccaagtcgtccacaagatctgcgagctggtggagaaatatgacgccgtgattgcgctcgaagacttaaattctgggttcaagaactcccgcgtgaaggtggaaaaacaggtgtatcagaaattcgagaaaatgctgatcgacaaactcaattatatggtggataagaagtccaacccgtgtgccacagggggcgcgctgaagggctatcagatcaccaacaagttcgagagcttcaagagcatgagcacccagaacgggtttattttctacatcccggcgtggctc acctccaagattgacccgagcaccggcttcgtgaacctcctgaagacaaagtatacctccattgccgacagcaagaagtttatctcctccttcgaccgcattatgtatgtgccggaggaggacctcttcgagttcgccctcgactacaaaaacttcagccgcacagatgcggattacatcaagaagtggaagctgtactcctacgggaacaggatccgcatcttcaggaatccaaaaaaaaataacgtctttgactgggaggaagtgtgcctgacatccgcctacaaggaactgttcaataaatacggcatcaattaccagcagggcgacattcgcgccctcctctgtgagcagtccgacaaagcgttttactccagcttcatggccctcatgtccctgatgctccaaatgaggaatagcatcacagggcgcaccgacgtcgacttcctcatcagcccggtgaagaactccgacgggatcttttacgactcccgcaactatgaggcgcaagagaatgcgatcctcccgaagaacgccgatgcgaacggggcctataatatcgccaggaaagtgctctgggccatcgggcagttcaaaaaggcggaggatgagaagctcgacaaggtgaaaattgccatttccaacaaggagtggctggagtacgcgcagacctccgtgaagcacaaaaggccggcggccacgaaaaaggccggccaggcaaaaaagaaaaagggatcctacccatacgatgttccagattacgcttatccctacgacgtgcctgattatgcatacccatatgatgtccccgactatgcctaa

Description of drawings

Figure 1-1 to Figure 1-4 are the nucleotide sequence alignment of plantLbCpf1 modified by codon phytization and original LbCpf1. Wherein, Optimized is the nucleotide sequence of the optimized plantLbCpf1, and Original is the nucleotide sequence of the original LbCpf1.

Figure 2 is the alignment of the amino acid sequences of the protein encoded by plantLbCpf1 and the original LbCpf1 gene after codon phytization. Optimized is the amino acid sequence encoded by the optimized plantLbCpf1 gene, and Original is the amino acid sequence encoded by the original LbCpf1.

Fig. 3 is a schematic diagram of the PHUN611 (LbCpf1) vector plasmid.

Fig. 4 is the electrophoresis diagram of PCR detection of transgenic plants. The amplified fragment is a part of the LbCpf1 gene, and the fragment size is 496bp. M is DL2kbmarker; PC is positive control; NC is negative control; 1-8 are randomly selected 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 DavidRussell, Molecular Cloning: A Laboratory Manual, 3rd ed., Vols 1, 2; CharlesNeal 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——Phytochemical modification of codons of LbCpf1 gene

According to the information in the GENBANK database, the inventors of the present application collected 92188 CDS sequences of rice, with a total of 34132283 codons, and analyzed the distribution of these codons in rice on the website http://www.kazusa.or.jp/codon/ Usage. For the same amino acid in the same species, different codons are used differently. According to the usage of each codon in rice, the inventors replaced the original codons with a combination of self-designed codons. The LbCpf1 gene from Escherichia coli Carry out plant transformation, obtain a new DNA sequence, and add rice preferred stop codon TGA to the end of this DNA sequence to form a new gene, which is named plantLbCpf1, and the sequence is shown in SEQ ID NO: 1, See Figure 1 for the sequence alignment with LbCpf1.

Further analysis of its base composition, the results are shown in Table 2. It is known from Table 2 that the GC content of plantLbCpf1 is as high as 53.06%, significantly higher than that of LbCpf1, which is 50.30%. 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 plant LbCpf1 and LbCpf1.

The amino acid sequences of proteins encoded by plant LbCpf1 and LbCpf1 genes were analyzed, and the comparison results are shown in Figure 2. It can be seen from Figure 2 that the amino acid sequences of the two are completely consistent.

The designed plantLbCpf1 gene was sent to Suzhou Jinweizhi Biotechnology Co., Ltd. for synthesis, then connected to the PUC57-AMP vector to form the PUC57-AMP-plant LbCpf1 vector, and loaded into E. coli XL-blue strain.

Example 2—Construction of plant targeting vector containing plant LbCpf1 gene

From Escherichia coli XL-blue containing the PUC57-AMP-plant LbCpf1 vector, the plasmid was extracted with the Axygen plasmid extraction kit, digested with NotI/SacI, and the plantLbCpf1 fragment was recovered. At the same time, the NotI/SacI enzyme was used to linearize pHUN600, recover pHUN600, and connect the above-mentioned plantLbCpf1 fragment and pHUN600 fragment with T4 ligase (purchased from TaKaRa Company) to obtain the plant expression vector pHUN600-plant LbCpf1 (Fig. 3). Named pHUN 611.

The nucleotide sequence TTTGGAAGAGAGTGACTGCGCTAGCAATC at position 2463-2487 in the rice BEL gene (LOC_Os03g55240) was selected as the targeting site. The target site sequence was fused to pHUN611 to form pHUN611-BEL. 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 pHUN611-BEL 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 pHUN611-BEL 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 Agrobacterium suspension medium, evenly disperse the blotted callus 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, the callus was transferred to the screening medium (see Table 1 for ingredients), and 25 callus were connected to each culture dish, and cultured 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 got 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 plantLbCpf1 codon phytochemically modified were 5'-TTCACAACCGCGTTTACC-3' and 5'-TCACCACCGCCTTCTTCT-3', and a fragment with a length of 492bp was generated. 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 which were positive after identification, and the positive rate reached 100% (see Figure 4).

Leaf DNA was extracted from all 34 transgenic plants, and the obtained genomic DNA samples were used for PCR analysis. The PCR primers used to amplify the BEL gene fragment were 5'-GTGGAGGTCGACATGACTGAAG-3' and 5'-TTGCACATTCATACAAATTGGT-3', resulting in a 416bp 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 14 of the 34 transgenic plants tested; the mutation efficiency was 41.2%. It shows that the modified Lbcpf1 can cut the rice genome and has a high mutation efficiency.

sequence listing

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

<120> A codon-modified LbCpf1 gene and its application

<130> HCI20160265

<160> 3

<170> PatentIn version 3.5

<210> 1

<211> 3822

<212>DNA

<213> backbone plasmid vector

<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 atggggggct gggacaagga taaagagaca 1620

gactaccgcg 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> 22

<212>DNA

<213> Primer

<400> 2:

gtggaggtcg acatgactga ag 22

<210> 3

<211> 22

<212>DNA

<213> Primer

<400> 3:

ttgcacattc atacaaattg gt 22

Claims (7)

1. the LbCpf1 genes that a kind of codon vegetalization is transformed, it is characterised in that the LbCpf1 genes are including at least such as sequence Nucleotide sequence in list shown in SEQ ID NO.1.

2. the LbCpf1 genes that a kind of codon vegetalization according to claim 1 is transformed, it is characterised in that described LbCpf1 genes are made up of the nucleotide sequence in sequence table shown in SEQ ID NO.1.

3. a kind of expression cassette, it is characterised in that comprising the LbCpf1 genes described in claim 1 in the expression cassette.

4. a kind of expression vector, it is characterised in that the expression vector includes the LbCpf1 genes or right described in claim 1 Require the expression cassette described in 2.

5. the carrier described in the gene described in a kind of claim 1, the expression cassette described in claim 3 or claim 4 should With, it is characterised in that the application includes being realized to paddy gene using the LbCpf1 genes of codon vegetalization transformation The shearing of group, obtains the genetically modified plants containing mutational site or plant part.

6. it is a kind of using containing described in claim 1 codon vegetalization transformation LbCpf1 genes expression vector establishment Specific gene 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 with produce secondary heal Injured tissue；

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

(3) callus obtained in step (2) is contacted 15 minutes with Agrobacterium, wherein, institute is introduced in the Agrobacterium Targeting vector is stated, the LbCpf1 genes of the codon vegetalization transformation are carried in the targeting vector；

(4) callus after step (3) process 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) callus after step (4) process is placed on front screening and culturing medium and is cultivated 5-7 days；

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

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

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

7. the expression vector establishment specific gene of the LbCpf1 genes of codon vegetalization transformation according to claim 1 is beaten Targeting vector, by the method for targeting vector Introduced into Rice cell, it is characterised in that the paddy rice is Jing rice.