CN115948451A - Application of LsARF3 protein or coding gene thereof in regulation and control of high-temperature bolting performance of leaf lettuce - Google Patents
Application of LsARF3 protein or coding gene thereof in regulation and control of high-temperature bolting performance of leaf lettuce Download PDFInfo
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Abstract
The invention relates to the technical field of plant genetic engineering, in particular to application of LsARF3 protein or an encoding gene thereof in regulating and controlling the high-temperature bolting performance of leaf lettuce. The research of the invention finds that the LsARF3 protein has a great influence on the high-temperature bolting performance of the leaf lettuce, and the regulation and control of the high-temperature bolting performance of the leaf lettuce can be realized by regulating the expression of the LsARF3 gene, wherein the high-temperature bolting performance is the bolting performance of the leaf lettuce after the treatment at 33 +/-2 ℃. The LsARF3 gene provided by the invention can be applied to the high-temperature bolting performance of the leaf lettuce, can effectively adjust the high-temperature bolting performance of the leaf lettuce, and has great significance for discussing a high-temperature bolting mechanism, making scientific management measures and creating excellent bolting-resistant germplasm.
Description
Technical Field
The invention relates to the technical field of plant genetic engineering, in particular to application of LsARF3 protein or an encoding gene thereof in regulating and controlling the high-temperature bolting performance of leaf lettuce.
Background
Leaf lettuce (Lactuca sativa L.) commonly called lettuce prefers cold climate, and is an important worldwide fresh-eating vegetable. The vegetable is popular with consumers due to good taste and excellent nutritional quality, and the cultivation area is rapidly expanded in recent years, so that the vegetable is one of important types of green vegetables. However, when the temperature in summer exceeds 30 ℃, early bolting of the leaf lettuce is easily caused, and then the yield is reduced, and the edible quality and the commodity value of the leaf lettuce are seriously influenced.
At present, certain results have been obtained in the research on the cultivation technology and physiological level of the high-temperature bolting of the leaf lettuce, and the research on the protein and gene level is very deficient. Therefore, the method has important biological significance for researching the bolting of the leaf lettuce by combining with a gene engineering technology, has important theoretical guidance significance and application prospect for subsequently formulating scientific measures to regulate bolting, solving the core problems of improving yield and quality and ensuring annual production in industrial development.
Disclosure of Invention
In order to solve the technical problems in the prior art, the invention provides application of LsARF3 protein or a coding gene thereof in regulating and controlling the high-temperature bolting performance of leaf lettuce.
In a first aspect, the invention provides an application of LsARF3 protein or a coding gene thereof, or a biological material containing the coding gene thereof in regulating and controlling the high-temperature bolting performance of leaf lettuce.
The invention further provides application of the LsARF3 protein or the coding gene thereof, or a biological material containing the coding gene thereof in improvement of high-temperature-resistant bolting germplasm resources of the leaf lettuce.
Further, the high-temperature bolting resistance performance is as follows:
the bolting performance of the leaf lettuce after the treatment at 31-35 ℃.
Further, the biological material is an expression cassette, a vector or a transgenic cell.
Further, the LsARF3 protein comprises an amino acid sequence shown as SEQ ID NO. 1.
In a second aspect, the invention provides a method for improving bolting resistance of leaf lettuce, which is used for inhibiting expression of LsARF3 protein in the leaf lettuce; the LsARF3 protein comprises an amino acid sequence shown as SEQ ID NO. 1.
Further, the inhibition of the expression of the LsARF3 protein in the leaf lettuce is as follows:
regulating and controlling the expression of the LsARF3 gene in the leaf lettuce by methods of transgenosis, gene knockout, RNA interference, hybridization, backcross, selfing or asexual propagation;
the LsARF3 gene comprises a nucleotide sequence shown as SEQ ID NO. 2.
Further, the transgenesis comprises introducing a recombinant expression vector containing the LsARF3 gene into a plant by using a Ti plasmid, a plant virus vector, direct DNA transformation, microinjection, a gene gun, conductance or an agrobacterium-mediated method to obtain a transgenic plant strain; and/or the presence of a gas in the gas,
the gene knockout comprises the step of knocking out the LsARF3 gene by utilizing a DNA homologous recombination technology, a Cre/LoxP technology or a CRISPR/Cas9 technology to obtain a transgenic plant line.
Further, the present invention further provides a gRNA comprising a nucleotide sequence shown as SEQ ID No.3 or SEQ ID No. 4. The gRNA can be applied to gene knock-out.
The invention has the following beneficial effects:
the research of the invention finds that the LsARF3 protein has a great influence on the high-temperature bolting performance of the leaf lettuce, and the regulation and control of the high-temperature bolting performance of the leaf lettuce can be realized by regulating the expression of the LsARF3 gene. The LsARF3 gene provided by the invention can be applied to the high-temperature bolting performance of the leaf lettuce, can effectively adjust the bolting performance of the leaf lettuce at high temperature, and has great significance for discussing the high-temperature bolting mechanism, making scientific management measures and creating excellent bolting-resistant germplasm.
Drawings
In order to more clearly illustrate the technical solutions of the present invention or the prior art, the drawings needed for the description of the embodiments or the prior art will be briefly described below, and it is obvious that the drawings in the following description are some embodiments of the present invention, and those skilled in the art can also obtain other drawings according to the drawings without creative efforts.
FIG. 1 shows the results of electrophoresis detection of a gRNA1-pCBC-DT1T2-g RNA2 fragment provided in example 1 of the present invention, with parallel lanes.
FIG. 2 shows the result of the linearization of pKSE401 vector detected by electrophoresis in example 1 of the present invention, and the multiple lanes are parallel.
FIG. 3 shows the result of detecting PCR products of E.coli colonies by electrophoresis as provided in example 1 of the present invention, wherein the multiple lanes are parallel.
FIG. 4 shows the electrophoresis result of the PCR product when the primer CRISPR-DNA-F/R is used for amplifying the positive seedling provided by the embodiment 1 of the invention.
FIG. 5 is a schematic diagram of editing types of lsarf3 homozygous mutant lines provided in example 1 of the present invention; in the figure, wild Type (WT), lsarf3-4, lsarf3-6, and lsarf3-9, respectively; wherein, the 7 th base after Target 2 of ls arf3-4 is mutated, one base T is inserted into the first 82 bases of Target 1 of ls arf3-6, lsarf3-9 is a chimera, 3 base mutations are generated, C-G, A-G and A-T.
Fig. 6 is a photograph of phenotype of WT and lsarf3 homozygous mutant plants at 32, 70 days of high temperature provided in example 1 of the present invention, with ruler =6cm.
FIG. 7 shows the flower bud differentiation status of WT and lsarf3 homozygous mutant plants at 32 days under high temperature as provided in example 1 of the present invention; in the figure, 1 represents WT,2 represents lsarf3-4,3 represents lsarf3-6,4 represents lsarf3-9, and scale =200 μm.
Fig. 8 shows the statistical results of bolting, budding and flowering time of the wild type and mutant plants at high temperature provided in example 1 of the present invention (p <0.05, p < 0.01).
FIG. 9 shows the results of electrophoresis detection of the LsARF 3-added homologous arm fragment provided in example 2 of the present invention, wherein the multiple lanes show parallel results.
FIG. 10 shows the result of detecting the linearization of the PRI101 vector by electrophoresis according to example 2 of the present invention, where 1 is the result of cleavage of PRI101, 2 is the PRI101 plasmid, and multiple lanes are parallel.
FIG. 11 shows the results of the electrophoresis detection of PCR products from E.coli colonies provided in example 2 of the present invention, and the multiple lanes show parallel results.
FIG. 12 shows the electrophoresis results of PCR products obtained by amplifying positive seedlings with the primers G418-F/G418-R provided in example 2 of the present invention, and the results are shown in parallel in the multiple lanes.
FIG. 13 is a photograph of phenotype of WT and over-expressed plants at 60 days and 108 days under normal growth conditions as provided in example 2 of the present invention; WT, OE-1, OE-7, OE-6, respectively, are shown, with scale =6cm.
Fig. 14 shows the relative expression levels of LsARF3 gene in WT and over-expressed plants at 60 days of normal growth (p <0.05, p < 0.01) provided in example 2 of the present invention.
FIG. 15 shows flower bud differentiation status of WT and overexpressed plants at 60 days under normal growth conditions as provided in example 2 of the present invention; in the figure, 1 represents WT,2 represents OE-1,3 represents OE-7,4 represents OE-6, and the scale =200 μm.
Fig. 16 shows statistical results of bolting, budding, and flowering time of wild type and over-expressed plants under normal growth conditions (p <0.05, p < 0.01) provided in example 2 of the present invention.
Detailed Description
In order to make the objects, technical solutions and advantages of the present invention clearer, the technical solutions of the present invention will be clearly and completely described below with reference to the accompanying drawings, and it is obvious that the described embodiments are some, but not all embodiments of the present invention. All other embodiments, which can be derived by a person skilled in the art from the embodiments given herein without making any creative effort, shall fall within the protection scope of the present invention.
Example 1CRISPR/Cas9-LsARF3 vector construction
1.LsARF3 gene targeting site (gRNA) design
According to the CRISPR/Cas9 system original spacer sequence adjacent motif recognition (PAM) principle and gene knockout target site guide RNAs (gRNAs) design principle, a CRISPR-gRNA design website (http:// CRISPR. Hzau.edu.cn/CRISPR2 /) and NCBI-Blast potential off-target site detection are used for selecting two most suitable gRNAs.
2. Design of four primers
Four primers were designed based on the two gRNA sequences, and the final primer sequences were as follows:
DT1-BsF(SEQ ID NO.5)：
5’-GCTAGAGTCGAAGTAGTGATTGCTCGAACAGCTACAATCCAGCGG-3’；
DT1-F0(SEQ ID NO.6)：5’-CTCGAACAGCTACAATCCAGCGGGTTTTAGAGCTAGAAATAGC-3’；
DT1-R0(SEQ ID NO.7)：5’-GTGGGTGCTGGTATCGGAGGCGGCAATCTCTTAGTCGACTCTAC-3’；
DT1-BsR(SEQ ID NO.8)：5’-CTTGCTATTTCTAGCTCTAAAACGTGGGTGCTGGTATCGGAGGCGG-3’。
3. add homology arm
pCBC-DT1T2 vector plasmid is used as a template, and Novozam high-fidelity enzyme is used
Max Master Mix (Dye Plus), four primers DT1-BsF + DT1-F0/D DT1-BsR + DT1-R0 amplification to get gRNA1 and gRNA2 series repeated expression cassette fragment with added homologous arm. Detection was performed by 1% agarose gel electrophoresis (see FIG. 1 for results).
4. Vector linearization
BsaI-HFv2 restriction enzyme (NEB) linearized pKSE401 vector, and 1% agarose gel electrophoresis was used to determine whether the cleavage was complete (see FIG. 2 for results).
5. Homologous recombination
Homologous recombination PCR products (gRNA 1-pCBC-DT1T2-gRNA2 fragments) with the linearized vector (the cleaved products obtained in Step 4) were obtained from ClonexpressIIOne Step Cloning Kit (Novozam). The reaction procedure was 30min at 37 ℃ and 5min at 4 ℃. The reaction system is shown in table 1 below.
TABLE 1 recombination reaction System (5. Mu.L)
6. Competent transformation of Escherichia coli
5ul of transformed E.coli competence was taken and screened on Kan plates.
Adding 5 μ l of the homologous recombination product obtained in step 5 into 50 μ l of ice, mixing, and standing on ice for 30min; the mixture was heat-shocked in a water bath at 42 ℃ for 30s, and rapidly transferred to an ice bath for 2min.
The following work is performed in the clean bench:
add 500. Mu.l of blank LB liquid medium to the centrifuge tube, mix well and shake at 37 ℃ for 1h on a shaker at 200 rpm. The activated bacterial suspension was pipetted 200. Mu.l and spread on LB solid medium containing Kan, and the plate was inverted overnight in an incubator at 37 ℃ for culture (see the general formula gold Trans1-T1 Phage resist chemical composition Cell Specification).
7. Detection of Positive clones
KANG is Taq enzyme (2 xTaq MasterMix (Dye)) and primer U626-IDF/U629-IDR for colony PCR with reaction program of 94 deg.C for 2min;35 × (94 ℃,30s, 59 ℃,30s, 72 ℃,30 s); 72 ℃ for 2min.
The reaction system is shown in Table 2. The PCR product was detected on a 1% agarose gel and the band size was approximately 750bp (see FIG. 3 for results). The positive bacteria liquid is sent to the company for sequencing, and the sequencing primer is U626-IDF/U629-IDR.
Colony PCR and sequencing primers:
U626-IDF(SEQ ID NO.9)：5’-TGTCCCAGGATTAGAATGATTAGGC-3’；
U629-IDR(SEQ ID NO.10)：5’-AGCCCTCTTCTTTCGATCCATCAAC-3’。
TABLE 2 colony PCR reaction System (25. Mu.L)
8. Agrobacterium infection transformation
Sequencing results show that 2 plasmids of gRNA can be subjected to agrobacterium-competent transformation.
Add 50. Mu.l GV3101 competence to 5ng plasmid, standing on ice for 5min, liquid nitrogen for 5min, water bath at 37 ℃ for 5min, ice bath for 5min. Add 700. Mu.l of blank LB to the centrifuge tube. Shaking at 28 deg.C with a shaker 200r for 2-3h. The strain was enriched by centrifugation at 6000rmp for 1min, leaving about 100. Mu.l of supernatant resuspended, and the inoculum was spread on solid LB medium containing Kan and Rif. The cells were cultured in an incubator at 28 ℃ for 2 to 3 days in an inverted manner (see GV3101 chemical company Cell Specification, only).
9. Genetic transformation of leaf lettuce
9.1 Medium formulation
MS liquid medium (1L): m519 4.43g + sucrose 30g, pH =5.8, 121 ℃,20min sterilization;
MS solid medium (1L): m519 4.43g + sucrose 30g + agar 7g, pH =5.8, sterilization at 121 ℃ for 20 min;
1/2MS solid Medium (1L): m519 2.215g + sucrose 30g + agar 7g, PH =5.8, 121 ℃,20min sterilized;
preculture/cocultivation medium (1L): MS +0.6 mg/L6-BA +0.06mg/L NAA, PH =5.8;
differentiation medium (1L): MS +0.06mg/L NAA +0.6 mg/L6-BA + Tim + Kan, PH =5.8;
rooting medium (1L): 1/2MT (Beijing cool stroke) 2.32g +1mg/L IBA +0.1mg/L IAA + activated carbon, pH =5.8.
9.2 genetic transformation procedure
In the test, the genetic transformation system of the leaf lettuce mediated by agrobacterium is used for the cotyledon as an explant, the culture temperature is 25 +/-3 ℃, the photoperiod is 16/8h, and the illumination intensity is 12000lux.
The method comprises the following specific steps:
(1) Cultivation of sterile seedlings
Sterilizing with 50% 84 disinfectant for 15min, washing with sterile water for 4 times (each time for 1 min); the sterilized seeds were placed in a petri dish with 1 layer of sterile filter paper (wetted with sterile water) and placed in an illuminated incubator. After 1-2 days, the seeds germinate, and two cotyledons with the age of 4-7 days are taken as experimental materials.
(2) Preculture
The cotyledon cut was cut at the petiole (no growth point was needed) and placed on pre-culture medium (MS +0.5mg/L KT +0.05mg/L NAA) spread with filter paper. Preculture for 1 day (dark culture)
(3) Infection by infection
Streaking the bacterial liquid before infection at 28 deg.c for culture, selecting single colony in MS infection liquid after the bacterial plate grows out, shaking at 28 deg.c for 3 hr, and measuring OD value in 0.1-0.3 after the bacterial liquid is turbid. The cut cotyledons are placed in a sterilized dish or bottle, the prepared bacterial liquid is poured, gently shaken, and infected for 7 minutes. Taking out the infected cotyledon explants, putting the explants on filter paper, and sucking off the redundant staining solution on the surface. Then transferred to a co-cultivation medium (MS +0.5mg/L KT +0.05mg/L NAA). After sealing with a sealing film, the cells were cultured in the dark for about 30 hours (30 hours or more).
(4) Differential culture
Transferring to a differentiation culture medium MS +0.5mg/L KT +0.05mg/L NAA +300mg/L Tim +50mg/L Kan, placing 20 cotyledons in each dish, and replacing with a new culture medium after two to three weeks to determine whether seedlings are differentiated.
(5) Bud cutting and rooting
Preparing a sterile rooting medium in advance: 1/2MS +0.05mg/L NAA +300mg/L timentin, sterilized forceps, scissors, knife and filter paper. After the callus developed a sterile seedling, it was removed and placed on filter paper, and the sterile bud with the growing point was carefully excised.
(6) Transplanting and acclimatizing
After the regenerated seedlings grow to be strong enough, the regenerated seedlings are carefully taken out, the culture medium is cleaned in water and transplanted into nutrient soil, the water is poured, the tissue culture seedlings are moisturized, the moisture-preserving film is uncovered a little after the seedling culture is adapted to about one week, and the seedlings continue to grow in a greenhouse.
10. Positive seedling identification and homozygous mutant acquisition
On a lettuce genome DNA sequence (LOC 111884676), primers CRISPR-DNA-F/R are designed about 200bp before and after two gRNAs, a positive seedling target sequence is amplified (the result is shown in figure 4), the amplification enzyme is Novozam high-fidelity enzyme, and the reaction program is as follows: at 95 ℃ for 3min;35 × (95 ℃,15s, 60 ℃,15s, 72 ℃,60 s); 72 ℃ for 5min. The reaction system is shown in Table 3. The PCR product is sequenced to identify the editing type. The editing types mainly include four types, i.e., unedited, homozygous, heterozygous and chimeric. Wherein, the unedited and homozygous editing types can be judged directly through the sequencing result of the PCR product, the sequencing result of the heterozygous and chimeric PCR product is bimodal, after the PCR product is connected to a Blunt-Zero cloning vector, at least 10 monoclones are sent to each sample for sequencing, and then the judgment is carried out. F of 3 strains is finally obtained 2 Generation homozygous mutant. The lsarf3 gene was edited as follows (see FIG. 5 for results).
Identifying a primer:
Cas9-F(SEQ ID NO.11)：5’-GAACAGCGACAAGCTCATCG-3’；
Cas9-R(SEQ ID NO.12)：5’-GAGCTGAGACAGGTCGATGC-3’；
CRISPR-DNA-F(SEQ ID NO.13)：5’-CTCGAACAGCTACAATCCAGCGG-3’；
CRISPR-DNA-R(SEQ ID NO.14)：5’-GTGGGTGCTGGTATCGGAGGCGG-3’。
TABLE 3 DNA sequence amplification PCR reaction System (50. Mu.L)
11. Bolting phenotype identification of lettuce lsarf3 homozygous mutant used for leaves at high temperature
Planting leaf lettuce wild type and the obtained homozygous mutant plant in a computer greenhouse, wherein the culture conditions are as follows: light/dark: 14h/10h; light intensity 12000lux; the relative humidity is 65-75%. At normal temperature, day temperature/night temperature: 20 plus or minus 2 ℃/13 plus or minus 2 ℃. When the seedlings grow to six leaves and one heart, carrying out continuous high-temperature treatment, wherein the day temperature/night temperature: 33 +/-2 ℃/25 +/-2 ℃. After the lsarf3 mutant strain was treated at high temperature, the plants showed significantly delayed bolting (see results in FIG. 6). The paraffin section result of the stem tip of the plant 32 days after high temperature shows that the growth cone at the top end of the knockout mutant is obvious in protrusion, is coated in the middle of a leaf primordium, is in a semicircular shape, is still in a vegetative growth phase and does not start flower bud differentiation; the wild type plants had flat and round growing points at the top and had entered the bud differentiation initiation stage (see FIG. 7 for results). Particularly, the bolting, budding and flowering time is counted, the result shows that the bolting time is about 20 days later than that of the wild type, the budding time was 32 days later and the flowering time was 48 days later (see figure 8 for results).
Example 2OE-LsARF3 vector construction
1. Cloning of LsARF3 Gene
The PCR material sequences were as follows:
LsARF3-F：5’-ATGATGTGTGGTTTAATCGATCTGAACACC-3’；
LsARF3-R：5’-TCACAATCCTTGTACACAACCATCATTTGATC-3’。
2. add homology arm
Using PRI101 vector plasmid as template and Novozam high fidelity enzyme
Max Master Mix (Dye Plus), two primers LsARF3-F/R amplification to add the homologous arm of the expression cassette fragment. Detection was performed by 1% agarose gel electrophoresis (see FIG. 9 for results).
3. Vector linearization
The PRI101 vector was linearized with SmaI-HFv2 restriction enzyme (NEB) and checked for completion by 1% agarose gel electrophoresis (see FIG. 10 for results).
4. Homologous recombination
The Clonexpress II One Step Cloning Kit (Novozan) homologous recombination PCR product and the linearized vector (the cleavage product obtained in Step 4). The reaction sequence was 37 deg.C, 30min,4 deg.C, 5min. The reaction system is shown in Table 4 below.
TABLE 4 recombination reaction System (10. Mu.L)
6. Competent transformation of Escherichia coli
The procedure is as in example 1, step 6
7. Detection of Positive clones
KANG is Taq enzyme (2 xTaq MasterMix (Dye)) and primer G418-F/G418-R, used for colony PCR, and the reaction program is 94 ℃ for 2min;35 × (94 ℃,30s, 59 ℃,30s, 72 ℃,30 s); 72 ℃ for 2min. The reaction system is shown in Table 5. The PCR product was detected on a 1% agarose gel and the band size was approximately 363bp (see FIG. 11 for results). Positive bacteria were sent to the company for sequencing. The sequencing primer is G418-F/G418-R.
Colony PCR and sequencing primers:
G418-R：5’-AAAAGCGGCCATTTTCCACC-3’；
G418-R：5’-AAAAGCGGCCATTTTCCACC-3’。
TABLE 5 colony PCR reaction System (25. Mu.L)
8. Agrobacterium infection transformation
The procedure is as in step 8 of example 1
9. Genetic transformation
The procedure is as in example 1, step 9
10. Positive seedling identification
Extracting transgenic seedling leaf DNA, taking DNA as a template and 418-F/418-R as a primer, and amplifying to obtain T when a bright band appears 0 Transgenic positive shoots (see FIG. 12 for results), the amplification enzyme was 2X Phanta Max Master Mix (Dye Plus) of Novozam. Will T 0 Sowing the transgenic seeds and wild seeds simultaneously, and picking when the seeds grow to six leaves and one heartPositive seedling detection was performed by real-time fluorescence quantification (see FIG. 13 for results), with primers LsARF3-qF/LsARF3-qR.
Detection primers:
LsARF3-qF：5’-TGGTTGGAGTGCGTTCGTGAC-3’；
LsARF3-qR：5’-TGCTTGTGTCGCTCTTCGGATTC-3’。
11. plant phenotype result identification
Leaf lettuce wild type and positive over-expressed plants were grown in a computer greenhouse under light/dark conditions: 14h/10h; light intensity 12000lux; the relative humidity is 65-75%. At normal temperature, day temperature/night temperature: 20 plus or minus 2 ℃/13 plus or minus 2 ℃. Compared with wild plants, transgenic plants overexpressing LsARF3 showed obvious bolting phenomenon (see the result in FIG. 14). The expression level is about 4 times, 8 times and 16 times of that of wild plants (see the result in FIG. 15). The flower bud differentiation results showed that it started bolting at day 60. The specific bolting, budding and flowering time is counted, and the over-expressed plants are found to be about 48 days earlier than the wild plants in bolting time, about 33 days earlier and about 40 days earlier (see the results in FIG. 16).
Finally, it should be noted that: the above examples are only intended to illustrate the technical solution of the present invention, and not to limit it; although the present invention has been described in detail with reference to the foregoing embodiments, it will be understood by those of ordinary skill in the art that: the technical solutions described in the foregoing embodiments may still be modified, or some technical features may be equivalently replaced; and such modifications or substitutions do not depart from the spirit and scope of the corresponding technical solutions of the embodiments of the present invention.
Claims (10)
- The LsARF3 protein or the coding gene thereof or the biological material containing the coding gene thereof is applied to the regulation of the high-temperature bolting performance of the leaf lettuce.
- The application of LsARF3 protein or coding gene thereof, or biological material containing the coding gene thereof in the improvement of high-temperature-resistant bolting germplasm resources of leaf lettuce.
- 3. The use according to claim 1 or 2, wherein the high temperature bolting resistance of the leaf lettuce is modulated by regulating the expression level of the LsARF3 protein.
- 4. The use of any one of claims 1-3, wherein the high temperature bolting resistance is:the bolting performance of the leaf lettuce after the treatment at 31-35 ℃.
- 5. The use according to any one of claims 1 to 4, wherein the biological material is an expression cassette, a vector or a transgenic cell.
- 6. The use of any one of claims 1 to 5, wherein the LsARF3 protein comprises the amino acid sequence shown as SEQ ID No. 1.
- 7. A method for improving bolting resistance of leaf lettuce is characterized in that expression of LsARF3 protein in the leaf lettuce is inhibited; the LsARF3 protein comprises an amino acid sequence shown as SEQ ID NO. 1.
- 8. The method of claim 7, wherein the inhibiting expression of LsARF3 protein in leaf lettuce is:regulating and controlling the expression of the LsARF3 gene in the leaf lettuce by methods of transgenosis, gene knockout, RNA interference, hybridization, backcross, selfing or asexual propagation;the LsARF3 gene comprises a nucleotide sequence shown as SEQ ID NO. 2.
- 9. The method according to claim 8, wherein the transgene comprises introducing a recombinant expression vector comprising the LsARF3 gene into a plant using Ti plasmid, plant viral vector, direct DNA transformation, microinjection, gene gun, conductance, or agrobacterium-mediated methods to obtain a transgenic plant line; and/or the presence of a gas in the gas,the gene knockout comprises the step of knocking out the LsARF3 gene by utilizing a DNA homologous recombination technology, a Cre/LoxP technology or a CRISPR/Cas9 technology to obtain a transgenic plant line.
- 10. A gRNA comprising a nucleotide sequence set forth in SEQ ID No.3 or SEQ ID No. 4.
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Non-Patent Citations (3)
| Title |
|---|
| LI YUNFENG等: "LsARF3 mediates thermally induced bolting through promoting the expression of LsCO in lettuce (Lactuca sativa L.)", FRONT. PLANT SCI, 8 September 2022 (2022-09-08), pages 1 - 14 * |
| WANG YUBO等: "Application of exogenous auxin and gibberellin regulates the bolting of lettuce (Lactuca sativa L.)", OPEN LIFE SCIENCES, vol. 17, 29 April 2022 (2022-04-29), pages 438 * |
| 李艳君: "草莓ARF4 基因沉默载体构建及功能缺失分析", 硕士电子期刊库, no. 02, 15 February 2020 (2020-02-15), pages 1 - 64 * |
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