CN114958869B

CN114958869B - Hybrid tulip tree meristem growth key gene LhWOX4 and application thereof

Info

Publication number: CN114958869B
Application number: CN202210565985.7A
Authority: CN
Inventors: 郝兆东; 邱晨; 刘思芹; 鲁路; 施季森; 陈金慧; 黄世清; 陈英
Original assignee: Nanjing Forestry University
Current assignee: Nanjing Forestry University
Priority date: 2022-05-23
Filing date: 2022-05-23
Publication date: 2023-06-02
Anticipated expiration: 2042-05-23
Also published as: CN114958869A

Abstract

The application discloses a hybrid tulip tree meristem growth key gene LhWOX4 and application thereof. The nucleotide sequence of the key gene LhWOX4 for the growth of the meristem of the hybrid tulip tree is shown as SEQ ID NO.1, and the amino acid sequence of the expression protein is shown as SEQ ID NO. 2. The LhWOX4 gene is extracted from the hybrid tulip tree, the gene is constructed to an over-expression vector and introduced into arabidopsis thaliana to study the functions of the gene, and the result shows that the arabidopsis thaliana plant over-expressing the hybrid tulip tree LhWOX4 gene has the phenotypes of vigorous plant top meristem division, slow root system growth and the like, so that the gene can be widely applied to the production and breeding of tulip tree and other plants.

Description

Hybrid tulip tree meristem growth key gene LhWOX4 and application thereof

Technical Field

The invention belongs to the technical field of plant genetic engineering, and particularly relates to a key gene LhWOX4 for growth of a hybrid tulip tree meristem and application thereof.

Background

Liriodendron is old and wiggles of Magnoliaceae (Magnoliaceae), 23 species are original, most species are extinct in the fourth glacier after being subjected to glacier for several times, and only two wild species exist in the world, one species is Liriodendron (Liriodendron chinense (hemsl.) Sargent.) distributed in Asia, and is mainly located in the middle and southern mountain areas of China and distributed in star dispersion; the other is liriodendron tulipifera (Liriodendron tulipifera linn.) distributed in north america, located in the middle-west of the united states to the southeast of canada. Both the tulip tree and the liriodendron tulipifera are excellent precious broad-leaved wood species, the trunk is round and straight, the wood structure is fine, the quality is light and soft, the strength is weak, the drying is easy, the deformation is avoided, and the tulip tree is a high-quality raw material for industries of buildings, furniture and paper pulp. The flower is bright and the leaf shape is peculiar, so that the flower is one of the preferred tree species of the forestation tree species. The introduction of a small amount of liriodendron tulipifera in China in the 30 th century of 20, and the introduction of resources after the 90 th year are relatively systematic, thus providing possibility for the subsequent development of cross breeding work.

In the sixties of the twentieth century, a famous forest breeder She Peizhong in China taught to hybridize tulip trees and Liriodendron tulip trees, and bred new species hybrid tulip trees (lirioodendron×sinoamericanum p.c.yieh ex c.b.shang & Zhang r.wang) with characters superior to the parents. Compared with tulip trees and liriodendron tulipifera, the hybrid tulip trees have obvious growth advantages, and the growth quantity and the adaptability of the hybrid tulip trees are obviously better than those of parent tree species. The introduction and cultivation test of the hybrid tulip tree shows that: the hybridized tulip tree shows very strong heterosis, and the height, diameter and individual standing tree volume of the hybridized tulip tree are obviously higher than those of a control seed. The hybridized tulip tree has the characteristics of fast growth, straight trunk, beautiful and leaf shape, big and beautiful flowers, light yellow and yellow color, less plant diseases and insect pests, strong adaptability and the like, and the survival rate can reach 85 to 90 percent by adopting grafting propagation, thereby being an excellent greening afforestation tree species. The goose palm is a hermaphrodite homoflower plant, and as the pistil is mature before the petals are unfolded, the pistil is not mature at the moment, the male and female flowers are not in the same period, the male and female gamete abortion phenomenon is common, so that the germination rate of seeds is low, the reproduction of the tulip tree is seriously influenced, and the tulip tree is classified as a national second-level important rare endangered protection plant. At present, the hybridized tulip tree becomes an important tree species for use in the southern area of China.

Several transcription factors (Transcription factor, TF) have been shown to play a critical role in regulating SAM and RAM maintenance, for example single mutations in TF encoding genes, such as WUSCHEL (WUS) or SHOOT MERISTEMLESS (STM) in SAM and SHORT-ROOT (SHR) or SCARECROW (SCR) in RAM, can lead to termination of apical growth. Several transcription factors required for cambium meristem activity have been found, including the WUSCHEL-RELATED homologous genes WUSCHEL-RELATED HOMEOBOX4 (WOX 4), WUSCHEL-RELATED HOMEOBOX 14 (WOX 14), AINTEGUMENTA (ANT), ETHYLENE RESPONSE FACTOR 018 (ERF 018) and ETHYLENE RESPONSE FACTOR (ERF 109), and the auxin response factor (AUXINRESPONSEFACTORS, ARF). Members of the WOX gene family are expressed almost in all organs of plants, with the main site of expression being in the cell population where division of each organ is vigorous. The family of genes play an important regulatory role in the process of critical developmental stages of plants such as embryo formation, stem cell stability and organ formation. These functions are indispensible from their ability to promote cell division or prevent premature differentiation of immature cells.

Meristem is the stem cell tissue of a plant and is critical to the growth and development of the plant. The vascular cambium is a secondary meristem, originating from primary meristems, stem Apical Meristems (SAM) and Root Apical Meristems (RAM), and is critical for the production of wood biomass. The developmental process is regulated by a number of genetic and other endogenous factors, of which transcription factors play an extremely critical regulatory role. Although some transcription factors have been shown to regulate cambium activity, the phenotype of the corresponding loss-of-function mutants is not obvious, which limits our understanding of vascular tissue-related transcriptional regulatory mechanisms, and thus transcriptional regulatory mechanisms for cambium activity have yet to be studied intensively.

The WOX4 transcription factor has important regulation function in the growth and development process of plants, mainly participates in the development of stem, root, leaf and apical meristem, and also has certain function in embryo development and corresponding drought stress. In arabidopsis, expression patterns of AtWOX4 in inflorescences, leaves, stems and roots were observed by tissue section and confocal imaging. The regulatory network, TDIF/CLE41/CLE44-TDR/PXY-WOX4, is present in Arabidopsis to maintain cambium activity. WOX4 regulates cambium cell activity by mediating ligand-receptor signaling pathways. In WOX4 mutants, cambium activity was inhibited. In populus trichocarpa, ptrWOX4 gene is a key gene for regulating and controlling the development of vascular cambium, and is involved in regulating and controlling the development of apical meristem. In phyllostachys pubescens, pheWOX4c is actively expressed in phyllostachys pubescens stem apical tissues (including shoot apical tissues and whip apical tissues), possibly involved in the development of stem apical meristems. WOX4 also plays an important role in root development. In blueberries, vcWOX4b inhibits adventitious root formation by reducing IAA/Cks and IAA/ABA values, so that the xylem area of the root system is enlarged, and the number of layers of a cambium is increased. In walnut, the overexpression of JrWOX4 leads to shortening and thickening of adventitious roots and increase of adventitious roots.

Disclosure of Invention

Aiming at the defects in the prior art, the invention aims to provide the key gene LhWOX4 for the growth of the meristem of the hybrid tulip tree. The invention aims to provide a new application of the hybrid tulip tree meristem growth key gene LhWOX4.

In order to solve the technical problems, the invention adopts the following technical scheme:

the nucleotide sequence of the gene LhWOX4 which is a key gene for the growth of the meristem of the hybrid tulip tree is shown as SEQ ID NO. 1.

The amino acid sequence of the expression protein of the WOX4 gene of the key growth gene of the tulip tree meristem is shown as SEQ ID NO. 2.

The application of the hybrid tulip tree meristem growth key gene WOX4 gene in promoting the division of the plant top meristem.

The application of the gene can over-express the LhWOX4 gene of the hybridized tulip tree in plants, and promote the meristem at the top of the transgenic plants to divide vigorously.

The application comprises the following steps:

1) Constructing a carrier of a hybridized tulip tree LhWOX4 gene;

2) Transforming the constructed carrier of the hybridized tulip tree LhWOX4 gene into plant cells;

3) And (3) cultivating the transgenic plant to promote the meristem at the top of the plant to divide vigorously.

The carrier of the LhWOX4 gene of the hybridized tulip tree in the step 1) is LhWOX4+pBI121.

The transformation described in step 2) is performed using Agrobacterium tumefaciens GV 3101.

The application of the hybrid tulip tree meristem growth key gene WOX4 gene in constructing a plant root growth retardation model.

The application of the WOX4 gene which is a key gene for the growth of the meristem of the hybrid tulip tree in the plant growth delay is provided.

The beneficial effects are that: compared with the prior art, the LhWOX4 gene is extracted from the hybrid tulip tree, and is constructed into an over-expression vector to be introduced into arabidopsis thaliana for researching the functions, and the result shows that the arabidopsis thaliana plant over-expressing the hybrid tulip tree LhWOX4 gene has the phenotypes of vigorous plant top meristem division, slow root system growth and the like, so that the gene can be widely applied to the production and breeding of tulip tree and other plants.

Description of the drawings:

FIG. 1 is a 1% agarose gel electrophoresis chart of a full-length PCR product of a hybridized tulip tree LhWOX4 gene; in the figure, a, b: the full length of LhWOX4 gene obtained by taking cDNA as a template through PCR; m:2000bp Marker;

FIG. 2 is a 1% agarose gel electrophoresis of single colony PCR of pBI121-LhWOX4 Agrobacterium; in the figures, a-f: transferring LhWOX4 gene agrobacterium; g: ddH ₂ O；M：2000bp Marker；

FIG. 3 is a diagram of a constructed hybrid tulip tree LhWOX4 overexpression vector;

FIG. 4 is a graph of PCR detection results of T1 generation positive plants of transgenic Arabidopsis thaliana over-expressing LhWOX 4; in the figures, a-h: transferring LhWOX4 gene arabidopsis plant DNA; mock: transferring the empty Arabidopsis plant DNA of pBI 121; ck-: wild type arabidopsis plant DNA; ck+: plasmid DNA; h ₂ O：ddH ₂ O；M：2000bp Marker；

FIG. 5 is a control phenotype map of T1 generation plants overexpressing LhWOX4 transgenic Arabidopsis with WT and MOCK;

FIG. 6 is a phenotype map of T1 generation plants of transgenic Arabidopsis that overexpress LhWOX4.

Detailed Description

The invention will be further illustrated with reference to specific examples.

Example 1

The hybridized tulip tree plant is used as material to extract total RNA, reverse transcribed into cDNA, designed corresponding primer for PCR, agarose gel electrophoresis detection, recovered target band, connected with pClone007 carrier, transferred into colibacillus, sequenced and analyzed. Selecting positive clones for plasmid extraction, transferring the extracted plasmids into an agrobacterium strain GV3101 by a heat shock method, after the arabidopsis is suitable for age, transforming the arabidopsis inflorescence by an agrobacterium-mediated method to obtain transgenic arabidopsis, observing the difference between the transgenic positive plants and wild arabidopsis on phenotypes such as leaves, stems, flowers and the like, and carrying out statistics.

1. Extraction of Total RNA

Taking the stem tip of the hybridized tulip tree as a material, extracting RNA according to the operation steps of a FastPure Plant Total RNA Isolation Kit kit (Vazyme), and carrying out 1% agarose gel electrophoresis on the total RNA of the stem tip of the hybridized tulip tree to obtain complete and clear bands; the absorbance of total RNA is measured by ultraviolet spectrophotometry, and the result shows that the RNA has better integrity and high purity, and can be used for reverse transcription.

2. cDNA acquisition

cDNA was obtained by reverse transcription using the extracted RNA as a template, using Vazyme Corp

III 1st Strand cDNA Synthesis Kit (+gDNA wind). The maximum amount of RNA used in the experiment is 5 mug according to the instruction, and the specific steps are carried out according to the instruction of a kit and mainly comprise: RNA template denaturation, genomic DNA removal, preparation of first strand cDNA synthesis reaction solution, and first strand cDNA synthesis reaction (37 ℃ C. 45min,85 ℃ C. 5sec,4 ℃ C. Infinity), and the product can be immediately used for PCR reaction or stored at-20 ℃.

3. Homologous cloning to obtain target gene

And designing a primer by utilizing oligo7.0, performing PCR (polymerase chain reaction) to obtain a target gene, performing transformation sequencing, and finally performing comparison analysis. The primer sequences were as follows:

LhWOX4-F：5’-ATGAAGGTGCACCAGCTCACTCGTGGC-3’

LhWOX4-R：5’-TCATCTGCCTTCCGGGTGTAAAGGGAACAG-3’

1) PCR amplification system: using Vazyme Corp

Max Super-Fidelity DNA Polymerase reagent, preparing PCR reaction liquid according to the specification, 50uL reaction system: ddH ₂ O18 uL,2 XPhantaMaxBuffera 25uL, dNTPMmix (10 mMeach) luL, upstream primer (10. Mu.M) 2uL, downstream primer (10. Mu.M) 2uL,cDNA 1uL,PhantaMaxSuper-FidelityDNAPolyase 1uL.

2) PCR reaction conditions: 3min at 95 ℃;95℃15sec,61℃15sec,72℃30sec,35cycles;72 ℃ for 5min;4 ℃ is infinity.

The PCR product was detected by electrophoresis on a 1% agarose gel (FIG. 1) to obtain the desired fragment, and the desired fragment was recovered and purified using a DNA gel recovery kit from TSINGKE, as follows:

1) In the adsorption column EC, 250ul Buffer BL,12000g is added and centrifuged for 1min, and the silica gel film is activated. 2) Under 365nm long wave ultraviolet lamp, cutting the DNA band to be recovered with clean blade, cutting the gel without DNA as far as possible to obtain gel with smaller gel volume, and placing the gel with target DNA band into 2mL centrifuge tube. 3) 500uL Buffer GL was added. 4) Air bath at 65deg.C for 6min, mixing every 2min, and reversing until gel is completely melted, and making the solution light yellow (if gel block volume is large, adding Buffer GL to the solution to make the solution light yellow). 5) Transferring the solution into an adsorption column EC, centrifuging for 1min at 12000g, discarding the waste liquid, and placing the adsorption column EC back into an empty collecting tube. 6) Adding 700ul Buffer W2 and 12000g into an adsorption column EC, centrifuging for 1min, and discarding the waste liquid; repeating once. 7) The adsorption column EC was put back into the empty collection tube, and 12000g was centrifuged for 2min. 8) Taking out the adsorption column EC, putting into a clean 1.5mL centrifuge tube, uncovering and standing for 2min at 20-25 ℃, adding 40ul of Eluent preheated at 65 ℃ into the central part of the adsorption column membrane, standing for 2min at 20-25 ℃, centrifuging for 2min at 12000g, and eluting once again to improve the yield.

The target gene fragment was ligated with the cloning Vector pClone007Blunt Vector Kit (TSINGKE TSV-007B). 10uL reverse reactionThe reaction system is as follows: PCR purification of product 2uL,pClone007BluntVector 1uL,10 ×TopoMix lu, ddH ₂ O6 uL. After all the solutions are directly added into the bottom of the tube, the solution is sucked and beaten by a pipette and uniformly mixed, and the reaction is completed after the reaction is carried out for 5min at room temperature (22-30 ℃).

Transfer of ligation products into Trelief in E.coli strains ^TM 5α Chemically Competent Cell (TSINGKE TSC). The method comprises the following specific steps: (1) Taking out 100u1 competent cells from the ultralow temperature refrigerator at-80 ℃, placing the competent cells on ice for melting, adding 10ul of a connecting product, gently mixing, and standing on the ice for 5min; (2) Heat shock is conducted in a water bath at the temperature of 42 ℃ for 45s, the mixture is quickly transferred into an ice bath, and the mixture is stood for 2min; (3) Adding 900ul of liquid culture medium without antibiotics, and resuscitating for 1h at 37 ℃ by a shaking table; (4) And (3) directly coating a proper volume of bacterial liquid on an LB solid medium containing Amp, and culturing the bacterial liquid in an inverted incubator at 37 ℃ overnight.

And (3) selecting independent colonies for PCR verification, detecting the recombinant plasmid, and sequencing and analyzing the obtained positive clones. As a result, the length of the coding region of the WOX4 gene is 726bp, the coding region comprises a complete Open Reading Frame (ORF), the sequence is shown as SEQ ID NO.1, and the coded protein sequence is shown as SEQ ID NO.2 and comprises 242 amino acids.

Example 2

1. Gene functional analysis

First, a hybrid tulip tree 35S is constructed: the LhWOX4 over-expression vector is transferred into an agrobacterium strain, a wild type arabidopsis inflorescence Columbia Col (Columbia) is transformed by an agrobacterium-mediated method, a positive transgenic plant over-expressing the LhWOX4 gene is obtained, the phenotype difference of the transgenic positive plant and the phenotype of the wild type plant is observed, and the function of the hybridized tulip tree LhWOX4 is researched and analyzed.

2. Construction of vectors

The E.coli strain used in this example was DH 5. Alpha (Tiangen CB 101-02); the expression vector was pBI121.

The specific process is as follows:

1) The XbaI and SacI double enzyme cutting sites are respectively added on the upstream and downstream of the LhWOX4 gene fragment by PCR, the PCR system and the reaction conditions are used for isogenic full-length amplification, and the primers are respectively as follows:

LhWOX4+XbaI-F：5′-agagaacacgggggactctagaATGAAGGTGCACCAGC-3′；

LhWOX4+SacI-R：5′-gatcggggaaattcgagctctcaTCTGCCTTCCGGGT-3′；

2) And (3) carrying out double enzyme digestion reaction by using corresponding endonuclease after the recombinant plasmid is sequenced correctly. Double cleavage with XbaI and SacI restriction enzymes gave a LhWOX4 gene fragment containing a cohesive end and covering the entire ORF. The empty pBI121 expression vector was treated with the same cleavage reaction.

The 20uL double cleavage reaction system is as follows: recovery of product 1ug,10 XMbus buffer 2uL,XbaI 1uL,SacI 1uL,ddH ₂ O up to20uL。

And (3) enzyme cutting: heat inactivation method. PCR reaction conditions: 30min at 37 ℃; 30min at 65 ℃.

And separating and detecting the double enzyme digestion products by 1% agarose gel electrophoresis, and judging that the LhWOX4 gene and the pBI121 expression vector are correctly cut according to the size of the band.

The target gene fragment, the double enzyme-digested product of the empty expression vector PBI121, was recovered and purified by using a DNA gel recovery kit (TSINGKE GE 0101), and dissolved in 40. Mu.L of ddH ₂ O.

3) And detecting the concentration and purity of the recovered enzyme-cleaved product, and adding each reagent according to a connection system. Homologous recombination ligation Using Vazyme Co

II One Step Cloning Kit C112。/>

The amount of the optimal cloning vector used in the recombination reaction system was 0.03pmol, and the amount of the optimal insert was 0.06pmol (molar ratio of vector to insert was 1:2).

The 20uL ligation reaction system was: 5 XCE II Buffer 4ul, PBI121 1ul, target fragment 4ul,Exnase II 2ul,ddH ₂ O 9ul。

PCR reaction conditions: 30min at 37 ℃;4 ℃ is infinity.

4) E.coli DH5a competent cells (Tiangen CB 101-02) were transformed with the ligation product, single colonies were picked up and inoculated into LB liquid medium, shake cultured at 37℃and bacterial liquid PCR was performed using full length primers to screen positive clones and sequence, and plasmids were extracted with plasmid miniprep kit (Tiangen DP 103-03) with correct sequencing. The construction of the expression vector is shown in FIG. 3, and includes the length and position of the promoter, the target gene and the terminator.

3. Transformation of Agrobacterium GV3101

The agrobacterium strain used in this example was GV3101, and the constructed Lh WOX4 overexpression vector was transferred into agrobacterium by liquid nitrogen freeze-thawing. The specific process is as follows: 1) The agrobacteria competent cells were thawed in ice bath, 1-10uL (100 ng) of the purified recombinant plasmid was recovered per tube, gently blotted and mixed, placed on ice, and left to stand in ice bath for 30min. 2) Quick freezing with liquid nitrogen for 1min, heat-shock at 37deg.C for 1-5min, and rapidly placing on ice for 1-2min. 3) 700ul of LB liquid medium without antibiotics is added, and the culture is carried out at 28 ℃ and 220rpm under slow shaking for 2-4 hours. 4) Centrifuge at 6000rpm for 1min and aspirate part of the supernatant. 5) A proper amount of bacterial liquid is left, mixed gently and spread on a solid LB medium containing 50mg/L kanamycin. 6) Culturing at 28deg.C for 30-48 hr to obtain single colony. 7) The bacterial liquid PCR detects positive clone and is preserved at 4 ℃ for standby.

4. Transformation of Arabidopsis thaliana

The arabidopsis to be planted grows to bloom, and the healthy state of the arabidopsis is maintained. Positive clones detected by PCR were shaken to OD ₆₀₀ 0.6-0.8, and transferring the target gene into wild Arabidopsis thaliana by an inflorescence soaking method. The specific process is as follows: 1) Centrifuging the bacterial liquid at 5000rpm for 5min, collecting bacterial cells, and suspending with 1/2MS solution containing 5% sucrose; 2) Before soaking, adding Silwet L-77 with concentration of 0.05%, shaking out foam; 3) Soaking the aerial parts of Arabidopsis thaliana in an agrobacterium suspension solution for 15-30 sec, and slightly shaking during the soaking; 4) Placing the soaked arabidopsis thaliana on a tray horizontally, covering the tray with a preservative film for moisturizing, and sealing the tray with tinfoil paper for 24 hours in a dark place; 5) And taking down the preservative film, culturing under normal conditions until the seeds are mature, and stopping watering after the seeds are mature.

5. Phenotype observation of transgenic plants

1) The dried seeds were harvested and T1 generation seeds were screened with 50mg/L of 1/2MS medium of calicheamicin and positive plants were found to still grow normally while other negative, non-calicheamicin resistant plants had died.

2) Transplanting the screened possible transgenic positive plants into nutrient soil, and culturing normally. And selecting transgenic positive plant leaves, extracting DNA as a template, and carrying out PCR detection to determine the positive plants.

The PCR detection primers were as follows:

35S seq-F：5′-CTTCGTCAACATGGTGGAG-3′；

PBI121WOX4-R：5′-ATCTGTTGCTGTCCTCTTCC-3′；

the detection result is shown in FIG. 4, which shows the negative control ddH ₂ O and wild type are free of bands, and the PCR target bands of the transgenic plants are consistent with positive control plasmids, so that positive plants are determined.

3) Phenotypic observation of T1 transgenic plants overexpressing LhWOX4 gene. Transgenic plants in the background of the wild Col show slow growth compared with the wild Col control, vigorous division of meristems at the top of the plants and slow growth of root systems. The results are shown in FIGS. 5 and 6.

4) Phenotype statistics of T1 generation transgenic plants overexpressing LhWOX4 gene. At 20 days of growth, 15 wild-type Col (WT), empty transgenic (MOCK) and LhWOX4 overexpressing transgenic (LhWOX 4) plants were taken, respectively, and their surface areas were calculated and compared. The average values of the measurements are shown in Table 1.

TABLE 1 surface area results

	WT	MOCK	LhWOX4
				Area average value/mm ²	39.88	24.02	12.27

Sequence listing

<110> university of Nanjing forestry

<120> hybrid tulip tree meristem growth key gene LhWOX4 and application thereof

<130> 100

<160> 8

<170> SIPOSequenceListing 1.0

<210> 1

<211> 726

<212> DNA

<213> Liriodendron × sinoamericanum P.C. Yieh ex C.B. Shang & Zhang R. Wang

<400> 1

atgaaggtgc accagctcac tcgtggcttt tgggagcatg agccctcttc tctcagctgc 60

aagcgcctcc gcccacttgc acccaagctc tccagcggtg tcggtgacgg tgtcacccct 120

atagatttga agagtttcat caagcctgag agtggcccta ggagtgttgc atccatggat 180

gacaagcgag atacaactca ggcagaagcg cactctggag gcacacggtg gaacccgacc 240

caagaacaga ttggaatcct ggagatgctc tataggggtg gcatgagaac gccaaacgca 300

caacagattg agcaaatcac ggcccagctt gggaagttcg ggaagataga agggaagaat 360

gtgttttact ggttccagaa tcacaaagca cgggaacggc agaagcaaag gcgtaattca 420

ctcggcctcg gcaattctcc aagagcccca gccattgcca tttccagcag taactctaac 480

gtaggatgcg atgaggcccc accggatgat atatcaagtg ggatctccac aatgggacca 540

gccatgggag aagtgggaaa ggatgatgat agttactgta agcgcaaatg caggagttgg 600

ggcgtttata gcatggaaga ggacagcaac agattgtgtg gtggtttcac attccaagaa 660

gaagaagaag aagaagaggg tgatagaact ctaatgctgt tccctttaca cccggaaggc 720

agatga 726

<210> 2

<211> 241

<212> PRT

<213> Liriodendron × sinoamericanum P.C. Yieh ex C.B. Shang & Zhang R. Wang

<400> 2

Met Lys Val His Gln Leu Thr Arg Gly Phe Trp Glu His Glu Pro Ser

1 5 10 15

Ser Leu Ser Cys Lys Arg Leu Arg Pro Leu Ala Pro Lys Leu Ser Ser

20 25 30

Gly Val Gly Asp Gly Val Thr Pro Ile Asp Leu Lys Ser Phe Ile Lys

35 40 45

Pro Glu Ser Gly Pro Arg Ser Val Ala Ser Met Asp Asp Lys Arg Asp

50 55 60

Thr Thr Gln Ala Glu Ala His Ser Gly Gly Thr Arg Trp Asn Pro Thr

65 70 75 80

Gln Glu Gln Ile Gly Ile Leu Glu Met Leu Tyr Arg Gly Gly Met Arg

85 90 95

Thr Pro Asn Ala Gln Gln Ile Glu Gln Ile Thr Ala Gln Leu Gly Lys

100 105 110

Phe Gly Lys Ile Glu Gly Lys Asn Val Phe Tyr Trp Phe Gln Asn His

115 120 125

Lys Ala Arg Glu Arg Gln Lys Gln Arg Arg Asn Ser Leu Gly Leu Gly

130 135 140

Asn Ser Pro Arg Ala Pro Ala Ile Ala Ile Ser Ser Ser Asn Ser Asn

145 150 155 160

Val Gly Cys Asp Glu Ala Pro Pro Asp Asp Ile Ser Ser Gly Ile Ser

165 170 175

Thr Met Gly Pro Ala Met Gly Glu Val Gly Lys Asp Asp Asp Ser Tyr

180 185 190

Cys Lys Arg Lys Cys Arg Ser Trp Gly Val Tyr Ser Met Glu Glu Asp

195 200 205

Ser Asn Arg Leu Cys Gly Gly Phe Thr Phe Gln Glu Glu Glu Glu Glu

210 215 220

Glu Glu Gly Asp Arg Thr Leu Met Leu Phe Pro Leu His Pro Glu Gly

225 230 235 240

Arg

<210> 3

<211> 27

<212> DNA

<213> LhWOX4-F sequence (Artificial Sequence)

<400> 3

atgaaggtgc accagctcac tcgtggc 27

<210> 4

<211> 30

<212> DNA

<213> LhWOX4-R sequence (Artificial Sequence)

<400> 4

tcatctgcct tccgggtgta aagggaacag 30

<210> 5

<211> 38

<212> DNA

<213> LhWOX4+XbaI-F sequence (Artificial Sequence)

<400> 5

agagaacacg ggggactcta gaatgaaggt gcaccagc 38

<210> 6

<211> 37

<212> DNA

<213> LhWOX4+SacI-R sequence (Artificial Sequence)

<400> 6

gatcggggaa attcgagctc tcatctgcct tccgggt 37

<210> 7

<211> 19

<212> DNA

<213> 35S seq-F sequence (Artificial Sequence)

<400> 7

cttcgtcaac atggtggag 19

<210> 8

<211> 20

<212> DNA

<213> PBI121 WOX4-R sequence (Artificial Sequence)

<400> 8

atctgttgct gtcctcttcc 20

Claims

1. The nucleotide sequence of the gene LhWOX4 which is a key gene for the growth of the meristem of the hybrid tulip tree is shown as SEQ ID NO. 1.

2. The expression protein of the gene LhWOX4 which is a key gene for the growth of the meristem of the hybrid tulip tree according to claim 1, wherein the amino acid sequence of the expression protein is shown as SEQ ID NO. 2.

3. The use of the gene LhWOX4, a key gene for growth of the meristem of the hybrid tulip tree according to claim 1, for promoting the division of the apical meristem of arabidopsis thaliana; the application is that the LhWOX4 gene of the hybrid tulip tree is overexpressed in the arabidopsis thaliana, so that the meristem at the top of the transgenic arabidopsis thaliana is promoted to divide vigorously.

4. The use according to claim 3, characterized by the following steps:

1) Constructing a carrier of a hybridized tulip tree LhWOX4 gene;

2) Transforming the constructed carrier of the hybridized tulip tree LhWOX4 gene into an Arabidopsis cell;

3) The transgenic arabidopsis thaliana is cultivated, and the apical meristem of the arabidopsis thaliana is promoted to divide vigorously.

5. The use according to claim 4, wherein the vector of the LhWOX4 gene of the hybrid tulip tree in step 1) is LhWOX4+pBI121.

6. The use according to claim 4, wherein the transformation in step 2) is performed using Agrobacterium tumefaciens GV 3101.

7. The application of the hybridized tulip tree meristem growth key gene LhWOX4 gene in the construction of an arabidopsis root system growth retardation model according to claim 1; the application is that the LhWOX4 gene of the hybrid tulip tree is overexpressed in the arabidopsis thaliana, so that the growth retardation of the root system of the transgenic arabidopsis thaliana is promoted.

8. The application of the hybrid tulip tree meristem growth key gene LhWOX4 gene in delaying the growth of arabidopsis thaliana according to claim 1; the application is that the LhWOX4 gene of the hybrid tulip tree is overexpressed in the arabidopsis thaliana, so that the transgenic arabidopsis thaliana is promoted to delay growth.