CN114990132A - Application of citrus CcLFY gene in regulation and control of shoot development - Google Patents
Application of citrus CcLFY gene in regulation and control of shoot development Download PDFInfo
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Abstract
The invention provides an application of a citrus CcLFY gene or a protein coded by the citrus CcLFY gene in regulation and control of plant shoot development, wherein the sequence of the citrus CcLFY gene is shown as SEQ ID NO. 1. The citrus CcLFY gene is functionally identified for the first time, an excessive expression vector of the gene is constructed, stable transformation of the lemon is carried out by utilizing an agrobacterium-mediated genetic transformation method, a transgenic plant is obtained, phenotype observation and analysis show that the transgenic lemon has a phenotype of axillary branch increase, and the citrus CcLFY gene cloned by the method has the function of regulating and controlling branch tip development. The discovery of the gene function provides a research foundation and new genetic resources for the regulation and improvement of the development of citrus and woody fruit trees branches.
Description
Technical Field
The invention relates to the technical field of plant genetic engineering, in particular to application of a citrus CcLFY gene in shoot development regulation.
Background
Citrus is the first fruit in the world, belongs to perennial woody fruit trees, and has a very important position in the gardening economy of China. For woody fruit trees such as citrus, a reasonable tree structure is very important for achieving the goals of high quality and high yield and simplifying cultivation, and branch tip regulation of the tree body is the basis for achieving an ideal tree shape. Therefore, it is necessary to research the branch tip regulation mechanism of woody fruit trees such as citrus, and a foundation is laid for cultivating new varieties of fruit trees with ideal plant types.
Disclosure of Invention
Therefore, the application of the citrus CcLFY gene in regulation and control of shoot development is needed, and a foundation can be laid for cultivating a new variety of fruit trees in an ideal tree form.
The invention adopts the following technical scheme:
the invention provides an application of a citrus CcLFY gene or a protein coded by the citrus CcLFY gene in regulation and control of plant shoot development, wherein the sequence of the citrus CcLFY gene is as follows: a) the nucleotide sequence is shown as SEQ ID NO. 1; b) the sequence shown in SEQ ID NO.1 is a sequence which is obtained by substituting, deleting and/or adding one or more nucleotides and can encode a protein with the function of regulating shoot development.
In some embodiments, the amino acid sequence of the protein encoded by the CcLFY gene is as follows: a) the amino acid sequence is shown as SEQ ID NO. 2; b) the amino acid sequence of which the sequence is substituted, deleted and/or added with one or more amino acids and has the function of regulating and controlling the development of shoot tips.
In some of these embodiments, the plant is lemon.
In some of these embodiments, shoot outgrowth is increased using overexpression of the CcLFY gene. Preferably, the method for over-expressing the CcLFY gene is as follows: PBI121 is used as an expression vector, fusion of a target gene segment and the vector is carried out by a homologous recombination method to construct a super-expression vector, and agrobacterium-mediated genetic transformation is utilized.
In some embodiments, the cloning method of the target gene fragment is as follows: taking total cDNA obtained by reverse transcription of the Klimenberger leaf tissue RNA as a template to carry out PCR amplification, wherein the sequence of a forward primer is shown as SEQ ID NO.3, and the sequence of a reverse primer is shown as SEQ ID NO. 4.
In some embodiments, the sequences of PCR amplification primers used for construction of the overexpression vector are respectively shown as SEQ ID NO.5, and the sequence of a reverse primer is shown as SEQ ID NO. 6.
In some of these embodiments, the method step of using agrobacterium tumefaciens mediated genetic transformation: preparing an epicotyl of the lemon seedling as a transformation material; dip-dyeing the epicotyl of the lemon seedling by using the activated agrobacterium dip-dyeing solution, transferring the epicotyl into a co-culture medium, and continuously culturing in dark conditions to obtain a stem section sprouting material; placing the stem bud-producing material in a screening culture medium containing kanamycin for subculture screening culture to obtain a stem elongation material; placing the stem elongation material in a rooting culture medium for continuous culture, identifying the positive transgenic material, and observing the shoot phenotype.
The beneficial effects of the invention are:
compared with the prior art, the invention discovers for the first time that the citrus CcLFY gene and the protein coded by the same have the regulation and control functions of axillary branches. The discovery of the new function of the gene provides a research foundation and new genetic resources for the regulation and improvement of the development of citrus and woody fruit trees branches.
Drawings
FIG. 1 shows the comparison result of the development of common lemon (WT) and lemon axillary buds overexpressing CcLFY gene.
FIG. 2 shows the comparison result of the development states of common lemon (WT) and lemon branches overexpressing CcLFY gene, wherein S1, S2 and S3 are three stages of the development of CcLFY overexpressing lemon 1# axillary bud.
Fig. 3 is a graph comparing the phenotype of the lemon-positive materials 1#, 2# overexpressing the CcLFY gene in example 3 with conventional normal lemon (WT) material.
Detailed Description
The present invention is further described in detail below with reference to specific examples so that those skilled in the art can more clearly understand the present invention.
The following examples are provided only for illustrating the present invention and are not intended to limit the scope of the present invention. All other embodiments obtained by a person skilled in the art based on the specific embodiments of the present invention without any inventive step are within the scope of the present invention.
In the examples of the present invention, all the raw material components are commercially available products well known to those skilled in the art unless otherwise specified; in the examples of the present invention, unless otherwise specified, all technical means used are conventional means well known to those skilled in the art.
Key raw material sources and composition description:
the krementanus leaf is prepared from the material in a specimen garden of Huazhong agriculture university in Wuhan, Hubei, wherein the Wild Type (WT) krementanus plant type is single-stem type and has less branches: immediately preserving with liquid nitrogen after collection, directly grinding for use, or preserving in a refrigerator at-80 deg.C for use.
MT basic culture medium: trace, ferric salt, glycine, inositol, VB and VC are 10mL respectively, macroelements are 100mL, sucrose is 40g/L, and agar is 8 g/L.
The citrus tube seeding culture medium comprises 25g/L of MT + sucrose and 8.0g/L of Agar.
Suspension medium (LM) MT + malt extract powder 0.5g/L + glutamine 1.5 g/L.
Germination culture medium (SY), MT +6BA 0.5mg/L + NAA 0.1mg/L + KT 0.5mg/L + Agar 8 g/L;
Co-Culture Medium (CM) SY + AS 50mg/L + Agar 8 g/L.
The Screening Medium (SM) is SY + Cef 400mg/L + Km50mg/L + Agar 8 g/L.
Elongation Medium (SEM): MT +6BA 0.1mg/L + GA30.5mg/L + IAA 0.5mg/L + Cef 400mg/L + Km50mg/L + Agar 8 g/L.
Rooting medium (RIM): 1/2MT + NAA 0.5mg/L + IBA 0.1mg/L + active carbon 0.5g/L + Agar 8g/L + Km 25mg/L + Cef 400 mg/L.
The sequence of the citrus CcLFY gene is as follows:
>Ciclev10033942m.g
ATGGACCCGGAAGCTTTCACGGCGAGTTTGTTCAAGTGGGACCCACGAGTGGTCGTGGCACCACCACCGGCCAGGGTGCAGCTGGAACAGGTGTCACAACCACCAGCAGTGCCGCTTGGTGCTGCGGCGGCGGCAGCCTACTCGGCGCTGGTGCGTCCAAGGGAGTTGGGTGGGCTGGAGGAGCTGTTCCAGGCTTATGGGATAAGATACCACACGGCAGCGAAGATAGCGGAGCTCGGGTTCACGGTGAACACGCTGTTGGACATGAAAGACGAGGAGCTCGATGAGATGATGAACAGCTTGGGTCACTTGTTCAGGTGGGAGCTGCTCGTTGGAGAGAGATACGGCATCAAAGCTGCTGTTAGGGCTGAGAGGAGAAGGCTTGACGAAGATGATTTACGACGGCGTCACTTTCTGTCTAGTGATACTACTACCAACGCTGTTGATGCTTTGTCCCAAGAAGGTATGTGGTCACATTCTTTCTGCCAGAGCAAAATAGTATATATAGTAGTTGATTTTTAATGTCAATTTGGTTTTCTTGTTATTTTTTTTTCTTTTGAAGTTATTGATAAGAAAGGAAATGAAGTTCCAACTCCAAGAATTATAGACTTGAACTCAAGATTTTTAGGTTAGTGCCCTAGTGGATCACAGCTTGTTTGAGCCTATATAGTTAGTATCCATAGTCAAAACATTGCATTGTACCAGCACTTCTAGGAGTTTTTTTTTTTATTTGGTCTATGGTTGCTTGCTTTTGCCTCTAACCCCAAGTCCGTGACTACTGTATACTTGTAGTAGCGTGTAATAACTTTGTAATAATAATGTGTATATTTATTTTGTTTTCTGTGGGTGGGGTTGTGATTTTTTGGTGGGTAGGGTTATCGGAGGAGCCGGTGCAGCAAGATAAGGAGGCGGCAGGGAGCGGTGGAGGGGGACCGTGGGAGGTGGCGGAGAAGAAGAAGAAACAGCGGGGGAGGAAGGGACCGAGACCGAGGAAGGTGGTGGATGTTAATCTTGAAGACGAGAATGATGACGATGAATATGGTGGTGGTTGCGGTGGCATGGAGAGACAGCGAGAGCACCCATTCATTGTGACCGAACCTGGTGAGGTGGCACGTGGCAAAAAGAACGGCCTCGATTACCTCTTCCATCTCTACGAGCAATGCCGTGATTTCTTGATCCAGGTCCAGAACATCGCCAAGGAGCGCGGCGAAAAATGCCCAACCAAGGTAATTTAATTTGGACTTTTAATCCAAACCGTTCGATTCATTTATTGAATTGGCCGGATACAATGCAACTATAAGTCTCCATACGTTCACATTTAGCATTTGTGGTGACTCAGTGTTAATTGACTGACTCGTGAGTCGTGAAAGAATAACGATGACTCCCTTAATGGTGGATAGCTCGCGCCATTAGAAAATGTGATATGCTGTGGTTAAAAGTTCTTGCAGAAAATATGACTGGTCAAGTAAGTACAGGCTGGTGGAGGCCTGTCAATTGTGGGCTAGAAATTGCATGGTTAAAATTTAATACTGCGCTCGAAGTCCTTTTTACCAGTCCATACATGGAGTTGGCTTATCCTGGCTGTGATAAAAAAAATTTGCAAATAGCACATGACTACTGAGGACCCCCAAAAAAAAATAAATAAATTGAATTTAGTATATGATTTTAGAAATATGGCAAAATTTAGCTTGTCATGCAAATTGCAGGTGACTAATCAGGTGTTTAGGTATGCGAAGAAGGCCGGAGCGAGCTATATTAACAAGCCAAAAATGCGGCACTATGTGCATTGCTACGCATTGCACTGCCTGGACGAGGAAGCATCGAATGCACTGAGGAGGGCTTTCAAGGAGAGAGGGGAGAATGTTGGGGCATGGAGACAGGCATGCTACAAGCCTCTTGTAGCCATTGCTGCCCGCCAAGGTTGGGATATTGATGCCATTTTCAATGCTCATCCCCGTCTTGGCATTTGGTACGTGCCCACCAGGCTTCGCCAACTTTGTCATGCTGAGCGCAATGGTGCTGGCGCTGCCGCTTCTAGCTCTGTTTCTGCCGGGGCTGAACATTCCGTCATTTTGAAGCCATACTGA(SEQ ID NO.1)
the amino acid sequence of the protein encoded by the citrus CcLFY gene is as follows:
>Ciclev10033942m
MDPEAFTASLFKWDPRVVVAPPPARVQLEQVSQPPAVPLGAAAAAAYSALVRPRELGGLEELFQAYGIRYHTAAKIAELGFTVNTLLDMKDEELDEMMNSLGHLFRWELLVGERYGIKAAVRAERRRLDEDDLRRRHFLSSDTTTNAVDALSQEGLSEEPVQQDKEAAGSGGGGPWEVAEKKKKQRGRKGPRPRKVVDVNLEDENDDDEYGGGCGGMERQREHPFIVTEPGEVARGKKNGLDYLFHLYEQCRDFLIQVQNIAKERGEKCPTKVTNQVFRYAKKAGASYINKPKMRHYVHCYALHCLDEEASNALRRAFKERGENVGAWRQACYKPLVAIAARQGWDIDAIFNAHPRLGIWYVPTRLRQLCHAERNGAGAAASSSVSAGAEHSVILKPY*(SEQ ID NO.2)
example 1
The embodiment provides a cloning method of a CcLFY gene for regulating and controlling citrus shoot development, which comprises the following steps:
(1) RNA extraction of Crimangt orange leaves
Fully grinding fresh plant tissues (leaves of the Krementanus trifoliate) in liquid nitrogen or directly and quickly grinding the cut plant tissues in TRIpure, adding 1mL of TRIpure into every 50-100 mg of plant tissues, uniformly mixing, and putting on ice for operation in the whole process. Pre-cooled 0.2mL chloroform was added per 1mL TRIPURE. And covering the pipe cover tightly, shaking for 15s violently, and standing for 2-3 min. High speed refrigerated centrifugation at 12000rpm centrifugal force at 4 ℃ for 15 min. The supernatant (ca. 500. mu.L) was transferred to a clean 1.5mL RNAase free centrifuge tube, pre-cooled iso-propanol was added, the mixture was inverted and mixed, and then placed in a refrigerator at-20 ℃ for 30 minc. Centrifuging at 12000rpm at 4 deg.C for 10min, and discarding the supernatant. The precipitate was washed by adding 1mL of 75% ethanol which had been precooled, centrifuged at 12000rpm for 5min at 4 ℃ and the supernatant was discarded. The remaining small amount of liquid can be centrifuged briefly and then aspirated off with a lance tip, taking care not to discard the pellet. And (5) placing on ice for 2-3 min, and drying. mu.L of RNase free water was added to dissolve the RNA sufficiently. The extraction quality was checked by electrophoresis using 3. mu.L of RNA solution, and the remaining RNA was used directly for reverse transcription or stored at-80 ℃ to prevent degradation.
(2) Reverse transcription
Reverse transcription kit adopting Nanjing NuoZan biology companyII Q RT SuperMixfor qPCR (+ gDNA wiper) reverse transcription reaction. All the operation processes are carried out in a clean bench, and the reaction steps are as follows:
a) genomic DNA removal
The following mixture was prepared in an RNase-free centrifuge tube
RNase-free ddH 2 O | to 16μL |
4×gDNA wiper Mix | 4μL |
Template RNA | Total RNA:1pg-1μg |
Gently pipetting and mixing. 42 ℃ for 2 min.
b) Preparing a reverse transcription reaction system
Directly adding 5 XHiScript II qRTSupermix II into the reaction tube in the step a
5×HiScript II qRTSuperMix II | 4μL |
The reaction solution of the step a | 16μL |
Gently pipetting and mixing.
c) The reverse transcription procedure was carried out as follows:
50℃ | 15min |
85℃ | 5sec |
after the reaction is finished, the mixture is stored at 4 ℃ for standby and is stored in a refrigerator at minus 80 ℃ for a long time.
(3) CcLFY Gene CDS region cloning
Taking total cDNA obtained by reverse transcription of Crimemann orange leaf tissue RNA as a template, and utilizing high-fidelity DNA polymerase of Nanjing Novozam biological companyMax Super-FidelityDNApolymerase kit for PCR amplification.
Wherein, the forward primer is 5'-atggacccggaagctttcac-3' (SEQ ID NO. 3);
the reverse primer was 5'-tcagtatggcttcaaaatga-3' (SEQ ID NO. 4).
All operations should be carried out on ice, the components should be fully mixed after being thawed, and the components should be put back to-20 ℃ for storage after being used up. The amplification reaction system is as follows:
components | Volume of |
2×Phanta Max Buffer | 25μL |
dNTP Mix(10mM each) | 1μL |
Forward primer (10. mu.M) | 2μL |
Reverse primer (10. mu.M) | 2μL |
Phanta Max Super-Fidelity DNA Polymerase | 1μL |
Template cDNA | 1μL |
ddH 2 O | 18μL |
The PCR amplification procedure was as follows:
the step of circulation | Temperature of | Time | Number of cycles | |
Pre-denaturation | 95 | 3min | 1 | |
Denaturation of the material | 95℃ | 15sec | 35 | |
Annealing of | 55℃ | 15sec | 35 | |
Extension | 72℃ | 1min | 35 | |
Extend completely | 72 | 10min | 1 | |
Keep warm/preserve | 16 | 10min | 1 |
And taking out the product after the PCR amplification reaction is finished, placing the product in a refrigerator at 4 ℃ for short-term storage after gel electrophoresis detection, and subsequently recovering the target fragment.
Example 2 overexpression vector construction and Agrobacterium transformation
The construction method of the overexpression vector in this example is to use pBI121 as the expression vector, and to perform fusion construction of the target gene fragment and the vector by using homologous recombination method, and all the reagents used are the same as those of Nanjing Novozam Bio IncIIOne Step Cloning Kit, the specific experimental operation steps are as follows:
selecting restriction enzymes Xba I and Xho I to perform double enzyme digestion on the PBI121 vector plasmid, performing enzyme digestion at 37 ℃ for 1 hour, and then recovering the linearized vector;
designing a recombinant primer, and carrying out PCR amplification by using a CDS region cloning product of a CcLFY gene as a template. Wherein the recombination forward primer is
5'-gagaacacgggggactctagaatggacccggaagctttcac-3'(SEQ ID NO.5)
The recombinant reverse primer is
5’-ggggatccgcggccgctcgaggtatggcttcaaaatgacgg-3'(SEQ ID NO.6)
The amplification reaction system is consistent with the CDS region amplification system of the CcLFY gene, and the recombinant amplification product is recovered.
Preparing a linearization vector and a target gene fragment, measuring the recovery concentration, adjusting the proportion of the linearization vector and the target gene fragment, and performing ligation reaction at 37 ℃, wherein the specific recombination ligation reaction system is as follows:
components | Recombination reactions |
Linearized vector | 150ng |
Inserting a target gene fragment | 50ng |
5×CE II Buffer | 2μL |
Exnase II | 1μl |
ddH 2 O | to 10μl |
5 minutes before the end of ligation, the competent large intestine DH 5. alpha. was removed from the freezer at-80 ℃ and thawed on ice. And adding the ligation products into DH5 alpha competent cells, gently sucking and mixing the ligation products by using a gun head, carrying out ice bath for 30min, carrying out heat shock for 90s at 42 ℃ in a mixing instrument, and then placing the mixture on ice for 1 min. 400 μ L of blank LB liquid medium was added and incubated for 1h at 37 ℃ on a shaker at 220 r/min. Centrifuging at 12000rpm for 1min, sucking off supernatant, sucking 100 μ L bacterial liquid, mixing, spreading on LB solid culture dish with kanamycin, and culturing in 37 deg.C incubator.
And selecting monoclonal bacterial plaques for PCR detection, sending detected positive clones to a company for sequencing, then carrying out sequence comparison analysis, and finally determining that the vector is successfully constructed.
And (3) carrying out plasmid extraction on the coliform liquid with correct sequencing, wherein the method for extracting the plasmids refers to the specification of a large intestine plasmid extraction kit of the Edley organism company. The extracted plasmid is transferred into agrobacterium tumefaciens competence EHA105, and the specific operation steps are as follows:
taking the agrobacterium-infected EHA105 out of a refrigerator at the temperature of-80 ℃, freezing and thawing the agrobacterium-infected EHA105 on ice, adding 0.1-1 mu g of plasmid, placing the agrobacterium-infected EHA on ice for 5min, quickly freezing the agrobacterium-infected EHA in liquid nitrogen for 5min, quickly taking the agrobacterium-infected EHA out, placing the agrobacterium-infected EHA on ice for 5min, and then placing the agrobacterium-infected EHA on ice for 5min after warm bath in a thermostat at the temperature of 37 ℃. Adding 800 μ L blank liquid LB culture medium into the competent cells, and recovering bacteria by shaking at 220r/min on a shaking table at 28 ℃ for 3 h. Centrifuging for 1min by a high-speed centrifuge at 12000rpm, sucking and removing supernatant, reserving 100 mu L of bacterial liquid, sucking, uniformly mixing, coating the mixture on an LB solid culture dish added with kanamycin and rifampicin, and inversely culturing for 2-3 days in a constant-temperature incubator at 28 ℃.
Selecting monoclonal bacterial plaque to carry out PCR detection, uniformly mixing the positive clone bacterial liquid and 50% glycerol in a ratio, storing in a refrigerator at the temperature of minus 80 ℃ for later use, and then activating to be used for plant transformation.
Example 3 genetic transformation and phenotypic analysis of lemon
The method for genetic transformation of lemon in the embodiment comprises the following steps:
(1) preparing lemon seedlings, and adopting the epicotyls of the lemon seedlings as transformation materials
The preparation method comprises the following specific steps: healthy, fresh and full-fleshed lemon seeds are taken out of the fruit and the pulp attached to the surface is washed away. Soaking in 1moL/L NaOH solution, stirring for 15min to remove pectin substances, pouring out NaOH solution, and washing the seeds with distilled water for 4-5 times. And then, operating in an ultra-clean workbench, sterilizing the seeds by using 3-4% NaClO solution, and treating for 15-20 min by using a shaking table. Pouring out the NaClO solution, washing the seeds with sterile water for 4-5 times until the sterile water is clear, and finally soaking the seeds with sterile water for storage for later use. Placing the lemon seeds to be sown on filter paper sterilized at high temperature, peeling off the inner and outer seed coats, inserting the lemon seeds into a citrus test tube sowing culture medium, paying attention to the direction of the radicle growing downwards, carrying out dark culture for about 25-30 days, taking out, placing in illumination culture for 7-10 days, and turning green to be used for subsequent transformation.
(2) Preparation of agrobacterium transformation staining solution
Before genetic transformation of the lemons, agrobacterium tumefaciens needs to be activated, and preparation of transformation staining solution is carried out under the condition that thalli are kept in a better activity state, and the method comprises the following specific operation steps:
strain activation: inoculating CcLFY agrobacterium liquid connected with a PBI121 overexpression vector into a solid LB culture medium added with kanamycin and rifampicin, and carrying out inverted culture in a constant temperature incubator at 28 ℃ for 2 days, namely carrying out first activation.
And (3) propagation of colonies: selecting agrobacterium, carrying out monoclonal detection to determine positive, shaking, mixing, inoculating to a solid LB culture medium added with kanamycin and rifampicin, and carrying out inverted culture in a constant-temperature incubator at 28 ℃ for 2 days to carry out secondary activation.
Preparing a staining solution: scraping all thalli into an LM suspension medium, and placing in a constant temperature shaking table at the temperature of 28 ℃ for 1 hour at 220r/min to completely disperse the thalli. Measuring the concentration of the suspension by ultraviolet spectrophotometer, and adjusting OD 600 Adding acetosyringone (AS, 50mg/L) at a value of 0.6-0.8, and placing in a constant temperature incubator at 28 deg.C for use.
(3) Infection and co-cultivation process
The method is characterized in that the lemon seedling epicotyl bias cut stem section and the prepared infection solution are used for co-culture, and the specific steps of the process are as follows:
a) and (3) infection process: cutting the epicotyl of the lemon seedling with good growth state and turning green for 7-10 days on sterile filter paper into a trapezoidal stem section with the length of about 1cm by using a sterile scalpel in a super-clean workbench, inclining the cut as far as possible to increase the cut area to promote conversion, and immediately putting the stem section into an LM suspension culture medium to keep the cut wet, so that the cut of the stem section is prevented from being air-dried and dehydrated and the conversion efficiency is prevented from being influenced. Soaking all cut stem segments in the prepared agrobacterium-mediated staining solution, sealing the opening of the triangular flask with a sealing film, and placing in a constant temperature shaking table at 28 ℃ for 20 minutes at 220 r/min.
b) And (3) co-culture process: in a clean bench, the bacterial liquid is poured off, the residual agrobacterium infection liquid on the surface of the stem segment is sucked up by using sterile filter paper, the stem segment is placed in a co-culture medium (MT + AS, 50mg/L) with a layer of sterile filter paper paved on the surface in a state that the cut is upward, and the stem segment is cultured for 3 days in a 21 ℃ incubator in the dark.
(4) Screening culture
And (3) primarily screening shoot buds of stem segments by using a screening culture medium (SM culture medium: SY + Cef 400mg/L + Km50mg/L + Agar 8g/L) added with kanamycin, so that the influence on subsequent positive identification and detection work due to excessive shoot buds and high false positive ratio is avoided. The method comprises the following specific steps:
a) cleaning: transferring the stem segments of the lemon after 3 days of co-culture to a conical flask filled with sterile water in an ultra-clean workbench, cleaning for 3-5 times by using the sterile water, sealing, placing in a constant-temperature shaking table at 28 ℃ at 220r/min for 5-10min each time, and then cleaning and replacing by using the sterile water for multiple times until the water becomes transparent. The sterile water was decanted and the residual moisture on the surface of the stem sections was blotted with sterile filter paper.
b) Screening and culturing: and (3) flatly paving the washed lemon stem section with the cut upwards in a screening culture medium added with kanamycin and cephalosporin, carrying out dark culture for 7-10 days at the temperature of 26 ℃, then transferring to illumination culture, keeping the illumination for 16/8 hours, and carrying out subculture once about 25 days.
c) Cutting buds: the stem segments of the lemons are screened and cultured for about 1-2 months, new buds can grow at the cut, then the buds growing at the cut are cut off by a sterile scalpel in a super clean workbench, and are transferred to an elongation culture medium to continue to grow, and the subculture is carried out once every 25 days.
d) Rooting culture: after the buds grow for 1-2 months, the buds are transferred to a rooting culture medium and subcultured once in about 25 days.
(5) Positive identification and phenotypic observation analysis
And (4) carrying out positive identification on the finally obtained material, finally determining a positive transgenic material, and further carrying out phenotype observation analysis on the positive transgenic material. The method comprises the following specific steps:
and (3) carrying out DNA extraction on the obtained lemon material, and identifying the positive condition by utilizing PCR amplification, namely determining that the target gene fragment is successfully transferred into the plant material.
Then extracting the RNA of the transgenic lemon material, carrying out reverse transcription to obtain cDNA, and determining the successful transformation of the target gene segment through PCR amplification.
Then, the successful expression of the fused GFP protein in the vector in the transgenic lemon material is determined by using an in vivo imaging technology.
And finally, determining successful expression of the target gene in the transgenic lemon material by using Western detection and a GFP antibody.
Phenotypic observation and analysis: the results are shown in FIGS. 1 to 3. Compared with wild lemon, the CcLFY over-expression lemon material has the advantages that axillary branches are increased, and the axillary bud development is promoted. Therefore, the cloned citrus CcLFY gene has the function of regulating and controlling the development of shoot tips.
It should be noted that the above examples are only for further illustration and description of the technical solution of the present invention, and are not intended to further limit the technical solution of the present invention, and the method of the present invention is only a preferred embodiment, and is not intended to limit the protection scope of the present invention. Any modification, equivalent replacement, or improvement made within the spirit and principle of the present invention should be included in the protection scope of the present invention.
Sequence listing
<110> university of agriculture in Huazhong
Application of citrus CcLFY gene in regulation and control of shoot development
<160> 6
<170> SIPOSequenceListing 1.0
<210> 1
<211> 2087
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 1
atggacccgg aagctttcac ggcgagtttg ttcaagtggg acccacgagt ggtcgtggca 60
ccaccaccgg ccagggtgca gctggaacag gtgtcacaac caccagcagt gccgcttggt 120
gctgcggcgg cggcagccta ctcggcgctg gtgcgtccaa gggagttggg tgggctggag 180
gagctgttcc aggcttatgg gataagatac cacacggcag cgaagatagc ggagctcggg 240
ttcacggtga acacgctgtt ggacatgaaa gacgaggagc tcgatgagat gatgaacagc 300
ttgggtcact tgttcaggtg ggagctgctc gttggagaga gatacggcat caaagctgct 360
gttagggctg agaggagaag gcttgacgaa gatgatttac gacggcgtca ctttctgtct 420
agtgatacta ctaccaacgc tgttgatgct ttgtcccaag aaggtatgtg gtcacattct 480
ttctgccaga gcaaaatagt atatatagta gttgattttt aatgtcaatt tggttttctt 540
gttatttttt tttcttttga agttattgat aagaaaggaa atgaagttcc aactccaaga 600
attatagact tgaactcaag atttttaggt tagtgcccta gtggatcaca gcttgtttga 660
gcctatatag ttagtatcca tagtcaaaac attgcattgt accagcactt ctaggagttt 720
ttttttttat ttggtctatg gttgcttgct tttgcctcta accccaagtc cgtgactact 780
gtatacttgt agtagcgtgt aataactttg taataataat gtgtatattt attttgtttt 840
ctgtgggtgg ggttgtgatt ttttggtggg tagggttatc ggaggagccg gtgcagcaag 900
ataaggaggc ggcagggagc ggtggagggg gaccgtggga ggtggcggag aagaagaaga 960
aacagcgggg gaggaaggga ccgagaccga ggaaggtggt ggatgttaat cttgaagacg 1020
agaatgatga cgatgaatat ggtggtggtt gcggtggcat ggagagacag cgagagcacc 1080
cattcattgt gaccgaacct ggtgaggtgg cacgtggcaa aaagaacggc ctcgattacc 1140
tcttccatct ctacgagcaa tgccgtgatt tcttgatcca ggtccagaac atcgccaagg 1200
agcgcggcga aaaatgccca accaaggtaa tttaatttgg acttttaatc caaaccgttc 1260
gattcattta ttgaattggc cggatacaat gcaactataa gtctccatac gttcacattt 1320
agcatttgtg gtgactcagt gttaattgac tgactcgtga gtcgtgaaag aataacgatg 1380
actcccttaa tggtggatag ctcgcgccat tagaaaatgt gatatgctgt ggttaaaagt 1440
tcttgcagaa aatatgactg gtcaagtaag tacaggctgg tggaggcctg tcaattgtgg 1500
gctagaaatt gcatggttaa aatttaatac tgcgctcgaa gtccttttta ccagtccata 1560
catggagttg gcttatcctg gctgtgataa aaaaaatttg caaatagcac atgactactg 1620
aggaccccca aaaaaaaata aataaattga atttagtata tgattttaga aatatggcaa 1680
aatttagctt gtcatgcaaa ttgcaggtga ctaatcaggt gtttaggtat gcgaagaagg 1740
ccggagcgag ctatattaac aagccaaaaa tgcggcacta tgtgcattgc tacgcattgc 1800
actgcctgga cgaggaagca tcgaatgcac tgaggagggc tttcaaggag agaggggaga 1860
atgttggggc atggagacag gcatgctaca agcctcttgt agccattgct gcccgccaag 1920
gttgggatat tgatgccatt ttcaatgctc atccccgtct tggcatttgg tacgtgccca 1980
ccaggcttcg ccaactttgt catgctgagc gcaatggtgc tggcgctgcc gcttctagct 2040
ctgtttctgc cggggctgaa cattccgtca ttttgaagcc atactga 2087
<210> 2
<211> 418
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 2
Met Asp Pro Glu Ala Phe Thr Ala Ser Leu Phe Lys Trp Asp Pro Arg
1 5 10 15
Val Val Val Ala Met Asp Pro Glu Ala Phe Thr Ala Ser Leu Phe Lys
20 25 30
Trp Asp Pro Arg Val Val Val Ala Pro Pro Pro Ala Arg Val Gln Leu
35 40 45
Glu Gln Val Ser Gln Pro Pro Ala Val Pro Leu Gly Ala Ala Ala Ala
50 55 60
Ala Ala Tyr Ser Ala Leu Val Arg Pro Arg Glu Leu Gly Gly Leu Glu
65 70 75 80
Glu Leu Phe Gln Ala Tyr Gly Ile Arg Tyr His Thr Ala Ala Lys Ile
85 90 95
Ala Glu Leu Gly Phe Thr Val Asn Thr Leu Leu Asp Met Lys Asp Glu
100 105 110
Glu Leu Asp Glu Met Met Asn Ser Leu Gly His Leu Phe Arg Trp Glu
115 120 125
Leu Leu Val Gly Glu Arg Tyr Gly Ile Lys Ala Ala Val Arg Ala Glu
130 135 140
Arg Arg Arg Leu Asp Glu Asp Asp Leu Arg Arg Arg His Phe Leu Ser
145 150 155 160
Ser Asp Thr Thr Thr Asn Ala Val Asp Ala Leu Ser Gln Glu Gly Leu
165 170 175
Ser Glu Glu Pro Val Gln Gln Asp Lys Glu Ala Ala Gly Ser Gly Gly
180 185 190
Gly Gly Pro Trp Glu Val Ala Glu Lys Lys Lys Lys Gln Arg Gly Arg
195 200 205
Lys Gly Pro Arg Pro Arg Lys Val Val Asp Val Asn Leu Glu Asp Glu
210 215 220
Asn Asp Asp Asp Glu Tyr Gly Gly Gly Cys Gly Gly Met Glu Arg Gln
225 230 235 240
Arg Glu His Pro Phe Ile Val Thr Glu Pro Gly Glu Val Ala Arg Gly
245 250 255
Lys Lys Asn Gly Leu Asp Tyr Leu Phe His Leu Tyr Glu Gln Cys Arg
260 265 270
Asp Phe Leu Ile Gln Val Gln Asn Ile Ala Lys Glu Arg Gly Glu Lys
275 280 285
Cys Pro Thr Lys Val Thr Asn Gln Val Phe Arg Tyr Ala Lys Lys Ala
290 295 300
Gly Ala Ser Tyr Ile Asn Lys Pro Lys Met Arg His Tyr Val His Cys
305 310 315 320
Tyr Ala Leu His Cys Leu Asp Glu Glu Ala Ser Asn Ala Leu Arg Arg
325 330 335
Ala Phe Lys Glu Arg Gly Glu Asn Val Gly Ala Trp Arg Gln Ala Cys
340 345 350
Tyr Lys Pro Leu Val Ala Ile Ala Ala Arg Gln Gly Trp Asp Ile Asp
355 360 365
Ala Ile Phe Asn Ala His Pro Arg Leu Gly Ile Trp Tyr Val Pro Thr
370 375 380
Arg Leu Arg Gln Leu Cys His Ala Glu Arg Asn Gly Ala Gly Ala Ala
385 390 395 400
Ala Ser Ser Ser Val Ser Ala Gly Ala Glu His Ser Val Ile Leu Lys
405 410 415
Pro Tyr
<210> 3
<211> 20
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 3
atggacccgg aagctttcac 20
<210> 4
<211> 20
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 4
tcagtatggc ttcaaaatga 20
<210> 5
<211> 41
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 5
gagaacacgg gggactctag aatggacccg gaagctttca c 41
<210> 6
<211> 41
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 6
ggggatccgc ggccgctcga ggtatggctt caaaatgacg g 41
Claims (8)
1. The application of the citrus CcLFY gene or the protein coded by the citrus CcLFY gene in regulating and controlling the development of plant shoot tips, wherein the sequence of the citrus CcLFY gene is as follows:
a) the nucleotide sequence is shown as SEQ ID NO. 1;
b) the sequence shown in SEQ ID NO.1 is substituted, deleted and/or added with one or more nucleotides and can encode a sequence with a function of regulating shoot development.
2. The use according to claim 1, wherein the amino acid sequence of the protein encoded by the CcLFY gene is as follows:
a) the amino acid sequence is shown as SEQ ID NO. 2;
b) the amino acid sequence of which the sequence is substituted, deleted and/or added with one or more amino acids and has the function of regulating and controlling the development of shoot tips.
3. Use according to claim 1 or 2, wherein the plant is lemon.
4. Use according to claim 3, wherein overexpression of the CcLFY gene is used to increase shoot height.
5. The use according to claim 4, wherein the method for overexpression of the CcLFY gene is as follows: PBI121 is used as an expression vector, a homologous recombination method is used for fusing a target gene segment with the vector to construct a super-expression vector, and agrobacterium-mediated genetic transformation is utilized.
6. The use of claim 5, wherein the cloning method of the target gene fragment comprises: taking total cDNA obtained by reverse transcription of Crimemann orange leaf tissue RNA as a template to perform PCR amplification, wherein the sequence of a forward primer is shown as SEQ ID NO.3, and the sequence of a reverse primer is shown as SEQ ID NO. 4.
7. The application of claim 5, wherein the PCR amplification primer sequences adopted for constructing the overexpression vector are respectively shown as SEQ ID NO.5, and the reverse primer sequences are shown as SEQ ID NO. 6.
8. The use according to claim 5, characterized in that the method step of using Agrobacterium tumefaciens mediated genetic transformation:
preparing an epicotyl of the lemon seedling as a transformation material;
dip-dyeing the epicotyl of the lemon seedling by using the activated agrobacterium-mediated dip-dyeing solution, transferring the epicotyl into a co-culture medium, and continuously culturing under dark conditions to obtain a stem section germination material;
placing the stem bud-producing material in a screening culture medium containing kanamycin for subculture screening culture to obtain a stem elongation material;
placing the stem section elongation material in a rooting culture medium for continuous culture, identifying a positive transgenic material, and observing a shoot phenotype.
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Citations (3)
Publication number | Priority date | Publication date | Assignee | Title |
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CN110819607A (en) * | 2019-12-05 | 2020-02-21 | 西南大学 | Application of CsLYK gene and coding protein thereof in improving citrus canker resistance |
CN112851781A (en) * | 2021-02-01 | 2021-05-28 | 华中农业大学 | Application of citrus bZIP transcription factor in shortening plant childhood |
CN113150091A (en) * | 2021-02-18 | 2021-07-23 | 华中农业大学 | CsHD2 protein and gene for promoting plant lateral bud growth and application thereof |
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CN110819607A (en) * | 2019-12-05 | 2020-02-21 | 西南大学 | Application of CsLYK gene and coding protein thereof in improving citrus canker resistance |
CN112851781A (en) * | 2021-02-01 | 2021-05-28 | 华中农业大学 | Application of citrus bZIP transcription factor in shortening plant childhood |
CN113150091A (en) * | 2021-02-18 | 2021-07-23 | 华中农业大学 | CsHD2 protein and gene for promoting plant lateral bud growth and application thereof |
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