CN111118033B - Hybrid liriodendron LhAP2L gene and application thereof - Google Patents

Abstract

The invention discloses a hybrid liriodendron LhAP2L gene and application thereof, belonging to the technical field of plant molecular biology and genetic engineering. The invention obtains the LhAP2L gene of the hybrid liriodendron by cloning, and observes the phenotype discovery of LhAP2L gene-transferred tobacco: the number of petals is increased by the expression of the LhAP2L gene, and the number of stigma divisions is changed from 2 to 3; the result of the floral organ mutation is smaller than that of the wild type, the number of the floral organs is small, and the floral organs are shriveled; the average height of 30 transgenic positive plants is 127.1cm, while the average height of wild type is 118.2 cm. The LhAP 2L-transgenic Arabidopsis thaliana was observed, and it was found that the calyx and pistil of the flower organ were increased as compared with the wild type. Therefore, the hybrid liriodendron LhAP2L gene provided by the application has the effects of promoting vegetative growth, inhibiting reproductive growth and changing the development form of flower organs, and can be used for flower cultivation and application of promoting vegetative growth of plants and inhibiting reproductive growth.

Description

Hybrid liriodendron LhAP2L gene and application thereof

Technical Field

The invention belongs to the technical field of plant molecular biology and genetic engineering, and particularly relates to a hybrid liriodendron LhAP2L (APETALA2-like) gene and application thereof.

Background

AP2 is one of a series of important genes for regulating flower development of Arabidopsis, and the gene does not contain MADS box structure and has unique structural characteristics. Jofuku et al first isolated the AP2 gene from Arabidopsis thaliana, and identified for the first time the AP2/EREBP domain of the AP2 gene in Arabidopsis thaliana, containing 2 stretches of a particularly conserved amino acid sequence, the YRG and RAYD elements, respectively. The AP2/EREBP transcription factors encoded by AP2 are mainly characterized by at least one highly conserved DNA binding region consisting of about 60 to 70 amino acid residues, which is called AP2 binding domain. In Arabidopsis, the AP2/ERF family, including AP2 (containing 2 AP2 domains), RAV (Related-to-ABI3/VP1), DREB (depression responsive element binding protein), ERF (ethylene responsive factor) and other subfamilies (containing a single AP2 domain).

Transcription factors containing AP2/EREBP binding domains widely exist in plants, and play multiple roles in the life cycle of plants, such as participating in the growth and development of plants, regulating the specialization of floral organ characteristics, controlling the identity of leaf epidermal cells, participating in signal transduction of various physiological and biochemical reactions, responding to various biological stresses, environmental stresses and the like. The AP2/ERF transcription factor is involved in a plurality of signal transduction pathways such as salicylic acid, abscisic acid, ethylene, jasmonic acid and the like, and is a connecting factor in an adversity signal crossing pathway.

Some AP2 homologous genes are found in woody plants, and the AP2 homologous gene in different plants has different functions with the AP2 gene in Arabidopsis, which indicates that the AP2 homologous gene has certain difference in function among different plants. The analysis of the functions of the AP2 homologous genes on different plants is of great significance for understanding the functional characteristics and further utilization of the genes.

Disclosure of Invention

In view of the above problems in the prior art, the technical problem to be solved by the present invention is to provide a hybrid liriodendron LhAP2L gene, which has the effects of promoting vegetative growth, inhibiting reproductive growth and changing the development morphology of flower organs. The invention also aims to provide application of the LhAP2L gene of the hybrid liriodendron.

In order to solve the technical problems, the technical scheme adopted by the invention is as follows:

a hybrid liriodendron LhAP2L gene has a nucleotide sequence shown in SEQ ID NO. 1.

The amino acid sequence of the expression protein of the hybrid liriodendron LhAP2L gene is shown in SEQ ID NO. 2.

A vector, a recombinant bacterium or a host cell containing the LhAP2L gene of the hybrid liriodendron.

The vector containing the hybrid liriodendron LhAP2L gene is a plant recombinant expression vector.

The host cell containing the hybrid liriodendron LhAP2L gene is agrobacterium tumefaciens EHA 105.

The application of the hybrid liriodendron LhAP2L gene in changing the development morphology of flower organs, promoting vegetative growth of plants or inhibiting reproductive growth.

The application comprises the following steps:

1) constructing a vector of the LhAP2L gene of the hybrid liriodendron;

2) transforming the constructed vector of the LhAP2L gene of the hybrid liriodendron into plants or plant cells;

3) and culturing and screening to obtain the plants with the changed development forms of the flower organs, the enhanced vegetative growth or the inhibited reproductive growth.

In the application, the plant is tobacco.

Has the advantages that: compared with the prior art, the invention has the advantages that:

the invention obtains the hybrid liriodendron LhAP2L gene by cloning technology, and the observation of the phenotype of the transgenic tobacco obtained by genetic transformation of the tobacco by the gene shows that: the expression of the LhAP2L gene in tobacco influences the development of flowers, so that the number of petals is increased, and the division of stigma of the flowers is from 2 fission to 3 fission; compared with the wild tobacco fruit, the capsule formed after the floral organ mutation has smaller volume, less seed quantity and the phenomenon of seed shriveling; the height of the 30 transgenic positive plants is measured, the average height of the transgenic plants is 127.1cm, and the average height of the wild plants is 118.2 cm. In flower organs of LhAP 2L-transgenic Arabidopsis, sepals and pistils are enlarged. Therefore, the hybrid liriodendron LhAP2L gene provided by the application has the effects of promoting vegetative growth, inhibiting reproductive growth and changing the development form of flower organs, and can be used for flower cultivation and application of promoting vegetative growth of plants and inhibiting reproductive growth.

Drawings

FIG. 1 is a schematic diagram showing the structure of a plasmid vector containing the LhAP2L gene of a hybrid liriodendron;

FIG. 2 is a graph showing the real-time quantitative results of LhAP2L gene in different organs;

FIG. 3 is a diagram showing the results of PCR detection electrophoresis of genomic DNA of a partial plant obtained from LhAP 2L-transgenic tobacco; 1-11 in the figure: transgenic tobacco plant DNA; CK-: wild type tobacco plant DNA; CK +: plasmid DNA; h₂O：ddH₂O；M：2000bp Mark；

FIG. 4 is a diagram showing the results of reverse transcription PCR detection electrophoresis of LhAP2L transgenic tobacco plants; 1-5 in the figure: transferring LhAP2L gene PCR positive tobacco plant (line number: 1, 9, 10, 11, 21) plant cDNA; m: 2000bp Mark; CK-: wild type tobacco cDNA; h₂O：ddH₂O; CK +: a plasmid;

FIG. 5 is a phenotypic picture of LhAP2L transgenic tobacco plants, flowers and fruits; in the figure, a, c, d and g are phenotypes of mature plants, flowers and fruits of LhAP2L transgenic tobacco; b. e, f: the phenotype of mature plants, flowers and fruits of wild-type tobacco, respectively.

FIG. 6 is a comparison of the floral organs of the T1 generation LhAP2L transgenic Arabidopsis line with the wild type; in the figure, left: wild type arabidopsis floral organ; and (3) right: transgenic LhAP2L Arabidopsis floral organ;

Detailed Description

The invention is further described with reference to specific examples.

Experimental materials: the experimental material used in the experiment is a tobacco sterile seedling with the age of about 3 weeks of subculture, and young leaves with the size, the character and the color of the top of the plant and good growth state are taken as the transformation material.

Reagent: kanamycin is purchased from Sigma, cefamycin is purchased from Shanghai Biotechnology, Inc., RNA extraction Kit RNAprep Pure Plant Kit is purchased from TIANGEN, and reverse transcription Kit BioTeke supermoIII RT Kit is purchased from BioTeke; taq enzyme was purchased from Takara. Solution I: 50mmol/L glucose, 25mmol/L Tris-Cl (pH8.0), 10mmol/L LEDTA (pH8.0) were autoclaved and stored at 4 ℃. Solution II: 0.2moL/L NaOH, 1% SDS (freshly prepared). Solution III: 60mL of 5moL/L potassium acetate, 11.5mL of glacial acetic acid and 28.5mL of water.

Culture medium:

tobacco differentiation medium: MS + BA2.0mg/L + IAA0.5mg/L + sucrose 30g/L + agar 6.8g/L pH7.0

Tobacco screening culture medium: MS + BA2.0mg/L + IAA0.5mg/L + sucrose 30g/L + agar 6.8g/L + Kan100mg/L + Cef400mg/LpH7.0

Tobacco rooting culture medium: MS + sucrose 30g/L + agar 6.8g/L pH7.0

Tobacco rooting screening culture medium: MS + sucrose 30g/L + agar 6.8g/L + Kan100mg/L + Cef400mg/LpH7.0

LB liquid medium: tryptone 10g/L, yeast extract 5g/L, sodium chloride 10g/L, pH7.0

LB solid medium: 10g/L of tryptone, 5g/L of yeast extract, 10g/L of sodium chloride, 15g/L of agar and pH7.0

Plasmids and strains: the Agrobacterium strain used in the present application is Agrobacterium tumefaciens (Agrobacterium tumefaciens) EHA105 strain. The plasmid vector LhAP2L of the hybrid liriodendron tulipifera used in the present application is shown in FIG. 1.

Instruments and equipment: superclean bench, constant temperature shaking table, constant temperature incubator, low temperature refrigerator, ultra-low temperature refrigerator, PCR amplification appearance, electrophoresis tank, electrophoresis apparatus.

Example 1: cloning to obtain LhAP2L gene

1. Extraction of Total RNA

Taking young leaves of hybrid liriodendron as a material, extracting RNA according to the operation steps of a TIANGEN plant total RNA extraction kit (DP432), treating used reagents and consumables with 0.1% DEPC water overnight, and autoclaving for 40min to make the young leaves free of RNase. The result of 1% agarose gel electrophoresis of the total RNA of the hybridized liriodendron tulipifera shows that 28S, 18S and 5.8S bands are all clearly visible, the fluorescence brightness of 28S is twice of that of 18S, and the RNA quality is better.

The specific process of RNA extraction is as follows:

1) and (3) homogenizing treatment: cut about 100mg of fresh leaves into powder rapidly in liquid nitrogen, transfer to 450 μ LRL (Add beta-mercaptoethanol before use), vortex vigorously and shake well.

2) All solutions were transferred to a filter column (filter column placed in a collection tube), centrifuged at 1,2000rmp for 4min, and the supernatant from the collection tube was carefully pipetted out into the RNase-free centrifuge tube, with the tip being kept free from contact with the pellet of cell debris in the collection tube.

3) Slowly adding 0.5 times of the volume of the supernatant of anhydrous ethanol, mixing, transferring the obtained solution and precipitate into an adsorption column, centrifuging at 12000rmp for 1min, pouring off the waste liquid in the collection tube, and putting the adsorption column back into the collection tube.

4) Adding 350 μ L deproteinized solution RW1, centrifuging at 12000rmp for 1min, pouring off waste liquid in the collection tube, and placing the adsorption column back into the collection tube.

5) Preparation of a Dnase1 working solution: 10uL of Dnasel stock solution was put into a new RNase-free centrifuge tube, 70. mu.L of RDD solution was added, and gently mixed.

6) 80 μ L of DNase1 working solution was added to the center of the adsorption column and left at room temperature for 15 min.

7) Adding 350 μ L deproteinized solution RW1 to the center of the adsorption column, centrifuging at 12000rmp for 1min, pouring off the waste liquid in the collection tube, and returning the adsorption column to the collection tube.

8) Adding 500 μ L of rinsing solution RW (ethanol before use), standing at room temperature for 2min, centrifuging at 12000rmp for 1min, removing waste liquid from the collection tube, and placing the adsorption column back into the collection tube.

9) And repeating the step 8.

10) Centrifuging at 12000rmp for 2min, pouring off waste liquid, placing the adsorption column at room temperature for several minutes, and completely drying the residual rinsing liquid in the adsorption material.

11) Putting the adsorption column into a new RNase-free centrifuge tube, suspending and dropwise adding 30-50 mu L of RNase-free ddH into the middle part of the adsorption column₂O, standing at room temperature for 2min, centrifuging at 12000rmp for 2min to obtain RNA solution, and immediately storing at-80 deg.C for use.

2. Obtaining of cDNA

The total RNA of the hybrid goose-paw is used as a template, cDNA is obtained through reverse transcription, and oligo (dT) is used as an anchor primer, and first strand cDNA is synthesized through reverse transcription.

III First-Strand Synthesis Kit (Invitrogen 18080-:

1) prepare reaction solution (10 μ L): mu.L of RNA (. ltoreq.5. mu.g), 1. mu.L of Primer (Oligo dT), 1. mu.L of 10mM dNTP mix, DEPC-Treated Water up to 10. mu.L. And (4) centrifuging at a low speed for a short time, and immediately placing on ice for 1-2 min after 5min at 65 ℃.

2) Adding corresponding reagents into the PCR tube in the previous step in the following sequence: mu.L 10 × RT Buffer, 4. mu.L 25mM MgCl2, 2. mu.L 0.1M DTT, 1. mu.L RNase OUT (40U/. mu.L), 1. mu.L SuperScript IIIRT. After being gently mixed, the mixture is placed on a PCR instrument, and the reaction program is 50min at 50 ℃ and 5min at 85 ℃.

3) After low speed centrifugation, 1 μ L of RNase H was added to each tube, and after digestion at 37 ℃ for 20min, cDNA was collected and stored at-20 ℃ for further use.

3. Homologous cloning of a Gene of interest

1) Designing a primer by utilizing Oligo7.0, carrying out PCR (polymerase chain reaction) so as to obtain a target gene, carrying out transformation sequencing, and finally carrying out comparison analysis. The designed primer sequences are as follows:

LhAP2L-F：5’-AGCTCTGATGTTCGATCTCAATGT-3’；

LhAP2L-R：5’-TTGTCGCGTGTGAAGTCTCCAAGT-3’。

2) PCR amplification System:

a50. mu.L reaction system was prepared by preparing a PCR reaction solution according to the following components: mu.L 10 XPCR Buffer, 4. mu.L 2mM dNTPs, 4. mu.L 25mM MgSO4, 2. mu.L Forward Primer, 2. mu.L Reverse Primer, 3. mu.L cDNA, 1. mu.L KOD-Plus polymerase, 29. mu.L ddH₂O。

3) And (3) PCR reaction conditions: 94 ℃ for 5 min; 94 ℃, 30sec, 57 ℃, 30sec, 72 ℃, 1.5min, 36 cycles; 10min at 72 ℃; 4 ℃ and Forever.

4. Obtaining the full Length of the Gene

(1) Recovery of target Gene fragment

The PCR reaction product is detected by electrophoresis in 1% agarose gel, and the PCR product is purified and recovered by using a DNA gel recovery kit of AXYGEN company, and the steps are as follows:

1) the agarose gel containing the desired DNA was cut under an ultraviolet lamp, and the surface of the gel was blotted with a paper towel and minced. Calculate gel weight (record 1.5mL centrifuge tube weight in advance) as one gel volume (e.g. 100mg to 100 mL);

2) adding 3 gel volumes of Buffer DE-A, uniformly mixing, heating at 75 ℃, and intermittently mixing (every 2-3 min) until the gel blocks are completely melted (about 6-8 min);

3) adding 0.5 gel volume of Buffer DE-B, and mixing uniformly; when the separated DNA fragment is less than 400bp, adding 1 gel volume of isopropanol;

4) sucking the mixed solution in the previous step, transferring the mixed solution into a DNA preparation tube (placed in a 2ml centrifuge tube), centrifuging the mixed solution at 12000 Xg for 1min, and removing the filtrate;

5) the preparation tube is placed back into the 2mL centrifuge tube again, 0.5mL Buffer W1 is added, 12000 Xg is centrifuged for 30s, and the filtrate is discarded;

6) the preparation tube was replaced in the 2mL centrifuge tube, 0.7mL Buffer W2 was added, and the tube was centrifuged at 12000 Xg for 30s, and the filtrate was discarded. Washing with 0.7mL Buffer W2 once in the same way, and centrifuging at 12000 Xg for 1 min;

7) the prepared tube is placed back into the 2mL centrifuge tube again, and is centrifuged for 1min at 12000 Xg;

8) placing the preparation tube in a clean 1.5mL centrifuge tube, and adding 25-30 muL ddH to the midpoint of the DNA preparation membrane₂O or Eluent buffer, and standing for 1min at room temperature. The DNA was eluted by centrifugation at 12000 Xg for 1 min.

(2) Tailing of target fragment

KOD PLUS high fidelity enzyme is blunt end enzyme, and TA cloning can be carried out only by adding A tail, and the specific method is as follows:

reaction system (20 μ L): 10 μ L of DNA, 2 μ L of 10 XPCR Buffer, 0.4 μ L of 10mM dNTPs, 1.6 μ L of 25mM MgCl₂，0.2μL rTaq polymerase，5.8μL ddH₂O。

Reaction procedure: 72 ℃ for 30 min; 4 ℃ and Forever.

The ligation reaction is carried out by adopting TaKaRa pMD19-T Vector (Takara D102A) Vector system, and the specific steps are as follows: in a freshly sterilized 0.5mL centrifuge tube, the following ingredients were added in order: mu.L Solution I, 1. mu.L PMD19-T Vector, 4. mu.L PCR purified product (50 ng). The mixture was pipetted and incubated overnight at 16 ℃.

(3) Conversion of ligation products

The ligation product was transformed into E.coli strain DH5 alpha. The method comprises the following specific steps: before the experiment, LB solid medium (added with Amp + IPTG + X-gal) (each medium plate is coated with 40. mu.L each of 50mg/mL IPTG and 20mg/mL X-gal, and is left to stand at room temperature for use);

1) taking out the competent cells from an ultra-low temperature refrigerator at minus 80 ℃, putting the competent cells on ice for melting, adding 5 mu L of the ligation product into 100 mu L of the competent cells, gently mixing the competent cells and the ligation product uniformly, and carrying out ice bath for 30 min;

2) thermally shocking at 42 deg.C for 90sec, immediately standing on ice for 2 min;

3) adding 800 μ L LB liquid culture medium (without Amp antibiotics), shaking culturing at 37 deg.C and 180rpm for 1-2 h;

4) centrifuging at 4000rpm for 3min, washing off 800 μ L of supernatant, and sucking and resuspending the residual bacteria liquid;

5) taking a proper amount of heavy suspension, uniformly coating the heavy suspension on an LB solid culture medium, and carrying out inverted culture at 37 ℃ for 12-16 h.

(4) Screening of recombinant plasmid and PCR verification of bacterial liquid

Screening positive clones for strains: a single white positive colony from an LB plate was picked and inoculated into 750. mu.L of LB liquid medium containing Amp antibiotics. The cultivation temperature was 37 ℃ and the cultivation temperature was 220rpm overnight.

PCR verification of bacterial liquid:

20 μ L of the PCR amplification system was: 10 XPCR Buffer 2. mu.L, dNTP mix (2.5 mM each) 1.6. mu.L, MgCl₂(25mM) 1.2. mu.L, M13-F1. mu.L, M13-R1. mu.L, bacterial liquid 2. mu.L, rTaq (5U/. mu.L) 0.2. mu.L, ddH₂O 11μL。

And (3) PCR reaction conditions: 5min at 94 ℃; 94 ℃ 30sec, 55 ℃ 30sec, 72 ℃ 1min 40sec, 36 Cycles; 10min at 72 ℃; 4 ℃ forever.

And detecting the PCR product by agarose gel electrophoresis. The obtained positive clones were subjected to sequencing analysis. As a result, the total length of the AP2L gene is 1616 bp. The sequence analysis result shows that the length of the ORF of the LhAP2L gene is 1476bp, the total length is 492 amino acids, and the LhAP2L gene has 2 AP2L structural domains. The sequence of the Open Reading Frame (ORF) is shown as SEQ ID NO.1, and the sequence of the coded protein is shown as SEQ ID NO. 2.

The sequence obtained by sequencing is analyzed by a BLAST tool in NCBI, and the sequence is compared with the family gene homology of AP2 such as arabidopsis thaliana and the like to reach more than 90 percent.

Example 2: expression analysis of LhAP2L gene in hybrid liriodendron

The root, stem, leaf, bud, flower quilt, stamen and pistil of the hybridized liriodendron are selected as materials, RNA is extracted and then reverse transcribed into cDNA, and the LhAP2L expression conditions in different organs are analyzed. The specific process is as follows:

real-time quantitative PCR was performed on ABI 7500 Real time PCR Systems (Applied Biosystems) using Power SYBR Green PCR Master Mix kit from ABI, Inc., with three replicates of each sample reaction, and data extraction analysis using 7500System SDS software (Applied Biosystems).

1) Designing RT-PCR primers: according to the requirements of RT-PCR, 3 pairs of PIN gene primers are designed, and through analysis of a dissolution curve, 18S rRNA in the hybridized liriodendron is finally selected as an internal reference, wherein the primers are as follows:

F-RT：5′-CCAACTTCAGTACCCCAATGC-3′；

R-RT：5′-AGCAGCATGGCTCTACTCCAT-3′

2) RT-PCR reaction system: mu.L of 2 XPower SYBR Green PCR Master Mix, 1. mu.L Forward Primer, 1. mu.L Reverse Primer, 1. mu.L cDNA, 7. mu.L ddH 2O.

3) qRT-PCR reaction conditions: at 95 ℃ for 10 min; 95 ℃, 15sec, 60 ℃, 1min, 40 Cycles.

4) And (3) test results: as shown in FIG. 2, the LhAP2L gene was expressed in all of roots, stems, shoots, leaves, integuments and pistils. The expression level is highest in roots and buds, the expression level is lowest in stems, flower covers and pistils, the expression level is lowest in leaves, and the expression level is absent in stamens.

Example 3: genetic transformation of tobacco by LhAP2L gene

(1) Preparation of bacterial liquid

Picking and storing on LB solid mediumInoculating the single colony of the agrobacterium containing the target gene into 5mL of LB liquid culture medium added with corresponding screening antibiotics Kan50mg/L and Str30mg/L, carrying out shake culture at 28 ℃ and 220rpm for 24h, carrying out amplification inoculation in the LB liquid culture medium containing the corresponding antibiotics according to the proportion of 1: 50, carrying out shake culture at 28 ℃ and 250rpm for 4-6h, and then measuring the OD value (the OD value is 0.3-0.4) by using an ultraviolet spectrophotometer. Centrifuging at 4000rpm for 10min, removing supernatant, and collecting thallus. Suspending the bacteria with certain volume of MS or 1/2MS liquid culture medium, diluting the bacteria liquid to OD₆₀₀And (5) setting the value to be 0.2 for standby.

(2) Preculture

Tissue culturing sterile test-tube plantlets of 3-4 weeks old, taking 1 st to 4 th leaves with basically consistent size, character and color and good growth state from the top of the test-tube plantlets, shearing off the edges of the leaves by using scissors, removing main veins, and shearing the leaves into 0.5cm²-1.0cm²The left and right small square leaves, the distal ends of which are spread downward on a differentiation medium without any antibiotic and cultured in dark for 2 days.

(3) Infection and co-culture

Immersing the pre-cultured leaf blades to the prepared OD₆₀₀The bacterial solution is infected for 10min, and the bacterial solution is gently shaken during the infection, so that the leaves can be in full contact with the thallus. Taking out, drying the bacterial liquid on the surface of the leaf by using sterile filter paper, and placing on a filter paper-paved differentiation culture medium without antibiotics for co-culture for 2 days, wherein the co-culture is dark culture.

(4) Screening and rooting

The co-cultured leaf blades are washed 2-3 times (5 min each time) with MS liquid culture medium added with Cef400mg/L, washed 3 times with sterile water, cultured on tobacco screening culture medium, and the culture medium is replaced every two weeks. When the bud grows to about 1cm, the bud is transferred to a tobacco screening rooting culture medium.

Example 4: screening of positive plants of LhAP2L gene transformed tobacco

1. Detection of tobacco plant genomic DNA

(1) Extraction of tobacco genome DNA by CTAB method

1) Opening the constant-temperature water bath kettle, adjusting the temperature to 65 ℃ and preheating the lysate CTAB;

2) respectively placing transgenic and control fresh young leaves in a 2mL centrifuge tube, adding sterile glass beads, thoroughly grinding plant materials by using a vibration ball mill, placing the centrifuge tube on ice after full grinding, rapidly adding 700 mu L CTAB, violently shaking and uniformly mixing;

3) placing the material in a mixing apparatus at a constant temperature of 65 deg.C for 30min, and shaking the centrifuge tube for several times;

4) taking out the centrifuge tube after 30min, cooling to room temperature, adding chloroform/isoamylol with the same volume (24: 1), shaking vigorously and mixing uniformly, and standing for 10 min;

5) placing the mixed solution in a centrifuge for centrifugation at 14000rpm for 10min, transferring the supernatant into a clean 1.5mL centrifuge tube, adding chloroform/isoamylol (24: 1) with the same volume, and extracting once again;

6) transferring the supernatant into a new centrifuge tube, adding isopropanol with the same volume, uniformly mixing, standing for 10min, and centrifuging for 10min at 14000 rpm;

7) carefully pouring out the supernatant, adding 1mL of 70% alcohol, blowing, washing and precipitating, centrifuging at 14000rpm for 2min, pouring out the alcohol, and repeating the steps once;

8) centrifuging at 10000r for 2min, sucking residual liquid with a gun head, drying DNA, and dissolving in 50 μ L of TE or ddH preheated at 65 deg.C₂In O, the mixture is stored at 4 ℃.

(2) Extraction of Agrobacterium plasmid

1) Taking 1.5mL of bacterial liquid, carrying out centrifugation at 12000r/min for 30s, discarding supernatant, and collecting thalli;

2) adding 300 mu L of solution I to resuspend the thallus precipitate, and violently shaking (vortex);

3) adding 300 μ L of newly prepared solution II, quickly and gently inverting for tens of times, standing for 5min

4) Adding 300 μ L of pre-cooled solution III, inverting the centrifuge tube several times, ice-cooling for 5min, and centrifuging at 12000rpm and 4 deg.C for 10-15min

5) Transferring the supernatant into a clean centrifuge tube, adding 500 μ L isopropanol, turning upside down, mixing, standing for 2min, centrifuging at 12000rpm for L0min, and removing the supernatant;

6) adding 700 μ L70% ethanol, washing twice, centrifuging at 12000rpm for 5min

7) The supernatant was discarded and the residue was discarded,centrifuging again, sucking to dry the waste liquid, drying the precipitate at room temperature for 10min, adding appropriate amount of TE or ddH₂O lysis of nucleic acids, storage in a freezer at-20 ℃;

8) the quality of the extracted plasmid was checked by electrophoresis on a 1% agarose gel.

(3) PCR detection of genomic DNA

The primers used for PCR detection of genome DNA of LhAP 2L-transgenic tobacco plants are 35S promoter primer 35S-F and LhAP2L gene downstream primer, and corresponding plasmid vector is used as positive control (CK +), wild type tobacco DNA and ddH are used₂O was used as a negative control (CK-). The PCR detection primers for LhAP2L transgenic tobacco plant genome DNA are as follows

Primer 1: 35S-F: 5'-TGAAGATAGTGGAAAAGGAAGGTG-3'

Primer 2: 35S-LhAP 2L-R: 5'-GCGACGAGGATGATGACGAAGAAG-3'

And (3) PCR reaction system: 2.0 μ L10 XPCR Buffer (Mg)²⁺Free)，1.2μLMgCl₂(25mmol/L), 0.4. mu.L dNTPs (10mmol/L), 1.0. mu.L primer 1 (10. mu.M), 1.0. mu.L primer 2 (10. mu.M), 2.0. mu.L rTaq (5U/. mu.L), 2.0. mu.L LDNA, ddH₂Make up to 20. mu.L of O.

PCR reaction procedure: 5min at 95 ℃; 95 ℃ for 45s, 57 ℃ for 45s, 72 ℃ for 45s, 35 Cycles; 10min at 72 ℃; 4 ℃ forever.

During the PCR detection of the genomic DNA of the T1 generation plant and the wild type plant which are transformed with the LhAP2L gene, the wild type tobacco plant DNA and ddH are used₂And O is used as a negative control, plasmid DNA is used as a positive control, and PCR detection is carried out on the transgenic tobacco plant by using a 35S promoter primer 35S-F and a target gene LhAP2L primer 35S-LhAP 2L-R. 1.2% of the DNA fragments were separated by agarose gel electrophoresis, and the length of the amplified fragment obtained using the primers was 666 bp. As can be seen in FIG. 3, the negative control ddH₂No corresponding specific segment is amplified in the O and wild plants, and the target segment with the same size as the positive control band is amplified in the resistant plant. Through PCR molecular detection, the exogenous LhAP2L gene can be proved to be integrated into the tobacco genome. 30 positive plants are obtained through Kan screening and PCR detection.

2. Detection of expression of LhAP2L gene in tobacco

(1) Total RNA extraction

In the experiment, the total RNA extraction kit of the RNAprep Pure plant is applied to the total RNA extraction. The method comprises the following operation steps: 1) and (3) homogenizing treatment: 50-100mg of fresh plant leaves are quickly ground into powder in liquid nitrogen, 450 mu L of lysis solution RL (1 percent of beta-mercaptoethanol is added before use) is added, and the mixture is vortexed and vigorously shaken and mixed evenly.

2) All solutions were transferred to the filtration column CS (filtration column CS placed in the collection tube), centrifuged at 12000rpm for 2-5min, and the supernatant from the collection tube carefully pipetted into the RNase-Free centrifuge tube.

3) Equal volume of chloroform was added: isoamyl alcohol (24: 1) is mixed evenly, centrifuged at 12000rpm for 10min, and the supernatant in the centrifuge tube is carefully sucked into the RNase-Free centrifuge tube. Slowly adding 0.5 times of the volume of the supernatant of anhydrous ethanol, mixing, transferring the obtained solution and the precipitate into an adsorption column CR3, centrifuging at 12000rpm for 30-60sec, pouring off the waste liquid in the collection tube, and returning the adsorption column CR3 to the collection tube.

4) 350 μ L of deproteinizing solution RW1 was added to adsorption column CR3, centrifuged at 12000rpm for 30-60sec, the waste liquid in the collection tube was discarded, and adsorption column CR3 was returned to the collection tube.

5) Preparing DNaseI working solution: 10 μ L of DNase I stock solution was put into a new RNase-Free centrifuge tube, and 70 μ L of LRDD solution was added and gently mixed.

6) 80. mu.L of DNase I working solution was added to the center of the adsorption column CR3, and the mixture was left at room temperature for 15 min.

7) 350 μ L of deproteinizing solution RW1 was added to adsorption column CR3, centrifuged at 12000rpm for 30-60sec, the waste liquid in the collection tube was discarded, and adsorption column CR3 was returned to the collection tube.

8) Adding 500 μ L of rinsing solution RW (adding anhydrous ethanol before use) into adsorption column CR3, standing at room temperature for 2min, centrifuging at 12000rpm for 30-60sec, removing waste liquid from the collection tube, and returning adsorption column CR3 to the collection tube.

9) And repeating the step 8.

10) Centrifuging at 12000rpm for 2min, and discarding waste liquid. The adsorption column CR3 was left at room temperature for several minutes to thoroughly dry the residual rinse solution from the adsorption material.

11) Placing the adsorption column CR3 into a new RNase-Free centrifuge tube, and dripping 30-100 μ L RNase-Free ddH into the middle part of the adsorption membrane₂O, standing at room temperature for 2min, and centrifuging at 12000rpm for 2min to obtain an RNA solution.

(2) Synthesis of Single-stranded cDNA

Single-stranded cDNA was prepared by reverse transcription using BioTeke SupermoiiRT Kit from BioTeke.

The reverse transcription procedure was as follows:

1) the reverse transcription reaction system is as follows: 0.2-2. mu.g Total RNA or Poly (A) RNA, 1. mu.L Oligo (dT) or random primer (50. mu.M), 1. mu.L dNTP mix (10mM each), 4. mu.L 5 Xfirst-strand Buffer, 1. mu. L M-MLV Reverse transcript (200U/. mu.L), 1. mu.L RNase Inhibitor (40U/. mu.L), RNase-free H₂Make up to 20. mu.L of O.

2) The reverse transcription reaction was performed on a PCR instrument under the following conditions: 45-60min at 50 ℃ and 10min at 70 ℃. After the reaction is finished, the cDNA is immediately placed on ice for cooling, and the obtained cDNA can be directly used for subsequent reactions such as PCR and the like, and can also be frozen at 20 ℃ for later use.

(3) Reverse transcription RT-PCR assay

Designing specific primers of LhAP2L gene, carrying out reverse transcription RT-PCR detection on tobacco PCR positive plants of transgenic LhAP2L gene, taking corresponding plasmid vector as positive control (CK +), taking wild tobacco cDNA and ddH₂O was used as a negative control (CK-). Further, it was determined whether or not the LhAP2L gene was expressed in tobacco. The reverse transcription PCR detection primer of LhAP2L transgenic tobacco plant RNA is as follows

Primer 1: LhAP 2L-F: 5'-CAATGGTCCGAAGGAGAGTGAG-3'

Primer 2: LhAP 2L-R: 5'-GGAAATGGTGGTTGGTAGTGGG-3'

The reverse transcription PCR reaction system is as follows: 2.0 μ L10 XPCRBuffer (Mg)²⁺Free)，1.2μLMgCl₂(25mmol/L), 0.4. mu.L dNTPs (10mmol/L), 1.0. mu.L primer 1 (10. mu.M), 1.0. mu.L primer 2 (10. mu.M), 0.2. mu.L rTaq (5U/. mu.L), 2.0. mu.L cDNA, ddH₂Make up to 20. mu.L of O.

PCR reaction procedure: 5min at 94 ℃; 94 ℃ for 30s, 56 ℃ for 30s, 72 ℃ for 30s, 27 Cycles; 10min at 72 ℃; 4 ℃ forever.

(3) The PCR products were detected by 2.0% agarose gel electrophoresis and photographed.

Detecting the transcription level of cDNA of LhAP2L transgenic tobacco plants which are positive in PCR detection by using wild type tobacco cDNA and ddH₂O as negative control (CK-), and the corresponding plasmid DNA as positive control (CK +). The target fragment was amplified using primers specific to the LhAP2L gene itself. As the influence of GelRed dye and other factors causes the stripe hysteresis, as can be seen from FIG. 4, no specific segment is amplified in the negative control, the target segment with the same stripe size as the positive control is amplified in the resistant plant, and the amplified segment is between 250bp and 500bp and basically accords with the expected size of 377 bp. Through PCR molecular detection, the exogenous LhAP2L gene can be proved to be expressed in tobacco.

Example 5: observation of LhAP2L transgenic tobacco T1 representative form

Hardening the transgenic positive plants for 2-3d, and cleaning the root culture medium during transplanting to prevent the later potted seedlings from rotting roots. Transplanting the plant into a greenhouse at 25 ℃, continuously culturing, and watering once every 2-3 days.

The phenotype change of the transgenic tobacco plants is regularly observed, such as the structure, the plant height and the fruit phenotype of the transgenic LhAP2L gene-transferred tobacco positive plant T1 florescence generation.

As can be seen from FIG. 5, the division of the stigma of the LhAP2L transgenic tobacco is abnormal, namely 3 divisions; FIG. 5d shows that the number of petals in tobacco partially transformed with LhAP2L gene was 6. As can be seen in FIG. 5-e, the number of wild type tobacco petals is 5 and the stigma 2 is split. The experimental results show that the floral organs of the transgenic tobacco have variation, so that the expression of the LhAP2L gene in the tobacco influences the development of flowers, the number of petals is changed, and the LhAP2L gene has certain correlation with the development of the floral organs. Observation of floral organ variant lines in transgenic lines revealed that capsules formed after floral organ variation (fig. 5g) were smaller in size, smaller in number of seeds, and shriveled in seeds, compared to wild-type tobacco fruits (fig. 5 f). The height of the 30 obtained transgenic positive plants is measured, the average height of the transgenic plants is 127.1cm, and the average height of wild plants is 118.2 cm. The experimental results show that the plant height of tobacco is obviously increased and the fruit becomes smaller after the LhAP2L is over-expressed, and the reason for the result is presumed to be that the plant height is increased after the LhAP2L is over-expressed, so that more nutrition is used for vegetative growth, and the reproductive growth causes the fruit volume to be smaller due to the lack of nutrition supply.

Example 6: arabidopsis thaliana transformed with LhAP2L gene

(1) Genetic transformation of Arabidopsis thaliana

The planted healthy arabidopsis thaliana grows to flower. The positive clone detected by PCR is shaken to OD₆₀₀When the number is 0.8, the flower organ of arabidopsis thaliana is subjected to dipping transformation. The specific process is as follows: centrifuging the bacterial liquid at 5000rpm for 5min, collecting thallus, and suspending with 5% sucrose solution; before soaking, Silwet L-77 is added, the concentration is 0.05% (500 mu L/L), and foam is shaken out; soaking the overground part of the arabidopsis thaliana in the agrobacterium suspension solution for 15-30 sec, slightly shaking the overground part of the arabidopsis thaliana, flatly lying the soaked arabidopsis thaliana in a tray, covering the tray with a preservative film for preserving moisture, and sealing tin foil paper for keeping out of the sun for 24 hours; the tinfoil paper is uncovered, cultured under normal conditions, and watering is stopped when the seeds are mature.

(2) Positive plant screening and phenotype observation of transgenic Arabidopsis

Seeds of dried Arabidopsis were harvested and 50mg.L of seeds of T1 generation were used^-1Screening was performed in 1/2MS medium with kanamycin. It was found that negative plants did not have kanamycin resistance and died, except that positive plants still grew normally.

Transplanting the selected possible transgenic positive plants into soil for continuous culture. Extracting total DNA of the transgenic arabidopsis as a template to carry out PCR detection, and determining the transgenic arabidopsis as a positive plant. Observing the flower organs of the transformed positive plants, as shown in FIG. 6, the pistils in the flower organs of the transgenic plants are obviously elongated, and sepals also have an increased phenotype. The gene can influence the flower organ morphology of the plant to a certain extent.

Sequence listing

<110> Nanjing university of forestry

<120> hybrid liriodendron LhAP2L gene and application thereof

<130> 100

<160> 2

<170> SIPOSequenceListing 1.0

<210> 1

<211> 1476

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<213> Liriodendron chinense × tulipifera

<400> 1

atgttcgatc tcaatgtcgt ctcggaacct gcctgtgaag aacaactggt gaccggtaag 60

cttctcgaca ctcccggcgg cccgtaccat atggatgatt ccggcacgtc gaattcctcc 120

gtggtcaatg ccgaggcatc aagcaacggc ggcggtgacg acgattcgtg ctcgacccat 180

gttgccaagc gcagcaatat cttcgccttc agcttcgata tcttgaagaa gaggggtgat 240

gatgtcgttg ccgttgaaga tgacaatgac gatgatgaag aagacaatgg tgtttctccg 300

cagttgggtt ttgtcaccag gcagctcttt ccggcacctc ccagcggagg cggcttctgc 360

tcgtcggctt cttcgtcatc atcctcgtcg cggccacatt gggcagatat tactttctgc 420

cgtgccgatg ctgctgggca ggcggaggtt aaggtggtgc agccaccacc attgccgcag 480

cagcagcagg tgaagaagag ccgccgtggg cctcggtcaa gaagctctca gtaccgagga 540

gttacattct accgaagaac aggaagatgg gaatcgcata tctgggattg tgggaagcaa 600

gtttatttgg gggggttcga cactgctcat gctgctgctc gggcttacga tcgagctgcc 660

attaagttca ggggagttga tgcagacatc aatttcaacc ttagtgatta tgatgaagat 720

ctgaaacaga tgaggaacct taccaaggaa gaattcgtgc acatcctccg tcgtcagagc 780

actggattct caagggggag ttcaaaatac agaggtgtaa cactgcacaa atgcggccga 840

tgggaagctc gtatggggca gttccttggc aagaagtaca tatatcttgg gctattcgac 900

agcgaagtag aagctgcaag ggcctatgac aaggcggcta taaaatgcaa tggaagggaa 960

gctgtgacca atttcgaacc gagtacatat gaaggagaga tagctgtgga ggctgattct 1020

ggaggaactg ccgacaatct tgatctgaac ttggggattt cccccccttc ttttgccaat 1080

ggtccgaagg agagtgagag ttcggctggc ttccattttc attgcagccc ttttgaggtg 1140

cctgaaaaca ggaaaactag gactgagaac ccaacttcag taccccaatg cttgaccatg 1200

gcgcctgagc atcctcctgt gtggactggt ctctatcctg gtttctttcc caacttcgag 1260

ggaagagtga tagagaagag ggtggaagtt ggttcgccga tcttttctaa ctgggcttgg 1320

cagcagcatg gctctactcc atcaatgcca ctcttctctt ctgcagcatc atcaggattc 1380

tctaccgcga ctaccactac aacaacagct gcccttcctc ctccgccccc tcccactacc 1440

aaccaccatt tcccgcctca ctactattac aggagc 1476

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<213> Liriodendron chinense × tulipifera

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Met Phe Asp Leu Asn Val Val Ser Glu Pro Ala Cys Glu Glu Gln Leu

1 5 10 15

Val Thr Gly Lys Leu Leu Asp Thr Pro Gly Gly Pro Tyr His Met Asp

20 25 30

Asp Ser Gly Thr Ser Asn Ser Ser Val Val Asn Ala Glu Ala Ser Ser

35 40 45

Asn Gly Gly Gly Asp Asp Asp Ser Cys Ser Thr His Val Ala Lys Arg

50 55 60

Ser Asn Ile Phe Ala Phe Ser Phe Asp Ile Leu Lys Lys Arg Gly Asp

65 70 75 80

Asp Val Val Ala Val Glu Asp Asp Asn Asp Asp Asp Glu Glu Asp Asn

85 90 95

Gly Val Ser Pro Gln Leu Gly Phe Val Thr Arg Gln Leu Phe Pro Ala

100 105 110

Pro Pro Ser Gly Gly Gly Phe Cys Ser Ser Ala Ser Ser Ser Ser Ser

115 120 125

Ser Ser Arg Pro His Trp Ala Asp Ile Thr Phe Cys Arg Ala Asp Ala

130 135 140

Ala Gly Gln Ala Glu Val Lys Val Val Gln Pro Pro Pro Leu Pro Gln

145 150 155 160

Gln Gln Gln Val Lys Lys Ser Arg Arg Gly Pro Arg Ser Arg Ser Ser

165 170 175

Gln Tyr Arg Gly Val Thr Phe Tyr Arg Arg Thr Gly Arg Trp Glu Ser

180 185 190

His Ile Trp Asp Cys Gly Lys Gln Val Tyr Leu Gly Gly Phe Asp Thr

195 200 205

Ala His Ala Ala Ala Arg Ala Tyr Asp Arg Ala Ala Ile Lys Phe Arg

210 215 220

Gly Val Asp Ala Asp Ile Asn Phe Asn Leu Ser Asp Tyr Asp Glu Asp

225 230 235 240

Leu Lys Gln Met Arg Asn Leu Thr Lys Glu Glu Phe Val His Ile Leu

245 250 255

Arg Arg Gln Ser Thr Gly Phe Ser Arg Gly Ser Ser Lys Tyr Arg Gly

260 265 270

Val Thr Leu His Lys Cys Gly Arg Trp Glu Ala Arg Met Gly Gln Phe

275 280 285

Leu Gly Lys Lys Tyr Ile Tyr Leu Gly Leu Phe Asp Ser Glu Val Glu

290 295 300

Ala Ala Arg Ala Tyr Asp Lys Ala Ala Ile Lys Cys Asn Gly Arg Glu

305 310 315 320

Ala Val Thr Asn Phe Glu Pro Ser Thr Tyr Glu Gly Glu Ile Ala Val

325 330 335

Glu Ala Asp Ser Gly Gly Thr Ala Asp Asn Leu Asp Leu Asn Leu Gly

340 345 350

Ile Ser Pro Pro Ser Phe Ala Asn Gly Pro Lys Glu Ser Glu Ser Ser

355 360 365

Ala Gly Phe His Phe His Cys Ser Pro Phe Glu Val Pro Glu Asn Arg

370 375 380

Lys Thr Arg Thr Glu Asn Pro Thr Ser Val Pro Gln Cys Leu Thr Met

385 390 395 400

Ala Pro Glu His Pro Pro Val Trp Thr Gly Leu Tyr Pro Gly Phe Phe

405 410 415

Pro Asn Phe Glu Gly Arg Val Ile Glu Lys Arg Val Glu Val Gly Ser

420 425 430

Pro Ile Phe Ser Asn Trp Ala Trp Gln Gln His Gly Ser Thr Pro Ser

435 440 445

Met Pro Leu Phe Ser Ser Ala Ala Ser Ser Gly Phe Ser Thr Ala Thr

450 455 460

Thr Thr Thr Thr Thr Ala Ala Leu Pro Pro Pro Pro Pro Pro Thr Thr

465 470 475 480

Asn His His Phe Pro Pro His Tyr Tyr Tyr Arg Ser

485 490

Claims (7)

1. Hybrid liriodendronLhAP2LThe nucleotide sequence of the gene is shown in SEQ ID NO. 1.

2. The hybrid tulip tree of claim 1LhAP2LThe amino acid sequence of the gene expression protein is shown in SEQ ID NO. 2.

3. Comprises the hybrid tulip tree of claim 1LhAP2LA vector or recombinant bacterium of the gene.

4. The method of claim 3, comprising hybridizing trembling poplarLhAP2LThe gene vector is characterized in that the vector is a plant recombinant expression vector.

5. The hybrid tulip tree of claim 1LhAP2LThe application of the gene in changing the development form of flower organs, promoting the vegetative growth of plants or inhibiting the reproductive growth.

6. Use according to claim 5, characterized in that it comprises the following steps:

1) construction of hybrid LiriodendronLhAP2LA vector for the gene;

2) the constructed hybrid Chinese tulip tree is usedLhAP2LTransforming a vector of the gene into a plant or plant cell;

3) and culturing and screening to obtain the plants with the changed development forms of the flower organs, the enhanced vegetative growth or the inhibited reproductive growth.

7. Use according to claim 5 or 6, wherein the plant is tobacco.

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