CN114990132B - 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 gene in regulating and controlling plant shoot development, wherein the sequence of the citrus CcLFY gene is shown as SEQ ID NO. 1. According to the invention, the function of the citrus CcLFY gene is identified for the first time, the transgenic plant is obtained by constructing an over-expression vector of the gene and performing stable transformation of lemon by using an agrobacterium-mediated genetic transformation method, and the phenotype observation and analysis show that the transgenic lemon has a phenotype of increased axillary branches, so that the cloned citrus CcLFY gene has the function of regulating and controlling shoot development. The discovery of the gene function provides a research basis and a new genetic resource for the regulation, the improvement and the like of the branch development of citrus and woody fruit trees.

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 an application of citrus CcLFY genes in shoot development regulation.

Background

Citrus is the first fruit in the world, belongs to perennial woody fruit trees, and has very important roles in gardening economy in China. For woody fruit trees such as citrus, a reasonable tree structure is extremely important for achieving the aims of high quality, high yield and light and simplified cultivation, and the shoot regulation of the tree body is a foundation for achieving ideal tree shape. Therefore, the branch tip regulation mechanism aiming at woody fruit trees such as citrus is necessary to be studied, and a foundation is laid for cultivating new varieties of fruit trees with ideal plant types.

Disclosure of Invention

Based on the above, it is necessary to provide an application of the citrus CcLFY gene in regulating and controlling shoot development, which can lay a foundation for cultivating new varieties of fruit trees with ideal tree shapes.

The invention adopts the following technical scheme:

the invention provides an application of a citrus CcLFY gene or a protein coded by the gene in regulating and controlling 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 substituted, deleted and/or added with one or more nucleotides and can code a sequence with a function of regulating shoot development.

In some of these 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 sequence is an amino acid sequence with the functions of regulating shoot development by substituting, deleting and/or adding one or more amino acids.

In some embodiments, the plant is lemon.

In some of these embodiments, shoot growth is achieved by over-expression of the CcLFY gene. Preferably, the method of using overexpression of the CcLFY gene is as follows: and (3) taking PBI121 as an expression vector, carrying out fusion of a target gene fragment and the vector by using a homologous recombination method to construct an overexpression vector, and carrying out genetic transformation by using agrobacterium tumefaciens.

In some embodiments, the cloning method of the target gene fragment comprises the following steps: and (3) performing PCR amplification by using total cDNA obtained by reverse transcription of RNA of the tissue of the Criman Ding Ju leaf as a template, wherein the forward primer sequence is shown as SEQ ID NO.3, and the reverse primer sequence is shown as SEQ ID NO. 4.

In some embodiments, the sequence of PCR amplification primers used for constructing the super-expression vector is shown in SEQ ID NO.5, and the sequence of reverse primers is shown in SEQ ID NO. 6.

In some of these embodiments, the method step of utilizing agrobacterium tumefaciens-mediated genetic transformation comprises: preparing an epicotyl of a lemon seedling serving as a transformation material; the activated agrobacterium tumefaciens dip-dyeing liquid is used for dip-dyeing the epicotyl of the lemon seedling, and the epicotyl is transferred into a co-culture medium, and the culture is continued in a dark condition, so that a stem bud growing material is obtained; placing the stem bud material in a screening culture medium containing kanamycin for secondary screening culture to obtain a stem elongation material; the stem segment elongation material is placed in rooting culture medium for continuous culture, positive transgenic material is identified, and shoot phenotype is observed.

The beneficial effects of the invention are as follows:

compared with the prior art, the invention discovers that the citrus CcLFY gene and the protein coded by the gene have the regulation and control functions of axillary branches for the first time. The discovery of new functions of the gene provides a research basis and new genetic resources for the regulation, improvement and the like of branch development of citrus and woody fruit trees.

Drawings

FIG. 1 shows the results of comparative development of axillary buds of common lemon (WT) and lemon overexpressing the CcLFY gene.

FIG. 2 shows the results of comparison of the development states of common lemon (WT) and lemon branches overexpressing the CcLFY gene, wherein S1, S2 and S3 are three stages of development of the axillary bud of CcLFY overexpressing lemon # 1.

FIG. 3 is a graph comparing phenotypes of lemon positive materials # 1, # 2 overexpressing the CcLFY gene in example 3 with conventional normal lemon (WT) material.

Detailed Description

The present invention will be described in further detail with reference to specific examples so as to more clearly understand the present invention by those skilled in the art.

The following examples are given for illustration of the invention only and are not intended to limit the scope of the invention. All other embodiments obtained by those skilled in the art without creative efforts are within the protection scope of the present invention based on the specific embodiments of the present invention.

In the examples of the present invention, all raw material components are commercially available products well known to those skilled in the art unless specified otherwise; in the embodiments of the present invention, unless specifically indicated, all technical means used are conventional means well known to those skilled in the art.

The key raw material sources and the composition are as follows:

the leaves of Kleman Ding Ju, material from the university of China farm in Wuhan, hubei, were obtained from a specimen garden, and the Wild (WT) Kleman Ding Ju strain was single-dry and less branched: the obtained product can be stored in liquid nitrogen immediately after collection, and can be directly ground for use or stored in a refrigerator at-80deg.C for use.

MT minimal medium: trace, ferric salt, glycine, inositol, VB and VC 10mL each, macroelement 100mL, sucrose 40g/L and agar 8g/L.

Citrus test tube sowing culture medium is MT+sucrose 25g/L+Agar 8.0g/L.

Suspension medium (LM) MT+malt extract 0.5 g/L+glutamine 1.5g/L.

Bud culture medium (SY) MT+6BA 0.5mg/L+NAA 0.1 mg/L+KT0.5 mg/L+agar 8g/L;

Co-Culture Medium (CM) SY+AS 50mg/L+Agar 8g/L.

Screening Medium (SM) SY+Cef400 mg/L+Km50mg/L+Agar 8g/L.

Elongation Medium (SEM): MT+6BA 0.1mg/L+GA3 0.5mg/L+IAA 0.5 mg/L+Cef400 mg/L+Km50mg/L+Agar 8g/L.

Rooting medium (RIM): 1/2MT+NAA 0.5mg/L+IBA 0.1 mg/L+activated carbon 0.5g/L+Agar 8g/L+Km 25mg/L+Cef 400mg/L.

The sequence of the citrus CcLFY gene of the invention 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 citrus branch tip development regulation CcLFY gene, which comprises the following steps:

(1) RNA extraction from Criman Ding Ju leaves

Fresh plant tissues (Kleman Ding Ju leaves) are taken and fully ground in liquid nitrogen or cut into pieces and then are directly and rapidly ground in a TRIPURE, 1mL of TRIPURE is added into each 50-100 mg of plant tissues, and the plant tissues are uniformly mixed and are placed on ice for operation in the whole process. 0.2mL of pre-chilled chloroform was added per 1mL of TRIpure. The tube cover is tightly covered, and the tube cover is vigorously vibrated for 15s and placed for 2-3 min. High-speed refrigerated centrifugation was performed at a centrifugal force of 12000rpm at 4℃for 15min. The supernatant (about 500. Mu.L) was transferred to a clean 1.5mL RNAase free centrifuge tube, pre-chilled equal volume of isopropyl alcohol was added, mixed upside down and placed in a-20deg.C refrigerator for 30minc. Centrifuge at 12000rpm for 10min at 4deg.C, discard supernatant. The precipitate was washed with 1mL of pre-chilled 75% ethanol, centrifuged at 12000rpm at 4℃for 5min, and the supernatant was discarded. The remaining small amount of liquid may be centrifuged briefly and then sucked out with the gun head, taking care not to suck out the pellet. Placing on ice for 2-3 min, and airing. 30 mu L RNase free water was added to dissolve RNA sufficiently. The quality of extraction was checked by electrophoresis on 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 for Nanjinouzan biological companyII Q RT SuperMixfor qPCR (+gDNA wind) reverse transcription reaction was performed. All the operation processes are carried out in an ultra-clean workbench, and the reaction steps are as follows:

a) Genomic DNA removal

The following mixture was prepared in a centrifuge tube of RNase-free

RNase-free ddH 2 O	to 16μL
		4×gDNA wiper Mix	4μL
Template RNA	Total RNA:1pg-1μg

Gently beating and mixing by a pipette. 42 ℃ for 2min.

b) Preparation of reverse transcription reaction System

Directly adding 5X HiScript II qRTSuperMix II into the reaction tube of the step a

5×HiScript II qRTSuperMix II	4μL
		Reaction solution of step a	16μL

Gently beating and mixing by a pipette.

c) The reverse transcription reaction procedure was performed as follows:

50℃	15min
		85℃	5sec

after the reaction is finished, the mixture is preserved at 4 ℃ for standby and stored in a refrigerator at-80 ℃ for a long time.

(3) Cloning of CDS region of CcLFY Gene

Total cDNA obtained by reverse transcription of RNA of tissue of Criman Ding Ju leaf is used as a template, and high-fidelity DNA polymerase of Nanjinouzan biological company is usedPCR amplification was performed using the Max Super-FidelityDNAPolyase kit.

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 are carried out on ice, the components are fully and uniformly mixed after thawing, and the components are put back to-20 ℃ for preservation after being used up. The amplification reaction system is as follows:

component (A)	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:

circulation step	Temperature (temperature)	Time	Cycle number
				Pre-denaturation	95℃	3min	1
Denaturation (denaturation)	95℃	15sec	35
				Annealing	55℃	15sec	35
Extension	72℃	1min	35
				Extend thoroughly	72℃	10min	1
Thermal insulation/preservation	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 method for constructing the overexpression vector of the embodiment uses pBI121 as an expression vector, and utilizes a homologous recombination method to fusion construct a target gene fragment and the vector, wherein all reagents are Nanjinozan biological companyIIOne Step Cloning Kit, the specific experimental procedure is as follows:

double enzyme digestion is carried out on the PBI121 vector plasmid by using restriction enzymes XbaI and XhoI, and after enzyme digestion is carried out for 1 hour at 37 ℃, the linearization vector is recovered;

and designing a recombinant primer, and performing PCR amplification by using a cloning product of the CDS region of the CcLFY gene as a template. Wherein the recombinant forward primer is

5'-gagaacacgggggactctagaatggacccggaagctttcac-3'(SEQ ID NO.5)

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.

After preparing linearization vector and target gene fragment, measuring recovery concentration, adjusting the proportion between them, and making ligation reaction at 37 deg.C, the specific recombination ligation reaction system is as follows:

component (A)	Recombination reactions
		Linearization carrier	150ng
Insertion of a Gene fragment of interest	50ng
		5×CE II Buffer	2μL
Exnase II	1μl
		ddH 2 O	to 10μl

The large intestine competent DH 5. Alpha. Was removed from the-80℃refrigerator 5 minutes before the end of ligation and thawed on ice. The whole ligation product was added to DH 5. Alpha. Competent cells, and mixed by gently sucking with a gun head, and after ice bath for 30min, heat-shocked at 42℃for 90s in a mixer, and then placed on ice for 1min. 400. Mu.L of blank LB liquid medium was added and incubated for 1h at 220r/min on a shaker at 37 ℃. Centrifuging at 12000rpm for 1min, removing supernatant, collecting 100 μl of bacterial liquid, mixing, spreading on LB solid culture dish containing kanamycin, and culturing in 37 deg.C incubator overnight.

And (3) selecting monoclonal bacterial plaques for PCR detection, sequencing the detected positive clones by a company, and then carrying out sequence comparison analysis to finally determine that the vector is successfully constructed.

Plasmid extraction is carried out on the coliform bacteria liquid with correct sequencing, and the plasmid extraction method is referred to the specification of a coliform plasmid extraction kit of Edley biological company. The extracted plasmid is transferred into agrobacterium competent EHA105, and the specific operation steps are as follows:

the agrobacteria competent EHA105 is taken out from a refrigerator at the temperature of minus 80 ℃ and then frozen and thawed on ice, 0.1-1 mug of plasmid is added, the ice is placed on the ice for 5min, the ice is frozen in liquid nitrogen for 5min, and the agrobacteria competent EHA105 is quickly taken out and then is placed on the ice for 5min after being subjected to a medium-temperature bath in a thermostat at the temperature of 37 ℃. 800 mu L of blank liquid LB culture medium is added into competent cells, and shaking bacteria is carried out on a shaking table at 28 ℃ for 3 hours at 220 r/min. Centrifuging at 12000rpm for 1min by a high-speed centrifuge, sucking and removing the supernatant, reserving 100 mu L of bacterial liquid, sucking and beating uniformly, coating the supernatant in an LB solid culture dish added with kanamycin and rifampicin, and culturing in an inverted manner in a constant-temperature incubator at 28 ℃ for 2-3 days.

And (3) selecting monoclonal bacterial plaques for PCR detection, uniformly mixing positive clone bacterial liquid with 50% glycerol one by one, and storing in a refrigerator at the temperature of minus 80 ℃ for standby, and then activating to obtain the plant transformation.

Example 3 genetic transformation and phenotypic analysis of lemon

The genetic transformation method of lemon of the embodiment comprises the following steps:

(1) Preparing lemon seedlings, and taking the epicotyl of the lemon seedlings as a transformation material

The preparation method comprises the following specific steps: healthy fresh and full lemon seeds are taken out of the fruits and the pulp attached to the surface is rinsed off. The pectic substance is removed by soaking and stirring for 15min with NaOH solution of 1moL/L, the NaOH solution is poured off, and the seeds are washed 4-5 times with distilled water. Then operating in an ultra-clean workbench, sterilizing seeds by using 3-4% NaClO solution, and carrying out shaking table treatment for 15-20 min. Pouring out NaClO solution, washing the seeds with sterile water for 4-5 times until the sterile water is clear, and finally soaking and preserving with sterile water for standby. Placing the lemon seeds to be sown on the filter paper sterilized at high temperature, peeling off the inner and outer seed coats, inserting the citrus test tube sowing culture medium, taking the direction of the radicle growing downwards, culturing for about 25-30 days in a dark state, taking out, culturing for 7-10 days in a light state, and turning green to be used for subsequent transformation.

(2) Preparation of agrobacteria transformation invasion solution

Before genetic transformation of lemon, agrobacterium is required to be activated, and preparation of a transformation invasion solution is carried out under the condition that thalli are kept in a better active state, and the specific operation steps are as follows:

strain activation: inoculating the CcLFY agrobacterium solution connected with the PBI121 overexpression vector into a solid LB culture medium added with kanamycin and rifampicin, and culturing in an incubator at a constant temperature of 28 ℃ for 2 days in an inversion way, thus performing the first activation.

Propagation of colonies: and (3) selecting agrobacterium monoclonal detection to determine positive, shaking, mixing, re-inoculating in a solid LB culture medium added with kanamycin and rifampicin, and performing inversion culture in a constant temperature incubator at 28 ℃ for 2 days to perform secondary activation.

Preparing an aggressive dyeing liquid: all the thalli are scraped in an LM suspension culture medium and placed in a shaking table with constant temperature of 220r/min and 28 ℃ for 1 hour, so that the thalli are completely dispersed. Measuring suspension concentration by ultraviolet spectrophotometer, and adjusting OD ₆₀₀ The value is between 0.6 and 0.8, acetosyringone (AS, 50 mg/L) is added, and the mixture is placed in a constant temperature incubator at 28 ℃ for standby.

(3) Infection and co-cultivation process

The stem section is obliquely cut by utilizing the epicotyl of the lemon seedling and is co-cultured with the prepared infection liquid, and the specific steps of the process are as follows:

a) The infection process comprises the following steps: cutting the epicotyl of the lemon seedling with good growth state after turning green for 7-10 days into a trapezoid stem section with about 1cm on sterile filter paper by using a sterile scalpel in an ultra-clean workbench, enabling the incision to incline as much as possible to increase the incision area so as to promote transformation, and immediately placing the stem section incision into an LM suspension culture medium to keep the incision moist, so that the air drying and water loss of the stem section incision are avoided, and the transformation efficiency is influenced. Soaking all the cut stems in prepared agrobacterium infection solution, sealing the triangular flask mouth with a sealing film, and placing in a shaking table at a constant temperature of 28 ℃ for 20 minutes at 220 r/min.

b) Co-cultivation: pouring out bacterial liquid in an ultra-clean workbench, sucking out residual agrobacterium infection liquid on the surface of the stem section by using sterile filter paper, keeping the incision upward, placing in a co-culture medium (MT+AS, 50 mg/L) with a layer of sterile filter paper laid on the surface, and co-culturing for 3 days under the dark condition of a constant temperature oven at 21 ℃.

(4) Screening culture

The screening culture medium (SM culture medium: SY+Cef400 mg/L+Km50mg/L+Agar 8 g/L) added with kanamycin is used for carrying out primary screening on the buds of the stem segments, so that excessive buds are avoided, the false positive proportion is large, and the follow-up positive identification and detection work is influenced. The method comprises the following specific steps:

a) Cleaning: transferring the lemon stem segments after co-culture for 3 days into a conical flask filled with sterile water in an ultra-clean workbench, cleaning for 3-5 times by using the sterile water, sealing and placing in a constant-temperature shaking table at the temperature of 28 ℃ at 220r/min for 5-10min each time, and cleaning and replacing with the sterile water for a plurality of times until the water becomes transparent. The sterile water was poured off and the residual water on the surface of the stem sections was blotted with sterile filter paper.

b) Screening and culturing: spreading the cleaned lemon stem incision upwards in a screening culture medium added with kanamycin and cephalosporin, culturing in dark at 26 ℃ for 7-10 days, transferring into illumination culture, maintaining 16/8h illumination, and subculturing for about 25 days.

c) Cutting buds: the lemon stem is screened and cultured for about 1-2 months, new buds grow at the incision, then the buds sprouting at the incision are cut off by a sterile scalpel in an ultra-clean workbench, and transferred to an elongation culture medium for continuous growth for about 25 days.

d) Rooting culture: after the buds grow for 1-2 months, transferring the buds into rooting culture medium, and carrying out secondary culture for about 25 days.

(5) Positive identification and phenotypic observation analysis

And (3) carrying out positive identification on the finally obtained material, finally determining positive transgenic material, and further carrying out phenotype observation analysis on the positive transgenic material. The method comprises the following specific steps:

and extracting DNA of the obtained lemon material, and identifying positive conditions by utilizing PCR amplification, namely determining that the target gene fragment is successfully transferred into plant materials.

And then extracting transgenic lemon material RNA, carrying out reverse transcription to obtain cDNA, and determining successful transformation target gene fragments through PCR amplification.

The fusion GFP protein in the vector was then successfully expressed in transgenic lemon material using in vivo imaging techniques.

And finally, determining successful expression of the target gene in the transgenic lemon material through GFP antibody by using Western detection.

Phenotypic observation analysis: the results are shown in fig. 1 to 3. Compared with wild lemon, the CcLFY over-expression lemon material has the advantages of increasing axillary branches and promoting axillary bud development. Therefore, the cloned citrus CcLFY gene has the function of regulating and controlling shoot development.

It should be noted that the above examples are only for further illustrating and describing the technical solution of the present invention, and are not intended to 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 scope of the present invention. Any modification, equivalent replacement, improvement, etc. 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 China

Application of <120> 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 (6)

1. Over-expressing citrusCcLFYApplication of gene in regulating and controlling development of lemon branch tip, and citrusCcLFYThe nucleotide sequence of the gene is shown as SEQ ID NO. 1.

2. The use according to claim 1, wherein,the saidCcLFYThe amino acid sequence of the protein coded by the gene is shown as SEQ ID NO. 2.

3. The use according to claim 1, characterized in that overexpression is usedCcLFYThe method of the gene comprises the following steps: PBI121 is used as an expression vector, a target gene fragment and the vector are fused by a homologous recombination method to construct a super-expression vector, and agrobacterium tumefaciens mediated genetic transformation is utilized.

4. The use according to claim 3, wherein the cloning method of the gene fragment of interest is: and (3) performing PCR amplification by using total cDNA obtained by reverse transcription of RNA of the tissue of the Criman Ding Ju leaf as a template, wherein the forward primer sequence is shown as SEQ ID NO.3, and the reverse primer sequence is shown as SEQ ID NO. 4.

5. The use according to claim 3, wherein the sequences of the PCR amplification primers used in the construction of the super-expression vector are shown in SEQ ID NO.5 and SEQ ID NO.6, respectively.

6. The use according to claim 3, characterized in that the method step of using agrobacterium tumefaciens mediated genetic transformation is:

preparing an epicotyl of a lemon seedling serving as a transformation material;

the activated agrobacterium tumefaciens dip-dyeing liquid is used for dip-dyeing the epicotyl of the lemon seedling, and the epicotyl is transferred into a co-culture medium, and the culture is continued in a dark condition, so that a stem bud growing material is obtained;

placing the stem bud material in a screening culture medium containing kanamycin for secondary screening culture to obtain a stem elongation material;

the stem segment elongation material is placed in rooting culture medium for continuous culture, positive transgenic material is identified, and shoot phenotype is observed.