CN112746079B - Liriodendron transcription factor LcbHLH52 gene and application thereof - Google Patents

Abstract

The invention discloses a transcription factor LcbHLH52 gene of liriodendron tulipifera and application thereof, belonging to the technical field of plant genetic engineering. The invention analyzes the sequence of the liriodendron bHLH family through a bioinformatics tool, and successfully clones the LcbHLH52 transcription factor gene through sequencing and homologous comparison. Constructing an overexpression vector for the cloned LcbHLH52 transcription factor gene, and transforming Arabidopsis thaliana to obtain seeds of the T1 generation; the LcbHLH52 transgenic Arabidopsis T2 generation plant and the wild Arabidopsis plant are simultaneously put into 4 ℃ low temperature treatment for 3d, the phenotype observation shows that the transgenic plant is less affected by low temperature stress, the wild plant is inhibited from growing, and leaves have wilting phenomenon. The result shows that the liriodendron LcbHLH52 gene can enhance the tolerance capability of arabidopsis plants to low-temperature stress, and has important application value in improving the molecular breeding of the low-temperature stress of plants.

Description

Liriodendron transcription factor LcbHLH52 gene and application thereof

Technical Field

The invention belongs to the technical field of plant genetic engineering, and particularly relates to a transcription factor LcbHLH52 gene of liriodendron tulipifera and application thereof.

Background

Chinese tulip tree (Liriodendron Chinese) as an important rare tree species (national secondary protective plant) in China has values in various aspects such as scientific research, economy, appreciation, medicine, ecology and the like. The main body is as follows: 1. the method has important scientific research value for the research of the ancient botany; 2. straight trunk end, umbrella-shaped crown, peculiar leaf shape and large and beautiful flower, and is an excellent ornamental tree species; 3. the wood has straight texture, less cracks when being dried, moderate light and soft, easy processing, less deformation and no worm damage, and is a high-quality material for indoor decoration and furniture manufacture (Liujian Ping, 2020). Chinese tulip trees are mainly distributed in the south of Yangtze river basin in China, are warm and moist (Fangyanming, 1994; Hechiming et al, 1995), and belong to cold-sensitive plants. Therefore, the low temperature limits the large-area popularization of the Chinese tulip tree in northern areas of China. Therefore, the cultivation of low temperature resistant varieties has important practical and theoretical significance (Zhang Xin, 2010). The rapid development of current genome sequencing technologies has driven the deciphering of more and more plant genomes. The release of the liriodendron genome lays a foundation for analyzing the basic biological problems and molecular mechanisms of important traits of the liriodendron genome, and provides possibility for analyzing and mining genes for controlling the important traits of the liriodendron (Chen et al, 2019).

Due to the relatively conserved and complex and diversified structural domain characteristics of sequences of plant bHLH transcription factors, different members of the plant bHLH transcription factor family are endowed with similar and different biological functions. The interpretation of the tulip tree genome provides a premise for performing genome-wide analysis of the bHLH transcription factor family (Chen et al, 2019). Therefore, the clone and function analysis of the transcription factor of the liriodendron bHLH can help to analyze the biological action and breed stress-resistant germplasm.

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 transcription factor LcbHLH52 gene of Liriodendron tulipifera. Another technical problem to be solved by the present invention is to provide the application of the transcription factor LcbHLH52 gene of Liriodendron tulipifera.

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

a transcription factor LcbHLH52 gene of Liriodendron tulipifera has a nucleotide sequence shown in SEQ ID NO. 1.

The amino acid sequence of the expression protein of the transcription factor LcbHLH52 of the liriodendron tulipifera is shown in SEQ ID NO. 2.

A carrier containing the transcription factor LcbHLH52 gene of the liriodendron.

The application of the transcription factor LcbHLH52 gene of the liriodendron tulipifera in enhancing the tolerance of plants to low-temperature stress.

Further, the application comprises the following steps:

1) constructing a vector of the transcription factor LcbHLH52 gene of the liriodendron;

2) transforming the constructed vector of the transcription factor LcbHLH52 gene into a plant or a plant cell;

3) and culturing and screening to obtain the plant with enhanced low-temperature stress tolerance.

Further, in the application, the plant is arabidopsis thaliana.

Compared with the prior art, the invention has the beneficial effects that:

the invention analyzes the sequence of the liriodendron bHLH family through a bioinformatics tool, and selects LcbHLH52 transcription factor genes from the liriodendron bHLH gene family members for cloning. By sequencing and homology comparison, 52 genes of LcbHLH were successfully cloned. Constructing an overexpression vector for the cloned LcbHLH52 gene, transforming Arabidopsis thaliana, and obtaining a T1 generation seed from the gene; the LcbHLH52 transgenic Arabidopsis T2 generation plant and the wild Arabidopsis plant are simultaneously put into 4 ℃ low temperature treatment for 3d, the phenotype observation shows that the transgenic plant is less affected by low temperature stress, the wild plant is inhibited from growing, and leaves have wilting phenomenon. The results show that the liriodendron LcbHLH52 gene can enhance the tolerance of arabidopsis plants to low-temperature stress.

Drawings

FIG. 1 is a diagram of PCR amplification products of LcbHLH52 gene, in which: m: marker DL 2000; 1-4: PCR products, wherein 18250 refers to LcbHLH52 gene;

FIG. 2 is a graph showing the PCR electrophoresis results of Liriodendron LcbHLH52 bacterial liquid, wherein: m: marker DL 2000; 1-8: PCR products of bacterial liquid;

FIG. 3 is a map of the pCambia2301-ky plasmid;

FIG. 4 is a diagram of the growth of transgenic Arabidopsis and wild type plants;

FIG. 5 is a phenotypic graph of wild type Arabidopsis thaliana and LcbHLH52 transgenic Arabidopsis thaliana under cold stress treatment.

Detailed Description

The invention is further described with reference to specific examples. The experimental procedures in the following examples are conventional unless otherwise specified. The test materials used in the following examples were purchased from a conventional biochemical reagent store unless otherwise specified.

Plant material: the plant material, Liriodendron tulipifera, is obtained from university campus of Nanjing forestry, young and fresh leaf of Liriodendron tulipifera is taken in 2018 and stored at-80 deg.C after frozen in liquid nitrogen for use.

Arabidopsis thaliana used for genetic transformation is the wild type Col (Arabidopsis thaliana).

Strains and carriers: coli (Escherichia coli, e. coli) DH5 α competent cells, agrobacterium EHA105 competent cells, pMD-19t (simple) cloning vector, purchased from dalibao bioengineering, ltd; the Pcambia2301-ky eukaryotic expression vector is stored in the laboratory.

The main reagents are as follows: the main reagent is total RNA extraction reagent TRIZOL which is purchased from Tiangen Biochemical technology limited company; the reverse transcription kit, the high fidelity enzyme and the 2 XTaq Master Mix enzyme are all purchased from Vazyme company; the gel recovery kit was purchased from OMEGA; t4 ligase was purchased from Thermo Fisher Scientific; other reagents were analytical grade reagents.

Culture medium: LB solid liquid culture medium, LB/Amp/X-Gal/IPTG plate culture medium, Amp screening culture medium, Kan screening culture medium, 1/2MS solid culture medium, etc., the specific preparation method refers to Sujiang (2015).

Instruments and equipment: a high-temperature high-pressure sterilization pot, a common PCR amplification instrument, a gel electrophoresis imaging instrument, a vortex oscillation instrument, a high-speed low-temperature centrifuge, a 4 ℃ refrigerator, a minus 80 ℃ ultra-low temperature refrigerator, a constant-temperature incubator, a shaking table, an ultramicro spectrophotometer and the like.

Example 1: liriodendron bHLH family analysis and LcbHLH52 gene cloning

Download model the plant Arabidopsis thaliana (https:// www, arabidopsis. org) and rice (http:// rapdb. dna. affrc. go. jp) databases obtained the bHLH family sequences as 177 and 135 strips. Then, a model is constructed by using protein domain alignment software HMMER3.0, potential bHLH family sequences of the liriodendron are found, and candidate bHLH family sequences of the liriodendron are aligned by using BLASTP. The LcbHLH52 transcription factor was finally selected for cloning and sequencing.

1. Extraction and quality detection of total RNA

Before extracting total RNA, all containers used in the extraction process need to be subjected to RNase-free treatment in order to remove the interference of genomic DNA. Soaking plastic vessel such as centrifuge tube, grinding tool such as mortar and pestle, and tweezers in 0.1% DEPC water overnight, draining water, wrapping with newspaper, and sterilizing at high temperature and high pressure; all reagents were prepared with RNase free water. Extracting total RNA by a TRIZOL method:

(1) adding 50-100 mu g of leaves (stored at minus 80 ℃) into a mortar fully cooled by liquid nitrogen, grinding the leaves into powder, transferring the powder into a pre-cooled 1.5mL centrifuge tube, adding 1mL TRIZOL, shaking, uniformly mixing and standing for 5 min;

(2) adding 200 μ L chloroform into the centrifuge tube, mixing, standing for 5min, and preparing a refrigerated centrifuge in advance;

(3) centrifuging at 12000rpm at 4 deg.C for 10min to obtain precipitate at the bottom of the tube, and transferring the upper aqueous phase to another tube to obtain about 400 μ L. Adding isovolumetric isopropanol, mixing uniformly, standing for 10min, 12000rpm, and centrifuging at 4 deg.C for 10 min;

(4) and (4) sucking supernatant liquid, rinsing the supernatant liquid for 2 times by using 75% ethanol, uniformly mixing and standing for 5 min. Centrifuge at 12000rpm at 4 ℃ for 1 min. Discarding the supernatant, and drying for 10min on a super clean bench;

(5) addition of H₂O (treated by DEPC) to 30 μ L, and mixing;

(6) the total RNA was detected by agarose gel electrophoresis (0.5 XTBE electrophoresis buffer, 1.5% agarose gel), and the two bands were clear and showed no degradation. And simultaneously, detecting OD values of 260nm and 280nm by using a Nanodrop 2000 spectrophotometer, wherein the purity and the quality of the RNA meet the experimental requirements. The extracted RNA can be used immediately or stored at-20 deg.C for a short period.

2. First Strand cDNA Synthesis

cDNA synthesis of Liriodendron tulipifera was performed using a reverse transcription extraction kit from Vazyme. The preparation steps are as follows:

(1) denaturation of RNA template

The following mixture was placed in an RNase-free centrifuge tube (Table 1). The prepared mixed solution is heated at 65 ℃ for 5min, quickly placed on ice for quenching, and kept stand on the ice for 2 min.

TABLE 1 Mixed solution preparation system

Reagent	Dosage of
		RNase-free ddH2O	To 8.0μL
Oligo(dT)23VN(50μm)	1.0μL
		Total RNA	50ng

(2) Reverse transcription to synthesize the first strand of cDNA

First strand cDNA synthesis reaction solution (Table 2) was prepared, and the reaction was carried out at 50 ℃ for 45min and at 85 ℃ for 5 min. The synthesized cDNA can be used immediately or stored for a short period at-20 ℃.

TABLE 2 Synthesis reaction solution preparation System

3. Design and Synthesis of PCR primers

According to arabidopsis thaliana and rice bHLH family protein sequences searched in a database, after sequence comparison, a specific primer for synthesizing a gene is designed as follows:

LcbHLH52-F：5′-GGGGTACCATGGAAATAGATGAACATGG-3′，

LcbHLH52-R：5′-CGGGATCCCTACATGCATCTCCCTCC-3′。

4. PCR amplification of target gene sequence fragment

The first chain of cDNA of the liriodendron leaf is used as a template (the dosage is 1-5 mu L and is not more than 1/10 of the total volume of PCR reaction), the specific primers of the gene are used as upstream and downstream primers to carry out PCR amplification according to a PCR reaction system (table 3) and a PCR reaction program (table 4), and the target gene is amplified by adopting high-fidelity enzyme of vazyme company.

TABLE 3 PCR reaction System

TABLE 4 PCR reaction procedure

5. Detecting and recovering the DNA fragment of interest by electrophoresis

The amplification products were detected by electrophoresis on a 1.5% agarose gel and recovered according to the gel recovery kit procedure of the OMEGA company. The recovered DNA target fragment can be used immediately or stored at-20 deg.C for a short period of time.

The 1.5% agarose gel electrophoresis detection result shows that a single band is successfully amplified and is consistent with the expected size compared with the Marker, and if a DNA fragment of about 1000bp is amplified by the LcbHLH52 (figure 1), the PCR product is purified and recovered.

6. Target DNA fragment ligation 19-T vector

After the PCR product of the high fidelity enzyme amplification is purified and recovered, Taq enzyme is added to realize an A adding reaction, an ATP adaptor is added on two sides of the product, and then the product is connected with a commercial T vector pMD19 by T4 ligase of Thermofisiher company (Table 5).

And (3) putting the reaction system into a constant-temperature water bath kettle at the temperature of 22 ℃ for reacting for more than 60min or overnight at the temperature of 4 ℃, and placing the centrifugal tube on ice after the reaction is finished.

TABLE 5 ligation reaction System

Reagent	Dosage of
		10×T4 DNA Ligase buffer	2.0μL
PMD-19T vector	20-100ng
		DNA of a target gene	1∶1 to 5∶1 molar ratio over vector
T4 DNA Ligase	1.0μL
		ddH2O	to 20μL

7. Transformation of competent cells of E.coli with ligation products

(1) mu.L of the ligation product was added to 100. mu.L of E.coli competent cells, ice-cooled for 30min, heat-shocked in a water bath at 42 ℃ for 90s, and ice-cooled for 2 min. Adding 800 μ LLB liquid culture medium, and shake culturing;

(2) centrifuging, removing supernatant, and coating on LB solid medium containing Amp, IPTG and X-Gal;

(3) white colonies were picked by inverted culture at 37 ℃ and 100. mu.L of LB liquid medium containing Amp was added to each well in a 96-well plate;

(4) adding the selected bacterial colony into a liquid culture medium for shake cultivation;

(5) taking 1 mu L of bacterial liquid to perform PCR positive determination, and storing at-20 ℃ for a short time.

8. Positive colony PCR detection

The bacterial solution was subjected to PCR amplification using 2 XTaq enzyme system (Vazyme) according to the reaction system (Table 6) and the reaction program (Table 7).

And taking out the amplified bacterial liquid PCR for gel electrophoresis detection, sampling the bacterial liquid with the band and the target fragment in the electrophoretogram, namely the positive clone bacterial liquid, and sending the bacterial liquid to a company for sequencing by a universal primer M13-47/M13-48. The sequencing result was compared with BLAST (http:// BLAST. ncbi. nlm. nih. gov/BLAST. cgi) software to see if the correct target gene was obtained. And (3) constructing an overexpression vector with the consistent TA clone sequencing sequence and the reference sequence.

TABLE 6 colony PCR reaction System

Reagent	Dosage of
		ddH2O	7.0μL
2×Taq Master Mix	10μL
		Bacterial liquid	1.0μL
Primer 1 (10. mu.M)	1.0μL
		Primer 2 (10. mu.M)	1.0μL
Total Volume	20μL

TABLE 7 colony PCR reaction procedure

And (3) detecting and analyzing positive colonies by PCR:

the bacteria detection result shows that the size of the PCR product of the bacteria liquid is basically consistent with that of the target fragment, and has no impurity band, the PCR product is preliminarily identified as a recombinant, and the LcbHLH52 is a DNA fragment of about 1500bp (figure 2). Sequencing the No.1 positive bacteria liquid.

TA cloning sequencing results and analysis

And (3) sequencing the TA clone, wherein the sequencing result shows that the 3 TA clone sequences of the LcbHLH52 gene have base difference with the reference sequence, and the translated amino acid of clone No.1 is consistent with the reference sequence. After confirmation, clone No.1 is taken for overexpression vector construction. The CDS sequence of the TA clone sequencing No.1 used for vector construction is shown as SEQ ID NO.1, and the amino acid sequence of the expression protein is shown as SEQ ID NO. 2. By searching the plant protein database (blast, https:// blast.ncbi. nlm. nih. gov/blast. cgi), the gene has 72.93% similarity to sassafras bHLH93(NCBI accession No.: RWR77887.1) and 64.20% similarity to grape indecer of CBF expression 4. Therefore, the cloned liriodendron belongs to the ICE subfamily of the bHLH transcription factor family.

Example 2: construction of Liriodendron LcbHLH52 gene expression vector

1. Design of enzyme digestion primer

The Primer containing the cleavage site of the LcbHLH52 gene ORF was designed using software Primer premier 5.0 based on the Pcambia2301 plasmid map (FIG. 3) and the characteristics of the LcbHLH52 gene coding region. The enzyme cutting sites of the LcbHLH52 gene ORF are searched by software, so that no KpnI and BamHI enzyme cutting sites exist in the LcbHLH52 gene ORF, and KpnI and BamHI double enzyme cutting primers can be designed as follows:

LcbHLH52-KpnI-F：5′-GGggtaccATGGAAATAGATGAACATGG-3′，

LcbHLH52-BamHI-R：5′-CGggatccCTACATGCATCTCCCTCC-3′。

2. PCR amplification of enzyme-digested fragment of target gene

The same procedure as in "4, PCR amplification of the target Gene sequence fragment" in example 1 "

3. Electrophoretic detection and recovery of amplification product

In the same manner as in "5, detection by electrophoresis and recovery of the objective DNA fragment" in example 1 "

4. The amplification product is connected with a vector and transformed into Escherichia coli

The E.coli competent cells were transformed with "6, the objective DNA fragment ligated 19-T vectors" and "7" in example 1 "

5. Positive colony PCR detection

The same procedure as in "8, positive colony PCR detection" in example 1 was repeated.

6. Double enzyme digestion target gene and carrier plasmid

According to the principle of vector construction, double enzyme digestion vector pCambia2301-KY and XbaI and BamHI enzyme digestion sites in the primer are selected for double enzyme digestion of the selected TA cloning target gene (Table 8) and the vector pCambia2301-KY (Table 9) respectively. Mixing the reaction system, standing at 37 deg.C for 2h, detecting with 1.5% agarose gel electrophoresis, performing 180v electrophoresis for 20min, photographing the gel in a gel imager, cutting the target fragment on an ultraviolet gel cutter, and purifying and recovering with gel recovery kit of OMEGA company.

TABLE 8 double enzyme digestion of the Gene reaction System of interest

TABLE 9 Dual enzyme digestion vector reaction System

Reagent	Dosage of
		10×K Buffe	2μL
Vector plasmid	5μL
		Cleavage site
1	2μL
		Cleavage site
2	2μL
		ddH2O	To 40μL

7. Ligation of expression vectors

Recovering the TA cloning target fragment and pCambia2301-KY vector fragment which are correctly digested in the previous step, connecting the target gene fragment and the vector fragment, and connecting the reaction system with the reaction system shown in the table 5. And putting the reaction system into a constant-temperature water bath kettle at the temperature of 22 ℃ for reaction for more than 60min or overnight at the temperature of 4 ℃.

8. Transformation of expression vectors

(1) Adding 10 mu L of expression vector plasmid DNA with target gene segment into melted 100 mu L of escherichia coli DH5 alpha competent cells;

(2) performing ice bath for 30 min;

(3) carrying out water bath heat shock at 42 ℃ for 90 s;

(4) then ice bath is carried out for 2 min;

(5) adding 800 μ L LB liquid medium;

(6) shaking-culturing at 37 deg.C for 30 min;

(7) centrifuging at 6000rpm for 3min, discarding supernatant, transforming into LB solid culture medium containing Kana25mg/L Escherichia coli resistance screening culture medium, and culturing at 37 deg.C for 2 h;

(8) taking 1 mu L of bacterial liquid to carry out PCR positive determination, sequencing and detecting whether the expression vector is constructed successfully.

9. Extraction of plant expression vector plasmid

The extraction steps of pCambia2301-LcbHLH52 plant expression vector plasmid are the same as the cloning and functional analysis of genes of Della protein family Bsgai1 and Bsgai2 of Huanghao, Pearl boxwood [ D ]. Nanjing university of forestry, 2017 ] "

Vector construction sequencing results and analysis:

according to the vector construction principle, the LcbHLH52 gene selects two enzyme cutting vectors pCambia2301 and KpnI and BamHI enzyme cutting sites, respectively carries out double enzyme cutting reaction on the target gene cloned by TA and the vector pCambia2301, connects the two reaction systems, transforms the two reaction systems, carries out electrophoresis PCR detection, selects PCR positive transformants, shakes the bacteria to culture and extracts plasmids and carries out sequencing. The sequencing result shows that the constructed over-expression vector No.1 cloning sequencing sequence is consistent with the sequence shown in SEQ ID NO.1, which indicates that the target gene is inserted into the vector and the construction of the expression vector is accurate.

Example 3: agrobacterium-mediated genetic transformation of LcbHLH52 gene into Arabidopsis thaliana

1. Freeze thawing method for transferring agrobacterium

(1) Melting Agrobacterium EHA105 competent cells on ice, adding 2 μ L of the extracted plant expression vector plasmid, and blowing and stirring the gun head uniformly;

(2) ice-cooling for 30min, freezing with liquid nitrogen for 1min, and water-bathing at 37 deg.C for 5 min;

(3) adding 700 μ L LB liquid culture medium, shaking and culturing at 28 deg.C and 200rpm for 3 h;

(4) centrifuging at 4000rpm for 3min, and collecting a little supernatant;

(5) uniformly mixing the residual bacteria liquid after conversion, and coating the mixture on an LB solid culture medium containing Kan;

(6) inverted culture at 28 ℃ for 3d

(7) And (5) identifying positive clones.

Positive clone identification and analysis: transferring the successfully constructed LcbHLH52 gene expression vector into agrobacterium EHA105 competent cells, and selecting a positive colony for sequencing. The gene comparison software DNAMAN is used for carrying out sequence comparison, whether the sequence of the constructed expression vector is consistent with the reference sequence or not is judged, and the result shows that the two sequences are the same, so that subsequent tests can be carried out.

2. Cultivation of infested material

(1) Placing Arabidopsis seeds into a sterilized centrifugal tube, immersing for 30s by using 75% ethanol, and then performing disinfection treatment for 15min by using 1% sodium hypochlorite;

(2) removing the supernatant after standing, washing with sterile water for 4-5 times, adding 0.1% agarose, and spotting the resuspended Arabidopsis seeds on 1/2MS solid culture medium containing kanamycin by using a pipette gun for culture;

(3) vernalizing at 4 ℃ for 48h, transferring to a light incubator for culture for 7d, transplanting to a nutrition pot for culture until the flower buds are more when the arabidopsis thaliana grows to a proper size, and preparing for infection.

3. Preparation of agrobacterium infection liquid

(1) Taking out the agrobacterium liquid with correct target gene from-80 ℃ and activating;

(2) after being coated with a flat plate, the plate is placed in a dark culture at 28 ℃ for 24h, and then monoclonal bacteria liquid is selected to fall into a 1.5mL centrifuge tube for shake culture for 12 h;

(3) drawing 100 mu L of shake culture bacterial liquid, carrying out expansion culture to 250 mu L of conical flask, and carrying out shake culture until the OD value of the bacterial liquid is between 0.6 and 0.8;

(4) centrifuging at 5000rpm for 10min, discarding the supernatant, collecting Agrobacterium, and adding 1/2MS liquid culture medium containing 5% sucrose to resuspend the bacteria solution;

(5) 0.05% Silwet L-77 was added to the above solution, and the solution was shaken well to dissolve the colonies for use.

4. Transformation by infection with Agrobacterium

(1) Cutting off open flowers on the day before infection, and immersing unopened inflorescences of arabidopsis thaliana in the agrobacterium infection liquid for 30s, wherein the inflorescences at the leaf bases cannot be ignored;

(2) after infection, horizontally placing the arabidopsis thaliana in a tray, wrapping the tray with a black plastic bag, sealing the tray in the dark, and tying the bag to keep the air permeability;

(3) after dark culture is carried out for 24h, removing the black plastic bag, and placing the arabidopsis thaliana in a light incubator for culture, wherein more water is needed;

(4) stopping watering after the arabidopsis flowers, numbering the screened positive T1 generation plants after the fruits are mature, respectively obtaining more than 10 strains from each gene, respectively collecting seeds by using a sieve, and carrying out low-temperature treatment at 4 ℃ and storage.

5. Acquisition of T2-Generation transgenic lines

(1) Respectively putting numbered T1 generation seeds and wild type Arabidopsis seeds into sterilized centrifuge tubes, immersing for 30s with 75% ethanol, and then sterilizing with 1% sodium hypochlorite for 15 min;

(2) removing the supernatant after standing, washing with sterile water for 4-5 times, adding 0.1% agarose, and spotting the resuspended Arabidopsis seeds on 1/2MS solid culture medium containing kanamycin by using a pipette gun for culture;

(3) firstly, vernalizing at a low temperature of 4 ℃ for 48h, then transferring to a light incubator for culturing for 10d, and observing the growth conditions of an arabidopsis transgenic plant and an arabidopsis wild plant;

(4) selecting a positive transgenic seedling with dark green cotyledon and developed root system and an arabidopsis wild seedling, transplanting the positive transgenic seedling and the arabidopsis wild seedling into nutrient soil, and culturing to obtain a T2 generation transgenic plant.

6. Phenotypic observations of transgenic lines and wild-type Arabidopsis

And (3) sowing the Arabidopsis LcbHLH52 transgenic homozygous line and the wild type WT into nutrient soil at the same time, and culturing for 10d to observe the growth conditions of the transgenic line and the Arabidopsis wild type plant.

After the LcbHLH52 transgenic lines 2, 3, 7 and 12 and the wild type Arabidopsis plants are grown for 10 days in an incubator at 22 ℃, the observation of the phenotype growth condition shows that the growth vigor of the transgenic lines, such as the number and the size of leaves, are not completely the same, but the overall growth vigor is better than that of the wild type Arabidopsis plants (figure 4).

7. Phenotypic characteristics of transgenic lines under low temperature stress

Transplanting 4-5 leaf-old arabidopsis tissue culture seedlings which grow well in 1/2MS solid culture medium added with kanamycin into nutrient soil for 10 days, starting cold stress treatment, simultaneously placing LcbHLH52 transgenic arabidopsis 2, 3, 7 and 12 lines and wild type arabidopsis WT in a 4 ℃ refrigerator, photographing after low temperature stress for 3 days, observing phenotype difference of the transgenic plants and the arabidopsis plants, and preliminarily analyzing the effects of the two genes in response to low temperature.

The LcbHLH52 transgenic plant is placed in a low-temperature incubator at 4 ℃ for stress 3d, recorded and observed, and the growth of the LcbHLH52 transgenic plant (figure 5) is less affected by low-temperature stress, while the growth of a wild plant is inhibited, and leaves have a wilting phenomenon. Therefore, the liriodendron LcbHLH52 gene can enhance the tolerance of arabidopsis thaliana to low-temperature stress.

Sequence listing

<110> Nanjing university of forestry

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ggcgatacca tcgattacat gaaagagctg ctggatcgaa tcaagaagct gcaagatgaa 660

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Arg Ile Cys Lys Val Glu Met Ala Gln Ser Ala Glu Ile Pro Val Phe

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Thr Asp Thr Arg Ile Asp Ile Cys Cys Ala Thr Lys Pro Gly Leu Leu

260 265 270

Leu Ser Thr Ile Ala Thr Leu Glu Ala Leu Gly Leu Glu Ile Gln Gln

275 280 285

Cys Val Ile Ser Cys Phe Asn Asp Phe Gly Met Gln Ala Ser Cys Ser

290 295 300

Glu Asp Leu Glu Gln Arg Ser Val Ser Ser Ser Glu Glu Ile Lys Gln

305 310 315 320

Ala Leu Phe Arg Asn Ala Gly Tyr Gly Gly Arg Cys Met

325 330

<210> 3

<211> 28

<212> DNA

<213> LcbHLH52-F(Artificial)

<400> 3

ggggtaccat ggaaatagat gaacatgg 28

<210> 4

<211> 26

<212> DNA

<213> LcbHLH52-R(Artificial)

<400> 4

cgggatccct acatgcatct ccctcc 26

<210> 5

<211> 28

<212> DNA

<213> LcbHLH52-KpnI-F(Artificial)

<400> 5

ggggtaccat ggaaatagat gaacatgg 28

<210> 6

<211> 26

<212> DNA

<213> LcbHLH52-BamHI-R(Artificial)

<400> 6

cgggatccct acatgcatct ccctcc 26

Claims (6)

1. A transcription factor LcbHLH52 gene of Liriodendron tulipifera has a nucleotide sequence shown in SEQ ID NO. 1.

2. The expression protein of the transcription factor LcbHLH52 gene of liriodendron tulipifera as claimed in claim 1, wherein the amino acid sequence of the expression protein is shown as SEQ ID NO. 2.

3. A vector comprising LcbHLH52 gene of transcription factor of Liriodendron tulipifera as claimed in claim 1.

4. The use of the transcription factor LcbHLH52 gene of Liriodendron tulipifera as claimed in claim 1 for enhancing the tolerance of plants to low-temperature stress.

5. Use according to claim 4, characterized in that it comprises the following steps:

1) constructing a vector of the transcription factor LcbHLH52 gene of the liriodendron;

2) transforming the constructed vector of the transcription factor LcbHLH52 gene into a plant or a plant cell;

3) and culturing and screening to obtain the plant with enhanced low-temperature stress tolerance.

6. Use according to claim 4 or 5, wherein the plant is Arabidopsis thaliana.

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