Application of pevde gene in bamboo gene editing as gene editing screening tag

Abstract

The application belongs to the technical field of plant gene editing, and particularly relates to application of a PeVDE gene as a gene editing endogenous screening tag in bamboo gene editing. In the application, the PeVDE gene is used as a gene editing screening tag to avoid the influence of a resistance screening tag on normal growth of bamboo; and whether the gene edited bamboo is obtained can be directly judged through a chlorophyll fluorescence parameter, i.e. non-photochemical quenching (NPQ), and the judging method is simple and fast.

Description

TECHNICAL FIELD

[0001] The application belongs to the technical field of plant gene editing, and particularly relates to application of a PeVDE gene as a gene editing screening tag in bamboo gene editing. BACKGROUND

[0002] Bamboo is a green renewable biomass resource, and most of them are perennial woody bamboos, with long vegetative growth time, unpredictable flowering period, low hybrid seed setting rate, and short seed viability maintenance time, so it is extremely difficult to breed bamboo by conventional breeding. So far, only a few bamboo hybrid varieties such as 'Zhenlvteng No. 3', 'Zhenmateng No. 7' and 'Zhenmaqingteng No. 1' have been bred, which cannot meet the demand for multifunctional, multipurpose, high-performance and high-value special bamboo varieties in industrial utilization, and it is urgent to cultivate new bamboo varieties with high yield, high quality and stress resistance through gene editing technology.

[0003] However, the process of cultivating plants by gene editing technology has certain randomness, and after gene editing is completed, plants that have obtained gene editing need to be screened out. The existing screening method is usually to use common resistance screening markers such as antibiotics or herbicides for screening, but using resistance screening markers for screening will further reduce the low regeneration efficiency of plants that have obtained gene editing, affect the normal growth of gene editing plants, and the screening process needs to be screened and cultivated, which is relatively cumbersome. Therefore, a gene editing screening tag that is convenient and fast and does not affect the normal growth of gene editing plants is necessary. SUMMARY

[0004] In view of the above problems, one of the purposes of the present application is to provide a screening tag in bamboo gene editing, PeVDE (bamboo violaxanthin de-epoxidase) gene. The screening tag is an endogenous screening tag of bamboo, which does not affect the regeneration efficiency of gene editing bamboo and does not affect its normal growth. Moreover, the deletion or reduction of PeVDE gene in bamboo will cause obvious changes in traits, and whether the bamboo that has successfully undergone gene editing can be determined by chlorophyll fluorescence parameter non-photochemical quenching (NPQ), and the determination method is simple and fast.

[0005] In order to achieve the above purpose, the present application can adopt the following technical scheme:

[0006] In one aspect, the present application provides application of a PeVDE gene as a gene editing screening tag in bamboo gene editing.

[0007] In another aspect, the present application provides a bamboo gene editing reagent, which comprises a reagent for editing a PeVDE gene; and a reagent for editing a target gene or an exogenous gene.

[0008] In still another aspect of the present application, a CRISPR / Cas vector is provided, which comprises a gRNA recognizing a PeVDE gene, and the gRNA comprises a sequence as shown in SEQ ID NO: 2 and / or SEQ ID NO: 3.

[0009] In still another aspect of the present application, a method for genetically editing a bamboo is provided, which comprises: infecting a bamboo to be genetically edited with the CRISPR / Cas9 vector as described above by means of Agrobacterium mediation, culturing the genetically edited bamboo, and detecting the NPQ value in the genetically edited bamboo to determine whether the bamboo to be genetically edited has been successfully genetically edited.

[0010] The present application has at least the following advantages:

[0011] (1) In the present application, the PeVDE gene is used as a genetic editing screening tag to avoid the influence of a resistance screening tag on the normal growth of a bamboo;

[0012] (2) In the present application, the PeVDE gene is used as a genetic editing screening tag, and whether the genetically edited bamboo is obtained can be determined directly by the chlorophyll fluorescence parameter non-photochemical quenching (NPQ), and the determination method is simple and fast. BRIEF DESCRIPTION OF DRAWINGS

[0013] Figure 1 Figure 1 shows the structure of the Phyllostachys edulis PeVDE gene and the design of the genetic editing gRNA target point in Example 1, wherein the triangle represents the primer position;

[0014] Figure 2 Figure 2 shows the chlorophyll fluorescence parameter NPQ analysis of the Agrobacterium-mediated CRISPR / Cas9 genetic editing vector containing the PeVDE gene gRNA transformed into the leaves of Phyllostachys edulis in Example 2, wherein A: visible light image, B: NPQ imaging, and the value is the NPQ value in the dashed box;

[0015] Figure 3 Figure 3 shows the target one PCR fragment of the PeVDE gene in the genetically edited region of the Phyllostachys edulis leaves digested (+) and not digested (-) by XbaI endonuclease in Example 2, wherein lane 1: DNA molecular weight marker, lanes 2-3: PeVDE fragment of uninfected leaves, lanes 4-5: PeVDE fragment of leaves after 5 days of infection, lanes 6-7: PeVDE fragment of leaves after 10 days of infection;

[0016] Figure 4 Figure 4 shows the target two PCR fragment of the PeVDE gene in the genetically edited region of the Phyllostachys edulis leaves digested (+) and not digested (-) by AgeI endonuclease in Example 2, wherein lane 1: DNA molecular weight marker, lanes 2-3: PeVDE fragment of uninfected leaves, lanes 4-5: PeVDE fragment of leaves after 5 days of infection, lanes 6-7: PeVDE fragment of leaves after 10 days of infection;

[0017] Figure 5 Sequencing analysis of PeVDE gene mutation for the target one gene editing in Example 2;

[0018] Figure 6 Sequencing analysis of PeVDE gene mutation for the target two gene editing in Example 2;

[0019] Figure 7 Flow chart of PeDWF4 gene structure and gRNA target design for the bamboo in Example 3;

[0020] Figure 8 Structure diagram of PeDWF4 and PeVDE gene editing CRISPR / Cas9 expression vector in Example 3;

[0021] Figure 9 Chlorophyll fluorescence parameter NPQ of Agrobacterium-mediated transformation of bamboo leaves containing PeDWF4 and PeVDE gene editing CRISPR / Cas9 expression vector in Example 3; wherein A: visible light image, B: NPQ imaging, the area indicated by the arrow is the area with lower NPQ value;

[0022] Figure 10 PCR band digestion analysis of PeDWF4 gene target in the gene editing region of bamboo leaves in Example 3;

[0023] Figure 11 Forward and reverse sequencing analysis of PeDWF4 gene mutation in the gene editing region of bamboo leaves in Example 3. DETAILED DESCRIPTION

[0024] The examples are used to better illustrate the present application, but are not intended to limit the present application to only the examples. Therefore, those skilled in the art can make non-essential improvements and adjustments to the embodiments according to the above description, which still belong to the protection scope of the present application.

[0025] The terms used herein are used only to describe specific embodiments and are not intended to limit the present disclosure. Unless there is a clear different meaning in the context, the singular form includes the plural form. As used herein, it is understood that terms such as "include", "have", "contain", etc. are intended to indicate the presence of features, numbers, operations, components, parts, elements, materials or combinations. The terms of the present application are disclosed in the specification, and are not intended to exclude the possibility that one or more other features, numbers, operations, components, parts, elements, materials or combinations can exist or can be added. As used herein, " / " can be interpreted as "and" or "or" depending on the circumstances.

[0026] The term "gene editing" in the present application refers to introducing an exogenous gene into a plant by transgenic technology or knocking out or knocking down a gene in the plant or inserting a new exogenous gene into the plant by gene editing technology (such as CRISPR / Cas9 technology).

[0027] The embodiments of the present application provide an application of a PeVDE gene as a screening tag in gene editing of bamboo.

[0028] It should be noted that plants in nature will encounter complex and variable natural environments, such as strong light stress. When plants absorb excess light energy, a large number of active oxygen free radicals will be generated in photosynthetic organs, which will further damage the photosynthetic organs. In order to avoid the formation of light damage, the light energy absorbed by the plant is no longer used for photosynthesis, but is directly converted into heat energy and dissipated, thereby avoiding the damage of strong light to the plant. This protection mechanism is called non-photochemical quenching (NPQ). The violaxanthin de-epoxidase (VDE) generates antheraxanthin (A) and zeaxanthin (Z) by catalyzing violaxanthin (V) in turn, and Z triggers the formation of NPQ by forming a thylakoid transmembrane pH gradient. It can be seen that VDE plays an important role in the NPQ process under strong light stress. A large number of studies have shown that overexpression of various plant VDE genes can improve the NPQ ability of transgenic or gene edited plants under strong light stress; inhibition or mutation of the VDE gene can severely inhibit the NPQ ability of plants under strong light stress, while not affecting the growth state of plants in normal growth environment. The visual chlorophyll fluorometer [IMAGING-PAM (Walz, Effeltrich)] can monitor the NPQ value of the plant in real time, and can also image the NPQ value of the region. Therefore, the content of Z can be determined by detecting the NPQ value, and then the expression of the VDE gene can be analyzed, and finally it can be determined which region of the VDE gene in the bamboo leaf is edited. At the same time, after editing the VDE gene in the bamboo leaf, the phenotype of a significant decrease in the NPQ value appears in the leaf, so this phenotype can be used as a trait tag to determine the plant after gene editing is successful. Using the PeVDE gene as a gene editing screening tag avoids the influence of the resistance screening tag on the normal growth of the bamboo; and whether the gene edited bamboo is obtained can be directly determined by the chlorophyll fluorescence parameter non-photochemical quenching (NPQ), and the determination method is simple and fast.

[0029] In some specific embodiments, a PeVDE gene is provided for application as a gene editing screening tag in transient in situ gene editing of bamboo.

[0030] It should be noted that, due to the weak foundation of bamboo genetic breeding research and the imperfect genetic transformation system, although the existing technology has obtained gene edited plants in Dendrocalamus latiflorus and Phyllostachys edulis by transforming callus induced from young shoots or young embryos, there are high difficulty in obtaining transformation materials, low regeneration efficiency of bamboo, and long genetic transformation cycle, and under the condition of selection pressure of antibiotic tag, the regeneration efficiency is lower. In some embodiments of the present application, the bamboo is successfully in-situ gene edited, which uses bamboo seedling leaves or bamboo shoots as materials, does not need the de-differentiation process of tissue culture (about 1 year), and the whole process only needs 2-3 weeks, the method is convenient and short in cycle; and the PeVDE gene is used as a gene editing screening tag to avoid the influence of resistance screening tag on the normal growth of bamboo; in addition, for bamboo species for shoots, due to the long and unstable flowering cycle of bamboo, it is difficult to remove the plasmid fragment integrated into the chromosome of bamboo in a short period of time by hybridization and other methods, so the bamboo species for shoots has the safety hidden danger of transgene, and the present application uses transient in-situ editing to avoid the safety hidden danger of transgene of bamboo species for shoots. In summary, the present application first establishes an efficient in-situ gene editing system of bamboo leaves, and develops an endogenous screening tag PeVDE gene for transient in-situ gene editing of bamboo, which provides an effective way for future rapid verification of gene function in bamboo by using gene editing technology.

[0031] It should be understood that, in the above application, the PeVDE gene is a PeVDE gene known in the art, and when used as a gene editing screening tag, one of the exon genomic sequences can be selected, such as the gene sequence shown as SEQ ID NO: 1.

[0032] It should be further noted that, although the Agrobacterium transient expression mediated gene editing technology has been applied in dicotyledonous plants tobacco, there is no report on Agrobacterium transient expression mediated gene editing in monocotyledonous plants; at the same time, the method of tobacco transient expression is not applicable to monocotyledonous plants such as rice and bamboo.

[0033] Another embodiment of the present application provides a bamboo gene editing reagent, which comprises a reagent for editing a PeVDE gene; and a reagent for editing a target gene or an exogenous gene. It should be noted that the reagent for editing the PeVDE gene refers to a reagent for knocking down or knocking out the PeVDE gene, such as a reagent related to CRISPR / Cas technology, ZFNs (zinc-finger nucleases) technology, TALENs (transcription activator-like effector nucleases) technology, BE (base editing) technology, or PE (prime editing) technology for editing the PeVDE gene; the reagent for editing the target gene refers to a reagent for knocking down or knocking out a gene that needs to be edited in the bamboo, such as a reagent related to CRISPR / Cas9 technology, ZFNs (zinc-finger nucleases) technology, TALENs (transcription activator-like effector nucleases) technology, BE (base editing) technology, or PE (prime editing) technology for editing the target gene; and the exogenous gene refers to a gene fragment that needs to be inserted or transferred into the bamboo, such as a gene fragment that can change the characteristics of the bamboo, such as thin and hollow wall, large transverse variability, large sharpness, and uneven length of the node, and the habit of preferring wet and not tolerating cold.

[0034] Similarly, in the bamboo gene editing reagent described above, the PeVDE gene is a PeVDE gene known in the art, and when used as a gene editing screening tag, one of the exon genomic sequences can be selected, such as a gene fragment including SEQ ID NO: 1.

[0035] In another embodiment of the present application, a CRISPR / Cas9 vector for editing PeVDE gene is provided, which comprises a gRNA for recognizing PeVDE gene, and the gRNA comprises a sequence as shown in SEQ ID NO: 2 and / or SEQ ID NO: 3. Specifically, PeVDE gene is an endogenous gene of bamboo, and for the purpose of being used as a gene editing screening tag or other gene editing needs, it is necessary to use a gene editing tool to knock down or knock out PeVDE gene. In the embodiment of the present application, a CRISPR / Cas9 vector for editing PeVDE gene is designed, and a gRNA for two different target points is designed, and the sequence of the gRNA comprises a sequence as shown in SEQ ID NO: 2 or SEQ ID NO: 3. The editing efficiency of the CRISPR / Cas9 vector comprising the gRNA as shown in SEQ ID NO: 2 on PeVDE gene in bamboo can reach 15.4%, and the editing efficiency of the CRISPR / Cas9 vector comprising the gRNA as shown in SEQ ID NO: 3 on PeVDE gene in bamboo can reach 32.6%. Both of the editing efficiencies are relatively high.

[0036] In addition, it should be understood that, in addition to the gRNA, the CRISPR / Cas9 vector part of the above-mentioned CRISPR / Cas9 vector for editing PeVDE gene can be an existing CRISPR / Cas9 tool, such as a commonly used pC1300-Ubi::Cas9 vector. Of course, it can also be a CRISPR / Cas9 tool comprising other Cas9 enzymes. In some specific embodiments, a promoter can be connected to the CRISPR / Cas9 vector to enhance the expression. The promoter can be selected from promoters known in the art, such as rice OsU3 pro .

[0037] It should be further pointed out that, the gRNA for editing PeVDE gene as shown in SEQ ID NO: 2 or SEQ ID NO: 3 can be used alone or together. When used together, the gRNA as shown in SEQ ID NO: 2 or SEQ ID NO: 3 can be connected to the same existing CRISPR / Cas9 tool, and then agrobacterium is used for mediation of infection. Alternatively, the gRNA as shown in SEQ ID NO: 2 or SEQ ID NO: 3 can be connected to two different existing CRISPR / Cas9 tools, and then agrobacterium of the same system is used for mediation of infection.

[0038] In some embodiments, the CRISPR / Cas9 vector described above can comprise an exogenous gene. As described above, the exogenous gene refers to a gene fragment that needs to be inserted or transformed into bamboo, such as a gene fragment that can change the characteristics of bamboo, such as thin and hollow wall, large transverse variability, large sharpness, and uneven length of bamboo joints, and the habit of preferring wet and not tolerating cold; specifically, the exogenous gene fragment can be inserted into bamboo by CRISPR / Cas9 tool to obtain a gene edited bamboo; it should be noted that when the bamboo is genetically edited, the exogenous gene and the gRNA recognizing the PeVDE gene are loaded on the same CRISPR / Cas9 vector, and the gene edited bamboo is obtained after being mediated by Agrobacterium to obtain the gene edited bamboo. The editing of the exogenous gene can be directly determined according to the editing of the PeVDE gene, which is convenient and fast.

[0039] In some embodiments, the CRISPR / Cas9 vector described above can further comprise a gRNA recognizing a target gene. It should be noted that for the genetic editing of bamboo, in addition to the insertion of an exogenous gene, the knockout or knockdown of a specified gene of bamboo is also included, and the target gene refers to a gene that needs to be knocked out or knocked down in bamboo, which can be any gene in bamboo. As described above, when the bamboo is edited, the gRNA recognizing the target gene that needs to be knocked out or knocked down and the gRNA recognizing the PeVDE gene can be loaded on the same CRISPR / Cas9 vector, and the gene edited bamboo is obtained after being mediated by Agrobacterium, and the editing of the target gene can be directly determined according to the editing of the PeVDE gene, which is convenient and fast.

[0040] In some embodiments, the target gene of the gene that needs to be knocked out or knocked down is the PeDWF4 gene, and the gRNA recognizing the target gene comprises a sequence as shown in SEQ ID NO: 5. It should be noted that the PeDWF4 gene is a PeDWF4 gene known in the art, which comprises a sequence as shown in SEQ ID NO: 4, and the gRNA recognizing the target gene can be designed according to the method known in the art. In the embodiment of the present application, the gRNA with the sequence as shown in SEQ ID NO: 5 is designed, which can effectively recognize the PeDWF4 gene and accurately edit it.

[0041] Another embodiment of the present application provides a method for genetically editing bamboo, which comprises: infecting a bamboo to be genetically edited by Agrobacterium mediated by the CRISPR / Cas9 vector described above, culturing the bamboo to obtain a genetically edited bamboo, and detecting the NPQ value of the genetically edited bamboo to determine whether the bamboo to be genetically edited has been successfully genetically edited. Generally, the bamboo seedlings are under strong light (1200 μmol·m -2 ·s -1) After 2h treatment, the excess absorbed light energy will dissipate in the form of heat, resulting in the increase of NPQ value; at this time, the NPQ value of the leaf under the activation light (800 μmol·m -2 ·s -1 ) of the chlorophyll fluorometer will be higher than 0.3; and the area lower than 0.25 is identified as the area where the PeVDE gene is edited.

[0042] Specifically, the CRISPR / Cas9 vector described above can be used to edit the genes of bamboo, and it should be understood that, to edit the genes of the plant, Agrobacterium-mediated infection is needed, and the Agrobacterium is known in the art, such as Agrobacterium GV3101 or Agrobacterium AGL1; as described above, the deletion or reduction of the PeVDE gene in bamboo will result in the change of NPQ value, and the NPQ value can be detected by the existing equipment (such as a chlorophyll fluorometer). For example, for the DWF4 gene involved in the synthesis of brassinosteroids and affecting the growth of plant cells, if the same CRISPR / Cas9 vector includes gRNA recognizing the PeVDE gene and gRNA recognizing the target gene PeDWF4, after successful editing of the PeVDE gene, it indicates that the CRISPR / Cas9 vector successfully enters the bamboo cell and completes gene expression, and the gRNA recognizing the target gene PeDWF4 on the same vector will also be expressed, which indirectly indicates that PeDWF4 should also be successfully edited; the microscope observation result shows that the cell length in the region is shortened, which further indicates that the PeDWF4 gene is successfully edited; similarly, if the same vector CRISPR / Cas9 vector includes gRNA recognizing other target genes and exogenous genes, after successful editing of the PeVDE gene, it indicates that the target gene is also successfully edited. That is, the method for editing the genes of bamboo of the present application can use the expression of the PeVDE gene as a trait tag to simply and quickly determine whether the editing of the genes of bamboo is successful or not.

[0043] In order to better understand the present application, the content of the present application will be further illustrated in combination with specific examples below, but the content of the present application is not limited to the examples below.

[0044] In the following examples, the YEP culture medium includes: 1 g of beef extract, 1 g of yeast extract and 0.5 g of NaCl per 100 mL of distilled water.

[0045] In the following examples, the infection solution includes: 10 mmol of MES (pH 6.0) and 10 mmol of MgCl2 per 1000 mL of distilled water.

[0046] Example 1 Bamboo PeVDE gene structure and gene editing gRNA target point design

[0047] (1) Using Phyllostachys edulis genome DNA as a template, the genomic sequence containing the first exon of PeVDE gene was amplified, and the primer sequences were as follows:

[0048] Upstream primer: 5'-TGTGGCTTCTAAAGCTCTGCAATCT-3',

[0049] Downstream primer: 5'-TGTCAATGCTACAAGTCCTGGCA-3',

[0050] After amplification and sequencing, the sequence SEQ ID NO: 1 was obtained for target design;

[0051] (2) Two target points containing single enzyme cutting sites were designed on the first exon, wherein target point one contained XbaI and target point two contained AgeI enzyme cutting sites, and the target sequence information was seen in SEQ ID NO: 2 and SEQ ID NO: 3, and rice OsU3 pro was used as a promoter;

[0052] (3) The two target point sequences were respectively constructed into the published pC1300-Ubi::Cas9 vector to obtain expression vectors pC1300-Ubi::Cas9-OsU3 pro ::PeVDE gRNA1 and pC1300-Ubi::Cas9-OsU3 pro ::PeVDE gRNA2 (as shown in Figure 1 ).

[0053] Example 2 Gene editing of Phyllostachys edulis PeVDE gene

[0054] (1) The expression vectors pC1300-Ubi::Cas9-OsU3 pro ::PeVDE gRNA1 and pC1300-Ubi::Cas9-OsU3 pro ::PeVDE gRNA2 were respectively introduced into Agrobacterium tumefaciens GV3101 strain, and cultured in YEP medium until OD 600 reached 0.6-0.8;

[0055] (2) The bacteria were centrifuged and collected, washed once with the infection solution and resuspended in the infection solution until OD 600 reached 0.4-0.6, and the Agrobacterium containing pC1300-Ubi::Cas9-OsU3 pro ::PeVDE gRNA1 and pC1300-Ubi::Cas9-OsU3 pro ::PeVDE gRNA2 were mixed at a ratio of 1:1 and used for transformation of bamboo leaves;

[0056] (3) One-month-old Phyllostachys edulis seedlings sowed in plug trays were removed from the plug trays and the roots were wrapped with aluminum foil. A sharp needle (e.g. a syringe needle) was used to wound the apical meristem of the bamboo shoots;

[0057] (4) The aboveground parts of the bamboo shoots were quickly inverted and immersed in the Agrobacterium infection solution, and then placed in a vacuum device for negative pressure vacuuming, with a negative pressure of 25-27 inches Hg for 2 min;

[0058] (5) The infected bamboo shoots were removed from the vacuum device, the aluminum foil was removed and the bamboo shoots were moved into the plug trays for cultivation. On the first day, a transparent film was used to cover the plug trays to maintain humidity (air relative humidity of about 80%). On the second day, the film was removed and the cultivation was continued;

[0059] (6) On the 10th day, the bamboo shoots were subjected to a high light intensity of (1200 μmol·m -2 ·s -1 ) for 2 h using a chlorophyll fluorescence imaging instrument IMAGING-PAM (Walz, Effeltrich). The activation light was set to 800 μmol·m -2 ·s -1 , and the treatment duration was 315 s. The chlorophyll fluorescence images of the transformed leaves were obtained (as shown in Figure 2 );

[0060] (7) The leaf regions with low and high NPQ values shown in Figure 2 were selected, and the genomic DNA was extracted and amplified using the primer pair of step (1) in Example 1. Part of the amplified fragments were subjected to enzyme digestion, and part were ligated to the pGEM-T Easy vector (Promega, USA) for Sanger DNA sequencing.

[0061] The enzyme digestion results showed that part of the target point one amplified fragments were not cut by XbaI, indicating that part of the target point one had been successfully edited out (as shown in Figure 3 ); part of the target point two amplified fragments were not cut by AgeI, indicating that part of the target point two had been successfully edited out (as shown in Figure 4 ); and the proportion of uncut fragments after 10 days of infection was significantly higher than that after 5 days of infection, indicating that gene editing was still in effect.

[0062] The sequencing results after 10 days of infection showed that the PeVDE gene of Phyllostachys edulis was edited, with a gene editing efficiency of 15.4% for target point one (as shown in Figure 5 ) and a gene editing efficiency of 32.6% for target point two (as shown in Figure 6 ).

[0063] The above examples show that the method can quickly and efficiently edit the endogenous genes in the bamboo leaves in situ.

[0064] Example 3 PeVDE gene editing as a tag for detecting editing of other genes

[0065] Fast-growing is the most prominent feature of bamboo, and various internal and external factors affect the rapid growth of bamboo. Brassinosteroids have the function of regulating plant growth and development, such as promoting cell division and elongation; DWF4 is an important rate-limiting enzyme for brassinosteroid synthesis, which affects brassinosteroid synthesis and thus affects growth and development. In the embodiments of the present application, the Phyllostachys edulis DWF4 gene (PeDWF4) is selected as the target gene for gene editing.

[0066] (1) The genomic DNA of Phyllostachys edulis was used as a template to amplify the genomic sequence containing the first exon of PeDWF4 gene, and the primer sequences were as follows:

[0067] upstream primer: 5'-AGCACCGCAACAGACGGAGAG-3',

[0068] downstream primer: 5'-CATGGACCACTTGCCCAGGATG-3',

[0069] After amplification, the sequence SEQ ID NO: 4 was obtained for target design;

[0070] (2) A target was designed on the first exon of PeDWF4, and the target sequence was SEQ ID NO: 5 (as shown in Figure 7 ); and the target of PeVDE gene was constructed into the same pC1300-Ubi::Cas9 vector to form the expression vector pC1300-Ubi::Cas9-OsU3 pro ::PeDWF4gRNA-Ubi::Cas9-OsU3 pro ::PeVDEgRNA2 (as shown in Figure 8 );

[0071] (3) The pC1300-Ubi::Cas9-OsU3 pro ::PeDWF4gRNA-Ubi::Cas9-OsU3 pro ::PeVDEgRNA2 vector was transformed into Agrobacterium tumefaciens GV3101 strain, and cultured in YEP medium to OD 600 0.6-0.8;

[0072] (4) The bacterial cells were centrifuged and collected, washed once with the infection solution, and then resuspended in the infection solution to OD 600to 0.4-0.6;

[0073] (5) One-month-old Phyllostachys edulis seedlings sowed in plug trays were removed from the plug trays and the roots were wrapped with aluminum foil. A sharp needle (such as a syringe needle) was used to prick the apical meristem of the bamboo seedling;

[0074] (6) The aboveground part of the bamboo seedling was quickly inverted and immersed in the Agrobacterium infection solution, and then placed in a vacuum device for negative pressure vacuuming, with a negative pressure of 25-27 inches Hg for 2 min;

[0075] (7) The infected bamboo seedling was removed from the vacuum device, the aluminum foil was removed, and the bamboo seedling was moved into a plug tray for culture. On the first day, a transparent film was used to cover the plug tray to maintain humidity (air relative humidity about 80%), and on the second day, the film was removed, and the culture was continued;

[0076] (8) On the 10th day, the bamboo seedling was subjected to a high light treatment of 1200 μmol·m -2 ·s -1 for 2 h using a chlorophyll fluorescence instrument IMAGING-PAM (Walz, Effeltrich). The activation light was set to 800 μmol·m -2 ·s -1 for a period of 315 s, and a chlorophyll fluorescence image of the transformed leaf was obtained (as shown in Figure 9 );

[0077] (9) The leaf regions with low and high NPQ values shown in Figure 6 were selected, and genomic DNA was extracted therefrom. The extracted genomic DNA was amplified using the primer pair of step (1) in Example 1. Part of the amplified fragments were subjected to enzyme digestion, and part of the amplified fragments were subjected to Sanger DNA sequencing.

[0078] The enzyme digestion results showed that, after 10 days of infection, part of the PeDWF4 target amplification fragments of the leaf were not digested by Sac I, indicating that part of the target had successfully achieved gene editing (as shown in Figure 10 ). The sequencing results after 10 days of infection showed that the PeDWF4 gene of Phyllostachys edulis was edited, and the gene editing efficiency was 8.8% (as shown in Figure 11 ).

[0079] The above examples show that the leaves edited by the PeVDE gene have a low NPQ value, which can be used as a trait detection tag for mutation screening after editing of other genes.

[0080] Finally, it is to be explained that the above embodiments are only used to illustrate the technical solutions of the present application but not to limit the present application. Although the present application is described in detail with reference to the preferred embodiments, it should be understood by those skilled in the art that the technical solutions of the present application can be modified or equivalently replaced without departing from the purpose and scope of the technical solutions of the present application, and all of them should be covered in the scope of the claims of the present application.

Claims

1. Application of the PeVDE gene as a gene editing screening tag in bamboo gene editing: gRNAs were designed with the genomic sequence containing the first exon of the PeVDE gene as the target. The genomic sequence containing the first exon of the PeVDE gene is shown in SEQ ID NO:

1.

2. The application according to claim 1, characterized in that, The application of gene editing screening tags in bamboo gene editing specifically refers to the application of gene editing screening tags in in situ gene editing of bamboo.

3. The application according to claim 1, characterized in that, The application of gene editing screening tags in bamboo gene editing specifically refers to the application of gene editing screening tags in transient in situ gene editing of bamboo.

4. A bamboo gene-editing reagent, characterized in that, The invention includes reagents for editing the PeVDE gene; and reagents for editing the target gene; the reagents for editing the PeVDE gene are designed with gRNAs targeting the genomic sequence containing the first exon of the PeVDE gene, as shown in SEQ ID NO:1; the gRNAs include sequences as shown in SEQ ID NO:2 and / or SEQ ID NO:

3.

5. The bamboo gene-editing reagent according to claim 4, characterized in that, It also includes gRNAs that recognize the target gene.

6. The bamboo gene-editing reagent according to claim 5, characterized in that, The target gene is the PeDWF4 gene, and the gRNA that recognizes the target gene includes the sequence shown in SEQ ID NO:

5.

7. A method for gene editing in bamboo, characterized in that, include: The bamboo gene-editing reagent according to any one of claims 4 to 6 is used to infect bamboo to be gene-edited via Agrobacterium-mediated infection, and gene-edited bamboo is cultured. The NPQ value in the gene-edited bamboo is detected to determine whether the gene editing of the bamboo to be gene-edited has been successfully performed.

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