CN114480469B

CN114480469B - Gene editing carrier loading tuckahoe endogenous sequence, editing system and application

Info

Publication number: CN114480469B
Application number: CN202210139672.5A
Authority: CN
Inventors: 金剑; 钟灿; 谢珍妮; 刘晓柳; 王紫菱; 张水寒
Original assignee: HUNAN ACADEMY OF CHINESE MEDICINE
Current assignee: HUNAN ACADEMY OF CHINESE MEDICINE
Priority date: 2022-02-16
Filing date: 2022-02-16
Publication date: 2023-10-31
Anticipated expiration: 2042-02-16
Also published as: CN114480469A

Abstract

The invention discloses a gene editing carrier for loading tuckahoe endogenous sequences, an editing system and application, wherein the gene editing carrier comprises tuckahoe endogenous RNA polymerase III type U6 type promoter, an endogenous terminator PcTer sequence, an endogenous self-replication sequence PcOri and an endogenous interval sequence PcSse for starting the transcription of coding DNA of sgRNA; the poria cocos endogenous RNA polymerase III type U6 type promoter comprises any one or more of PcU6-1, pcU6-2 and PcU 6-3; the genome editing vector also comprises an expression frame for sgRNA transcription regulated by a tuckahoe endogenous RNA polymerase III type U6 type promoter; in an expression frame of sgRNA transcription regulated by a U6 type promoter of a tuckahoe endogenous RNA polymerase III type, designing a tuckahoe endogenous terminator PcTer sequence after the sgRNA scaffold sequence; the expression frame for sgRNA transcription regulated by the Poria endogenous RNA polymerase III type U6 type promoter can be composed of a plurality of expression frames connected in series, and Poria endogenous spacer sequences PcSse are added between adjacent expression frames. The gene editing system of the invention can be used for transforming and gene editing of poria cocos cells.

Description

Gene editing carrier loading tuckahoe endogenous sequence, editing system and application

Technical Field

The invention relates to the technical field of gene editing, in particular to a gene editing carrier loading tuckahoe endogenous sequences, an editing system and application.

Background

Poria is a large amount of Chinese medicinal raw materials, called as "Jiuling of ten prescriptions", and is also a medicinal and edible Chinese medicinal resource approved by Ministry of health. Modern pharmacological researches show that pachyman and triterpenes are one of the main active ingredients, and have the effects of inhibiting tyrosinase, preventing renal tubular fibrosis, resisting inflammation and tumor, etc. Poria cocos is called indian bread in north america, and is a variety with strong research and development value in china and japan, and is applied to foods and cosmetics in addition to pharmaceuticals. However, the wild resources of the poria cocos are exhausted, and strain degeneration is caused by long-term asexual propagation in the artificial cultivation process. Therefore, it is important to develop a gene editing system for Poria cocos and apply it in the fields of Poria cocos gene modification and germplasm innovation.

Disclosure of Invention

Based on the technical problems in the background technology, the invention provides a gene editing carrier loading tuckahoe endogenous sequences, an editing system and application.

The gene editing vector loaded with the tuckahoe endogenous sequence provided by the invention comprises a tuckahoe endogenous RNA polymerase III type U6 type promoter for starting transcription of coding DNA of sgRNA.

Preferably, the Poria cocos endogenous RNA polymerase III type U6 type promoter comprises any one or more of PcU6-1, pcU6-2, pcU6-3 combinations; wherein:

the nucleotide sequence of PcU-1 is shown in SEQ ID NO. 1;

the nucleotide sequence of PcU-2 is shown in SEQ ID NO. 2;

the nucleotide sequence of PcU-3 is shown as SEQ ID NO. 3.

Preferably, the genome editing vector further comprises an expression cassette for sgRNA transcription regulated by the tuckahoe endogenous RNA polymerase type III U6 promoter.

Preferably, in the expression frame of the sgRNA transcription regulated by the Poria cocos endogenous RNA polymerase III type U6 type promoter, an sgRNA insertion position is constructed after the promoter and before the sgRNA scaffold sequence through a sequence containing an AarI enzyme cutting site, and a Poria cocos endogenous terminator PcTer sequence is designed after the sgRNA scaffold sequence; wherein:

the AarI enzyme cutting site sequence is shown in SEQ ID NO. 4;

the sgRNA scaffold sequence is shown as SEQ ID NO. 5;

the sequence of the tuckahoe endogenous terminator PcTer is shown in SEQ ID NO. 6.

Preferably, the genome editing vector further comprises an expression cassette of Cas9 protein, and the amino acid sequence of the expression cassette of Cas9 protein is shown in SEQ ID No. 7.

Preferably, the genome editing vector further comprises one or more combined selection markers in a resistance gene, fluorescent protein expression cassette.

Preferably, the screening marker is a hygromycin expression cassette, and the nucleotide sequence of the screening marker is shown as SEQ ID NO. 8.

Preferably, the genome editing vector further comprises an endogenous sequence PcOri capable of self-replication in Poria cocos.

Preferably, the nucleotide sequence of the self-replicating endogenous sequence PcOri is shown in SEQ ID NO. 9.

Preferably, the expression cassette for sgRNA transcription regulated by the Poria cocos endogenous RNA polymerase type III U6 promoter is composed of a plurality of tandem.

Preferably, tuckahoe endogenous spacer sequence PcSse is also added between adjacent expression frames, and the nucleotide sequence of the tuckahoe endogenous spacer sequence PcSse is shown in SEQ ID No. 10.

The gene editing system provided by the invention comprises the gene editing vector loaded with the tuckahoe endogenous sequence.

The gene editing method provided by the invention adopts the gene editing system to transform and edit the poria cocos cells.

The invention provides application of the gene editing system in poria cocos cells.

Preferably, the genome editing system is introduced into the poria cells by transforming the protoplasts of the poria cells mediated by PEG, editing is performed at the target site to be edited, and the transformants are selected by using the selection markers to obtain the gene editing mutant.

The beneficial technical effects of the invention are as follows: the gene editing carrier and editing system loaded with the tuckahoe endogenous sequence can realize stable replication, transcription start, termination and gene targeting editing in tuckahoe hyphae, and is beneficial to promoting tuckahoe gene modification and germplasm innovation development.

Drawings

FIG. 1 is a schematic diagram of a gene editing system for loading tuckahoe endogenous sequences according to the present invention.

FIG. 2 shows the endogenous sequence PcU-1-AarI-scale-PcTer-CRISPR/Cas 9 gene editing vector of Poria cocos.

FIG. 3 is a diagram of a tuckahoe endogenous promoter-based PcU6-1 multi-target gene editing system according to the invention.

FIG. 4 shows a multi-target gene editing system based on tuckahoe endogenous promoters PcU-1 and PcU-2.

FIG. 5 shows the transformation of competent cells of E.coli with CRISPR/Cas9 gene editing vector of FC332-PcU 6-1-SgPcLn-scale-PcTer according to the present invention.

Fig. 6 is a microscopic view of tuckahoe cell protoplast according to the invention.

FIG. 7 is a schematic diagram of agarose gel electrophoresis for vector transformation verification according to the present invention; wherein A is the transfer verification of the vector sgRNA, and B is the transfer verification of the vector Cas 9.

FIG. 8 is a bimodal view of the sequencing of sgPcLn target sites according to the present invention.

Detailed Description

Example 1

(1) Screening of Poria endogenous sequences

The genome fasta file is used as a template by adopting TBtools software, 3 sections of upstream 300bp sequences of U6 snRNA gene sequences are respectively extracted from the genome of the poria cocos, and promoter analysis is carried out to find out that the sequence accords with the general characteristics of the promoter, and the sequence is respectively named PcU-1 (the nucleotide sequence is shown as SEQ ID NO. 1), pcU-2 (the nucleotide sequence is shown as SEQ ID NO. 2) and PcU-3 (the nucleotide sequence is shown as SEQ ID NO. 3) and is used as the endogenous promoter sequence of the poria cocos.

(2) Design of sgRNA sequence position

The insertion position of the sgRNA is constructed after the promoter and before the sgRNA scafold sequence by the sequence containing the AarI cleavage site, and the sequence containing the AarI cleavage site in the vector is shown as SEQ ID NO. 4.

(3) Design of sgRNA scaffold sequence

The sgRNA scaffold sequence is shown in SEQ ID NO. 5.

(4) Terminator selection

And respectively extracting 500bp downstream of 3U 6 snRNA gene sequences by using TBtools software and tuckahoe genome fasta files as templates, and taking the downstream 500bp as a potential terminator region. The sequences are compared in Mega, homologous sequences are screened, a terminator sequence of tuckahoe U6 endogenous homology is used as a template, and the sgRNA terminator PcTer sequence of the vector is designed, and the nucleotide sequence is shown as SEQ ID NO. 6.

(5) Vector construction

The plasmid vector pFC332 is taken as a framework, and the framework comprises an expression frame of Cas9 protein (the amino acid sequence of which is shown as SEQ ID NO. 7), an ampicillin expression frame Amp, a hygromycin expression frame Hyg (the nucleotide sequence of which is shown as SEQ ID NO. 8) and a self-replicating expression frame PcOri (the nucleotide sequence of which is shown as SEQ ID NO. 9) which are selected as markers. The BglII (GATC) and PacI (AT) are taken as enzyme cutting sites, and the tuckahoe endogenous promoter PcU 6-1-AarI-scale-PcTer sequence is assembled into a pFC332 framework to construct a CRISPR/Cas9 gene editing system for loading tuckahoe endogenous promoter pFC332-PcU 6-1-AarI-scale-PcTer. The system can utilize an AarI enzyme cutting site to simply insert any sgRNA sequence corresponding to the gene to be knocked out into the vector through single enzyme cutting so as to realize gene editing.

Example 2

(1) Screening of Poria cocos U6 endogenous promoter PcU6-1

The TBtools software is adopted, a genome fasta file is used as a template, a 300bp sequence at the upstream of a 1-section U6 snRNA gene sequence is extracted from a poria cocos genome, and promoter analysis is carried out to find out that the sequence accords with the general characteristics of a promoter, and the sequence is named as PcU-1 (the nucleotide sequence is shown as SEQ ID NO. 1) and is used as a poria cocos endogenous promoter sequence.

(2) Design of sgRNA sequence position

pFC332 vector information was analyzed by Snapgene software, and the construction of the sgRNA insertion site after the promoter and before the sgRNA scaffold sequence was selected by the sequence containing the AarI cleavage site, the sequence of which in the vector is shown in SEQ ID NO. 4.

(3) Design of sgRNA scaffold sequence

The sgRNA scaffold sequence is shown in SEQ ID NO. 5.

(4) Terminator selection

By analysis, the sgRNA expression cassette terminator PcTer sequence in example 1 was used, and the nucleotide sequence was shown in SEQ ID NO. 6.

(5) Construction of a second target site

The terminator sequence of step 4 of this example was followed by the design of an endogenous spacer sequence PcSse inserted into Poria cocos, the spacer nucleotide sequence being shown in SEQ ID NO. 10. Steps 1, 2, 3 and 4 of this example were repeated after the spacer PcSse, wherein step 2 selects the cleavage site sequence in NheI, bstXI, pmeI, stuI, asuII to construct the sgRNA insertion site, completing the expression cassette for constructing the second target site.

(6) Vector construction

The plasmid vector pFC332 is taken as a framework, and the framework comprises an expression frame of Cas9 protein (the amino acid sequence of which is shown as SEQ ID NO. 7), a hygromycin expression frame of which the nucleotide sequence is shown as SEQ ID NO.8 and a self-replication sequence PcOri (the nucleotide sequence of which is shown as SEQ ID NO. 9) as a screening mark. The BglII (GATC) and PacI (AT) are taken as enzyme cutting sites, and the tuckahoe endogenous promoter PcU6-1-sgRNA 1-scaled-PcTer-PcSse-PcU 6-1-sgRNA 2-scaled-PcTer sequence is assembled into a pFC332 framework to construct the CRISPR/Cas9 gene editing system loading tuckahoe endogenous promoter pFC332-PcU6-1-sgRNA 1-scaled-PcTer-PcSse-PcU 6-1-sgRNA 2-scaled-PcTer.

The system can utilize enzyme cutting sites, and insert sgRNA sequences of a plurality of genes to be knocked out corresponding to target sites into the vector so as to realize gene editing.

Similarly, the above-mentioned CRISPR/Cas9 multi-target site gene editing system loaded with the tuckahoe endogenous promoter pFC332-PcU6-1-sgRNA 1-scale-PcTer-PcSse-PcU-1-sgRNA 2-scale-PcTer can be constructed without adopting a cleavage site mode, and the designed sgRNA can be directly constructed into a gene editing vector through a total gene synthesis mode.

Example 3

(1) Screening of Poria cocos U6 endogenous promoter PcU6-1

From the genome sequence of Poria cocos, through annotation comparison, TBtools software is adopted, genome fasta files are used as templates, and upstream 300bp sequences PcU-1 (nucleotide sequences are shown as SEQ ID NO. 1) of 1 section U6 snRNA gene sequences are respectively extracted from the genome of Poria cocos as endogenous promoter sequences.

(2) Design of sgRNA sequence position

(3) Design of sgRNA scaffold sequence

The sgRNA scaffold sequence is shown in SEQ ID NO. 5.

(4) Terminator selection

By analysis, the endogenous terminator PcTer sequence of the sgRNA expression cassette in example 1 was used, and the nucleotide sequence was shown in SEQ ID NO. 6.

(5) Construction of PcU6-2 promoter and second target site

An insertion spacer sequence PcSse was designed after the terminator sequence of step 4 of this example above, and the spacer nucleotide sequence is shown in SEQ ID NO. 10. Steps 1, 2, 3 and 4 of this example were repeated after the spacer sequence PcSse, wherein step 1 selects the tuckahoe endogenous promoter PcU-2 (nucleotide sequence as set forth in SEQ ID No. 1) as the tuckahoe endogenous promoter sequence. And step 2, selecting an enzyme cutting site sequence in NheI, bstXI, pmeI, stuI, asuII to construct an sgRNA insertion position, and completing the construction of an expression frame of a second target site.

(6) Vector construction

The plasmid vector pFC332 is taken as a framework, and the framework comprises an expression frame of Cas9 protein (the amino acid sequence of which is shown as SEQ ID NO. 7), a hygromycin expression frame of which the nucleotide sequence is shown as SEQ ID NO.8 and a self-replicating expression frame (the nucleotide sequence of which is shown as SEQ ID NO. 9) of a screening mark. The BglII (GATC) and PacI (AT) are taken as enzyme cutting sites, and the tuckahoe endogenous promoter PcU6-1-sgRNA 1-scaled-PcTer-PcSse-PcU 6-2-sgRNA 2-scaled-PcTer sequence is assembled into a pFC332 framework to construct the CRISPR/Cas9 gene editing system loading tuckahoe endogenous promoter pFC332-PcU6-1-sgRNA 1-scaled-PcTer-PcSse-PcU 6-2-sgRNA 2-scaled-PcTer.

Similarly, the above-mentioned CRISPR/Cas9 multi-target site gene editing system loaded with the tuckahoe endogenous promoter pFC332-PcU6-1-sgRNA 1-scale-PcTer-PcSse-PcU-2-sgRNA 2-scale-PcTer can be constructed without adopting a cleavage site mode, and the designed sgRNA can be directly constructed into a gene editing vector through a total gene synthesis mode.

Furthermore, pcU6-2 in this embodiment can be replaced with PcU 6-3.

Example 4

Design and vector construction of PcLnc gene target locus sgRNA

(1) Design of sgRNA

Based on PcLn sequence and website analysis, different sites are selected as knockout targets, TBtools software is adopted, and off-target analysis is carried out on the designed sequence. Screening for appropriate target sites sgrnas were designed, whose sequence information is shown in table 1.

TABLE 1 Crispr-Cas9 vector construction of sgRNA sequence information

(2) Construction of sgRNA vectors

AarI is selected as restriction endonuclease, the restriction conditions are carried out according to the optimal restriction conditions of the specification, and the sgRNA is respectively inserted into CRISPR/Cas9 gene editing vectors loaded with tuckahoe endogenous promoters pFC332-PcU 6-1-SgPcLn-scaled-PcTer.

(3) Coli transformed with sgRNA vector

The CRISPR/Cas9 gene editing vector of pFC332-PcU 6-1-SgLn-scale-PcTer is used for transforming competent cells of Tretief5a escherichia coli, plating is carried out, positive transformants with Kna resistance are selected, colony PCR is carried out on the positive transformants, and sequencing is carried out to confirm that the vector construction is successful.

Vector construction validation PCR reaction conditions are shown in Table 2.

TABLE 2 construction of vector to verify PCR reaction conditions

Vector construction the verification PCR primers are shown in table 3.

TABLE 3 construction of vector verification PCR primers

Example 5

Gene editing vector conversion poria cocos implementation target site gene editing

(1) Preparation of Poria protoplast

The protoplast was prepared by enzymatic hydrolysis of Poria mycelium with muramidase (Guangdong microbiological institute). The protoplast preparation conditions were optimized according to the mycelium enzymolysis conditions observed by a microscope, 1.5% lywallzyme was used, at 28 ℃,80rmp, for 2.5 hours, the solution was centrifuged at 3500rpm to obtain a precipitate, the precipitate was resuspended to obtain a cleaner protoplast precipitate, and the protoplast precipitate was dissolved by a mannitol solution to obtain a Poria protoplast solution as shown in fig. 6.

(2) PEG-mediated gene editing vector conversion Poria cocos

The CRISPR/Cas9 gene editing vector of C332-PcU 6-1-SgPcLn-scale-PcTer which is successfully constructed is used for converting Poria cocos protoplast solution by a PEG mediated method, the Poria cocos protoplast solution is placed into a fungus culture medium for 2-3d culture, the Poria cocos protoplast solution is coated on the fungus culture medium, and a layer of fungus culture medium with hygromycin resistance (0.125 mg/ml) is covered after the fine mycelia grow out. After hypha grows out on the covering layer, extracting the poria cocos DNA transformed by the vector by using a CTAB method, and carrying out sequencing verification on Cas9 genes and sequences near the sgRNA of the vector by using a primer, so that Cas9 genes and the sgRNA sequences of the vector can be amplified in the poria cocos, and the result shows that the CRISPR/Cas9 gene editing vector of pC332-PcU 6-1-SgPcLn-scale-PcTer has successfully transformed the poria cocos.

Wherein: the Cas9 gene amplification verified PCR conditions are shown in table 4, the sgRNA gene amplification verified PCR conditions are shown in table 5, and the amplification verified PCR primers are shown in table 6.

TABLE 5Cas9 Gene amplification verification of PCR conditions

TABLE 5gRNA Gene amplification verification of PCR conditions

Table 6 amplification verification PCR primers

(3) Target site gene editing

The target gene sequence is subjected to PCR amplification and sequencing verification by using a CTAB method to extract the poria cocos DNA transformed by the vector, and a double peak appears at the sgPcLn target site of the PcLn sequence (figure 7), which shows that the targeted knockout is successfully realized in the genome of the poria cocos cell nucleus, but only because the poria cocos is a polynuclear cell, not all cell nuclei are knocked out simultaneously, thus the double peak phenomenon appears, and the result shows that the CRISPR/Cas9 gene editing vector of pC 332-PcU-1-SgPcLn-scale-PcTer successfully converts the poria cocos and carries out gene editing. The conditions for gene editing verification PCR are shown in Table 7; the gene editing verification PCR primers are shown in table 8.

TABLE 7 Gene editing validation of PCR reaction conditions

Table 8 Gene editing verification PCR primers

The foregoing description of the preferred embodiments of the invention is not intended to be limiting, but rather is intended to cover all modifications, equivalents, alternatives, and improvements that fall within the spirit and scope of the invention.

Sequence listing

<110> Hunan province of Chinese medicine institute

<120> Gene editing vector carrying Poria endogenous promoter, editing System and application

<160> 10

<170> SIPOSequenceListing 1.0

<210> 1

<211> 304

<212> DNA

<213> PcU6-1(PcU6-1)

<400> 1

gatccatgag ggcgcagaag atgaggacga agcggaggac ggtggctgct gaagagtgtc 60

aatcgccgtg gagcatgtct gcgacagaag gcgcaacgtc gttcgtgcta ttcactggga 120

gccgtcagca tgaactgcag gtagattggc tggcctcttg cctctgtcga tatctgaatc 180

ggccatagtg gctgcagaag ggctccggag aaagcgctcg cacgtgccgg aatggtgaca 240

acccgcgggc tgaacaaaat gcaaaaacgc gtcgacatgc ctcttgggct ggtaactata 300

attg 304

<210> 2

<211> 304

<212> DNA

<213> PcU6-2(PcU6-2)

<400> 2

gatctcgcta ccggctgctc aagcgctcaa tgcccgacac aaccctctct ggagtctgaa 60

gcgcaaactc gtgttcttgc tgcacatata gctacattct gggccacaac gtcccttatt 120

gagcttcgaa atgaggtcag ctgagaggaa gttgtgtaca tccaaagtcc gcatggccag 180

tcacaatcag gtcacaagga ctcacacggc ggtcactgcc catcgctggc gagcgctgtc 240

gagcgtctat ttgttggcag ggaaatcggt cgatatcggc ctatacaggc cccgcatgta 300

agtg 304

<210> 3

<211> 300

<212> DNA

<213> PcU6-3(PcU6-3)

<400> 3

agtttatcgc accataatat gctcacgcta cgcgcataaa gactactaca cgtcctattc 60

acctttttca agtgcatcgg ctgtggtaat cgcagctgtc ggcacgtctg cgtcggccat 120

acaattcggc gaggtaggac gatcatataa catgtgttga ccagtagcgt tctgatcgag 180

taatgacagg ccagagcagg ggggcgccta ttcgatcaag gaagcgccca tttacaaccc 240

gcggggtgca cacaatgcag aactttgccc atgatcatct agcttgaact cctttggttg 300

<210> 4

<211> 36

<212> DNA

<213> AarI cleavage site sequence ()

<400> 4

attggctcng caggtgaaca caancacctg cacacn 36

<210> 5

<211> 76

<212> DNA

<213> sgRNA scaffold sequence ()

<400> 5

gttttagagc tagaaatagc aagttaaaat aaggctagtc cgttatcaac ttgaaaaagt 60

ggcaccgagt cggtgc 76

<210> 6

<211> 45

<212> DNA

<213> terminator PcTer sequence ()

<400> 6

ttttttttgt ttttgcctct gaactccttg cttttgaagt ccttg 45

<210> 7

<211> 1367

<212> PRT

<213> expression cassette of Cas9 protein ()

<400> 7

Met Asp Lys Lys Tyr Ser Ile Gly Leu Asp Ile Gly Thr Asn Ser Val

1 5 10 15

Gly Trp Ala Val Ile Thr Asp Glu Tyr Lys Val Pro Ser Lys Lys Phe

20 25 30

Lys Val Leu Gly Asn Thr Asp Arg His Ser Ile Lys Lys Asn Leu Ile

35 40 45

Gly Ala Leu Leu Phe Asp Ser Gly Glu Thr Ala Glu Ala Thr Arg Leu

50 55 60

Lys Arg Thr Ala Arg Arg Arg Tyr Thr Arg Arg Lys Asn Arg Ile Cys

65 70 75 80

Tyr Leu Gln Glu Ile Phe Ser Asn Glu Met Ala Lys Val Asp Asp Ser

85 90 95

Phe Phe His Arg Leu Glu Glu Ser Phe Leu Val Glu Glu Asp Lys Lys

100 105 110

His Glu Arg His Pro Ile Phe Gly Asn Ile Val Asp Glu Val Ala Tyr

115 120 125

His Glu Lys Tyr Pro Thr Ile Tyr His Leu Arg Lys Lys Leu Val Asp

130 135 140

Ser Thr Asp Lys Ala Asp Leu Arg Leu Ile Tyr Leu Ala Leu Ala His

145 150 155 160

Met Ile Lys Phe Arg Gly His Phe Leu Ile Glu Gly Asp Leu Asn Pro

165 170 175

Asp Asn Ser Asp Val Asp Lys Leu Phe Ile Gln Leu Val Gln Thr Tyr

180 185 190

Asn Gln Leu Phe Glu Glu Asn Pro Ile Asn Ala Ser Gly Val Asp Ala

195 200 205

Lys Ala Ile Leu Ser Ala Arg Leu Ser Lys Ser Arg Arg Leu Glu Asn

210 215 220

Leu Ile Ala Gln Leu Pro Gly Glu Lys Lys Asn Gly Leu Phe Gly Asn

225 230 235 240

Leu Ile Ala Leu Ser Leu Gly Leu Thr Pro Asn Phe Lys Ser Asn Phe

245 250 255

Asp Leu Ala Glu Asp Ala Lys Leu Gln Leu Ser Lys Asp Thr Tyr Asp

260 265 270

Asp Asp Leu Asp Asn Leu Leu Ala Gln Ile Gly Asp Gln Tyr Ala Asp

275 280 285

Leu Phe Leu Ala Ala Lys Asn Leu Ser Asp Ala Ile Leu Leu Ser Asp

290 295 300

Ile Leu Arg Val Asn Thr Glu Ile Thr Lys Ala Pro Leu Ser Ala Ser

305 310 315 320

Met Ile Lys Arg Tyr Asp Glu His His Gln Asp Leu Thr Leu Leu Lys

325 330 335

Ala Leu Val Arg Gln Gln Leu Pro Glu Lys Tyr Lys Glu Ile Phe Phe

340 345 350

Asp Gln Ser Lys Asn Gly Tyr Ala Gly Tyr Ile Asp Gly Gly Ala Ser

355 360 365

Gln Glu Glu Phe Tyr Lys Phe Ile Lys Pro Ile Leu Glu Lys Met Asp

370 375 380

Gly Thr Glu Glu Leu Leu Val Lys Leu Asn Arg Glu Asp Leu Leu Arg

385 390 395 400

Lys Gln Arg Thr Phe Asp Asn Gly Ser Ile Pro His Gln Ile His Leu

405 410 415

Gly Glu Leu His Ala Ile Leu Arg Arg Gln Glu Asp Phe Tyr Pro Phe

420 425 430

Leu Lys Asp Asn Arg Glu Lys Ile Glu Lys Ile Leu Thr Phe Arg Ile

435 440 445

Pro Tyr Tyr Val Gly Pro Leu Ala Arg Gly Asn Ser Arg Phe Ala Trp

450 455 460

Met Thr Arg Lys Ser Glu Glu Thr Ile Thr Pro Trp Asn Phe Glu Glu

465 470 475 480

Val Val Asp Lys Gly Ala Ser Ala Gln Ser Phe Ile Glu Arg Met Thr

485 490 495

Asn Phe Asp Lys Asn Leu Pro Asn Glu Lys Val Leu Pro Lys His Ser

500 505 510

Leu Leu Tyr Glu Tyr Phe Thr Val Tyr Asn Glu Leu Thr Lys Val Lys

515 520 525

Tyr Val Thr Glu Gly Met Arg Lys Pro Ala Phe Leu Ser Gly Glu Gln

530 535 540

Lys Lys Ala Ile Val Asp Leu Leu Phe Lys Thr Asn Arg Lys Val Thr

545 550 555 560

Val Lys Gln Leu Lys Glu Asp Tyr Phe Lys Lys Ile Glu Cys Phe Asp

565 570 575

Ser Val Glu Ile Ser Gly Val Glu Asp Arg Phe Asn Ala Ser Leu Gly

580 585 590

Thr Tyr His Asp Leu Leu Lys Ile Ile Lys Asp Lys Asp Phe Leu Asp

595 600 605

Asn Glu Glu Asn Glu Asp Ile Leu Glu Asp Ile Val Leu Thr Leu Thr

610 615 620

Leu Phe Glu Asp Arg Glu Met Ile Glu Glu Arg Leu Lys Thr Tyr Ala

625 630 635 640

His Leu Phe Asp Asp Lys Val Met Lys Gln Leu Lys Arg Arg Arg Tyr

645 650 655

Thr Gly Trp Gly Arg Leu Ser Arg Lys Leu Ile Asn Gly Ile Arg Asp

660 665 670

Lys Gln Ser Gly Lys Thr Ile Leu Asp Phe Leu Lys Ser Asp Gly Phe

675 680 685

Ala Asn Arg Asn Phe Met Gln Leu Ile His Asp Asp Ser Leu Thr Phe

690 695 700

Lys Glu Asp Ile Gln Lys Ala Gln Val Ser Gly Gln Gly Asp Ser Leu

705 710 715 720

His Glu His Ile Ala Asn Leu Ala Gly Ser Pro Ala Ile Lys Lys Gly

725 730 735

Ile Leu Gln Thr Val Lys Val Val Asp Glu Leu Val Lys Val Met Gly

740 745 750

Arg His Lys Pro Glu Asn Ile Val Ile Glu Met Ala Arg Glu Asn Gln

755 760 765

Thr Thr Gln Lys Gly Gln Lys Asn Ser Arg Glu Arg Met Lys Arg Ile

770 775 780

Glu Glu Gly Ile Lys Glu Leu Gly Ser Gln Ile Leu Lys Glu His Pro

785 790 795 800

Val Glu Asn Thr Gln Leu Gln Asn Glu Lys Leu Tyr Leu Tyr Tyr Leu

805 810 815

Gln Asn Gly Arg Asp Met Tyr Val Asp Gln Glu Leu Asp Ile Asn Arg

820 825 830

Leu Ser Asp Tyr Asp Val Asp His Ile Val Pro Gln Ser Phe Leu Lys

835 840 845

Asp Asp Ser Ile Asp Asn Lys Val Leu Thr Arg Ser Asp Lys Asn Arg

850 855 860

Gly Lys Ser Asp Asn Val Pro Ser Glu Glu Val Val Lys Lys Met Lys

865 870 875 880

Asn Tyr Trp Arg Gln Leu Leu Asn Ala Lys Leu Ile Thr Gln Arg Lys

885 890 895

Phe Asp Asn Leu Thr Lys Ala Glu Arg Gly Gly Leu Ser Glu Leu Asp

900 905 910

Lys Ala Gly Phe Ile Lys Arg Gln Leu Val Glu Thr Arg Gln Ile Thr

915 920 925

Lys His Val Ala Gln Ile Leu Asp Ser Arg Met Asn Thr Lys Tyr Asp

930 935 940

Glu Asn Asp Lys Leu Ile Arg Glu Val Lys Val Ile Thr Leu Lys Ser

945 950 955 960

Lys Leu Val Ser Asp Phe Arg Lys Asp Phe Gln Phe Tyr Lys Val Arg

965 970 975

Glu Ile Asn Asn Tyr His His Ala His Asp Ala Tyr Leu Asn Ala Val

980 985 990

Val Gly Thr Ala Leu Ile Lys Lys Tyr Pro Lys Leu Glu Ser Glu Phe

995 1000 1005

Val Tyr Gly Asp Tyr Lys Val Tyr Asp Val Arg Lys Met Ile Ala Lys

1010 1015 1020

Ser Glu Gln Glu Ile Gly Lys Ala Thr Ala Lys Tyr Phe Phe Tyr Ser

1025 1030 1035 1040

Asn Ile Met Asn Phe Phe Lys Thr Glu Ile Thr Leu Ala Asn Gly Glu

1045 1050 1055

Ile Arg Lys Arg Pro Leu Ile Glu Thr Asn Gly Glu Thr Gly Glu Ile

1060 1065 1070

Val Trp Asp Lys Gly Arg Asp Phe Ala Thr Val Arg Lys Val Leu Ser

1075 1080 1085

Met Pro Gln Val Asn Ile Val Lys Lys Thr Glu Val Gln Thr Gly Gly

1090 1095 1100

Phe Ser Lys Glu Ser Ile Leu Pro Lys Arg Asn Ser Asp Lys Leu Ile

1105 1110 1115 1120

Ala Arg Lys Lys Asp Trp Asp Pro Lys Lys Tyr Gly Gly Phe Asp Ser

1125 1130 1135

Pro Thr Val Ala Tyr Ser Val Leu Val Val Ala Lys Val Glu Lys Gly

1140 1145 1150

Lys Ser Lys Lys Leu Lys Ser Val Lys Glu Leu Leu Gly Ile Thr Ile

1155 1160 1165

Met Glu Arg Ser Ser Phe Glu Lys Asn Pro Ile Asp Phe Leu Glu Ala

1170 1175 1180

Lys Gly Tyr Lys Glu Val Lys Lys Asp Leu Ile Ile Lys Leu Pro Lys

1185 1190 1195 1200

Tyr Ser Leu Phe Glu Leu Glu Asn Gly Arg Lys Arg Met Leu Ala Ser

1205 1210 1215

Ala Gly Glu Leu Gln Lys Gly Asn Glu Leu Ala Leu Pro Ser Lys Tyr

1220 1225 1230

Val Asn Phe Leu Tyr Leu Ala Ser His Tyr Glu Lys Leu Lys Gly Ser

1235 1240 1245

Pro Glu Asp Asn Glu Gln Lys Gln Leu Phe Val Glu Gln His Lys His

1250 1255 1260

Tyr Leu Asp Glu Ile Ile Glu Gln Ile Ser Glu Phe Ser Lys Arg Val

1265 1270 1275 1280

Ile Leu Ala Asp Ala Asn Leu Asp Lys Val Leu Ser Ala Tyr Asn Lys

1285 1290 1295

His Arg Asp Lys Pro Ile Arg Glu Gln Ala Glu Asn Ile Ile His Leu

1300 1305 1310

Phe Thr Leu Thr Asn Leu Gly Ala Pro Ala Ala Phe Lys Tyr Phe Asp

1315 1320 1325

Thr Thr Ile Asp Arg Lys Arg Tyr Thr Ser Thr Lys Glu Val Leu Asp

1330 1335 1340

Ala Thr Leu Ile His Gln Ser Ile Thr Gly Leu Tyr Glu Thr Arg Ile

1345 1350 1355 1360

Asp Leu Ser Gln Leu Gly Gly

1365

<210> 8

<211> 1026

<212> DNA

<213> hygromycin expression cassette ()

<400> 8

atgaaaaagc ctgaactcac cgcgacgtct gtcgagaagt ttctgatcga aaagttcgac 60

agcgtctccg acctgatgca gctctcggag ggcgaagaat ctcgtgcttt cagcttcgat 120

gtaggagggc gtggatatgt cctgcgggta aatagctgcg ccgatggttt ctacaaagat 180

cgttatgttt atcggcactt tgcatcggcc gcgctcccga ttccggaagt gcttgacatt 240

ggggagttca gcgagagcct gacctattgc atctcccgcc gtgcacaggg tgtcacgttg 300

caagacctgc ctgaaaccga actgcccgct gttctccagc cggtcgcgga ggccatggat 360

gcgattgctg cggccgatct tagccagacg agcgggttcg gcccattcgg accgcaagga 420

atcggtcaat acactacatg gcgtgatttc atatgcgcga ttgctgatcc ccatgtgtat 480

cactggcaaa ctgtgatgga cgacaccgtc agtgcgtccg tcgcgcaggc tctcgatgag 540

ctgatgcttt gggccgagga ctgccccgaa gtccggcacc tcgtgcatgc ggatttcggc 600

tccaacaatg tcctgacgga caatggccgc ataacagcgg tcattgactg gagcgaggcg 660

atgttcgggg attcccaata cgaggtcgcc aacatcctct tctggaggcc gtggttggct 720

tgtatggagc agcagacgcg ctacttcgag cggaggcatc cggagcttgc aggatcgccg 780

cgcctccggg cgtatatgct ccgcattggt cttgaccaac tctatcagag cttggttgac 840

ggcaatttcg atgatgcagc ttgggcgcag ggtcgatgcg acgcaatcgt ccgatccgga 900

gccgggactg tcgggcgtac acaaatcgcc cgcagaagcg cggccgtctg gaccgatggc 960

tgtgtagaag tactcgccga tagtggaaac cgacgcccca gcactcgtcc gagggcaaag 1020

gaatag 1026

<210> 9

<211> 332

<212> DNA

<213> self-replicating endogenous sequence PcOri ()

<400> 9

tattagattt aggaatgcaa gataaaacta aatttataca agaaattgct aagtctaaag 60

aaccttggca atatctggcg gctttctttg ctttatataa ttataaacaa gatcctacaa 120

ctataatcca tctacctatt ttatatttag caagttgttc cgggcttcaa catttatccg 180

caattactaa agaagtttct ttagctaaaa atactaatgt tatagcgtta tcagataacc 240

caagagaaga taaaccggct gatttctatt ctttagttct aaatagaacc aatttgaatt 300

tatctataga taaaaatgaa aacttaagaa at 332

<210> 10

<211> 135

<212> DNA

<213> Poria endogenous spacer sequence PcSse ()

<400> 10

cttttaacga tgatcgatga atcgacgaca ctcgggacgc agcattgcga ttcgaattat 60

caagtcaaaa tgatataatt ctacagtaca atacatatgg agagcaaccc aacgatctaa 120

catccaacca taaat 135

Claims

1. A gene editing vector carrying an endogenous sequence of tuckahoe, comprising an endogenous RNA polymerase type III U6 promoter of tuckahoe for initiating transcription of DNA encoding sgRNA;

the poria cocos endogenous RNA polymerase III type U6 type promoter comprises any one or more of PcU6-1, pcU6-2 and PcU 6-3; wherein:

the nucleotide sequence of PcU-1 is shown in SEQ ID NO. 1;

the nucleotide sequence of PcU-2 is shown in SEQ ID NO. 2;

the nucleotide sequence of PcU-3 is shown in SEQ ID NO. 3;

the genome editing vector also comprises an expression frame for sgRNA transcription regulated by the tuckahoe endogenous RNA polymerase III type U6 type promoter;

in an expression frame of the transcription of the sgRNA regulated by the Poria cocos endogenous RNA polymerase III type U6 type promoter, an sgRNA insertion position is constructed after the promoter and before the sgRNA scaffold sequence through a sequence containing an AarI enzyme cutting site, and a Poria cocos endogenous terminator PcTer sequence is designed after the sgRNA scaffold sequence; wherein:

the AarI enzyme cutting site sequence is shown in SEQ ID NO. 4;

the sgRNA scaffold sequence is shown as SEQ ID NO. 5;

the tuckahoe endogenous terminator PcTer sequence is shown in SEQ ID NO. 6;

the genome editing vector also comprises an expression frame of Cas9 protein, wherein the amino acid sequence of the expression frame of the Cas9 protein is shown as SEQ ID NO. 7;

the genome editing vector further comprises an endogenous sequence PcOri capable of self-replication in Poria cocos; the nucleotide sequence of the self-replicating endogenous sequence PcOri is shown in SEQ ID NO. 9.

2. The gene editing vector loaded with an endogenous sequence of tuckahoe of claim 1, wherein the genome editing vector further comprises one or more combined selectable markers of a resistance gene, a fluorescent protein expression cassette.

3. The gene editing vector loaded with tuckahoe endogenous sequences according to claim 2, wherein the screening marker is a hygromycin expression cassette, and the nucleotide sequence of the hygromycin expression cassette is shown as SEQ ID No. 8.

4. The gene editing vector loaded with tuckahoe endogenous sequence according to claim 1, wherein the expression cassette for transcription of sgRNA under the control of tuckahoe endogenous RNA polymerase type III U6 promoter is composed of a plurality of tandem.

5. The gene editing vector loaded with tuckahoe endogenous sequences according to claim 4, wherein tuckahoe endogenous spacer sequences PcSse are also added between adjacent expression cassettes, and the nucleotide sequence of the tuckahoe endogenous spacer sequences PcSse is shown as SEQ ID No. 10.

6. A gene editing system comprising a gene editing vector according to any one of claims 1-5 loaded with an endogenous sequence of Poria cocos.

7. A method of gene editing, wherein the gene editing system of claim 6 is used to transform and edit poria cells.

8. Use of the gene editing system of claim 6 in gene editing of tuckahoe cells.

9. The use of the gene editing system according to claim 8 in gene editing of tuckahoe cells, wherein the gene editing system is introduced into tuckahoe cells by transforming protoplasts of tuckahoe cells mediated by PEG, editing is performed at a target site to be edited, and transformants are selected by using a selection marker to obtain a gene editing mutant.