CN112266913B - Method for simultaneously knocking out 1-SST and 1-FFT genes of hevea brasiliensis by using CRISPR/Cas9 system - Google Patents

Abstract

The invention relates to the technical field of rubber grass gene editing, in particular to a method for simultaneously knocking out rubber grass 1-SST and 1-FFT genes by using a CRISPR/Cas9 system. The sgRNA of the specific targeting inulin synthase gene 1-SST and 1-FFT provided by the invention can efficiently guide the incision enzyme protein Cas9 to cut at a target position and generate double-stranded DNA break, and combines resistance screening and DNA sequencing to realize accurate and efficient simultaneous knockout of the inulin synthase gene 1-SST and 1-FFT. The invention provides a convenient and efficient method for creating the rubberberis inulin synthase gene mutant, effectively solves the problems of long period, uncertain mutation, high screening difficulty, high cost and the like in conventional mutation breeding, and provides a new method for breeding the excellent germplasm of the rubberberis.

Description

Method for simultaneously knocking out 1-SST and 1-FFT genes of kochia scoparia by using CRISPR/Cas9 system

Technical Field

The invention relates to the technical field of rubus koenigii gene editing, in particular to sgRNA, an expression cassette, a vector and a host cell for simultaneously targeted knockout of rubus koenigii inulin synthase genes 1-SST and 1-FFT by using a CRISPR/Cas9 system and a method for simultaneously targeted knockout of rubus koenigii inulin synthase genes 1-SST and 1-FFT by using a CRISPR/Cas9 system.

Background

Natural rubber is an important strategic material and industrial raw material, mainly produced from hevea brasiliensis. The Taraxacum kok-saghyz Rodin is perennial herb of Taraxacum of Compositae, the root of the Taraxacum can synthesize high-quality natural rubber, the structure and the performance of the Taraxacum kok-saghyz Rodin are similar to those of the natural rubber produced by Brazilian rubber tree, and the Taraxacum kok-saghyz Rodin is a gum-producing crop with great development prospect. In recent years, the rubber grass germplasm obtained through germplasm resource investigation and collection provides abundant materials for variety improvement of rubber grass, but the highest rubber content is only about 9 percent at present, and the cost for extracting natural rubber through rubber grass is still high. Therefore, the creation of new germplasm with high rubber content is the key of the industrialization of the rubber grass.

Inulin and natural rubber are two important metabolites of hevea brasiliensis, the synthetic common substrate of which is sucrose, but the content of inulin at the roots of hevea brasiliensis is much higher than that of natural rubber. The existing research shows that the accumulation of the inulin and the natural rubber at the root of the Hevea brasiliensis has competitiveness, and the inhibition of the synthesis of the inulin is probably an effective way for improving the content of the natural rubber at the root of the Hevea brasiliensis. Sucrose is synthesized into natural rubber (cis-1, 4-polyisoprene) in milk duct cells through a mevalonate pathway (MVA), inulin is specifically accumulated at the roots of the hevea brasiliensis, and the synthesis is completed by taking sucrose as a substrate and catalyzing by sucrose-1-fructosyltransferase (1-SST, EC2.4.1.99) and fructan, namely fructan 1-fructosyltransferase (1-FFT, EC 2.4.1.100). Therefore, the 1-SST and 1-FFT gene of the rubberberis inulin synthase are knocked out, a new germplasm deficient in the synthesis of the rubberberis inulin can be obtained, the supply of sucrose can be improved, and the synthesis of natural rubber is promoted.

The CRISPR/Cas9 system is a high-efficiency genome DNA editing technology, the principle of realizing gene editing is to use a target gene sequence-specific sgRNA to guide Cas9 endonuclease to cut and edit the DNA of a target gene, and the mutation can be stably inherited. Compared with mutagenesis technologies such as chemical mutagenesis, physical mutagenesis, DNA insertion mutation and the like, the CRISPR/Cas9 technology has the advantages of clear target, high mutation efficiency, simultaneous mutation of multiple target points and the like, and is a powerful tool for germplasm innovation. The specificity of CRISPR/Cas9 editing is determined by artificially designed sgrnas, and precisely targeted sgrnas are key to achieving site-directed mutations. However, the simultaneous mutation of multiple target sites by using CRISPR/Cas9 technology has not been reported in the prior art.

Disclosure of Invention

The invention aims to provide sgRNA, an expression cassette, a vector and a host cell for simultaneously targeted knockout of 1-SST and 1-FFT of a rubberberis inulin synthase gene by using a CRISPR/Cas9 system. Another purpose of the invention is to provide a method for simultaneously knocking out 1-SST and 1-FFT genes of the kochia scoparia by using a CRISPR/Cas9 system.

The invention discloses a method for simultaneously knocking out 1-SST and 1-FFT in a targeted manner by using a CRISPR/Cas9 system by taking the 1-SST and 1-FFT genes of the synanthrin synthase as target genes. In the research and development process, multiple groups of targeting sgRNAs are designed aiming at 1-SST and 1-FFT, and the effect of different sgRNA sequences on the efficiency of simultaneous targeted knockout of the 1-SST and the 1-FFT is found to be large. In addition to reducing the off-target effect and improving the targeting efficiency as much as possible, the targeting efficiency of sgrnas targeting 1-SST and 1-FFT genes needs to be coordinated with each other to achieve efficient targeted knockout of both genes.

Specifically, the invention provides the following technical scheme:

in a first aspect, the invention provides sgrnas for simultaneously targeted knockout of rubberberised inulin synthase genes 1-SST and 1-FFT using a CRISPR/Cas9 system, the sgrnas including sgRNA-SST1 and sgRNA-SST2 targeting 1-SST genes and sgRNA-FFT1 and sgRNA-FFT2 targeting 1-FFT genes; the sgRNA-SST1, the sgRNA-SST2, the sgRNA-FFT1 and the sgRNA-FFT2 respectively comprise sequences shown as SEQ ID NO.1-4 in sequence.

The 4 targeted sgRNAs can be used for remarkably improving the efficiency of simultaneous targeted knockout of the 1-SST and 1-FFT genes.

In a second aspect, the invention provides an expression cassette comprising the sgRNA.

The expression cassette described above further comprises a promoter for driving transcription of the sgRNA, the promoter being any one or more selected from the group consisting of promoters AtU6-1, AtU6-29, AtU3b, and AtU3 d.

Preferably, the sgRNA-SST1 transcription is driven by the AtU6-1 promoter, the sgRNA-SST2 transcription is driven by the AtU6-29 promoter, the sgRNA-FFT1 transcription is driven by the AtU3b promoter, and the sgRNA-FFT2 transcription is driven by the AtU3d promoter.

The sequence of the AtU6-1 promoter is 7926-8248 bits of the sequence shown in SEQ ID NO.6, the sequence of the AtU6-29 promoter is 8382-8719 bits of the sequence shown in SEQ ID NO.6, the sequence of the AtU3b promoter is 8854-9197 bits of the sequence shown in SEQ ID NO.6, and the sequence of the AtU3d promoter is 9332-9452 bits of the sequence shown in SEQ ID NO. 6.

The strength of the promoter influences the transcription level of the sgRNAs, and the invention discovers that the combination mode of the promoter and the sgRNAs can enable the sgRNAs to have better coordination effect, and further improves the efficiency of simultaneous targeted knockout of the 1-SST and 1-FFT genes.

Specifically, the structure of the expression cassette is as follows: AtU6-1-sgRNA-SST1-AtU6-29-sgRNA-SST2-AtU3b-sgRNA-FFT1-AtU3d-sgRNA-FFT 2.

As a preferred embodiment of the invention, the sequence of the expression cassette is shown as SEQ ID NO. 5.

In a second aspect, the invention provides a vector for simultaneously targeted knockout of the indexate synzyme genes 1-SST and 1-FFT using the CRISPR/Cas9 system, the vector comprising the sgRNA or comprising the expression cassette.

The vector described above further comprises a Cas9 gene and a selection marker gene. Wherein the Cas9 gene encodes a Cas9 protein. The screening marker gene can be a screening marker gene commonly used in plant transgenic processes, and includes but is not limited to a bar gene and the like.

The vector can be obtained by connecting the expression cassette containing the sgRNA to a common CRISPR/Cas9 gene editing vector framework.

Preferably, the sequence of the vector is shown as SEQ ID NO. 6. The vector can realize efficient 1-SST and 1-FFT gene simultaneous targeted knockout in the kochia scoparia.

In a third aspect, the invention provides a host cell comprising the sgRNA or the expression cassette or the vector.

Preferably, the host cell is a microbial cell or a non-reproductive plant cell.

In a fourth aspect, the invention provides the use of the sgRNA or the expression cassette or the vector or the host cell for increasing the sucrose, fructose or natural rubber content of the roots of hevea brasiliensis, or for reducing the inulin content of the roots of hevea brasiliensis.

The invention also provides application of the sgRNA or the expression cassette or the vector or the host cell in rubber grass genetic breeding or germplasm resource improvement.

Preferably, the application is breeding of a rubberberis inulin synthesis defective germplasm or a high-yield natural rubber germplasm.

In a fifth aspect, the invention provides a method for simultaneously targeted knockout of 1-SST and 1-FFT of a rubberberis inulin synthase gene by using a CRISPR/Cas9 system, which comprises the following steps: and transferring the sgRNA or the expression cassette or the vector into a kok rubber plant, and screening to obtain a kok rubber plant with the inulin synthase gene 1-SST and the inulin synthase gene 1-FFT which are simultaneously knocked out.

Specifically, the method comprises the following steps:

(1) screening specific targeting sgRNAs in coding regions of inulin synthetase genes 1-SST and 1-FFT respectively, wherein the sgRNAs-SST 1(SEQ ID NO.1) and sgRNA-SST2(SEQ ID NO.2) target the 1-SST genes simultaneously, and the sgRNAs-FFT 1(SEQ ID NO.3) and the sgRNAs-FFT 2(SEQ ID NO.4) target the 1-FFT genes simultaneously;

(2) designing and synthesizing a primer for amplifying an expression cassette according to a targeted sgRNA by using an intermediate vector YLgRNA-AtU6/AtU3 as a template, and constructing a promoter, a target and the gRNA into a complete expression cassette through Overlapping PCR amplification;

(3) taking a CRISPR/Cas9 gene editing vector pYLCRISPR/Cas9 binary expression vector as a framework, connecting an expression cassette containing different target sgRNAs with the pYLCRISPR/Cas9 vector to obtain a recombinant expression vector SST-FFT-Cas9, and transferring the recombinant expression vector SST-FFT-Cas9 into an agrobacterium strain;

(4) infecting agrobacterium containing the recombinant expression vector with transformed hevea brasiliensis in agrobacterium-mediated mode, inducing differentiation and resistance screening to obtain regenerated plant;

(5) extracting positive plant DNA, respectively designing specific amplification primers of 1-SST (SEQ ID NO.7 and SEQ ID NO.8) and 1-FFT (SEQ ID NO.9 and SEQ ID NO.10) genes, amplifying a segment spanning a sgRNA site through PCR (polymerase chain reaction) and carrying out sequencing verification to identify a plant mutation site, and screening to obtain the hevea brasiliensis in which the 1-SST and 1-FFT genes are mutated simultaneously.

In the step (2), the intermediate vector YLgRNA-AtU6/AtU3 is used as a template, and primers SST1-gRT1(SEQ ID NO.11), SST1-AtU6-1T1(SEQ ID NO.12), SST2-gRT2(SEQ ID NO.13), SST 2-2-29T 2(SEQ ID NO.14), FFT 2-2 (SEQ ID NO.15), FFT 2-2 bT 2(SEQ ID NO.16), FFT 2-2 (SEQ ID NO.17), FFT 2-2 dT 2(SEQ ID NO.18) are used for amplification by using the primers SST 685 1-gRT1, SST 2-2 sgRNA, AtU 2-29-SST 685 4-sgRNA, AtU 3-2 sgRNA and AtU 2-2 sgRNA.

In the step (3), in the recombinant expression vector SST-FFT-Cas9, the AtU6-1 promoter is used for driving the sgRNA-SST1(SEQ ID NO.1), the AtU6-29 promoter is used for driving the sgRNA-SST2(SEQ ID NO.2), the AtU3b promoter is used for driving the sgRNA-FFT1(SEQ ID NO.3), the AtU3d promoter is used for driving the sgRNA-FFT2(SEQ ID NO.4), and the sequence of the recombinant expression vector SST-FFT-Cas9 is shown in SEQ ID NO. 6.

In the step (4), the root section of the tissue culture seedling of the kochia scoparia is adopted for infection transformation.

The invention has the beneficial effects that: the sgRNA of the specific targeting inulin synthase gene 1-SST and 1-FFT provided by the invention can efficiently guide the incision enzyme protein Cas9 to cut at a target position and generate double-stranded DNA break, and realizes the accurate and efficient simultaneous knockout of the inulin synthase gene 1-SST and 1-FFT by combining resistance screening and DNA sequencing. The invention provides a convenient and efficient method for creating the rubberberis inulin synthase gene mutant, effectively solves the problems of long period, uncertain mutation, high screening difficulty, high cost and the like in conventional mutation breeding, and provides a new method for breeding the excellent germplasm of the rubberberis.

Drawings

FIG. 1 is a structural diagram of CRISPR/Cas9 recombinant expression vector SST-FFT-Cas9 targeting inulin synthase gene 1-SST and 1-FFT simultaneously in example 1 of the invention.

FIG. 2 shows the DNA sequencing results of T0 generation plants in the target region of 1-SST and 1-FFT genes in example 3 of the invention, wherein 1-SST/WT and 1-FFT/WT are the partial sequences of 1-SST and 1-FFT genes of wild type control WT, respectively, mutant M2-2 is the partial sequence of the transformed plant gene, and mutant M2-2 has 2 base deletions in the 2 nd target (sgRNA-SST2) of the 1-SST gene, thereby leading to the change of the gene reading frame and the gene inactivation; meanwhile, compared with the wild control WT, the 1-FFT gene sequence has a large fragment deletion between a target 1(sgRNA-FFT1) and a target 2(sgRNA-FFT2), which deletes 504bp, thereby causing gene inactivation.

FIG. 3 shows the phenotype of T0 generation mutant plant and wild type plant in example 3 of the present invention, and the mutant M2-2 in T0 generation has obviously wider leaves and shorter leaf length compared with wild type control WT.

Detailed Description

The following examples are intended to illustrate the invention but are not intended to limit the scope of the invention. The primer information of each example of the present invention is shown in table 1.

TABLE 1 primer information

Example 1 sgRNA design of Merrea rubber inulin synthase genes 1-SST and 1-FFT and construction of CRISPR/Cas9 recombinant expression vector SST-FFT-Cas9 thereof

1. sgRNA targeting site screening

Because the hevea brasiliensis is a self-incompatible plant, gene sequences of different strains have differences, PAM sequences (NGG) are respectively searched in conserved regions of two genes by comparing 1-SST and 1-FFT gene sequences in different germplasm, a sequence of 20bp at the 5' end of the PAM position is a sgRNA sequence, and the specificity of a target is searched and analyzed in a published hevea brasiliensis genome sequence.

The invention determines 2 sgRNAs (sgRNA-SST1 and sgRNA-SST2) targeting 1-SST genes through a large amount of screening, wherein the sequences of the 2 sgRNAs are shown as SEQ ID NO.1 and SEQ ID NO.2 (both are positioned in a 4 th exon), and 2 sgRNAs (sgRNA-FFT1 and sgRNA-FFT2) targeting 1-FFT genes are shown as SEQ ID NO.3 and SEQ ID NO.4 (both are positioned in a 2 nd exon).

2. Construction of CRISPR/Cas9 recombinant expression vector SST-FFT-Cas9

The reference reports methods (Liu flare light topic group, Ma X, Zhang Q, Zhu Q, et al.. A robust CRISPR/Cas9 system for meeting, High-Efficiency multiplex gene editing in monocot and binary plants [ J]Molecular Plant,2015,8(8): 1274. su 1284.), expression vectors for AtU6-1 driving sgRNA-SST1, AtU6-29 driving sgRNA-SST2, AtU3b driving sgRNA-FFT1, AtU3d driving sgRNA-FFT2 and Cas9 proteins are constructed by using an overlappinging PCR and enzyme digestion connection method, and finally the 4 fragments are simultaneously connected into Bsa I digested pYLCRISPR/Cas9P _ubi -B vector. The specific process is as follows:

(1) construction of sgRNA expression cassette

Overlaying PCR first round PCR amplification: an intermediate vector YLgRNA-AtU6/AtU3 is used as a template, and an amplification primer pair of AtU6-1-SST1-sgRNA expression cassettes: U-F/SST1-AtU6-1T1 (reaction 1), gR-T/SST1-gRT1 (reaction 2); AtU6-29-SST2-sgRNA expression cassette amplification primer pair: U-F/SST2-AtU6-29T2 (reaction 1), gR-T/SST2-gRT2 (reaction 2); AtU3b-FFT1-sgRNA expression cassette amplification primer pair: U-F/FFT1-AtU3bT3 (reaction 1), gR-T/FFT1-gRT3 (reaction 2); AtU3d-FFT2-sgRNA expression cassette amplification primer pair: U-F/FFT2-AtU3dT4 (reaction 1), gR-T/FFT2-gRT4 (reaction 2). PCR amplification System: 2X Phanta Max Buffer 7.5. mu.L; 0.25 mu L of 10mmol/L dNTPs Mix; phanta Max Polymerase 0.2U; YLgRNA-AtU6/AtU 32-5 ng; 10. mu. mol/L U-F and U # -T # each 0.3. mu.L (reaction 1); 10. mu. mol/L gR-T # and gR-R each 0.3. mu.L (reaction 2); ddH ₂ O make up to 15. mu.L. The PCR amplification procedure was: amplification is carried out for 25-26 cycles at 95 ℃ for 10s,58 ℃ for 15s and 72 ℃ for 15 s.

Second round PCR: the products of the expression reactions 1 and 2 were diluted 10 times and mixed to serve as templates, and the primer pairs Pps-R/Pgs-2 (for AtU6-1-SST1-sgRNA expression cassettes), Pps-2/Pgs-3 (for AtU6-29-SST2-sgRNA expression cassettes), Pps-3/Pgs-4 (for AtU3b-FFT1-sgRNA expression cassettes), and Pps-4/Pgs-L (for AtU3d-FFT2-sgRNA expression cassettes) were used to perform the second round of PCR reaction. PCR amplification System: 2 × Phanta Max Buffer 15 μ L; 0.5 mu L of 10mmol/L dNTP Mix; phanta Max 0.4U; 0.5 mu L of 10 mu mol/L mixed universal primer; reaction 1+ reaction 2 diluent 1 μ L; ddH ₂ O to 30 μ L; the PCR amplification procedure was: amplifying at 95 ℃ for 10s,58 ℃ for 15s and 72 ℃ for 20s for 25-28 PCR cycles.

(2) Cloning of sgRNA expression cassette into pYLCRISPR/Cas9P _ubi -on a B support: assembling the sgRNA expression cassette to pYLCRISPR/Cas9P in the "side-by-side" method using the Bsa I-based enzymatic cleavage and ligation-based "gold gate" cloning method _ubi -B on a carrier. Reaction system: 10 × CutSmart Buffer 1.5 μ L; 1.5 μ L of 10mmol/L ATP; pYLCRISPR/Cas9P _ubi -60-80 ng of plasmid B; 10-15 ng of each expression cassette of the purified mixed sgRNA expression cassettes, and about 60-70 ng of 4 expression cassettes; bsa I-HF 10U; t4 DNA ligase 35U; ddH ₂ O make up to 15. mu.L. Carrying out edge cutting continuous reaction for 10-15 cycles (5 min at 37 ℃, 5min at 10 ℃ and 5min at 20 ℃) by using variable temperature circulation (a PCR instrument can be used); finally 5min at 37 ℃.

(3) And (3) conversion of a connecting product: the ligation product was dropped on Millipore VSWP04700 suspended on a 0.2 XTE dialysis membrane and dialyzed at 4 ℃ for 15-20 min. E.coli DH10B competent cells were transformed by electric stimulation with 1-1.5. mu.L of the dialyzed ligation product. After electric stimulation, 1mL of SOC medium was added and the culture was resumed at 37 ℃ for 1h, and the transformed cells were plated on LB plates containing kanamycin (25. mu.g/mL) overnight.

(4) Screening positive clones: a plurality of colonies are picked for culture and plasmid extraction, AscI enzyme digestion and electrophoresis confirmation are carried out, then sequencing verification is carried out, the success of construction is confirmed, and the structure of the recombinant vector SST-FFT-Cas9 is shown in figure 1.

3. The recombinant vector SST-FFT-Cas9 is introduced into agrobacterium: plasmids were extracted from the positive clones obtained in 2 above, and Agrobacterium (e.g., AGL1) was electrically transformed. Positive clones were verified by PCR amplification using all target adaptor forward and reverse primer pairs.

Example 2 transformation of Geranium strictipes with recombinant expression vector SST-FFT-Cas9 to obtain regenerated plants

1. Obtaining the sterile seedling of the Hemsleya amabilis: seeds of the hevea brasiliensis strain 20112 are taken as materials, sodium hypochlorite solution with the concentration of 10-15% (v/v) (the stock solution is used according to 100%), 1 drop (20 mu L)/200mL of the sodium hypochlorite solution is added with TritonX-100 and mixed uniformly to prepare disinfectant, the seeds are disinfected by the disinfectant for 10-15 minutes, the disinfectant is washed by the disinfectant for 3-5 times and then sowed in 1/2MS culture medium for accelerating germination, and after 5 days, the seeds are transferred to 1/2MS culture medium once for about 20 days to obtain aseptic seedlings with good root growth conditions.

2. The roots of aseptic seedlings in good growth state are taken, washed with aseptic water for 2-3 times, and then sucked dry by filter paper for infection transformation.

3. And (3) agrobacterium infection transformation: agrobacterium AGL1 containing recombinant plasmid SST-FFT-Cas9 was suspended with an infection solution (MS +:20g/L sucrose +1.5 mg/L6-BA +0.05mg/L NAA + 200. mu.M acetosyringone) to adjust OD ₆₀₀ The value is 0.5-0.8, the roots are cut into root segments of 0.5-1.0cm and are infected for 20-30 minutes.

4. The infected root segments are transferred to filter paper to be cultured for 1 to 3 days in the dark at the temperature of between 21 and 25 ℃, and then transferred to a differentiation medium (MS +20g/L of sucrose +1.5mg/L of 6-BA +0.05mg/L of NAA +500mg/L of timentin +3.95g/L of plant gel +5mg/L of Basta) to induce and differentiate adventitious buds, wherein the differentiation culture conditions are 21 to 25 ℃, the light cycle is 16 to 18 hours in the light, and the dark is 6 to 8 hours.

5. Rooting and resistance screening of the regenerated plants: transferring the differentiated adventitious bud to a rooting culture medium (1/2MS +10g/L sucrose +300mg/L timentin +4.0g/L plant gel +10mg/L Basta), transferring the same culture medium every 2-3 times, and co-transferring for 2-3 times for resistance screening to finally obtain a resistant regeneration plant.

6. Hardening and transplanting seedlings: and opening a seal of the regenerated plant with good rooting for 3-4 days, then transplanting the regenerated plant into a matrix, and covering a clean plastic film to keep humidity. And after 3 days, the opening of the holes is gradually opened to prevent the wind, so that the plants adapt to the external environment.

EXAMPLE 3 detection of mutations in transgenic plants and their phenotypes

1. Screening and detecting of transformed plants

DNA extraction: genomic DNA of T0-generation plants obtained in example 2 was extracted using a DNA extraction kit from Tiangen Biochemical technology (Beijing) Ltd.

2. PCR amplification of 1-SST and 1-FFT genes

The T0 generation plants were PCR amplified using KOD-FX (TOYOBO, cat. No. KFX-101, Shanghai) in the reaction scheme: 80-100ng of genomic DNA, 1.5 mu L of each of upstream and downstream primers (SST-390F/SST-1355R primer pair for amplifying 1-SST gene target site; and 10 mu mol/L of each of FFT-338F/FFT-1322R primer pair for amplifying 1-FFT gene target site), 2 XPCR Buffer 25 mu L, dNTP Mix 10 mu L, KOD polymerase1.0 mu L, and ddH ₂ O to 50. mu.L. The PCR amplification procedure was: denaturation at 98 deg.C for 2 min; denaturation at 98 ℃ for 10s, annealing at 55 ℃ for 30s, and extension at 68 ℃ for 30s for 32 cycles; finally, extending for 2min at 72 ℃; storing at 4 ℃.

3. Sample sequencing and sequence analysis thereof

PCR products of the above 1-SST and 1-FFT genes were recovered and purified, respectively, and then ligated to T-vector, and sent to Guangzhou Egyptian Biotechnology Ltd for sequencing verification. The sequencing result is shown in figure 2, 1-SST/WT and 1-FFT/WT are respectively the 1-SST and 1-FFT gene partial sequences of wild type control WT, mutant M2-2 is the transformed plant gene partial sequence, and it can be seen from the figure that mutant M2-2 has 2 base deletions in the 2 nd target (sgRNA-SST2) of 1-SST gene, thereby leading to gene reading frame change and gene inactivation; meanwhile, compared with a wild control WT, the 1-FFT gene sequence has large fragment deletion between a target 1(sgRNA-FFT1) and a target 2(sgRNA-FFT2), and 504bp is deleted, so that the gene is inactivated; the method provided by the invention is used for successfully realizing the simultaneous editing of the inulin synthase genes 1-SST and 1-FFT. 1 strain with simultaneous mutation of 1-SST and 1-FFT was detected in 17 resistant plants, and the mutation frequency of the double genes was 5.88%.

4. Phenotypic observation of mutants

As shown in fig. 3 and table 2, the mutant M2-2 of T0 generation has significantly wider leaves, shorter leaf length, significantly higher sucrose and fructose contents in roots, and significantly lower inulin content, compared with the wild type control WT. The result shows that the inulin content of the plant is reduced after the inulin synthetase genes 1-SST and 1-FFT are knocked out in the mutant, the homozygous inulin synthesis defective mutant is hopeful to be obtained through multi-generation purification, and an excellent material is provided for the later-stage breeding of the gum-producing type hevea brasiliensis strain.

TABLE 2 phenotypic statistics of mutant M2-2 and wild-type control WT

Name of plant	Ye Kuan	Leaf length	Sucrose content	Fructose content	Inulin content
						WT
20112	0.57	6.21	2.35％	1.64％	21.22％
						M2-2	1.48	4.15	8.83％	6.81％	6.77％

Although the invention has been described in detail hereinabove with respect to a general description and specific embodiments thereof, it will be apparent to those skilled in the art that modifications or improvements may be made thereto based on the invention. Accordingly, such modifications and improvements are intended to be within the scope of the invention as claimed.

Sequence listing

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aatagcaagt taaaataagg ctagtccgtt atcaacttga aaaagtggca ccgagtcggt 1380

gctttttttc aagagcttgg agtggatgga aaggactaca tggaatcggc agcaaaggaa 1440

taagcttatg atttcttttt tcttacgaat tttgcgtccc acatcggtaa gcgagtgaag 1500

aaataactgc tttatatatg gctacaaagc accattggtc ataagtggac cccggataac 1560

cgttttagag ctagaaatag caagttaaaa taaggctagt ccgttatcaa cttgaaaaag 1620

tggcaccgag tcggtgcttt ttttcaagag cttggagtgg atggatacgc g 1671

<210> 6

<211> 18075

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 6

ccagccagcc aacagctccc cgaccggcag ctcggcacaa aatcaccact cgatacaggc 60

agcccatcag tccgggacgg cgtcagcggg agagccgttg taaggcggca gactttgctc 120

atgttaccga tgctattcgg aagaacggca actaagctgc cgggtttgaa acacggatga 180

tctcgcggag ggtagcatgt tgattgtaac gatgacagag cgttgctgcc tgtgatcacc 240

gcggtttcaa aatcggctcc gtcgatacta tgttatacgc caactttgaa aacaactttg 300

aaaaagctgt tttctggtat ttaaggtttt agaatgcaag gaacagtgaa ttggagttcg 360

tcttgttata attagggaag gtgcgaataa gcggggaaat tcttctcggc tgactcagtc 420

atttcatttc ttcatgtttg agccgatttt ttctcccgta aatgccttga atcagcctat 480

ttagaccgtt tcttcgccat ttaaggcgtt atccccagtt tttagtgaga tctctcccac 540

tgacgtatca tttggtccgc ccgaaacagg ttggccagcg tgaataacat cgccagttgg 600

ttatcgtttt tcagcaaccc cttgtatctg gctttcacga agccgaactg tcgcttgatg 660

atgcgaaatg ggtgctccac cctggcccgg atgctggctt tcatgtattc gatgttgatg 720

gccgttttgt tcttgcgtgg atgctgtttc aaggttctta ccttgccggg gcgctcggcg 780

atcagccagt ccacatccac ctcggccagc tcctcgcgct gtggcgcccc ttggtagccg 840

gcatcggctg agacaaattg ctcctctcca tgcagcagat tacccagctg attgaggtca 900

tgctcgttgg ccgcggtggt gaccaggctg tgggtcaggc cactcttggc atcgacacca 960

atgtgggcct tcatgccaaa gtgccactga ttgcctttct tggtctgatg catctccgga 1020

tcgcgttgct gctctttgtt cttggtcgag ctgggtgcct caatgatggt ggcatcgacc 1080

aaggtgcctt gagtcatcat gacgcctgct tcggccagcc agcgattgat ggtcttgaac 1140

aattggcggg ccagttgatg ctgctccagc aggtggcgga aattcatgat ggtggtgcgg 1200

tccggcaagg cgctatccag ggataaccgg gcaaacagac gcatggaggc gatttcgtac 1260

agagcatctt ccatcgcgcc atcgctcagg ttgtaccaat gctgcatgca gtgaatgcgt 1320

agcatggttt ccagcggata aggtcgccgg ccattaccag ccttggggta aaacggctcg 1380

atgacttcca ccatgttttg ccatggcaga atctgctcca tgcgggacaa gaaaatctct 1440

tttctggtct gacggcgctt actgctgaat tcactgtcgg cgaaggtaag ttgatgactc 1500

atgatgaacc ctgttctatg gctccagatg acaaacatga tctcatatca gggacttgtt 1560

cgcaccttcc ttagcttctt ggggtatctt taaatactgt agaaaagagg aaggaaataa 1620

taaatggcta aaatgagaat atcaccggaa ttgaaaaaac tgatcgaaaa ataccgctgc 1680

gtaaaagata cggaaggaat gtctcctgct aaggtatata agctggtggg agaaaatgaa 1740

aacctatatt taaaaatgac ggacagccgg tataaaggga ccacctatga tgtggaacgg 1800

gaaaaggaca tgatgctatg gctggaagga aagctgcctg ttccaaaggt cctgcacttt 1860

gaacggcatg atggctggag caatctgctc atgagtgagg ccgatggcgt cctttgctcg 1920

gaagagtatg aagatgaaca aagccctgaa aagattatcg agctgtatgc ggagtgcatc 1980

aggctctttc actccatcga catatcggat tgtccctata cgaatagctt agacagccgc 2040

ttagccgaat tggattactt actgaataac gatctggccg atgtggattg cgaaaactgg 2100

gaagaagaca ctccatttaa agatccgcgc gagctgtatg attttttaaa gacggaaaag 2160

cccgaagagg aacttgtctt ttcccacggc gacctgggag acagcaacat ctttgtgaaa 2220

gatggcaaag taagtggctt tattgatctt gggagaagcg gcagggcgga caagtggtat 2280

gacattgcct tctgcgtccg gtcgatcagg gaggatatcg gggaagaaca gtatgtcgag 2340

ctattttttg acttactggg gatcaagcct gattgggaga aaataaaata ttatatttta 2400

ctggatgaat tgttttagta cctagaatgc atgaccaaaa tcccttaacg tgagttttcg 2460

ttccactgag cgtcagaccc cgtagaaaag atcaaaggat cttcttgaga tccttttttt 2520

ctgcgcgtaa tctgctgctt gcaaacaaaa aaaccaccgc taccagcggt ggtttgtttg 2580

ccggatcaag agctaccaac tctttttccg aaggtaactg gcttcagcag agcgcagata 2640

ccaaatactg tccttctagt gtagccgtag ttaggccacc acttcaagaa ctctgtagca 2700

ccgcctacat acctcgctct gctaatcctg ttaccagtgg ctgctgccag tggcgataag 2760

tcgtgtctta ccgggttgga ctcaagacga tagttaccgg ataaggcgca gcggtcgggc 2820

tgaacggggg gttcgtgcac acagcccagc ttggagcgaa cgacctacac cgaactgaga 2880

tacctacagc gtgagctatg agaaagcgcc acgcttcccg aagggagaaa ggcggacagg 2940

tatccggtaa gcggcagggt cggaacagga gagcgcacga gggagcttcc agggggaaac 3000

gcctggtatc tttatagtcc tgtcgggttt cgccacctct gacttgagcg tcgatttttg 3060

tgatgctcgt caggggggcg gagcctatgg aaaaacgcca gcaacgcggc ctttttacgg 3120

ttcctggcct tttgctggcc ttttgctcac atgttctttc ctgcgttatc ccctgattct 3180

gtggataacc gtattaccgc ctttgagtga gctgataccg ctcgccgcag ccgaacgacc 3240

gagcgcagcg agtcagtgag cgaggaagcg gaagagcgcc tgatgcggta ttttctcctt 3300

acgcatctgt gcggtatttc acaccgcata tggtgcactc tcagtacaat ctgctctgat 3360

gccgcatagt taagccagta tacactccgc tatcgctacg tgactgggtc atggctgcgc 3420

cccgacaccc gccaacaccc gctgacgcgc cctgacgggc ttgtctgctc ccggcatccg 3480

cttacagaca agctgtgacc gtctccggga gctgcatgtg tcagaggttt tcaccgtcat 3540

caccgaaacg cgcgaggcag ggtgccttga tgtgggcgcc ggcggtcgag tggcgacggc 3600

gcggcttgtc cgcgccctgg tagattgcct ggccgtaggc cagccatttt tgagcggcca 3660

gcggccgcga taggccgacg cgaagcggcg gggcgtaggg agcgcagcga ccgaagggta 3720

ggcgcttttt gcagctcttc ggctgtgcgc tggccagaca gttatgcaca ggccaggcgg 3780

gttttaagag ttttaataag ttttaaagag ttttaggcgg aaaaatcgcc ttttttctct 3840

tttatatcag tcacttacat gtgtgaccgg ttcccaatgt acggctttgg gttcccaatg 3900

tacgggttcc ggttcccaat gtacggcttt gggttcccaa tgtacgtgct atccacagga 3960

aagagaactt ttcgaccttt ttcccctgct agggcaattt gccctagcat ctgctccgta 4020

cattaggaac cggcggatgc ttcgccctcg atcaggttgc ggtagcgcat gactaggatc 4080

gggccagcct gccccgcctc ctccttcaaa tcgtactccg gcaggtcatt tgacccgatc 4140

agcttgcgca cggtgaaaca gaacttcttg aactctccgg cgctgccact gcgttcgtag 4200

atcgtcttga acaaccatct ggcttctgcc ttgcctgcgg cgcggcgtgc caggcggtag 4260

agaaaacggc cgatgccggg atcgatcaaa aagtaatcgg ggtgaaccgt cagcacgtcc 4320

gggttcttgc cttctgtgat ctcgcggtac atccaatcag ctagctcgat ctcgatgtac 4380

tccggccgcc cggtttcgct ctttacgatc ttgtagcggc taatcaaggc ttcaccctcg 4440

gataccgtca ccaggcggcc gttcttggcc ttcttcgtac gctgcatggc aacgtgcgtg 4500

gtgtttaacc gaatgcaggt ttctaccagg tcgtctttct gctttccgcc atcggctcgc 4560

cggcagaact tgagtacgtc cgcaacgtgt ggacggaaca cgcggccggg cttgtctccc 4620

ttcccttccc ggtatcggtt catggattcg gttagatggg aaaccgccat cagtaccagg 4680

tcgtaatccc acacactggc catgccggcc ggccctgcgg aaacctctac gtgcccgtct 4740

ggaagctcgt agcggatcac ctcgccagct cgtcggtcac gcttcgacag acggaaaacg 4800

gccacgtcca tgatgctgcg actatcgcgg gtgcccacgt catagagcat cggaacgaaa 4860

aaatctggtt gctcgtcgcc cttgggcggc ttcctaatcg acggcgcacc ggctgccggc 4920

ggttgccggg attctttgcg gattcgatca gcggccgctt gccacgattc accggggcgt 4980

gcttctgcct cgatgcgttg ccgctgggcg gcctgcgcgg ccttcaactt ctccaccagg 5040

tcatcaccca gcgccgcgcc gatttgtacc gggccggatg gtttgcgacc gctcacgccg 5100

attcctcggg cttgggggtt ccagtgccat tgcagggccg gcaggcaacc cagccgctta 5160

cgcctggcca accgcccgtt cctccacaca tggggcattc cacggcgtcg gtgcctggtt 5220

gttcttgatt ttccatgccg cctcctttag ccgctaaaat tcatctactc atttattcat 5280

ttgctcattt actctggtag ctgcgcgatg tattcagata gcagctcggt aatggtcttg 5340

ccttggcgta ccgcgtacat cttcagcttg gtgtgatcct ccgccggcaa ctgaaagttg 5400

acccgcttca tggctggcgt gtctgccagg ctggccaacg ttgcagcctt gctgctgcgt 5460

gcgctcggac ggccggcact tagcgtgttt gtgcttttgc tcattttctc tttacctcat 5520

taactcaaat gagttttgat ttaatttcag cggccagcgc ctggacctcg cgggcagcgt 5580

cgccctcggg ttctgattca agaacggttg tgccggcggc ggcagtgcct gggtagctca 5640

cgcgctgcgt gatacgggac tcaagaatgg gcagctcgta cccggccagc gcctcggcaa 5700

cctcaccgcc gatgcgcgtg cctttgatcg cccgcgacac gacaaaggcc gcttgtagcc 5760

ttccatccgt gacctcaatg cgctgcttaa ccagctccac caggtcggcg gtggcccata 5820

tgtcgtaagg gcttggctgc accggaatca gcacgaagtc ggctgccttg atcgcggaca 5880

cagccaagtc cgccgcctgg ggcgctccgt cgatcactac gaagtcgcgc cggccgatgg 5940

ccttcacgtc gcggtcaatc gtcgggcggt cgatgccgac aacggttagc ggttgatctt 6000

cccgcacggc cgcccaatcg cgggcactgc cctggggatc ggaatcgact aacagaacat 6060

cggccccggc gagttgcagg gcgcgggcta gatgggttgc gatggtcgtc ttgcctgacc 6120

cgcctttctg gttaagtaca gcgataacct tcatgcgttc cccttgcgta tttgtttatt 6180

tactcatcgc atcatatacg cagcgaccgc atgacgcaag ctgttttact caaatacaca 6240

tcaccttttt agacggcggc gctcggtttc ttcagcggcc aagctggccg gccaggccgc 6300

cagcttggca tcagacaaac cggccaggat ttcatgcagc cgcacggttg agacgtgcgc 6360

gggcggctcg aacacgtacc cggccgcgat catctccgcc tcgatctctt cggtaatgaa 6420

aaacggttcg tcctggccgt cctggtgcgg tttcatgctt gttcctcttg gcgttcattc 6480

tcggcggccg ccagggcgtc ggcctcggtc aatgcgtcct cacggaaggc accgcgccgc 6540

ctggcctcgg tgggcgtcac ttcctcgctg cgctcaagtg cgcggtacag ggtcgagcga 6600

tgcacgccaa gcagtgcagc cgcctctttc acggtgcggc cttcctggtc gatcagctcg 6660

cgggcgtgcg cgatctgtgc cggggtgagg gtagggcggg ggccaaactt cacgcctcgg 6720

gccttggcgg cctcgcgccc gctccgggtg cggtcgatga ttagggaacg ctcgaactcg 6780

gcaatgccgg cgaacacggt caacaccatg cggccggccg gcgtggtggt gtcggcccac 6840

ggctctgcca ggctacgcag gcccgcgccg gcctcctgga tgcgctcggc aatgtccagt 6900

aggtcgcggg tgctgcgggc caggcggtct agcctggtca ctgtcacaac gtcgccaggg 6960

cgtaggtggt caagcatcct ggccagctcc gggcggtcgc gcctggtgcc ggtgatcttc 7020

tcggaaaaca gcttggtgca gccggccgcg tgcagttcgg cccgttggtt ggtcaagtcc 7080

tggtcgtcgg tgctgacgcg ggcatagccc agcaggccag cggcggcgct cttgttcatg 7140

gcgtaatgtc tccggttcta gtcgcaagta ttctacttta tgcgactaaa acacgcgaca 7200

agaaaacgcc aggaaaaggg cagggcggca gcctgtcgcg taacttagga cttgtgcgac 7260

atgtcgtttt cagaagacgg ctgcactgaa cgtcagaagc cgactgcact atagcagcgg 7320

aggggttgga tcaaagtact ttgatcccga ggggaaccct gtggttggca tgcacataca 7380

aatggacgaa cggataaacc ttttcacgcc cttttaaata tccgattatt ctaataaacg 7440

ctcttttctc ttaggtttac ccgccaatat atcctgtcaa acactgatag tttaaactga 7500

aggcgggaaa cgacaatctg atccaagctc aagctgctct agcattcgcc attcaggctg 7560

cgcaactgtt gggaagggcg atcggtgcgg gcctcttcgc tattacgcca gctggcgaaa 7620

gggggatgtg ctgcaaggcg attaagttgg gtaacgccag ggttttccca gtcacgacgt 7680

tgtaaaacga cggccagtgc caagcttggc aaacagctat tatgggtgtc gacctgcagt 7740

cataacttcg tatagcatac attatacgaa gttatcagat ctaatactgc catttcatta 7800

cctctttctc cgcacccgac atagatgcaa taacttcgta taggctaatt tatacgatgt 7860

aggagatcga tgcatgcggc cgctagctcg agaggcgcgc caatgatacc gactagtcac 7920

gcgtatggaa tcggcagcaa aggagaaatc tcaaaattcc ggcagaacaa ttttgaatct 7980

cgatccgtag aaacgagacg gtcattgttt tagttccacc acgattatat ttgaaattta 8040

cgtgagtgtg agtgagactt gcataagaaa ataaaatctt tagttgggaa aaaattcaat 8100

aatataaatg ggcttgagaa ggaagcgagg gataggcctt tttctaaaat aggcccattt 8160

aagctattaa caatcttcaa aagtaccaca gcgcttaggt aaagaaagca gctgagttta 8220

tatatggtta gagacgaagt agtgattgat tgttccctcc acctggaggt tttagagcta 8280

gaaatagcaa gttaaaataa ggctagtccg ttatcaactt gaaaaagtgg caccgagtcg 8340

gtgctttttt tcaagagctt ggagtggatg gaccctgaca ctggaatcgg cagcaaagga 8400

aaatatcaga gatctcttac agttagtttc gttcttaatc caaactactg cagcctgaca 8460

gacaaatgag gatgcaaaca attttaaagt ttatctaacg ctagctgttt tgtttcttct 8520

ctctggtgca ccaacgacgg cgttttctca atcataaaga ggcttgtttt acttaaggcc 8580

aataatgttg atggatcgaa agaagagggc ttttaataaa cgagcccgtt taagctgtaa 8640

acgatgtcaa aaacatccca catcgttcag ttgaaaatag tagctctgtt tatatattgg 8700

tagagtcgac taagagattg tttctaccac gcacacgaag ttttagagct agaaatagca 8760

agttaaaata aggctagtcc gttatcaact tgaaaaagtg gcaccgagtc ggtgcttttt 8820

ttcaagagct tggagtggat ggagtgaaga ctttggaatc ggcagcaaag gatttacttt 8880

aaattttttc ttatgcagcc tgtgatggat aactgaatca aacaaatggc gtctgggttt 8940

aagaagatct gttttggcta tgttggacga aacaagtgaa cttttaggat caacttcagt 9000

ttatatatgg agcttatatc gagcaataag ataagtgggc tttttatgta atttaatggg 9060

ctatcgtcca tagattcact aatacccatg cccagtaccc atgtatgcgt ttcatataag 9120

ctcctaattt ctcccacatc gctcaaatct aaacaaatct tgttgtatat ataacactga 9180

gggagcaaca ttggtcactg tacattccac cagggatgtt ttagagctag aaatagcaag 9240

ttaaaataag gctagtccgt tatcaacttg aaaaagtggc accgagtcgg tgcttttttt 9300

caagagcttg gagtggatgg aaaggactac atggaatcgg cagcaaagga ataagcttat 9360

gatttctttt ttcttacgaa ttttgcgtcc cacatcggta agcgagtgaa gaaataactg 9420

ctttatatat ggctacaaag caccattggt cataagtgga ccccggataa ccgttttaga 9480

gctagaaata gcaagttaaa ataaggctag tccgttatca acttgaaaaa gtggcaccga 9540

gtcggtgctt tttttcaaga gcttggagtg gatggatacg cgtcgagcac tacggcgcgc 9600

cttaccggtg ctcccgatct agtaacatag atgacaccgc gcgcgataat ttatcctagt 9660

ttgcgcgcta tattttgttt tctatcgcgt attaaatgta taattgcggg actctaatca 9720

taaaaaccca cctcataaat aacgtcatgc attacatgtt aattattaca tgcttaacgt 9780

aattcaacag aaattatatg ataatcatcg caagaccggc aacaggattc aatcttaaga 9840

aactttattg ccaaatgttt gaacgatcgg gaggatccta cttctttttc ttagcctgtc 9900

cggccttttt ggtggcagca ggacgcttat caccaccaag ctgggaaagg tcgatacgag 9960

tctcgtaaag accggtgatg gactggtgga tgagagtagc gtcgagaacc tccttggtgg 10020

acgtgtaacg cttcctgtcg atggtggtgt cgaagtactt gaaagcagca ggggcgccga 10080

ggttcgtgag cgtgaagagg tggatgatgt tctcggcctg ctcgcggatg ggcttgtcgc 10140

ggtgcttgtt gtaggcggag aggaccttgt cgaggttagc gtcagcgagg atgacgcgct 10200

tggagaactc ggagatctgc tcgatgatct cgtcgaggta gtgcttgtgc tgctccacga 10260

agagctgctt ctgctcgtta tcctcagggg aacccttgag cttctcgtag tgggaggcga 10320

ggtagaggaa gttcacgtac ttggaaggaa gagcaagctc gttacccttc tggagctcac 10380

cagcggaagc cagcatcctc ttacgaccgt tctcgagctc gaaaagagag tacttgggga 10440

gcttgatgat gaggtccttc ttgacctcct tgtagccctt ggcctcgagg aagtcgatcg 10500

ggttcttctc gaaggaggag cgctccatga tggtgatgcc gaggagctcc ttgacggact 10560

tgagcttctt cgacttaccc ttctccacct tggcgaccac gaggacggag taggcgacag 10620

taggggagtc gaaaccaccg tacttcttag ggtcccaatc cttcttccta gcgatgagct 10680

tgtccgagtt tctctttgga aggatagact ccttggagaa gccaccggtc tggacctcgg 10740

tcttcttgac gatgttaacc tgaggcatag aaaggacctt gcgaacagta gcgaagtcgc 10800

gacccttgtc ccaaacgatc tcaccagtct caccgttcgt ctcgataaga gggcgcttgc 10860

ggatctcgcc gttggcgagg gtgatctcgg tcttgaagaa gttcatgatg ttggagtaga 10920

agaagtactt ggcggtggcc ttgccgatct cctgctcgga cttggcgatc atcttacgaa 10980

cgtcgtagac cttgtagtca ccgtagacga actcggactc aagcttaggg tacttcttga 11040

taagagcggt accaacgaca gcgttaaggt aagcatcgtg agcgtggtgg tagttgttga 11100

tctcgcggac cttgtagaac tggaagtcct tgcggaagtc ggagacgagc ttggacttga 11160

gggtgatcac cttgacctcg cggatgagct tgtcgttctc gtcgtacttg gtgttcatcc 11220

tagaatcgag gatctgagca acgtgcttgg taatctgcct cgtctcaaca agctgcctct 11280

tgatgaaacc agccttgtca agctcggaaa ggccacccct ctcagccttc gtgaggttgt 11340

cgaacttcct ctgggtaatg agcttagcgt tgagaagctg cctccagtag ttcttcatct 11400

tcttgacaac ctcctcggaa gggacgttgt ccgacttacc cctgttcttg tcggacctcg 11460

tgaggacctt gttgtcgatg gagtcatcct taaggaaaga ctgaggaaca atgtggtcga 11520

cgtcgtagtc agaaagcctg ttgatgtcga gctcctggtc aacgtacata tccctaccgt 11580

tctggaggta gtagaggtag agcttctcgt tctggagctg ggtgttctcg acagggtgct 11640

ccttaaggat ctgagaacca agctccttga taccctcctc aatcctcttc atgcgctccc 11700

tcgagttctt ctgacccttc tgggtagtct ggttctcacg agccatctcg atgacgatgt 11760

tctcaggctt gtgacgaccc ataaccttga caagctcatc gacaacctta acagtctgaa 11820

ggatgccctt cttgatagca ggggaaccag caaggttagc aatgtgctcg tggagagagt 11880

cgccctgacc ggacacctga gccttctgaa tatcctcctt gaaggtaaga gagtcatcgt 11940

ggatgagctg catgaagttc ctgttagcga aaccatcaga cttgaggaag tcgaggatag 12000

tcttgccgct ctgcttatcc ctgataccgt tgatgagctt gcgggagagc ctaccccaac 12060

cggtgtaacg gcgacgcttg agctgcttca taaccttgtc atcgaagaga tgagcgtaag 12120

tcttgagcct ctcctcgatc atctccctat cctcgaagag agtaagagtg aggacgatgt 12180

cctcgaggat gtcctcgttc tcctcgttgt cgaggaagtc cttgtccttg atgatcttga 12240

ggagatcgtg gtaggtaccg agagaagcgt tgaaacggtc ctcaacgccg ctgatctcga 12300

cggagtcgaa gcactcgatc ttcttgaagt agtcctcctt gagctgcttg acggtgacct 12360

tgcggttggt cttgaagagg aggtcaacga tagccttctt ctgctcgccg gagaggaagg 12420

caggcttgcg cataccctcg gtgacgtact tgaccttggt gagctcgttg tagaccgtga 12480

agtactcgta gaggagggag tgcttgggga ggaccttctc gttggggagg ttcttgtcga 12540

agttggtcat gcgctcgatg aaggactggg cggaagcacc cttgtcaaca acctcctcga 12600

agttccaagg ggtgatagtc tcctcggact tcctagtcat ccaagcgaaa cgggagttac 12660

cacgagcaag aggaccaacg tagtaaggaa tacggaaagt aaggatcttc tcgatcttct 12720

cacggttgtc cttgaggaaa gggtagaagt cctcctgcct acgaaggatg gcgtgaagct 12780

caccaaggtg gatctggtga gggatagagc cgttatcgaa agtcctctgc ttcctaagga 12840

ggtcctcacg gttaagctta acgagaagct cctcagtacc atccatcttc tcaaggattg 12900

gcttgatgaa cttgtagaac tcctcctggc tagctccacc gtcaatgtaa ccggcgtagc 12960

cgttcttgga ctggtcgaag aagatctcct tgtacttctc ggggagctgc tgacgaacaa 13020

gagccttgag gagggtgagg tcctggtggt gctcgtcgta gcgcttgatc atggaagcag 13080

aaagaggagc cttagtgatc tcagtgttaa ccctaaggat atccgaaagg aggatagcat 13140

cggagaggtt cttagcagca aggaagagat cagcgtactg atctccaatc tgagcgagga 13200

ggttgtcgag atcatcgtcg taggtatcct tggagagctg aagcttagcg tcctcagcga 13260

ggtcgaagtt ggacttgaag ttaggggtca gaccgaggga gagagcgatg aggttaccga 13320

aaagaccgtt cttcttctca ccagggagct gggcgatgag gttctcgaga cgcctggact 13380

tggagagcct agcggaaagg atcgccttag cgtcgacacc ggaagcgttg atagggttct 13440

cctcgaagag ctggttgtag gtctggacga gctggatgaa gagcttgtcc acgtcggagt 13500

tatcagggtt aaggtcaccc tcgataagga agtgaccacg gaacttgatc atgtgagcga 13560

gagcaaggta gatgagacga agatcagcct tatcagtaga gtcaacaagc ttcttacgaa 13620

ggtggtagat agtggggtac ttctcgtggt aggcgacctc gtcgacgatg ttgccgaaga 13680

tggggtggcg ctcgtgcttc ttgtcctcct ccacgaggaa ggactcctcg aggcggtgga 13740

agaaggagtc gtcgaccttc gccatctcgt tggagaagat ctcctggagg tagcagatgc 13800

ggttcttgcg gcgggtgtag cggcggcggg cggtgcgctt gaggcgggtc gcctccgccg 13860

tctcgccgga gtcgaagagg agggcgccga tgaggttctt cttgatggag tggcggtcgg 13920

tgttgcccag gaccttgaac ttcttggacg ggaccttgta ctcgtcggtg atcaccgccc 13980

agccgacgct gttggtgccg atgtcgaggc cgatggagta cttcttgtca gccgcaggca 14040

ccccgtgaat accaaccttc cgcttcttct taggagccat ctgcagaagt aacaccaaac 14100

aacagggtga gcatcgacaa aagaaacagt accaagcaaa taaatagcgt atgaaggcag 14160

ggctaaaaaa atccacatat agctgctgca tatgccatca tccaagtata tcaagatcaa 14220

aataattata aaacatactt gtttattata atagataggt actcaaggtt agagcatatg 14280

aatagatgct gcatatgcca tcatgtatat gcatcagtaa aacccacatc aacatgtata 14340

cctatcctag atcgatattt ccatccatct taaactcgta actatgaaga tgtatgacac 14400

acacatacag ttccaaaatt aataaataca ccaggtagtt tgaaacagta ttctactccg 14460

atctagaacg aatgaacgac cgcccaacca caccacatca tcacaaccaa gcgaacaaaa 14520

agcatctctg tatatgcatc agtaaaaccc gcatcaacat gtatacctat cctagatcga 14580

tatttccatc catcatcttc aattcgtaac tatgaatatg tatggcacac acatacagat 14640

ccaaaattaa taaatccacc aggtagtttg aaacagaatt ctactccgat ctagaacgac 14700

cgcccaacca gaccacatca tcacaaccaa gacaaaaaaa agcatgaaaa gatgacccga 14760

caaacaagtg cacggcatat attgaaataa aggaaaaggg caaaccaaac cctatgcaac 14820

gaaacaaaaa aaatcatgaa atcgatcccg tctgcggaac ggctagagcc atcccaggat 14880

tccccaaaga gaaacactgg caagttagca atcagaacgt gtctgacgta caggtcgcat 14940

ccgtgtacga acgctagcag cacggatcta acacaaacac ggatctaaca caaacatgaa 15000

cagaagtaga actaccgggc cctaaccatg gaccggaacg ccgatctaga gaaggtagag 15060

aggggggggg ggaggacgag cggcgtacct tgaagcggag gtgccgacgg gtggatttgg 15120

gggagatctg gttgtgtgtg tgtgcgctcc gaacaacacg aggttgggga aagagggtgt 15180

ggagggggtg tctatttatt acggcgggcg aggaagggaa agcgaaggag cggtgggaaa 15240

ggaatccccc gtagctgccg gtgccgtgag aggaggagga ggccgcctgc cgtgccggct 15300

cacgtctgcc gctccgccac gcaatttctg gatgccgaca gcggagcaag tccaacggtg 15360

gagcggaact ctcgagaggg gtccagaggc agcgacagag atgccgtgcc gtctgcttcg 15420

cttggcccga cgcgacgctg ctggttcgct ggttggtgtc cgttagactc gtcgacggcg 15480

tttaacaggc tggcattatc tactcgaaac aagaaaaatg tttccttagt ttttttaatt 15540

tcttaaaggg tatttgttta atttttagtc actttatttt attctatttt atatctaaat 15600

tattaaataa aaaaactaaa atagagtttt agttttctta atttagaggc taaaatagaa 15660

taaaatagat gtactaaaaa aattagtcta taaaaaccat taaccctaaa ccctaaatgg 15720

atgtactaat aaaatggatg aagtattata taggtgaagc tatttgcaaa aaaaaaggag 15780

aacacatgca cactaaaaag ataaaactgt agagtcctgt tgtcaaaata ctcaattgtc 15840

ctttagacca tgtctaactg ttcatttata tgattctcta aaacactgat attattgtag 15900

tactatagat tatattattc gtagagtaaa gtttaaatat atgtataaag atagataaac 15960

tgcacttcaa acaagtgtga caaaaaaaat atgtggtaat tttttataac ttagacatgc 16020

aatgctcatt atctctagag aggggcacga ccgggtcacg ctgcacccag cttgcggccg 16080

catggccggc cttgccctag ttcctatagc ctacattata ggatggaggg atatcctctc 16140

ttaaggtagc gagcggatcc ttcgctacct taggaccgtt atagttacgg taccgagctc 16200

gaattcgtaa tcatgtcata gctgtttcct gtgtgaaatt gttatccgct cacaattcca 16260

cacaacatac gagccggaag cataaagtgt aaagcctggg gtgcctaatg agtgagctaa 16320

ctcacattaa ttgcgttgcg ctcactgccc gctttccagt cgggaaacct gtcgtgccag 16380

ctgcattaat gaatcggcca acgcgcgggg agaggcggtt tgcgtattgg ctagagcagc 16440

ttgccaacat ggtggagcac gacactctcg tctactccaa gaatatcaaa gatacagtct 16500

cagaagacca aagggctatt gagacttttc aacaaagggt aatatcggga aacctcctcg 16560

gattccattg cccagctatc tgtcacttca tcaaaaggac agtagaaaag gaaggtggca 16620

cctacaaatg ccatcattgc gataaaggaa aggctatcgt tcaagatgcc tctgccgaca 16680

gtggtcccaa agatggaccc ccacccacga ggagcatcgt ggaaaaagaa gacgttccaa 16740

ccacgtcttc aaagcaagtg gattgatgtg aacatggtgg agcacgacac tctcgtctac 16800

tccaagaata tcaaagatac agtctcagaa gaccaaaggg ctattgagac ttttcaacaa 16860

agggtaatat cgggaaacct cctcggattc cattgcccag ctatctgtca cttcatcaaa 16920

aggacagtag aaaaggaagg tggcacctac aaatgccatc attgcgataa aggaaaggct 16980

atcgttcaag atgcctctgc cgacagtggt cccaaagatg gacccccacc cacgaagagc 17040

atcgtggaaa aagaagacgt tccaaccacg tcttcaaagc aagtggattg atgtgatatc 17100

tccactgacg taagggatga cgcacaatcc cactatcctt cgcaagaccc ttcctctata 17160

taaggaagtt catttcattt ggagaggaca cgctgaaatc accagtctct ctctacaaat 17220

ctatctctct cgagtctacc atgagcccag aacgacgccc ggccgacatc cgccgtgcca 17280

ccgaggcgga catgccggcg gtctgcacca tcgtcaacca ctacatcgag acaagcacgg 17340

tcaacttccg taccgagccg caggaaccgc aggagtggac ggacgacctc gtccgtctgc 17400

gggagcgcta tccctggctc gtcgccgagg tggacggcga ggtcgccggc atcgcctacg 17460

cgggcccctg gaaggcacgc aacgcctacg actggacggc cgagtcgacc gtgtacgtct 17520

ccccccgcca ccagcggacg ggactgggct ccacgctcta cacccacctg ctgaagtccc 17580

tggaggcaca gggcttcaag agcgtggtcg ctgtcatcgg gctgcccaac gacccgagcg 17640

tgcgcatgca cgaggcgctc ggatatgccc cccgcggcat gctgcgggcg gccggcttca 17700

agcacgggaa ctggcatgac gtgggtttct ggcagctgga cttcagcctg ccggtaccgc 17760

cccgtccggt cctgcccgtc accgagattt gactcgagtt tctccataat aatgtgtgag 17820

tagttcccag ataagggaat tagggttcct atagggtttc gctcatgtgt tgagcatata 17880

agaaaccctt agtatgtatt tgtatttgta aaatacttct atcaataaaa tttctaattc 17940

ctaaaaccaa aatccagtac taaaatccag atcccccgaa ttaattcggc gttaattcag 18000

tacattaaaa acgtccgcaa tgtgttatta agttgtctaa gcgtcaattt gtttacacca 18060

caatatatcc tgcca 18075

<210> 7

<211> 20

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 7

caatacaacc cggaatctgc 20

<210> 8

<211> 20

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 8

gtgtagctga gccaatctcg 20

<210> 9

<211> 20

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 9

attgttccac atgggttggt 20

<210> 10

<211> 20

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 10

tgcctatgtc gagtggaatg 20

<210> 11

<211> 37

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 11

attgttccct ccacctggag gttttagagc tagaaat 37

<210> 12

<211> 37

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 12

ctccaggtgg agggaacaat caatcactac ttcgtct 37

<210> 13

<211> 36

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 13

tttctaccac gcacacgaag ttttagagct agaaat 36

<210> 14

<211> 36

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 14

ttcgtgtgcg tggtagaaac aatctcttag tcgact 36

<210> 15

<211> 37

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 15

ctgtacattc caccagggat gttttagagc tagaaat 37

<210> 16

<211> 36

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 16

atccctggtg gaatgtacag tgaccaatgt tgctcc 36

<210> 17

<211> 37

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 17

taagtggacc ccggataacc gttttagagc tagaaat 37

<210> 18

<211> 37

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 18

ggttatccgg ggtccactta tgaccaatgg tgctttg 37

<210> 19

<211> 22

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 19

ctccgtttta cctgtggaat cg 22

<210> 20

<211> 20

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 20

cggaggaaaa ttccatccac 20

<210> 21

<211> 47

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 21

ttcagaggtc tctaccgact agtcacgcgt atggaatcgg cagcaaa 47

<210> 22

<211> 37

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 22

agcgtgggtc tcgtcagggt ccatccactc caagctc 37

<210> 23

<211> 38

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 23

ttcagaggtc tctctgacac tggaatcggc agcaaagg 38

<210> 24

<211> 38

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 24

agcgtgggtc tcgtcttcac tccatccact ccaagctc 38

<210> 25

<211> 38

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 25

ttcagaggtc tctaagactt tggaatcggc agcaaagg 38

<210> 26

<211> 38

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 26

agcgtgggtc tcgagtcctt tccatccact ccaagctc 38

<210> 27

<211> 38

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 27

ttcagaggtc tctgactaca tggaatcggc agcaaagg 38

<210> 28

<211> 40

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 28

agcgtgggtc tcgctcgacg cgtatccatc cactccaagc 40

<210> 29

<211> 19

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 29

atatcgaagg tgtcatgac 19

<210> 30

<211> 20

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 30

ctttaggagg gtcggtttca 20

<210> 31

<211> 20

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 31

ccaaccgaat ggtacgacat 20

<210> 32

<211> 20

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 32

taccacagtt cttccaacat 20

Claims (5)

1. A vector for simultaneously targeting and knocking out 1-SST and 1-FFT of a caerulein synthase gene by using a CRISPR/Cas9 system, which is characterized in that the vector comprises an expression cassette of sgRNA, the expression cassette comprises a promoter for driving the sgRNA to transcribe and the sgRNA,

the sgRNA comprises sgRNA-SST1 and sgRNA-SST2 which target 1-SST genes and sgRNA-FFT1 and sgRNA-FFT2 which target 1-FFT genes; the sequences of the sgRNA-SST1, the sgRNA-SST2, the sgRNA-FFT1 and the sgRNA-FFT2 are sequentially shown as SEQ ID No. 1-4;

the sequence of the vector is shown as SEQ ID NO. 6.

2. A host cell comprising the vector of claim 1.

3. Use of the vector of claim 1 or the host cell of claim 2 for increasing the sucrose, fructose or natural rubber content of the roots of hevea brasiliensis, or for reducing the inulin content of the roots of hevea brasiliensis.

4. Use of the vector of claim 1 or the host cell of claim 2 in the genetic breeding of kochia or in the improvement of germplasm resources;

the application is breeding of the synanthrin synthesis defective germplasm or the high-yield natural rubber germplasm.

5. A method for simultaneously knocking out 1-SST and 1-FFT of an inula kochiana synzyme gene in a targeted manner by using a CRISPR/Cas9 system is characterized in that the vector of claim 1 is transferred into the inula kochiana, and a hevea kochiana plant with the inula synzyme gene 1-SST and 1-FFT knocked out simultaneously is obtained through screening.

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