阅读说明：本技术 毛竹抗坏血酸过氧化物酶基因PeAPX4及其应用 (Phyllostachys pubescens ascorbic acid peroxidase gene PeAPX4 and application thereof ) 是由 李雪平 杨旸 宋笑龙 李夷骞 于 2021-07-20 设计创作，主要内容包括：本发明公开了毛竹抗坏血酸过氧化物酶基因PeAPX4及其应用。PeAPX4基因及其编码蛋白质的序列分别如SEQ ID NO:1和2所示。本发明首次从毛竹中克隆得到PeAPX4基因并通过在拟南芥中过表达该基因验证了其生物学功能。PeAPX4基因具有调控植物抗逆性的功能,能够为毛竹转基因研究提供有力支持,为毛竹分子育种提供有价值的候选基因。(The invention discloses a moso bamboo ascorbic acid peroxidase gene PeAPX4 and application thereof. The sequences of the PeAPX4 gene and the protein coded by the gene are respectively shown as SEQ ID NO. 1 and SEQ ID NO. 2. The invention clones the PeAPX4 gene from the moso bamboo for the first time and verifies the biological function by over-expressing the gene in arabidopsis thaliana. The PeAPX4 gene has the function of regulating and controlling plant stress resistance, can provide powerful support for moso bamboo transgenic research, and provides valuable candidate genes for moso bamboo molecular breeding.)

1. A Phyllostachys pubescens ascorbate peroxidase gene PeAPX4, which is a gene encoding the following protein (a) or (b):

(a) a protein consisting of an amino acid sequence shown as SEQ ID NO. 2;

(b) 2, protein which is derived from (a) and has the same function by substituting, deleting or adding one or more amino acids in the sequence shown in SEQ ID NO. 2.

2. The gene of claim 1, wherein the nucleotide sequence is represented by SEQ ID NO 1.

3. A biomaterial containing the gene of claim 1 or 2, which is a recombinant DNA, an expression cassette, a transposon, a plasmid vector, a viral vector or an engineered bacterium.

4. Use of the gene of claim 1 or 2 or the biomaterial of claim 3 for plant stress tolerance control; wherein, the stress resistance refers to drought resistance, salt resistance and low temperature resistance.

5. The use of claim 4, wherein the plant comprises Arabidopsis thaliana, Phyllostachys pubescens.

6. Use of the gene according to claim 1 or 2 or the biological material according to claim 3 for the preparation of transgenic plants.

7. A method of improving drought resistance in a plant, comprising: overexpressing the gene of claim 1 or 2 in a plant by genetic engineering means;

the mode of overexpression is selected from the following 1) to 5), or an optional combination:

1) by introducing a plasmid having the gene;

2) by increasing the copy number of the gene on the plant chromosome;

3) by altering the promoter sequence of said gene on the plant chromosome;

4) by operably linking a strong promoter to the gene;

5) by introducing an enhancer;

the plant includes Arabidopsis thaliana and Phyllostachys pubescens.

8. The method as claimed in claim 7, wherein Agrobacterium mediated method is adopted to transfer the Phyllostachys pubescens PeAPX4 gene into Arabidopsis thaliana plant to obtain transgenic plant with the gene over-expressed;

preferably, the Phyllostachys pubescens PeAPX4 gene is constructed on a plant expression vector pCAMBIA2300, agrobacterium is transformed, then an arabidopsis inflorescence is impregnated, and a transgenic plant is screened.

9. Use of a transgenic plant obtained according to the method of claim 7 or 8 in plant breeding.

10. Use according to claim 9, characterized in that the breeding method comprises transgenesis, crossing, backcrossing, selfing or asexual propagation.

Technical Field

The invention relates to the technical field of plant genetic engineering, in particular to a moso bamboo ascorbic acid peroxidase gene PeAPX4 and application thereof.

Background

Phyllostachys edulis is a kind of sporadic bamboo of Phyllostachys of Bambusoideae (Bambuseideae) of Bambusoideae (Gramminales) of Gramineae (Gramminales). The moso bamboo has the advantages of fast material forming, regeneration and the like, and has great potential in the aspects of biological energy development, water and soil loss prevention and the like. Among many factors affecting the growth of moso bamboos, moisture is one of the most significant factors affecting the normal growth and development thereof. In recent years, due to the influence of global warming, annual precipitation of the main growing areas of the moso bamboos is reduced year by year, the drought problem is frequent, the growth condition of the bamboos is greatly influenced, and the economic benefit of the moso bamboos is also reduced. In the north shift work of the south bamboos, the problems of less water in introduced land and high saline-alkali degree of land are difficult to overcome. Therefore, it is necessary to improve the bamboo species of moso bamboo and to cultivate new species of moso bamboo with early resistance and strong salt resistance. The research on the function of the related genes in the moso bamboo body under the adversity condition is the molecular basic work for breeding new varieties, and has important significance for revealing the adversity resisting molecular mechanism of the bamboo, breaking through the limitation of conventional breeding and accelerating the breeding process of the bamboo.

Disclosure of Invention

The invention aims to provide a moso bamboo ascorbic acid peroxidase gene PeAPX4 and application thereof.

In order to achieve the object of the present invention, in a first aspect, the present invention provides a phyllostachys pubescens ascorbate peroxidase gene PeAPX4, which is a gene encoding the following protein (a) or (b):

(a) a protein consisting of an amino acid sequence shown as SEQ ID NO. 2;

(b) 2, protein which is derived from (a) and has the same function by substituting, deleting or adding one or more amino acids in the sequence shown in SEQ ID NO. 2.

The nucleotide sequence of the Phyllostachys pubescens PeAPX4 gene is shown in SEQ ID NO. 1. The invention adopts the following method to clone and obtain the PeAPX4 gene:

the method comprises the steps of taking moso bamboo leaves and roots as materials, extracting total RNA, and carrying out reverse transcription on the extracted total RNA to obtain cDNA. In the present invention, the extraction of total RNA from Phyllostachys pubescens can be performed by a method for extracting total cellular RNA commonly used in the art, such as Trizol method.

And reversely transcribing the extracted total RNA of the moso bamboo into cDNA. In the present invention, the cDNA synthesis can be carried out by a method generally used in the art, for example, by using a cDNA synthesis kit available from Promega corporation.

(3) After obtaining the cDNA, PCR amplification of the PeAPX4 gene was performed to obtain the desired fragment.

In the present invention, the system for PCR amplification of PeAPX4 gene is preferably: 10 XPCR Buffer 2.0 uL, 2.5mM dNTP Mix 2.0 uL, upstream primer 1.0 uL, downstream primer 1.0 uL, cDNA template 2.0 uL, LA Taq DNA polymerase 0.2 uL, ddH₂O11.8. mu.L. The PCR amplification reaction procedure is preferably: pre-denaturation at 95 ℃ for 5 min; denaturation at 95 ℃ for 30s, annealing at 62 ℃ for 30s, extension at 72 ℃ for 53s, 35 cycles; 10min at 72 ℃; storing at 4 ℃.

Primers were designed using Oligo7 software to amplify the PeAPX4 gene. The primer sequences are as follows:

an upstream primer: 5'-ATGGCGGCTCCGGTGGTGG-3'

A downstream primer: 5'-TTACTTGCTCCTCTTGGAAG-3'

Fourth, after the target fragment is obtained by PCR amplification, the target fragment is sequenced to obtain the PeAPX4 gene. After PCR amplification, the target fragment is purified, and the purification method is not particularly limited, and may be performed using a DNA purification kit known to those skilled in the art.

After the purification is completed, the purified target fragment is connected to a pGEM-T Easy vector, introduced into escherichia coli DH5 alpha competent cells, verified to be positive clones by colony PCR, and then sequenced.

In a second aspect, the present invention provides a biological material containing the phyllostachys pubescens PeAPX4 gene, including, but not limited to, recombinant DNA, expression cassettes, transposons, plasmid vectors, viral vectors, engineered bacteria, transgenic cell lines, or non-regenerable plant parts.

In a third aspect, the invention provides an application of the phyllostachys pubescens PeAPX4 gene or a biological material containing the gene in plant stress resistance regulation (plant stress resistance improvement). Wherein, the stress resistance refers to drought resistance, salt resistance and low temperature resistance.

The plants of the invention include, but are not limited to, Arabidopsis thaliana and Phyllostachys pubescens.

In a fourth aspect, the invention provides the use of the Phyllostachys pubescens PeAPX4 gene or a biological material containing the gene in the preparation of transgenic plants.

In a fifth aspect, the invention also provides the use of the Phyllostachys pubescens PeAPX4 gene or the biological material containing the gene in plant breeding.

The breeding aim is to improve the stress resistance of plants. The PeAPX4 gene participates in drought, salt and low temperature stress response of moso bamboo, and the expression of the gene can be induced by adversity stress.

In a sixth aspect, the present invention provides a method for improving drought resistance and salt resistance of a plant, comprising: the Phyllostachys pubescens PeAPX4 gene is over-expressed in the plant by using a genetic engineering means.

The mode of overexpression is selected from the following 1) to 5), or an optional combination:

1) by introducing a plasmid having the gene;

2) by increasing the copy number of the gene on the plant chromosome;

3) by altering the promoter sequence of said gene on the plant chromosome;

4) by operably linking a strong promoter to the gene;

5) by introducing an enhancer.

In the present invention, the expression vector carrying the target gene can be introduced into plant cells by using conventional biotechnological methods such as Ti plasmid, plant virus vector, direct DNA transformation, microinjection, electroporation, and the like.

Furthermore, the Phyllostachys pubescens PeAPX4 gene can be transferred into an Arabidopsis plant by adopting an agrobacterium-mediated method to obtain a transgenic plant with the gene over-expressed.

Preferably, the Phyllostachys pubescens PeAPX4 gene is constructed on a plant expression vector pCAMBIA2300, agrobacterium is transformed, then an arabidopsis inflorescence is impregnated, and a transgenic plant is screened.

In one embodiment of the present invention, the plant expression vector pCAMBIA2300-PeAPX4 is constructed as follows:

carrying out PCR reaction by taking cDNA synthesized by reverse transcription as a template, and respectively introducing EcoRI restriction enzyme sites and KpnI restriction enzyme sites at the upstream and downstream of the PeAPX4 gene; connecting the amplified product to a pGEM-T Easy vector, transforming DH5 alpha competent cells, and carrying out sequence determination; extracting plasmid, connecting EcoRI and KpnI double-restriction enzyme PeAPX4 gene fragment with pCAMBIA2300-CaMV35S, transforming, extracting plasmid, sequencing, and constructing plant expression vector pCAMBIA2300-PeAPX 4.

The preparation method of the transgenic arabidopsis is as follows:

transforming the constructed plant expression vector pCAMBIA2300-PeAPX4 into agrobacterium strain GV3101 competence; selecting positive clone shake bacteria, dip-dyeing inflorescence and screening homozygous seeds; extracting Arabidopsis positive seedling leaf RNA, performing reverse transcription to form cDNA, and performing PCR identification by using primers PeAPX4-F (5'-CGGAATTCATGGCGGCTCCGGTGGTGG-3') and PeAPX4-R (5'-GGGGTACCTTACTTGCTCCTCTTGGAAG-3').

In a seventh aspect, the present invention provides the use of a transgenic plant obtained according to the above method in plant breeding.

Breeding methods include, but are not limited to, transgenic, hybrid, backcross, selfing, or vegetative propagation.

The invention discloses the biological function of the phyllostachys pubescens PeAPX4 gene for the first time, by constructing a PeAPX4 gene expression vector, combining an agrobacterium-mediated genetic transformation method and heterologously transforming Arabidopsis thaliana, investigating the influence of PeAPX4 on the stress resistance of transgenic Arabidopsis thaliana and simultaneously detecting the response of PeAPX4 to the drought and salt stress of the phyllostachys pubescens, a powerful tool is provided for the transgenic research of the phyllostachys pubescens, and valuable candidate genes are provided for the breeding of phyllostachys pubescens resistance molecules.

Drawings

FIG. 1 is an electrophoresis diagram of PCR products of PeAPX4 gene clone (A) and an electrophoresis diagram of PCR products of PCAMBIA2300-PeAPX4 enzyme digestion (B) in a preferred embodiment of the present invention; wherein, lanes 1-2 in A are PeAPX4 gene clone PCR products, lanes 1-2 in B are PCAMBIA2300-PeAPX4 enzyme digestion products, and M is DNAmarker.

FIG. 2 is a diagram showing the phenotype analysis of PeAPX4 transgenic Arabidopsis thaliana and wild Arabidopsis thaliana under drought stress in a preferred embodiment of the present invention. Wherein, A is transplanting arabidopsis seedlings with the size of 7d to mannitol culture media with different concentrations; b is the seedling growth to 14 days of seedling age in A.

FIG. 3 is the expression analysis of the PeAPX4 gene under salt stress in the root and leaf of Phyllostachys Pubescens in the preferred embodiment of the present invention.

FIG. 4 is the expression analysis of the PeAPX4 gene under drought stress in the root and leaf of Phyllostachys Pubescens in the preferred embodiment of the present invention.

FIG. 5 is the expression analysis of the PeAPX4 gene under low temperature stress in the root and leaf of Phyllostachys Pubescens in the preferred embodiment of the present invention.

Detailed Description

The following examples are intended to illustrate the invention but are not intended to limit the scope of the invention. Unless otherwise indicated, the examples follow conventional experimental conditions, such as the Molecular Cloning handbook, Sambrook et al (Sambrook J & Russell DW, Molecular Cloning: a Laboratory Manual,2001), or the conditions as recommended by the manufacturer's instructions.

Example 1 cloning of the Phyllostachys pubescens ascorbic acid peroxidase Gene PeAPX4

The method comprises the steps of taking moso bamboo leaves as a material, extracting total RNA of the leaves according to a Trizol RNA extraction kit (Tiangen Biochemical technology Co., Ltd.) instruction method, taking 1ng of RNA, carrying out reverse transcription according to a reverse transcription kit (Promega, USA) to obtain cDNA, and digesting a cDNA product by using RNase. Primers were designed to amplify the PeAPX4 gene using Oligo7 software according to the Phyllostachys pubescens genome database http:// www.forestrylab.org/db/PhePacBio/ExtractSeq/phe/index.

An upstream primer: 5'-ATGGCGGCTCCGGTGGTGG-3'

A downstream primer: 5'-TTACTTGCTCCTCTTGGAAG-3'

Polymerase chain reaction:

20 μ L reaction: 10 XPCR Buffer 2.0 uL, 2.5mM dNTP Mix 2 uL, upstream primer 1.0 uL, downstream primer 1.0 uL, cDNA template 2.0 uL, LA Taq DNA polymerase 0.2 uL, ddH₂O 11.8μL。

PCR reaction procedure: pre-denaturation at 95 ℃ for 5 min; denaturation at 95 ℃ for 30s, annealing at 62 ℃ for 30s, extension at 72 ℃ for 53s, 35 cycles; 10min at 72 ℃; storing at 4 ℃.

And connecting the recovered product to a pGEM-T Easy vector, transforming DH5 alpha competent cells, selecting positive clones to perform plaque PCR detection, and sequencing the positive clones (Shanghai Bioengineering Co., Ltd.) to obtain an accurate sequencing result. The nucleotide sequence of the PeAPX4 gene is shown in SEQ ID NO. 1.

Example 2 construction of plant expression vector pCAMBIA2300-PeAPX4

Designing primers (PeAPX4-F and PeAPX4-R) to carry out polymerase chain reaction, respectively introducing EcoRI and KpnI double enzyme cutting sites at the upstream and downstream of a target gene PeAPX4, connecting the product to a pGEM-T Easy vector (Promega company), transforming DH5 alpha competent cells, carrying out sequence determination, extracting a plasmid, connecting an EcoRI and KpnI double enzyme cut PeAPX4 gene fragment with a pCAMBIA2300-CaMV35S vector which is cut by the same enzyme, transforming, extracting the plasmid, and carrying out sequence determination.

The upstream primer PeAPX 4-F: 5'-CGGAATTCATGGCGGCTCCGGTGGTGG-3'

The downstream primer PeAPX 4-R: 5'-GGGGTACCTTACTTGCTCCTCTTGGAAG-3'

The method comprises the step of carrying out PCR reaction by taking Mao bamboo leaf cDNA as a template

20 μ L reaction: 10 XPCR Buffer 2.0 uL, 2.5mM dNTP Mix 2 uL, upstream primer 1.0 uL, downstream primer 1.0 uL, cDNA template 2.0 uL, LA Taq DNA polymerase 0.2 uL, ddH₂O 11.8μL。

PCR reaction procedure: pre-denaturation at 95 ℃ for 5 min; denaturation at 95 ℃ for 30s, annealing at 62 ℃ for 30s, extension at 72 ℃ for 53s, 35 cycles; 10min at 72 ℃; storing at 4 ℃.

Amplification product recovery and connection

The recovered fragment is connected to a pGEM-T Easy vector, DH5 alpha competent cells are transformed, sequence determination is carried out, and the sequencing result is accurate.

(3) Construction of expression vector pCAMBIA2300-CaMV35S-PeAPX4

A recombinant plasmid was constructed by double-digesting pGEM-T Easy (FIG. 1, A) ligated with the fragment of PeAPX4 with EcoRI and KpnI, and expression vector pCAMBIA2300-CaMV35S (Promega, USA) digested with EcoRI and KpnI, as follows (50. mu.L):

enzyme digestion is carried out for 4h at 37 ℃; the product was subjected to agarose gel electrophoresis, and the large fragment of plasmid pCAMBIA2300-CaMV35S and the small fragment of PeAPX4 were recovered using a gel recovery kit (Axygen). The two recovered products were ligated using T4 DNA ligase in the following ligation reaction (20. mu.L):

the ligation reaction was carried out overnight at 4 ℃. The ligation products were all transformed into DH5 α competent cells. Overnight culture at 37 ℃, selecting single clone, carrying out colony PCR verification, carrying out amplification culture, extracting plasmid pCAMBIA2300-PeAPX4, carrying out sequencing and enzyme digestion verification (figure 1, B), and successfully constructing the plant recombinant expression plasmid.

EXAMPLE 3 transformation of Arabidopsis thaliana with plant expression vector pCAMBIA2300-PeAPX4

Novel agrobacterium tumefaciens GV3101 strain transformed by freeze-thawing method

Adding 1ng of recombinant expression vector plasmid into 100 mu L of competent cells GV3101, carrying out ice bath for 10min, quickly freezing the competent cells in liquid nitrogen for 5min, quickly transferring the competent cells to a 37 ℃ constant-temperature water bath kettle for 5min, then placing the competent cells on ice for 5min, adding 600 mu L of LB liquid culture medium into a centrifugal tube, carrying out shake culture in a 28 ℃ shaking table for 2-3h, and recovering the thalli. 60 μ L of the suspension was applied to YEP solid medium containing Kan-resistant (50mg/mL) and Rif-resistant (50mg/mL) and plated upside down on a shaker at 28 ℃ for about 2-3 days until white colonies grew. After the monoclonal colony is picked and subjected to PCR detection, positive clone is selected and cultured by shaking.

Variegata inflorescence dip-dyeing method

The above-mentioned positive clones were inoculated into 10mL of YEP (containing 50. mu.g/mL rifampicin and 100. mu.g/mL kanamycin) liquid medium, shake-cultured at 28 ℃ for 12 hours in an incubator (160rpm), 2mL of the culture was transferred to 200mL of YEP (containing 50. mu.g/mL rifampicin and 100. mu.g/mL kanamycin) and subjected to mass culture, shake-cultured at 28 ℃ for 12 hours in an incubator (160rpm) at a culture concentration OD₆₀₀After reaching 1.8-2.2. mu.g/mL, 50mL of the culture solution was centrifuged at 5000rpm at 4 ℃ for 5min in a 50mL centrifuge tube, vigorously suspended in a transformation solution (2.2 g of MS medium containing 5% sucrose, adjusted to pH 5.8, mixed with 0.2% Silwet L-77), and the precipitate was diluted to 1.0. mu.g/mL to obtain an invader solution. Approximately 200mL of the insult solution was taken. Soaking the overground part of newly flowering Arabidopsis into the infection solution for 3min, wrapping the plant with a preservative film, and culturing in dark for 12-16hRemoving the preservative film, putting the plant into an incubator for culturing, and waiting for harvesting seeds.

(3) Screening of homozygotes

Seeds of T0 generation Arabidopsis thaliana are placed in a centrifuge tube, sterilized by adding 1mL of 70% alcohol for 5min, sterilized by using 1mL of 2.6% sodium hypochlorite solution for 10min, and then washed 5 times by using sterile water. Uniformly sowing seeds on a screening culture medium (1/2MS +100mg/L kanamycin), purifying at 4 ℃ for 2 days, then placing the seeds in a climatic incubator to culture until 4 cotyledons grow out, transplanting green and normally-growing positive plants into soil for cultivation, collecting T1 generation seeds by dividing single plants after maturation, screening T1 generation seedlings by the same method, counting the proportion of positive plants and non-positive plants of each strain of T1 generation, transplanting the positive plants of the strains with the proportion of 3:1 into the soil for cultivation, and obtaining the T2 generation seeds. T2 seedlings are screened by the same method to obtain T3 seeds.

PCR identification of positive plants

Extracting positive Arabidopsis thaliana leaf RNA, performing reverse transcription to form cDNA, and performing PCR identification by using a primer PeAPX4-F, PeAPX 4-R. The positive plants are all found to contain PeAPX4, which indicates that PeAPX4 is successfully transferred into Arabidopsis.

Example 4 PeAPX4 Gene transfer Arabidopsis thaliana stress resistance analysis

Under drought stress, the phenotype of the transgenic plants was observed. In a solution containing 50 mmol. L^-1And 100 mmol. L^-1After 7 days of growth on mannitol medium, the PeAPX4 transgenic plant and wild type did not significantly differ in growth, but contained 150 mmol.L^-1And 200 mmol. L^-1Compared with wild plants, the PeAPX4 transgenic plants on the mannitol culture medium have obviously higher lateral root number and root length than wild plants, and the overall growth vigor of the plants is better than that of the wild plants. Especially in the presence of 200 mmol. L^-1After 7 days of growth on mannitol medium, leaves of wild type plants almost completely lost green, and transgenic plants grew well (FIG. 2).

Example 5 analysis of the expression level of PeAPX4 Gene in Phyllostachys Pubescens under drought, salt and Low temperature stresses

A material processing

The moso bamboo seeds are collected in a Guangxi Zhuang autonomous region, are placed in a constant-temperature illumination incubator, the day and night temperature is 25 ℃/18 ℃, the photoperiod is 16h/8h in light/dark, are cultured for about three months, high salt, drought and low-temperature stress are simulated by 200mM NaCl, 20% PEG6000 and 4 ℃, young and tender main roots and fibrous roots and leaves at the same parts after treatment for 0h, 3h, 6h, 12h, 24h, 48h, 72h and 120h are rapidly frozen in liquid nitrogen, and are frozen and preserved at-80 ℃.

Synthesis of cDNA template

Extracting total RNA from root, young stem and leaf of moso bamboo seedling with Trizol Reagent, removing genome DNA with DNase I (TIANDZ) without RNase, and measuring A with ultraviolet spectrophotometer₂₆₀And A₂₈₀The brightness and integrity of the 28S rRNA, 18S rRNA and 5S rRNA amplified bands were checked by 1% agarose gel electrophoresis, the first strand of cDNA was synthesized by a reverse transcription kit from Promega corporation, and the synthesized product was stored in a refrigerator at-20 ℃.

(3) Real-time fluorescent quantitative PCR

And detecting the expression condition of the target gene by real-time fluorescent quantitative PCR (qRT-PCR). TIP41 gene (GenBank: FP092936.1) was used as an internal reference gene, TIP 41-F: 5'-AAAATCATTGTAGGCCATTGTCG-3', TIP 41-R: 5'-ACTAAATTAAGCCAGCGGGAGTG-3', respectively;

PeAPX4 5-F:5'-CTAAGGAGCCTCTGAAGTTTG-3'

PeAPX4 5-R:5'-CGTACAGGTAGCCCACGATA-3'。

the 10 μ L reaction was as follows:

reaction procedure: 1min at 95 ℃; 95 ℃ 10s, 62 ℃ 10s, 72 ℃ 20s, 45 cycles.

Other reaction parameters were system defaults, 3 biological replicates were set for each reaction, using Roche Light480 Analyzer data, utilize 2^-ΔΔCTMethod for analyzing 3 times of biological experimentsThe data were analyzed and plotted using Excel.

Fourth experiment result and analysis

After drought, salt and low-temperature stress treatment, the expression conditions of the PeAPX4 gene in the roots and leaves of the moso bamboo seedlings are respectively detected, and the results are shown in FIGS. 3-5: after salt treatment, the expression level of the PeAPX4 in leaves and roots is obviously up-regulated, and the expression level reaches a peak value after 72 hours of treatment, which is 7.1 times and 9.3 times of that of a control group respectively; after drought treatment, the expression level of the PeAPX4 in leaves and roots is approximately in a trend of ascending first and then descending, and the expression level reaches a peak value after 48 hours of treatment, which is 12.3 times and 22.3 times of that of a control group respectively; after the low temperature treatment, the expression of the PeAPX4 is obviously induced in the leaf, but the expression amount in the root is not changed greatly. The PeAPX4 gene is shown to be possibly involved in the drought, low temperature and salt stress response process of the moso bamboo.

Although the invention has been described in detail hereinabove with respect to a general description and specific embodiments thereof, it will be apparent to those skilled in the art that modifications or improvements may be made thereto based on the invention. Accordingly, such modifications and improvements are intended to be within the scope of the invention as claimed.

Sequence listing

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Met Ala Ala Pro Val Val Asp Ala Glu Tyr Leu Arg Gln Val Asp Arg

1 5 10 15

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

20 25 30

Ile Met Leu Arg Leu Ala Trp His Asp Ala Gly Thr Tyr Asp Val Ser

35 40 45

Thr Lys Thr Gly Gly Ala Asn Gly Ser Ile Arg Phe Glu Glu Glu Tyr

50 55 60

Thr His Gly Ser Asn Ala Gly Leu Lys Ile Ser Ile Asp Leu Leu Glu

65 70 75 80

Pro Ile Lys Ala Lys Asn Pro Arg Ile Thr Tyr Ala Asp Leu Tyr Gln

85 90 95

Leu Ser Gly Val Ile Ala Val Glu Val Thr Gly Gly Pro Thr Ile Glu

100 105 110

Phe Ile Pro Gly Arg Arg Asp Ser Ser Val Cys Pro Arg Glu Gly Arg

115 120 125

Leu Pro Asp Ala Lys Lys Gly Ala Pro His Leu Arg Asp Ile Phe Tyr

130 135 140

Arg Met Gly Leu Thr Asp Lys Asp Ile Val Ala Leu Ser Gly Gly His

145 150 155 160

Thr Leu Gly Arg Ala His Pro Glu Arg Ser Gly Phe Glu Gly Val Trp

165 170 175

Thr Lys Glu Pro Leu Lys Phe Asp Asn Ser Tyr Phe Leu Glu Leu Leu

180 185 190

Lys Gly Glu Ser Glu Gly Leu Leu Lys Leu Pro Thr Asp Lys Ala Leu

195 200 205

Leu Glu Asp Pro Glu Phe Arg Arg Tyr Val Glu Leu Tyr Ala Lys Asp

210 215 220

Glu Asp Ala Phe Phe Lys Asp Tyr Ala Glu Ser His Lys Lys Leu Ser

225 230 235 240

Glu Leu Gly Phe Thr Pro Arg Ser Ser Gly Pro Ala Ser Thr Lys Ser

245 250 255

Asp Leu Ser Thr Val Val Val Leu Ala Gln Ser Ala Val Gly Val Ala

260 265 270

Val Ala Ala Ala Val Val Ile Val Gly Tyr Leu Tyr Glu Ala Ser Lys

275 280 285

Arg Ser Lys

290