CN114990132A - Application of citrus CcLFY gene in regulation and control of shoot development - Google Patents

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

Application of citrus CcLFY gene in regulation and control of shoot development

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 company

II 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 company

Max 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 Inc

IIOne 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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