CN111748555A

CN111748555A - sgRNA for improving citrus and application and use method thereof

Info

Publication number: CN111748555A
Application number: CN202010711527.0A
Authority: CN
Inventors: 邹修平; 文庆利; 龙俊宏; 刘语诺; 龙琴; 何永睿; 彭爱红; 陈善春
Original assignee: Southwest University
Current assignee: Southwest University
Priority date: 2020-07-22
Filing date: 2020-07-22
Publication date: 2020-10-09

Abstract

The invention relates to the technical field of plant genetic engineering. The invention provides an sgRNA for modifying citrus and an application and a using method thereof, wherein the nucleotide sequence of the sgRNA is shown as SEQ ID No.1, SEQ ID No.2, SEQ ID No.3, SEQ ID No.4, SEQ ID No.5, SEQ ID No.6, SEQ ID No.7, SEQ ID No.8, SEQ ID No.9 or SEQ ID No. 10. The sgRNA of the invention has high score and low off-target efficiency, and the efficiency of guiding the Cas9 to cut the enzyme target sequence is high. The CsPP2B15 gene can be modified and expression regulated, the resistance of the transgenic citrus to the huanglongbing is obviously improved by the mutated CsPP2B15 gene, the symptom of the huanglongbing is obviously relieved, and the content of pathogenic bacteria is obviously reduced.

Description

sgRNA for improving citrus and application and use method thereof

Technical Field

The invention relates to the technical field of plant genetic engineering, in particular to sgRNA for improving citrus and an application and a using method thereof.

Background

Citrus yellow shoot is a systemic disease, the resulting hazard is devastating, and the pathogen can infect almost all citrus varieties. According to statistics, the citrus huanglongbing is spread in 19 provinces of China, the damaged area accounts for more than 80% of the total cultivation area of citrus, and the yield accounts for about 85% of the total yield. The causative agent of citrus greening disease is a gram-negative bacterium of the genus phloem, including asian species, african species, and south america species. At present, no technical measures for fundamentally solving the citrus yellow shoot disease exist in citrus production, and only a series of destructive measures such as destroying diseased plants or diseased orchards can be taken to thoroughly eliminate the citrus yellow shoot disease. For fruit growers, economic losses are very serious.

Because the problem of citrus greening disease cannot be completely solved by adopting the traditional means, a better method needs to be found. The method is the most economical and effective way for fundamentally solving the problem by utilizing resistant germplasm resources, excavating the disease-resistant genes by combining a plant genetic engineering means and cultivating disease-resistant varieties to enhance the resistance of the citrus to the yellow shoot.

Disclosure of Invention

The invention aims to provide an sgRNA for improving citrus and an application and a using method thereof, so that the threat of yellow shoot is fundamentally solved, and the yellow shoot of the citrus is thoroughly eliminated.

In order to achieve the above object, the present invention provides the following technical solutions:

the invention provides an sgRNA for modifying citrus, which has a nucleotide sequence shown as SEQ ID No.1, SEQ ID No.2, SEQ ID No.3, SEQ ID No.4, SEQ ID No.5, SEQ ID No.6, SEQ ID No.7, SEQ ID No.8, SEQ ID No.9 or SEQ ID No. 10.

Preferably, the sgRNA sequence acts on the citrus CsPP2B15 gene in modifying citrus.

The application also provides an application of the sgRNA of the modified citrus in modifying citrus greening disease resistance.

The application also provides a use method of the sgRNA of the modified citrus, which comprises the following steps:

(1) constructing an expression vector for CRISPR/Cas9 editing by using the sgRNA sequence;

(2) introducing the expression vector constructed in the step (1) into citrus, and screening out a transgenic plant;

(3) screening out a CsPP2B15 mutant plant from the transgenic plant obtained in the step (2);

(4) and (4) screening citrus yellow shoot resistant plants from the CsPP2B15 mutant plants obtained in the step (3).

Preferably, the vector used for constructing the vector in the step (1) is pCas9-CsPP2B15 sgRNA.

Preferably, the enzyme used in constructing the vector in step (1) is a BamHI/SalI enzyme.

Preferably, the introduction method is Agrobacterium tumefaciens mediated method or electroporation method.

Preferably, the screening method of the transgenic citrus plant in the step (2) is resistance screening, GUS histochemical staining and PCR verification.

Preferably, the method for screening mutant plants in step (3) is Sanger sequencing and re-sequencing technology.

Preferably, the screening method of the huanglongbing resistant plants in the step (4) comprises graft virus transmission, greenhouse symptom and section observation and quantitative PCR detection.

The invention provides an sgRNA for modifying citrus and an application and a using method thereof, wherein the nucleotide sequence of the sgRNA is shown as SEQ ID No.1, SEQ ID No.2, SEQ ID No.3, SEQ ID No.4, SEQ ID No.5, SEQ ID No.6, SEQ ID No.7, SEQ ID No.8, SEQ ID No.9 or SEQ ID No. 10. The sgRNA of the invention has high score and low off-target efficiency, and the efficiency of guiding the Cas9 to cut the enzyme target sequence is high. The CsPP2B15 gene can be modified and expression regulated, the resistance of the transgenic citrus to the huanglongbing is obviously improved by the mutated CsPP2B15 gene, the symptom of the huanglongbing is obviously relieved, and the content of pathogenic bacteria is obviously reduced.

Drawings

Fig. 1 is an electrophoretogram of in vitro detection of sgRNA activity;

FIG. 2 is a schematic structural diagram of a T-DNA of a plant expression vector pGN-CsPP2B 15;

FIG. 3 is a schematic diagram of the T-DNA structure of pCas9-PP2B15sgRNA vector;

FIG. 4 is a diagram of the PCR detection and plant phenotype of OEPP2B5 transgenic plants;

FIG. 5 is a PCR identification result diagram of a Cas9 transgenic plant;

FIG. 6 is the expression level of CsPP2B15 gene in plGN-CsPP2B15 transgenic line;

FIG. 7 is a Cas 9-mediated sequencing analysis of CsPP2B15 editing;

FIG. 8 is a graph of symptoms at 6 months after infection by a transgenic citrus that overexpresses CsPP2B 15;

FIG. 9 is a qPCR analysis of pathogen content at months 6 and 12 after infection of transgenic citrus overexpressing CsPP2B 15;

FIG. 10 is a graph of symptoms of mutant transgenic citrus at 6 months after infection;

fig. 11 is a qPCR analysis of pathogen content at 6 months and 12 months after transgenic citrus infection of the mutant.

Detailed Description

In the present invention, reagents and drugs which are not specifically described are generally commercially available, and materials and methods which are not specifically described are referred to molecular cloning protocols (Sambrook and Russell, 2001).

The technical solutions provided by the present invention are described in detail below with reference to examples, but they should not be construed as limiting the scope of the present invention.

Example 1 extraction of DNA

Selecting 0.1-1 g of citrus veins, extracting citrus genome DNA by using an Aidlab company kit (cat.No. DN15), and storing the extracted DNA at-20 ℃ for later use.

EXAMPLE 2 RNA extraction and cDNA Synthesis

Selecting 0.1g of citrus leaf vein, extracting total RNA of the leaf by using an EASYspin plant RNA rapid extraction kit (Aidlab, cat. No. RN09), and scanning by using non-denaturing agarose gel electrophoresis and an ultraviolet spectrophotometer to detect the quality of the RNA. First strand cDNA Synthesis was performed according to the BIO-RAD iScriptTM cDNA Synthesis Kit (BIO-RAD, cat. No.170-8891) instructions. The synthesized cDNA was stored at-20 for future use.

Example 3 PCR amplification of genomic sequences

(1)25 μ L of the amplification system was:

(2) the amplification procedure was: 94 ℃ for 5 min; 94 ℃, 30sec, 56 ℃, 30sec, 72 ℃, 1.5min, 35 cycles; extension at 72 ℃ for 10 min.

EXAMPLE 4 recovery of DNA fragments, ligation and transformation of E.coli DH5 alpha

The agarose gel block containing the target fragment of example 2 was cut off with a clean blade under an ultraviolet lamp, and the gel recovery method was performed with reference to the instruction manual of the kit (available from Omega). The recovered fragments were quantitated by electrophoresis on agarose gels.

The establishment of the enzyme digestion system and the reaction conditions refer to the instruction of a restriction enzyme kit of TaKaRa company. The recovered fragment obtained by enzyme digestion or the recovered fragment obtained by amplification is cloned on a pGEM-T/pGEM-T Easy (Promega) vector according to the instructions of a ligase kit. The ligation reaction system is as follows:

the molar ratio of the vector DNA fragment to the exogenous ligation product DNA fragment in the reaction system is 1: 3;

ligation was performed at 16 ℃ for 1h, after which the ligation products were transformed into E.coli DH5a competent cells and cultured at 37 ℃.

Example 5 cloning of CspP2B15 Gene

Cspp2B15 sequence from Huazhong NongsuanObtained in orange genomic database CAP (http:// citrus. hzau. edu. cn/cgi-bin/orange /) (Xu, et al, 2013), Gene ID Cs3g14680.1. The CsPP2B15 gene primers SEQ ID No.11 and SEQ ID No.12 are designed according to the gene sequence, and the CDS (SEQ ID No.13) and the genome sequence (SEQ ID No.14) of the coding sequence CsPP2B15 are amplified by PCR by respectively taking the citrus vein cDNA and DNA infected with the huanglongbing as templates. The PCR product was recovered, ligated with pGEM-T easy vector and transformed into E.coli DH5^α. Positive clones were picked for sequencing confirmation.

Example 6 sgRNA in vitro Activity assay

(1) Amplifying a target fragment for detecting sgRNA activity.

And (2) amplifying a target fragment by using late orange genome DNA as a template and primers CsPP2-f and CsPP2-r, wherein the sequence of CsPP2-f is SEQ ID No.15, the sequence of CsPP2-r is SEQ ID No.16, the length of the amplified fragment is 890bp, and the fragment contains a target sgRNA sequence. And (4) carrying out electrophoretic purification on the successfully amplified target fragments.

(2) sgRNA Activity detection

sgRNA activity assay was performed as follows.

20 μ L in vitro digestion reaction system was as follows:

complementing 20 mu L of enzyme digestion system by RNase Free Water;

the reaction is carried out at 37 ℃ for 1h, then 1 mu L of proteinase K is added to remove Cas9 proteinase, and the reaction is carried out at 37 ℃ for 20 min. The digested product was electrophoresed in 1.2% agarose gel at 120V for 30 min. Images of electropherograms were taken under UV conditions with Biospectrum300Imaging system, and the results are shown in fig. 1, which shows that ten sgrnas can guide Cas9 to cleave the target sequence.

EXAMPLE 7 construction of expression vector

Taking pGN vector (Zou et al, 2017) as a basic framework, cutting the CsPP2B15 gene from pGEM-T easy vector by BamH I and Spe I, connecting the cut CsPP2B15 gene with pGN vector subjected to the same enzyme digestion treatment, transforming escherichia coli, obtaining plant expression vector pGN-CsPP2B15 of over-expression CsPP2B15 through PCR and enzyme digestion verification, controlling the expression of CsPP2B15 by over-expression promoter 35S, wherein the structural diagram of T-DNA of the plant expression vector pGN-CsPP2B15 is shown in figure 2. Wherein 35S is a constitutive strong promoter; GUS, NPTII is a marker gene for screening transgenic citrus; nos is a transcription termination sequence; LB is the left arm of T-DNA; RB is the right arm of T-DNA

Four sgRNAs of SEQ ID No.2, SEQ ID No6, SEQ ID No9 and SEQ ID No10 are selected to construct a Cas9 expression vector. Firstly, designing primers according to a selected sgRNA sequence, sending the primers to a company for synthesis, diluting the primers to a concentration of 10 mmol.L < -1 > (namely a common PCR concentration), then taking 10 mu L of each pair of primers, uniformly mixing and annealing to form double-stranded DNA. The annealing procedure is as follows: denaturation at 65 ℃ for 30min, then placing to room temperature, annealing for 2h under the condition of keeping out of the sun, then connecting the annealing product into the vector puc119-sgRNA after BbsI enzyme digestion recovery, transforming escherichia coli, and verifying positive clone by PCR and sequencing. After obtaining a correct sgRNA vector, carrying out enzyme digestion on the sgRNA vector and a pGN-proCas9 vector by BamHI/SalI, recovering a sgRNA expression frame fragment, connecting the sgRNA expression frame fragment into a pGN-proCas9 vector, transforming escherichia coli, and confirming a positive clone by enzyme digestion and sequencing. The obtained pCas9-CspP2B15sgRNA, the pCas9-CspP2B15sgRNA is shown in FIG. 3. Wherein 35S is a constitutive strong promoter; GUS, NPTII is a marker gene for screening transgenic citrus; nos is a transcription termination sequence; AtU6 is Arabidopsis thaliana U6 promoter; TTTTT is a type III transcription termination sequence; LB is the left arm of T-DNA; RB is the right arm of T-DNA.

EXAMPLE 8 introduction of expression vector

1. The constructed plant expression vector plasmid was introduced into Agrobacterium EHA105 by electroporation, with reference to Bio-RAD MicroPulser user instructions.

2. The genetic transformation of citrus was carried out by the Agrobacterium tumefaciens-mediated method, and the medium components for the genetic transformation of citrus were as shown in Table 1.

TABLE 1 Agrobacterium tumefaciens-mediated culture medium for genetic transformation of citrus

MS:Murashige&Skoog,1962

3. The expression vector is introduced into the late orange by an agrobacterium-mediated epicotyl method. The specific method comprises the following steps:

(1) obtaining of epicotyl of golden orange seedling

Cleaning fresh Mallotus anomala, sterilizing with 70% ethanol, taking out seed under aseptic condition, peeling off seed coat, inoculating on MS solid culture medium for germination, culturing at 28 deg.C in dark for 2 weeks, and culturing under 16h/d photoperiod for 1 week. Taking the epicotyl of the germinated seedling, cutting the epicotyl into stem sections of about 1cm, and using the stem sections for agrobacterium-mediated genetic transformation.

(2) Preparation of transformed Agrobacterium

The agrobacterium liquid used for transfection is added with 30 percent of sterile glycerol and stored in an ultra-low temperature refrigerator at the temperature of 70 ℃ below zero. Before transfection, 50mg/L kanamycin-containing LB solid medium (0.5% sucrose (W/V), 0.1% yeast extract for bacteria (W/V), 1% tryptone for bacteria (W/V), 0.05% MgSO₄·7H₂O (W/V), agar powder (W/V) of 1.5% pH 7.0. Selecting single colony of Agrobacterium, inoculating in 5mL LB liquid culture medium containing the same antibiotic, shaking and culturing at 28 deg.C and 200r/min overnight. After single colonies grow out, picking the single colonies to an LB liquid culture medium with 50mg/ml kanamycin, and carrying out shaking culture at 28 ℃ for overnight; when the OD600 of the bacterial liquid reaches 1, diluting to 0.2 by using an LB liquid culture medium without kanamycin, and continuing shaking culture for about 3 hours; when the bacterial liquid reaches logarithmic phase (OD is 0.5), centrifuging at 5000r/min for 10min, discarding the supernatant, and suspending the agrobacterium in MS liquid culture medium with ph of 5.4 for transfection.

(3) Transformation of epicotyl stem of late orange

Soaking the stem of the epicotyl of the evening orange cut into about 1cm in agrobacterium for 10-15 min, and then sucking a bacterial solution indicated by the epicotyl by using sterile filter paper; the explants were transferred to co-culture medium and cultured in the dark at 26 ℃ for 2 d.

(4) Screening for transformants

After co-cultivation was complete, the epicotyls were transferred to selection medium and incubated in the dark at 28 ℃ for 7 d. The explants were then cultured at 28 ℃ for 16h under light, and subcultured every two weeks.

(5) Seedling culture of transformants

When the sprouts grow to be more than 1cm, grafting the sprouts to sterile golden orange seedlings in vitro, and culturing in a seedling culture medium; grafting the seedlings onto the immature bitter orange seedlings when the seedlings grow to about 5cm, and culturing in a greenhouse. The obtained transgenic citrus did not significantly differ in phenotype and growth development from the wild-type control.

Example 9 PCR detection of exogenous Gene integration

Taking 100mg of citrus leaves, extracting total DNA of transgenic plant leaves with GUS staining positive according to an operating method (cat.No. DN15) of a novel plant genome DNA rapid extraction kit of Aidlab company, carrying out PCR amplification on a target gene in the total DNA of the positive plant with primers Cas9-f and Cas9-r, wherein the sequence of Cas9-f is SEQ ID No.17, the sequence of Cas9-r is SEQ ID No.18, and identifying that a target gene amplified by the Cas9 transgenic plant is a Cas9 specific fragment with the size of about 900 bp; 35S-f and PP2-r are primers for identifying a transgenic plant of the over-expression CsPP2B15(OPP2B15), the length of an amplification fragment is 1520bp, the sequence of 5S-f is SEQ ID No.19, and the sequence of PP2-r is SEQ ID No. 20. The reaction volume is 50 mu L, and the PCR reaction condition is 94 ℃ for 3 min; 30s at 94 ℃, 30s at 58 ℃ and 1min at 72 ℃ for 35 cycles; 3min at 72 ℃. The results are shown in FIGS. 4 and 5. Obtaining 7 transgenic plants of over-expression CsPP2B15 and 6 transgenic plants of Cas 9. In FIG. 4, M is DNA marker, P is plasmid, WT is wild type control, and 1-7 are transgenic plants; in FIG. 5, M is DNA marker, P is plasmid, WT is wild type control, and 1-6 are transgenic plants.

Example 10 RT-qPCR analysis of CsPP2B15 expression in OEPP2B5 transgenic plants

The citrus leaves are taken, and then the total RNA of the veins is extracted by using an EASYspin plant RNA rapid extraction kit (Aidlab, cat. No. RN09). First strand cDNA Synthesis was performed according to the BIO-RAD iScriptTM cDNA Synthesis Kit (BIO-RAD, cat. No.170-8891) instructions. The expression of CsPP2B15 gene was quantitatively analyzed by using the Real-time PCR kit (BIO-RAD iQTM SYBR GreenSupermix, cat. No.170-8882 AP). The detected primers are qPP2B15-f and qPP2B15-r, the sequence of qPP2B15-f is SEQ ID No.21, the sequence of qPP2B15-r is SEQ ID No.22, and the internal standard gene actin table showsThe detection primers are qActin-f and qActin-r, the sequence of qActin-f is SEQ ID No.23, and the sequence of qActin-r is SEQ ID No. 24. The reaction volume was 20. mu.L. Reaction conditions are as follows: 95 ℃ for 3min and 94 ℃ for 10 s; at 56 ℃ for 10s, at 72 ℃ for 10s, and for 40 cycles; 10min at 72 ℃. The experiment was repeated 3 times and data were statistically analyzed using Bio-Rad CFX Manager 2.0 software. By using 2^-ΔΔCtThe method calculates the relative expression quantity of the antibacterial peptide gene in the transgenic plant: defining the water treated sample as reference factor, i.e. its expression level position 1 of antibacterial peptide gene, then calculating the expression multiple of 2 relative to reference factor gene of identical sample inoculated with xanthone pathogenic bacteria^-ΔΔCtRelative expression levels. The detection results are shown in FIG. 6. The result shows that the expression level of the CsPP2B15 of the transgenic plant is obviously higher than that of a wild control, and 3 transgenic plants (OPP2B15-3, OPP2B15-5 and OPP2B15-7) with the relative expression quantity more than 10 times are obtained.

Example 11 mutant identification of transgenic plants

Cas9 transgenic plant leaf DNA is extracted, primers PP2B15sgRNA-f and PP2B15sgRNA-r containing different sgRNAs are designed according to a genome DNA sequence of PP2B15, the sequence of the PP2B15sgRNA-f is SEQ ID No.25, and the sequence of the PP2B15sgRNA-r is SEQ ID No. 26. And recovering the target fragment amplified by PCR, connecting the target fragment to a T vector, sending the positive clone to a company for sanger sequencing, and screening the mutant. As shown in FIG. 7, 3 strains (mPP2B15-4, mPP2B15-5 and mPP2B15-6) among 6 strains exhibited higher editing efficiency: reaching 87.5 to 93.3 percent; furthermore, all three mutations were directed by S10 sgRNA.

Example 12 evaluation of transgenic plants for Huanglongbing resistance

According to the method of Wuliu et al (Wuliu et al, 2018), 3 plants are propagated in each transgenic line on average, the leaves of two-year-old healthy transgenic grafted seedlings are selected as 'rootstocks', the leaves of verified Huanglongbing disease plants are selected as 'scions', and the developed mature leaves with the same maturity are selected for leaf grafting. The two leaves are naturally dried after being washed by sterile water for several times, holes are drilled at the position about 1.5cm away from the leaf tips, the aperture is 7mm, the same hole puncher is used for punching a yellow dragon disease-containing leaf disc with the same size on the leaves of a diseased plant, the yellow dragon disease-containing leaf disc is grafted to the position of a transgenic grafted seedling leaf connector, the alignment of the middle vein of the 'scion' leaf disc and the middle vein of the 'stock' leaf is ensured, the side veins keep the consistent growth trend, and the front side and the back side are stuck and fixed by transparent adhesive tapes. The control group "scion" was the leaf of a two-year-old healthy wild type malorange graft, and the rest of the treatments were as above. The treated plants are placed in a greenhouse for culture, the morbidity is regularly observed, transgenic artery DNA is extracted, citrus 18S is used as an internal reference, the content of the 16S gene of the pathogenic bacteria is detected by quantitative PCR (qPCR), the content of the pathogenic bacteria is calculated by methods such as a Zou et al, 2017 and the like, and the proliferation and the diffusion are evaluated. The results are shown in fig. 8, 9, 10 and 11. Wherein WT was a control group, and 6MAI and 12MAI indicated months 6 and 12 after virus transmission.

And (4) conclusion: the inhibition of CsPP2B15 expression is an effective strategy for enhancing the resistance of citrus greening disease, and the overexpression of CsPP2B15 obviously reduces the resistance of transgenic citrus to the greening disease, so that the symptoms are aggravated, and the content of pathogenic bacteria is obviously increased; and the resistance of the transgenic citrus to the huanglongbing is obviously increased by the mutant CsPP2B15, the symptoms are obviously relieved, and the content of pathogenic bacteria is obviously reduced.

As can be seen from the above examples, the present invention provides a citrus-modified sgRNA, the nucleotide sequence of which is shown in SEQ ID No.1, SEQ ID No.2, SEQ ID No.3, SEQ ID No.4, SEQ ID No.5, SEQ ID No.6, SEQ ID No.7, SEQ ID No.8, SEQ ID No.9 or SEQ ID No.10, and applications and methods of use thereof. The sgRNA of the invention has high score and low off-target efficiency, and the efficiency of guiding the Cas9 to cut the enzyme target sequence is high. The CsPP2B15 gene can be modified and expression regulated, the resistance of the transgenic citrus to the huanglongbing is obviously improved by the mutated CsPP2B15 gene, the symptom of the huanglongbing is obviously relieved, and the content of pathogenic bacteria is obviously reduced.

The foregoing is only a preferred embodiment of the present invention, and it should be noted that, for those skilled in the art, various modifications and decorations can be made without departing from the principle of the present invention, and these modifications and decorations should also be regarded as the protection scope of the present invention.

Sequence listing

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<400>16

ggttctaagt tcggctactt 20

<210>17

<211>20

<212>DNA

<213> Artificial Sequence (Artificial Sequence)

<400>17

tcgtttcgct tggatgacca 20

<210>18

<211>20

<212>DNA

<213> Artificial Sequence (Artificial Sequence)

<400>18

agcaaggtta gcgatgtgct 20

<210>19

<211>20

<212>DNA

<213> Artificial Sequence (Artificial Sequence)

<400>19

cgacacgctt gtctactcca 20

<210>20

<211>20

<212>DNA

<213> Artificial Sequence (Artificial Sequence)

<400>20

tcattcttta ggtctaatct 20

<210>21

<211>22

<212>DNA

<213> Artificial Sequence (Artificial Sequence)

<400>21

gctctgagat ctagggtttc cg 22

<210>22

<211>20

<212>DNA

<213> Artificial Sequence (Artificial Sequence)

<400>22

cccctttcag atgctgaccc 20

<210>23

<211>18

<212>DNA

<213> Artificial Sequence (Artificial Sequence)

<400>23

catccctcag caccttcc 18

<210>24

<211>19

<212>DNA

<213> Artificial Sequence (Artificial Sequence)

<400>24

ccaaccttag cacttctcc 19

<210>25

<211>22

<212>DNA

<213> Artificial Sequence (Artificial Sequence)

<400>25

atgaacgtag atctgctgcc tg 22

<210>26

<211>21

<212>DNA

<213> Artificial Sequence (Artificial Sequence)

<400>26

tcattcttta ggtctaatct c 21

Claims

1. A sgRNA for modifying citrus is characterized in that the nucleotide sequence is shown as SEQ ID No.1, SEQ ID No.2, SEQ ID No.3, SEQ ID No.4, SEQ ID No.5, SEQ ID No.6, SEQ ID No.7, SEQ ID No.8, SEQ ID No.9 or SEQ ID No. 10.

2. The citrus-modified sgRNA of claim 1, wherein the sgRNA sequence is effective in modifying citrus CsPP2B15 gene.

3. Use of a citrus-modified sgRNA according to claim 1 to modify citrus greening disease resistance.

4. The method of using a citrus-modified sgRNA in accordance with claim 1, comprising the steps of:

5. The method for using the modified citrus sgRNA according to claim 4, wherein the vector used for constructing the vector in step (1) is pCas9-CsPP2B15 sgRNA.

6. The method for using the citrus modified sgRNA according to claim 4, wherein the enzyme used for constructing the vector in step (1) is BamHI/SalI enzyme.

7. The method for using the sgRNA of modified citrus fruit according to claim 4, wherein the introduction method is Agrobacterium tumefaciens mediated method and electroporation transformation method.

8. The method for using the sgRNA of modified citrus according to claim 4, wherein the transgenic citrus plant screening method in the step (2) is resistance screening, GUS histochemical staining and PCR verification.

9. The method for using the citrus modified sgRNA according to claim 4, wherein the method for screening mutant plants in step (3) is Sanger sequencing and re-sequencing technology.

10. The method for using the sgRNA of the modified citrus fruit as claimed in claim 4, wherein the screening method for the Huanglongbing-resistant plants in the step (4) is graft virus transmission, observation of greenhouse symptoms and sections, and quantitative PCR detection.