CN112779234B - Moso bamboo PeAPX5 gene and its application - Google Patents

Abstract

The invention discloses a moso bamboo PeAPX5 gene and application thereof. The sequences of the Phyllostachys pubescens PeAPX5 gene and the protein coded by the gene are respectively shown in SEQ ID NO. 1 and SEQ ID NO. 2. The invention clones the PeAPX5 gene from the moso bamboo for the first time and verifies the biological function by over-expressing the gene in arabidopsis thaliana. The PeAPX5 gene has the function of regulating and controlling plant stress resistance, can provide a powerful tool for moso bamboo transgenic research, and provides a valuable candidate gene for moso bamboo resistance molecule breeding.

Description

Translated fromChinese

毛竹PeAPX5基因及应用Moso bamboo PeAPX5 gene and its application

技术领域technical field

本发明涉及植物基因工程技术领域，具体地说，涉及毛竹PeAPX5基因及应用。The invention relates to the technical field of plant genetic engineering, in particular, to the Moso bamboo PeAPX5 gene and its application.

背景技术Background technique

毛竹(Phyllostachys edulis)是禾本科(Gramminales)竹亚科(Bambusoideae)刚竹属(Phyllostachys)散生竹种，在涵养水源、固土固沙、生物质能源开发等方面有巨大潜力，是重要的生态、经济和文化资源。然而，世界范围内的高温干旱问题是制约竹林生产的主要逆境因子。竹类植物在生长过程中需要充足的水分，尤其是笋的形成和生长，与水分供应十分密切。干旱严重影响了竹子的产量和质量。因此，进行竹种改良，培育抗旱性强的竹子新品种非常必要，也是扩大竹子分布范围，实现南竹北移的的一种重要的途径。而研究逆境条件下毛竹体内相关基因的功能则是进行新品种培育的分子基础工作，对于揭示竹子的抗逆分子机制，进而突破常规育种的局限性，加快竹子育种进程具有重要的现实意义。Phyllostachys edulis is a loose bamboo species belonging to the genus Phyllostachys in the subfamily Bambusoideae of the Gramminales family. , economic and cultural resources. However, the worldwide high temperature and drought is the main adverse factor restricting the production of bamboo forests. Bamboo plants need sufficient water during the growth process, especially the formation and growth of bamboo shoots, which are closely related to water supply. The drought severely affected the yield and quality of bamboo. Therefore, it is very necessary to improve bamboo species and cultivate new varieties of bamboo with strong drought resistance. However, studying the functions of related genes in Phyllostachys pubescens under adversity conditions is the molecular basis for the cultivation of new varieties.

发明内容SUMMARY OF THE INVENTION

本发明的目的是提供毛竹PeAPX5基因及应用。The purpose of the present invention is to provide Moso bamboo PeAPX5 gene and application.

为了实现本发明目的，第一方面，本发明提供毛竹PeAPX5基因，其为编码如下蛋白质(a)或(b)的基因：In order to achieve the object of the present invention, in the first aspect, the present invention provides the Moso bamboo PeAPX5 gene, which is a gene encoding the following protein (a) or (b):

(a)由SEQ ID NO:2所示的氨基酸序列组成的蛋白质；(a) a protein consisting of the amino acid sequence shown in SEQ ID NO: 2;

(b)SEQ ID NO:2所示序列经取代、缺失或添加一个或几个氨基酸且具有同等功能的由(a)衍生的蛋白质。(b) A protein derived from (a) having the sequence shown in SEQ ID NO: 2 substituted, deleted or added with one or several amino acids and having an equivalent function.

毛竹PeAPX5基因的核苷酸序列如SEQ ID NO:1所示。本发明采用如下方法克隆得到PeAPX5基因：The nucleotide sequence of the Moso bamboo PeAPX5 gene is shown in SEQ ID NO:1. The present invention adopts the following method to clone and obtain PeAPX5 gene:

⑴以毛竹叶片为材料提取总RNA，并将提取得到的总RNA反转录为cDNA。本发明中，毛竹总RNA的提取采用本领域常用的提取细胞总RNA的方法即可，本发明的实施例中具体可采用Trizol法。(1) Total RNA was extracted from bamboo leaves, and the extracted total RNA was reverse transcribed into cDNA. In the present invention, the extraction of the total RNA of Phyllostachys pubescens can be carried out by a method commonly used in the art for extracting the total RNA of cells. In the embodiment of the present invention, the Trizol method can be specifically used.

⑵在提取得到毛竹总RNA后，将所述总RNA反转录合成cDNA。在本发明中，cDNA的合成采用本领域常规的cDNA合成方法即可，无其他特殊要求；本发明的实施例中具体可采用Promega公司的cDNA合成试剂盒进行cDNA的合成。(2) After extracting the total RNA of Phyllostachys edulis, reverse transcribing the total RNA to synthesize cDNA. In the present invention, conventional cDNA synthesis methods in the art can be used for cDNA synthesis, and there are no other special requirements; in the embodiments of the present invention, cDNA synthesis kits from Promega Company can be specifically used for cDNA synthesis.

⑶在得到cDNA之后，进行PeAPX5基因的PCR扩增，得到目的片段。本发明中，PeAPX5基因PCR扩增的体系优选为20μL体系，包括：10×PCR Buffer 2.0μL，2.5mM dNTP Mix 2.0μL，10μM上、下游引物各1.0μL，cDNA模板2.0μL，5U/μL LA Taq DNA聚合酶0.2μL，ddH₂O 11.8μL。PCR扩增反应程序优选为：94℃预变性5min；94℃变性30s；94℃退火30s；72℃延伸45s，35个循环；72℃10min；4℃保存。(3) After the cDNA is obtained, PCR amplification of the PeAPX5 gene is performed to obtain the target fragment. In the present invention, the PeAPX5 gene PCR amplification system is preferably a 20 μL system, including: 10×PCR Buffer 2.0 μL, 2.5 mM dNTP Mix 2.0 μL, 10 μM upstream and downstream primers each 1.0 μL, cDNA template 2.0 μL, 5U/μL LA Taq DNA polymerase 0.2 μL, ddH₂ O 11.8 μL. The PCR amplification reaction program is preferably as follows: pre-denaturation at 94°C for 5 min; denaturation at 94°C for 30s; annealing at 94°C for 30s; extension at 72°C for 45s, 35 cycles; 72°C for 10 min; storage at 4°C.

用Oligo7软件设计引物扩增PeAPX5基因。引物序列如下：The primers were designed by Oligo7 software to amplify the PeAPX5 gene. The primer sequences are as follows:

上游引物：5′-ATGGCGAAGAACTACCCGGC-3′Upstream primer: 5'-ATGGCGAAGAACTACCCGGC-3'

下游引物：5′-TATGCATCAGCAAACCCCAGTTC-3′Downstream primer: 5'-TATGCATCAGCAAAACCCCAGTTC-3'

⑷PCR扩增得到目的片段后，将所述目的片段进行测序，得到PeAPX5基因。在PCR扩增后优选对目的片段进行纯化，对于纯化的方法没有特殊的限定，采用本领域技术人员熟知的DNA纯化试剂盒进行即可。(4) After PCR amplification to obtain the target fragment, the target fragment is sequenced to obtain the PeAPX5 gene. The target fragment is preferably purified after PCR amplification, and the purification method is not particularly limited, and can be carried out by using a DNA purification kit well known to those skilled in the art.

⑸纯化完成后，优选将所述纯化后的目的片段连接到pGEM-T Easy载体，导入大肠杆菌DH5α感受态细胞中，经菌落PCR验证为阳性克隆后进行测序。(5) After the purification is completed, the purified target fragment is preferably connected to the pGEM-T Easy vector, introduced into E. coli DH5α competent cells, and sequenced after the colony PCR is verified as a positive clone.

第二方面，本发明提供含有毛竹PeAPX5基因的生物材料，所述生物材料包括但不限于重组DNA、表达盒、转座子、质粒载体、病毒载体、工程菌或非可再生的植物部分。In a second aspect, the present invention provides biological materials containing the Moso bamboo PeAPX5 gene, including but not limited to recombinant DNA, expression cassettes, transposons, plasmid vectors, viral vectors, engineered bacteria or non-renewable plant parts.

第三方面，本发明提供毛竹PeAPX5基因或含有该基因的生物材料在植物抗逆调控(提高植物抗逆性)中的应用；其中，所述抗逆是指抗干旱、抗盐、耐低温。In a third aspect, the present invention provides the application of the Moso bamboo PeAPX5 gene or a biological material containing the gene in plant stress resistance regulation (improving plant stress resistance); wherein, the stress resistance refers to drought resistance, salt resistance, and low temperature resistance.

本发明所述植物包括但不限于拟南芥、毛竹。The plants of the present invention include, but are not limited to, Arabidopsis thaliana and Phyllostachys pubescens.

第四方面，本发明提供毛竹PeAPX5基因或含有该基因的生物材料在制备转基因植物中的应用。In a fourth aspect, the present invention provides the application of the Moso bamboo PeAPX5 gene or a biological material containing the gene in the preparation of transgenic plants.

第五方面，本发明提供本发明还提供毛竹PeAPX5基因或含有该基因的生物材料在植物育种中的应用。In a fifth aspect, the present invention provides the application of the Moso bamboo PeAPX5 gene or a biological material containing the gene in plant breeding.

所述育种目的是为提高植物抗旱能力。PeAPX5基因参与毛竹干旱、低温和盐胁迫应答。The purpose of the breeding is to improve the drought resistance of plants. PeAPX5 gene is involved in the response to drought, low temperature and salt stress in Phyllostachys pubescens.

第六方面，本发明提供一种提高植物抗旱能力的方法，包括：利用基因工程手段，在植物中过表达毛竹PeAPX5基因；In a sixth aspect, the present invention provides a method for improving the drought resistance of plants, comprising: overexpressing the Moso bamboo PeAPX5 gene in plants by means of genetic engineering;

所述过表达的方式选自以下1)～5)，或任选的组合：The overexpression mode is selected from the following 1) to 5), or an optional combination:

1)通过导入具有所述基因的质粒；1) by introducing a plasmid having the gene;

2)通过增加植物染色体上所述基因的拷贝数；2) by increasing the copy number of the gene on the plant chromosome;

3)通过改变植物染色体上所述基因的启动子序列；3) by changing the promoter sequence of the gene on the plant chromosome;

4)通过将强启动子与所述基因可操作地连接；4) by operably linking a strong promoter to the gene;

5)通过导入增强子。5) By introducing enhancers.

进一步地，采用农杆菌介导法将毛竹PeAPX5基因转入到拟南芥植株中，获得该基因过表达的转基因植株。Further, the Moso bamboo PeAPX5 gene was transferred into Arabidopsis thaliana plants by Agrobacterium-mediated method to obtain transgenic plants overexpressing the gene.

优选地，将毛竹PeAPX5基因构建到植物表达载体pCAMBIA2300上，转化农杆菌，然后浸染拟南芥花序，筛选转基因植株。Preferably, the Moso bamboo PeAPX5 gene is constructed into a plant expression vector pCAMBIA2300, transformed into Agrobacterium, and then dipped into Arabidopsis inflorescence to screen for transgenic plants.

在本发明的一个具体实施方式中，植物表达载体pCAMBIA2300-PeAPX5的构建方法如下：In a specific embodiment of the present invention, the construction method of plant expression vector pCAMBIA2300-PeAPX5 is as follows:

以反转录合成的cDNA为模板，进行PCR反应，在PeAPX5基因的上、下游分别引入EcoRI和HindIII酶切位点；将扩增产物连接到pGEM-T Easy载体，转化DH5α感受态细胞，进行序列测定；提取质粒，经EcoRI和HindIII双酶切的PeAPX5基因片段与pCAMBIA2300-CaMV35S连接，转化，提取质粒，进行序列测定，植物表达载体pCAMBIA2300-PeAPX5构建完成。Using the cDNA synthesized by reverse transcription as a template, a PCR reaction was performed, and EcoRI and HindIII restriction sites were introduced upstream and downstream of the PeAPX5 gene; the amplified product was connected to the pGEM-T Easy vector, transformed into DH5α competent cells, and carried out Sequence determination; plasmid extraction, the PeAPX5 gene fragment digested by EcoRI and HindIII was ligated with pCAMBIA2300-CaMV35S, transformed, and the plasmid was extracted, sequenced, and the plant expression vector pCAMBIA2300-PeAPX5 was constructed.

转基因拟南芥的制备方法如下：The preparation method of transgenic Arabidopsis is as follows:

将构建的植物表达载体pCAMBIA2300-PeAPX5转化农杆菌菌株GV3101感受态；选取阳性克隆摇菌，花序侵染及纯合子种子筛选；提取拟南芥阳性苗叶片RNA，反转录成cDNA，用引物PeAPX5-F、PeAPX5-R进行PCR鉴定。The constructed plant expression vector pCAMBIA2300-PeAPX5 was transformed into Agrobacterium strain GV3101 competent; the positive clones were selected for shaking bacteria, inflorescence infection and homozygous seed screening; the leaf RNA of Arabidopsis positive seedlings was extracted, reverse transcribed into cDNA, and the primer PeAPX5 was used. -F, PeAPX5-R were identified by PCR.

第七方面，本发明提供按照上述方法获得的转基因植物在植物育种中的应用。In a seventh aspect, the present invention provides the application of the transgenic plant obtained by the above method in plant breeding.

育种方法包括但不限于转基因、杂交、回交、自交或无性繁殖。Breeding methods include, but are not limited to, transgenic, crossing, backcrossing, selfing, or asexual reproduction.

本发明首次揭示了毛竹PeAPX5基因的生物学功能，通过构建PeAPX5基因表达载体，结合农杆菌介导的遗传转化法，异源转化拟南芥，考察PeAPX5对转基因拟南芥抗逆性的影响，并同时检测PeAPX5对毛竹干旱、低温和盐胁迫的响应，为毛竹转基因研究提供有力工具，为毛竹抗性分子育种提供有价值的候选基因。The invention discloses the biological function of Moso bamboo PeAPX5 gene for the first time. By constructing a PeAPX5 gene expression vector, combined with the genetic transformation method mediated by Agrobacterium, heterologously transforming Arabidopsis thaliana, and investigating the effect of PeAPX5 on the stress resistance of transgenic Arabidopsis, At the same time, the response of PeAPX5 to drought, low temperature and salt stress in Phyllostachys pubescens was detected, which provided a powerful tool for transgenic research of Phyllostachys pubescens and valuable candidate genes for molecular breeding of Phyllostachys pubescens.

附图说明Description of drawings

图1为本发明较佳实施例中PeAPX5基因克隆PCR产物电泳图(A)以及PCAMBIA2300-PeAPX5酶切产物电泳图(B)；其中，A中泳道1-2为PeAPX5 PCR产物，B中泳道1-2为PCAMBIA2300-PeAPX5酶切产物，M为DNA Marker。Fig. 1 is the electrophoresis image (A) of PeAPX5 gene clone PCR product and the electrophoresis image (B) of PCAMBIA2300-PeAPX5 digestion product in the preferred embodiment of the present invention; wherein, swimming lanes 1-2 in A are PeAPX5 PCR products, andswimming lane 1 in B -2 is PCAMBIA2300-PeAPX5 digestion product, M is DNA Marker.

图2为本发明较佳实施例中构建植物重组表达载体pCAMBIA2300-PeAPX5的流程图。Figure 2 is a flow chart of constructing a plant recombinant expression vector pCAMBIA2300-PeAPX5 in a preferred embodiment of the present invention.

图3为本发明较佳实施例中PeAPX5基因在干旱胁迫的毛竹根和叶中的相对表达量。Figure 3 shows the relative expression levels of PeAPX5 gene in the roots and leaves of Phyllostachys edulis under drought stress in a preferred embodiment of the present invention.

图4为本发明较佳实施例中PeAPX5基因在盐胁迫的毛竹根和叶中的相对表达量。Figure 4 is the relative expression level of PeAPX5 gene in salt-stressed Phyllostachys pubescens roots and leaves in a preferred embodiment of the present invention.

图5为本发明较佳实施例中PeAPX5基因在低温胁迫的毛竹根和叶中的相对表达量。Fig. 5 is the relative expression level of PeAPX5 gene in the roots and leaves of Phyllostachys edulis under low temperature stress in a preferred embodiment of the present invention.

图6为本发明较佳实施例中转PeAPX5基因拟南芥和野生型拟南芥在干旱胁迫下的表型差异。Figure 6 shows the phenotypic difference between Arabidopsis thaliana transgenic with PeAPX5 gene and wild-type Arabidopsis thaliana under drought stress in a preferred embodiment of the present invention.

图7为本发明较佳实施例中转PeAPX5基因拟南芥和野生型拟南芥在低温胁迫下的可溶性糖含量(A)、MDA含量(B)、抗氧化酶活性(C)、CAT含量(D)和APX活性(E)的变化。Fig. 7 is the soluble sugar content (A), MDA content (B), antioxidant enzyme activity (C), CAT content ( D) and changes in APX activity (E).

图8为本发明较佳实施例中转PeAPX5基因拟南芥和野生型拟南芥在盐胁迫下的可溶性糖含量(A)、MDA含量(B)、抗氧化酶活性(C)、CAT含量(D)和APX活性(E)的变化。Figure 8 shows the soluble sugar content (A), MDA content (B), antioxidant enzyme activity (C), CAT content ( D) and changes in APX activity (E).

具体实施方式Detailed ways

以下实施例用于说明本发明，但不用来限制本发明的范围。若未特别指明，实施例均按照常规实验条件，如分子克隆实验手册(Sambrook J&Russell DW，MolecularCloning:a Laboratory Manual，2001)，或按照制造厂商说明书建议的条件。The following examples are intended to illustrate the present invention, but not to limit the scope of the present invention. Unless otherwise specified, the examples are in accordance with conventional experimental conditions, such as Molecular Cloning Laboratory Manual (Sambrook J & Russell DW, Molecular Cloning: a Laboratory Manual, 2001), or in accordance with the conditions suggested by the manufacturer's instructions.

实施例1毛竹PeAPX5基因的克隆Example 1 Cloning of Moso bamboo PeAPX5 gene

以毛竹叶片为材料，按照Trizol RNA提取试剂盒(天根生化科技有限公司)说明书方法提取叶片总RNA，取1ng RNA按照反转录试剂盒(Promega，USA)反转录成cDNA，用RNase消化cDNA产物。根据毛竹基因组数据库http://www.forestrylab.org/db/PhePacBio/ExtractSeq/phe/index.php，用Prime Primer 5.0软件设计引物扩增PeAPX5基因。Using the leaves of Phyllostachys pubescens as material, the total RNA of leaves was extracted according to the instructions of the Trizol RNA extraction kit (Tiangen Biochemical Technology Co., Ltd.), and 1 ng of RNA was reversely transcribed into cDNA according to the reverse transcription kit (Promega, USA), and digested with RNase. cDNA product. According to the Moso bamboo genome database http://www.forestrylab.org/db/PhePacBio/ExtractSeq/phe/index.php, Prime Primer 5.0 software was used to design primers to amplify the PeAPX5 gene.

上游引物：5′-ATGGCGAAGAACTACCCGGC-3′Upstream primer: 5'-ATGGCGAAGAACTACCCGGC-3'

下游引物：5′-TATGCATCAGCAAACCCCAGTTC-3′Downstream primer: 5'-TATGCATCAGCAAAACCCCAGTTC-3'

聚合酶链式反应：polymerase chain reaction:

20μL反应体系：10×PCR Buffer 2.0μL，2.5mM dNTP Mix 2.0μL，10μM上、下游引物各1.0μL，cDNA模板2.0μL，5U/μL LA Taq DNA聚合酶0.2μL，ddH₂O 11.8μL。20 μL reaction system: 2.0 μL of 10×PCR Buffer, 2.0 μL of 2.5mM dNTP Mix, 1.0 μL of 10 μM upstream and downstream primers, 2.0 μL of cDNA template, 0.2 μL of 5U/μL LA Taq DNA polymerase, and 11.8 μL of ddH₂ O.

PCR反应程序：94℃预变性5min；94℃变性30s；94℃退火30s；72℃延伸45s，35个循环；72℃10min；4℃保存。PCR reaction program: pre-denaturation at 94 °C for 5 min; denaturation at 94 °C for 30 s; annealing at 94 °C for 30 s; extension at 72 °C for 45 s, 35 cycles; 72 °C for 10 min; storage at 4 °C.

回收产物连接到pGEM-T Easy载体，转化DH5α感受态细胞，挑选阳性克隆进行菌斑PCR检测，阳性克隆进行测序(上海生工生物工程公司)，测序结果准确无误。PeAPX5基因的核苷酸序列如SEQ ID NO:1所示。The recovered product was ligated to pGEM-T Easy vector, transformed into DH5α competent cells, positive clones were selected for plaque PCR detection, and positive clones were sequenced (Shanghai Sangon Bioengineering Company). The nucleotide sequence of the PeAPX5 gene is shown in SEQ ID NO:1.

实施例2植物表达载体pCAMBIA2300-PeAPX5的构建Example 2 Construction of plant expression vector pCAMBIA2300-PeAPX5

设计引物进行聚合酶链式反应，在目的基因PeAPX5的上下游分别引入EcoRI和HindIII双酶切位点，产物连接到pGEM-T Easy载体(Promega公司)，转化DH5α感受态细胞，进行序列测定，提取质粒，经EcoRI和HindIII双酶切的PeAPX5基因片段与经同样的酶酶切的pCAMBIA2300-CaMV35S载体连接，转化，提取质粒，进行序列测定。The primers were designed for polymerase chain reaction, and EcoRI and HindIII double enzyme cleavage sites were introduced into the upstream and downstream of the target gene PeAPX5 respectively. The product was connected to pGEM-T Easy vector (Promega Company), transformed into DH5α competent cells, and sequenced. The plasmid was extracted, the PeAPX5 gene fragment digested with EcoRI and HindIII was ligated with the pCAMBIA2300-CaMV35S vector digested with the same enzyme, transformed, and the plasmid was extracted and sequenced.

上游引物PeAPX5-F：5′-AGGAATTCATGGCGAAGAACTACCCGGCCG-3′Upstream primer PeAPX5-F: 5′-AGGAATTC ATGGCGAAGAACTACCCGGCCG-3′

下游引物PeAPX5-R：5′-CCGAAGCTTTTATGCATCAGCAAAC-3′Downstream primer PeAPX5-R: 5'-CCGAAGCTT TTATGCATCAGCAAAC-3'

⑴以毛竹叶片cDNA为模板进行PCR反应(1) PCR reaction with bamboo leaf cDNA as template

20μL反应体系：10×PCR Buffer 2.0μL，2.5mM dNTP Mix 2μL，10μM上、下游引物各1.0μL，cDNA模板2.0μL，5U/μL LA Taq DNA聚合酶0.2μL，ddH₂O 11.8μL。20 μL reaction system: 2.0 μL of 10×PCR Buffer, 2 μL of 2.5mM dNTP Mix, 1.0 μL of 10 μM upstream and downstream primers, 2.0 μL of cDNA template, 0.2 μL of 5U/μL LA Taq DNA polymerase, and 11.8 μL of ddH₂ O.

PCR反应程序：94℃预变性5min；94℃变性30s；94℃退火30s；72℃延伸45s，35个循环；72℃10min；4℃保存。PCR reaction program: pre-denaturation at 94 °C for 5 min; denaturation at 94 °C for 30 s; annealing at 94 °C for 30 s; extension at 72 °C for 45 s, 35 cycles; 72 °C for 10 min; storage at 4 °C.

⑵扩增产物回收及连接⑵Amplification product recovery and ligation

回收片段连接到pGEM-T Easy载体，转化DH5α感受态细胞，进行序列测定，测序结果准确无误。The recovered fragment was connected to the pGEM-T Easy vector, transformed into DH5α competent cells, and sequenced. The sequencing result was accurate.

⑶表达载体pCAMBIA2300-CaMV35S-PeAPX5的构建(3) Construction of expression vector pCAMBIA2300-CaMV35S-PeAPX5

用EcoRI和HindIII双酶切连接有PeAPX5片段的pGEM-T Easy(图1)，与EcoRI和HindIII双酶切的表达载体pCAMBIA2300-CaMV35S(Promega公司，美国)构建重组质粒，酶切体系如下(50μL)：The pGEM-T Easy (Figure 1) with the PeAPX5 fragment was double digested with EcoRI and HindIII, and the recombinant plasmid was constructed with the expression vector pCAMBIA2300-CaMV35S (Promega, USA) double digested with EcoRI and HindIII. The digestion system was as follows (50 μL) ):

37℃酶切4h；酶切产物经过琼脂糖凝胶电泳，用凝胶回收试剂盒(Axygen)回收质粒pCAMBIA2300-CaMV35S大片段和PeAPX5小片段。用T4 DNA连接酶连接两个回收产物，连接反应体系如下(20μL)：The digested products were subjected to agarose gel electrophoresis, and the plasmid pCAMBIA2300-CaMV35S large fragment and PeAPX5 small fragment were recovered by gel recovery kit (Axygen). The two recovered products were ligated with T4 DNA ligase, and the ligation reaction system was as follows (20 μL):

4℃过夜连接反应，将连接产物全部转化DH5α感受态细胞。37℃过夜培养，挑选单克隆，菌落PCR验证后，扩大培养，提取质粒pCAMBIA2300-PeAPX5，进行测序和酶切验证(图1)，植物重组表达质粒构建成功。植物重组表达载体pCAMBIA2300-PeAPX5构建流程见图2。The ligation reaction was performed overnight at 4°C, and all the ligation products were transformed into DH5α competent cells. After overnight cultivation at 37°C, single clones were selected, and after colony PCR verification, the culture was expanded, and the plasmid pCAMBIA2300-PeAPX5 was extracted for sequencing and enzyme digestion verification (Figure 1). The plant recombinant expression plasmid was successfully constructed. The construction flow of the plant recombinant expression vector pCAMBIA2300-PeAPX5 is shown in Figure 2.

实施例3植物表达载体pCAMBIA2300-PeAPX5转化拟南芥Example 3 Transformation of Arabidopsis thaliana with plant expression vector pCAMBIA2300-PeAPX5

⑴冻融法转化农杆菌GV3101菌株(1) Freeze-thaw method to transform Agrobacterium strain GV3101

将1ng重组表达载体质粒，加入到100μL感受态细胞GV3101中，冰浴10min后，将感受态细胞在液氮中速冻5min，迅速转移至37℃恒温水浴锅中水浴5min,后放置冰上5min，于离心管中加入600μL的LB液体培养基，在28℃摇床中震荡培养2-3h，复苏菌体。吸取60μL菌液涂布在含有Kan抗性(50mg/mL)和Rif(50mg/mL)抗性的YEP固体培养基中，于28℃恒温摇床中倒置平板培养2-3d左右，至长出白色菌落。挑取单克隆菌落PCR检测后，选取阳性克隆摇菌培养。Add 1 ng of recombinant expression vector plasmid to 100 μL of competent cells GV3101. After ice bathing for 10 minutes, the competent cells were snap-frozen in liquid nitrogen for 5 minutes, then quickly transferred to a constant temperature water bath at 37°C for 5 minutes, and then placed on ice for 5 minutes. Add 600 μL of LB liquid medium to the centrifuge tube, shake and culture in a shaker at 28°C for 2-3 hours, and recover the bacteria. Pipette 60μL of bacterial liquid and spread it on YEP solid medium containing Kan resistance (50mg/mL) and Rif (50mg/mL) resistance, and invert the plate in a constant temperature shaker at 28°C for about 2-3 days, until it grows out. White colonies. After picking up single clone colonies for PCR detection, select positive clones and shake them for culture.

⑵拟南芥花序浸染⑵ Arabidopsis inflorescence dip

将上述阳性克隆接种于10mL YEP(50μg/mL利福平+100μg/mL卡那霉素)液体培养基中，28℃培养箱震荡培养(160rpm)12h，取2mL培养液转移至200mL YEP(50μg/mL利福平+100μg/mL卡那霉素)中，进行大量培养，28℃培养箱震荡培养(160rpm)12h，培养液浓度OD₆₀₀达到1.8-2.2μg/mL，取50mL培养液，用50mL离心管，4℃5000rpm离心5min，用转化液(MS2.2g，5％蔗糖，调pH至5.8，加0.2％SilwetL-77混合)剧烈悬浮，将沉淀稀释至1.0μg/mL。制备200mL左右，以备浸染拟南芥。将刚开花的拟南芥地上部浸泡于转化液中3min，用保鲜膜包裹植株，暗培养12-16h后，去除保鲜膜，将植株置于培养箱中培养，待采收种子。The above-mentioned positive clones were inoculated into 10 mL of YEP (50 μg/mL rifampicin + 100 μg/mL kanamycin) liquid medium, cultured in a 28°C incubator with shaking (160 rpm) for 12 h, and 2 mL of the culture medium was transferred to 200 mL of YEP (50 μg/mL) /mL rifampicin + 100μg/mL kanamycin), carry out large-scale culture, 28 ℃ incubator shaking culture (160rpm) for 12h, the OD₆₀₀ of the culture solution reaches 1.8-2.2μg/mL, take 50mL culture solution, use 50mL centrifuge tube, centrifuge at 5000rpm at 4°C for 5min, use transformation solution (MS2.2g, 5% sucrose, adjust pH to 5.8, add 0.2% SilwetL-77 to mix) vigorously suspend, and dilute the precipitate to 1.0μg/mL. Prepare about 200mL for immersion in Arabidopsis. Immerse the shoots of the freshly bloomed Arabidopsis in the transformation solution for 3 minutes, wrap the plants with plastic wrap, and after culturing in the dark for 12-16 hours, remove the plastic wrap, and place the plants in an incubator for cultivation until the seeds are harvested.

⑶纯合子筛选(3) Homozygous screening

将T0代拟南芥种子置于离心管中，加1mL 70％酒精消毒5min后，再用2.6％次氯酸钠溶液1mL，消毒10min，然后用无菌水清洗5遍。将种子均匀播种于筛选培养基上1/2MS+100mg/L卡那霉素，经4℃低温纯化2d后置于人工气候培养箱中培养至长出4片子叶，将绿色的、正常生长的阳性植株移栽至土壤中栽培，待成熟后分单株收取T1代种子，应用同样的方法筛选T1代幼苗，统计T1代各株系阳性植株与非阳性植株的比例，并将比例约为3:1的株系的阳性植株移栽至土壤中培养，获得T2代种子。应用同样方法筛选T2代幼苗，获得T3代种子。Put the T0 generation Arabidopsis seeds in a centrifuge tube, add 1 mL of 70% alcohol to sterilize for 5 min, then use 1 mL of 2.6% sodium hypochlorite solution, sterilize for 10 min, and then wash with sterile water for 5 times. The seeds were evenly sown on the screening medium with 1/2MS+100mg/L kanamycin, purified at 4°C for 2 days, and then cultured in an artificial climate incubator until 4 cotyledons grew. The positive plants were transplanted into the soil for cultivation. After maturity, the T1 generation seeds were collected from individual plants. The same method was used to screen the T1 generation seedlings. The ratio of positive plants to non-positive plants in each T1 generation line was calculated, and the ratio was about 3 The positive plants of the lines with : 1 were transplanted into the soil and cultivated to obtain the T2 generation seeds. The T2 generation seedlings were screened by the same method, and the T3 generation seeds were obtained.

⑷阳性植株的PCR鉴定⑷ PCR identification of positive plants

提取阳性拟南芥叶片RNA，反转录成cDNA，用引物PeAPX5-F、PeAPX5-R进行PCR鉴定。发现阳性植株中均含有PeAPX5，表明PeAPX5成功转入拟南芥。The positive Arabidopsis leaf RNA was extracted, reverse transcribed into cDNA, and identified by PCR with primers PeAPX5-F and PeAPX5-R. It was found that all positive plants contained PeAPX5, indicating that PeAPX5 was successfully transferred into Arabidopsis thaliana.

实施例4干旱、盐、低温胁迫下毛竹PeAPX5基因表达量分析Example 4 Analysis of Moso bamboo PeAPX5 gene expression under drought, salt and low temperature stress

⑴材料处理⑴Material handling

毛竹种子采集于广西壮族自治区，置于恒温光照培养箱中，昼夜温度是25℃/18℃，光周期为光/暗16h/8h，培养至三个月左右，用200mM NaCl、20％PEG 6000、4℃分别模拟高盐、干旱和低温胁迫，分别取处理后0h、3h、6h、12h、24h、48h、72h、120h的幼嫩的主根和须根以及相同部位的叶片，在液氮中迅速冷冻，-80℃冷冻保存。Moso bamboo seeds were collected in Guangxi Zhuang Autonomous Region and placed in a constant temperature light incubator. The day and night temperature was 25°C/18°C, and the photoperiod was light/dark 16h/8h. The seeds were cultivated for about three months, using 200mM NaCl, 20% PEG 6000 , 4°C to simulate high-salt, drought and low temperature stress, respectively, take the young taproots and fibrous roots and leaves of the same parts at 0h, 3h, 6h, 12h, 24h, 48h, 72h, 120h after treatment, respectively, and rapidly in liquid nitrogen Freeze and store at -80°C.

⑵cDNA模板的合成(2) Synthesis of cDNA template

利用Trizol Reagent试剂提取毛竹实生苗根、幼茎及叶片中的总RNA，使用无RNA酶的DNase I(TIANDZ)去除基因组DNA，用紫外分光光度计测量A₂₆₀与A₂₈₀的比值及RNA浓度，并用1％琼脂糖凝胶电泳检测28S rRNA、18S rRNA和5S rRNA扩增条带的亮度和完整性，通过Promega公司的反转录试剂盒合成cDNA第一条链，合成产物置于-20℃冰箱保存。Utilize Trizol Reagent reagent to extract total RNA in Phyllostachys edulis seedling roots, young stems and leaves, use RNase-free DNase I (TIANDZ) to remove genomic DNA, measure the ratio of A₂₆₀ to A₂₈₀ and the RNA concentration with an ultraviolet spectrophotometer, The brightness and integrity of the amplified bands of 28S rRNA, 18S rRNA and 5S rRNA were detected by 1% agarose gel electrophoresis. The first strand of cDNA was synthesized by the reverse transcription kit of Promega Company, and the synthesized product was placed at -20°C. Store in refrigerator.

⑶实时荧光定量PCR(3) Real-time fluorescent quantitative PCR

通过实时荧光定量PCR(qRT–PCR)，检测目的基因表达情况。TIP41基因作为内参基因，TIP41-F：5'-AAAATCATTGTAGGCCATTGTCG-3'，TIP41-R：5'-ACTAAATTAAGCCAGCGGGAGTG-3'；PeAPX 5-F:5'-ACCCTCTGACATTTGACAACTCTT-3'，PeAPX 5-R:5'-ATCCGCAGCATATTTCTCCAC-3'。The expression of the target gene was detected by real-time quantitative PCR (qRT-PCR). TIP41 gene was used as an internal reference gene, TIP41-F: 5'-AAAATCATTGTAGGCCATTGTCG-3', TIP41-R: 5'-ACTAAATTAAGCCAGCGGGAGTG-3'; PeAPX 5-F: 5'-ACCCTCTGACATTTGACAACTCTT-3', PeAPX 5-R: 5' - ATCCGCAGCATATTTCTCCAC-3'.

10μL反应体系如下：The 10 μL reaction system is as follows:

反应程序：95℃1min；95℃10s，62℃10s，72℃20s，45个循环。Reaction program: 95°C for 1 min; 95°C for 10s, 62°C for 10s, 72°C for 20s, 45 cycles.

其他反应参数均为系统默认设置，每个反应设置3个生物学重复，用Roche

480仪分析数据，利用2^-ΔΔCT法分析3次生物学实验数据，用Excel进行作图。Other reaction parameters are the default settings of the system, each reaction is set to 3 biological replicates, using Roche

480 instrument to analyze the data, use the 2^-ΔΔCT method to analyze the data of three biological experiments, and use Excel to make graphs.

⑷实验结果与分析⑷Experimental results and analysis

干旱、盐、低温胁迫处理后，分别检测PeAPX5基因在毛竹实生苗根和叶片中的表达情况，结果如图3～图5所示：在干旱处理后，PeAPX5在叶片和根中的表达量呈先上升后下降趋势，处理48h表达水平达到峰值，分别为对照组的13.4倍和22.7倍；在盐和低温处理后，PeAPX5在根中的表达量也均有明显上调。表明PeAPX5基因可能参与毛竹干旱、低温和盐胁迫应答过程。After drought, salt and low temperature stress treatments, the expression of PeAPX5 gene in the roots and leaves of Phyllostachys pubescens seedlings was detected respectively. It first increased and then decreased, and the expression level reached a peak at 48h after treatment, which was 13.4 times and 22.7 times that of the control group, respectively. The expression levels of PeAPX5 in roots were also significantly up-regulated after salt and low temperature treatments. It indicated that PeAPX5 gene may be involved in the response process of Phyllostachys pubescens to drought, low temperature and salt stress.

实施例5转PeAPX5基因拟南芥抗旱性分析Example 5 Drought resistance analysis of Arabidopsis thaliana transgenic PeAPX5 gene

对转PeAPX5基因拟南芥T3代幼苗和野生型幼苗进行干旱胁迫处理，观察植株的表型差异。与野生型幼苗相比较，转PeAPX5基因的幼苗在干旱胁迫处理中有明显的胁迫响应特征。干旱处理前，转基因拟南芥和野生型拟南芥生长状态基本一致，叶片颜色为深绿色，表型差异不明显。自然干旱8d后，转基因拟南芥和拟南芥均出现明显的表型变化，表现为叶片干枯卷缩，花茎倒伏。但与野生型拟南芥相比，转基因拟南芥受干旱影响较小，未呈现死亡状态。说明转基因拟南芥的抗旱性比野生型增强(图6)。The T3 generation seedlings and wild-type seedlings of PeAPX5 transgenic Arabidopsis were treated with drought stress, and the phenotypic differences of the plants were observed. Compared with wild-type seedlings, the seedlings transfected with PeAPX5 gene had obvious stress response characteristics under drought stress treatment. Before drought treatment, the growth state of transgenic Arabidopsis and wild-type Arabidopsis was basically the same, the leaf color was dark green, and the phenotypic difference was not obvious. After 8 days of natural drought, both transgenic Arabidopsis and Arabidopsis showed obvious phenotypic changes, such as withered and curled leaves and lodging of flower stems. However, compared with wild-type Arabidopsis, transgenic Arabidopsis was less affected by drought and did not appear dead. It indicated that the drought resistance of transgenic Arabidopsis was enhanced compared with wild type (Fig. 6).

实施例6转PeAPX5基因拟南芥抗盐性分析Example 6 Salt tolerance analysis of Arabidopsis thaliana transgenic PeAPX5 gene

正常生长条件下，野生型拟南芥MDA含量和APX酶活性低于转基因拟南芥OE-1和OE-2，而可溶性糖含量高于转基因拟南芥，CAT含量和SOD活性无明显差异。200mM NaCl溶液处理24h后，转基因和野生型拟南芥MDA含量均上升，但转基因株系的增加量明显小于野生型，而转基因株系中可溶性糖和CAT含量及APX和SOD活性都明显高于野生型(图7)。Under normal growth conditions, the MDA content and APX activity of wild-type Arabidopsis were lower than those of transgenic Arabidopsis OE-1 and OE-2, while the soluble sugar content was higher than that of transgenic Arabidopsis. There was no significant difference in CAT content and SOD activity. After 24h treatment with 200 mM NaCl solution, the MDA content of transgenic and wild-type Arabidopsis increased, but the increase in transgenic lines was significantly smaller than that of wild-type, while the contents of soluble sugar and CAT and the activities of APX and SOD in transgenic lines were significantly higher than those of the wild type. Wild type (Figure 7).

实施例7转PeAPX5基因拟南芥耐低温分析Example 7 Analysis of low temperature resistance in Arabidopsis transgenic PeAPX5 gene

将转PeAPX5基因拟南芥T3代幼苗和野生型幼苗置于4℃进行低温胁迫，处理3d后，野生型拟南芥MDA含量增加，而转基因拟南芥MDA含量减少，且转基因株系中可溶性糖和CAT含量及APX和SOD活性均高于野生型(图8)。Transgenic Arabidopsis thaliana T3 seedlings and wild-type seedlings were placed under low temperature stress at 4°C. After 3 days of treatment, the MDA content of wild-type Arabidopsis increased, while the MDA content of transgenic Arabidopsis decreased, and the soluble soluble in transgenic lines Sugar and CAT contents and APX and SOD activities were higher than wild type (Fig. 8).

虽然，上文中已经用一般性说明及具体实施方案对本发明作了详尽的描述，但在本发明基础上，可以对之做一些修改或改进，这对本领域技术人员而言是显而易见的。因此，在不偏离本发明精神的基础上所做的这些修改或改进，均属于本发明要求保护的范围。Although the present invention has been described in detail above with general description and specific embodiments, some modifications or improvements can be made on the basis of the present invention, which will be obvious to those skilled in the art. Therefore, these modifications or improvements made without departing from the spirit of the present invention fall within the scope of the claimed protection of the present invention.

序列表sequence listing

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Claims (6)

Translated fromChinese

1.毛竹PeAPX5基因或含有毛竹PeAPX5基因的生物材料在植物抗逆调控中的应用；其中，所述抗逆是指抗干旱、抗盐、耐低温；1. the application of the biological material containing the Moso bambooPeAPX5 gene or the biological material containing the Moso bambooPeAPX5 gene in plant stress resistance regulation; Wherein, described stress resistance refers to drought resistance, salt resistance, low temperature resistance;所述毛竹PeAPX5基因编码的蛋白质的氨基酸序列如SEQ ID NO:2所示；The amino acid sequence of the protein encoded by the Moso bambooPeAPX5 gene is as shown in SEQ ID NO:2;所述植物为拟南芥。The plant is Arabidopsis.2.提高植物抗旱能力的方法，其特征在于，包括：利用基因工程手段，在植物中过表达毛竹PeAPX5基因；2. the method for improving plant drought resistance, is characterized in that, comprises: utilizes genetic engineering means, in plant overexpresses Phyllostachys pubescensPeAPX5 gene;所述过表达的方式选自以下1）~5），或任选的组合：The overexpression mode is selected from the following 1) to 5), or an optional combination:1）通过导入具有所述基因的质粒；1) by introducing a plasmid having the gene;2）通过增加植物染色体上所述基因的拷贝数；2) by increasing the copy number of said gene on the plant chromosome;3）通过改变植物染色体上所述基因的启动子序列；3) by changing the promoter sequence of the gene on the plant chromosome;4）通过将强启动子与所述基因可操作地连接；4) by operably linking a strong promoter to the gene;5）通过导入增强子；5) By importing enhancers;所述毛竹PeAPX5基因编码的蛋白质的氨基酸序列如SEQ ID NO:2所示；The amino acid sequence of the protein encoded by the Moso bambooPeAPX5 gene is as shown in SEQ ID NO:2;所述植物为拟南芥。The plant is Arabidopsis.3.根据权利要求2所述的方法，其特征在于，采用农杆菌介导法将毛竹PeAPX5基因转入到拟南芥植株中，获得该基因过表达的转基因植株。3 . The method according to claim 2 , wherein the Moso bambooPeAPX5 gene is transferred into an Arabidopsis thaliana plant by Agrobacterium-mediated method to obtain a transgenic plant overexpressing the gene. 4 .4.根据权利要求3所述的方法，其特征在于，将毛竹PeAPX5基因构建到植物表达载体pCAMBIA2300上，转化农杆菌，然后浸染拟南芥花序，筛选转基因植株。4. The method according to claim 3, wherein the Moso bambooPeAPX5 gene is constructed on the plant expression vector pCAMBIA2300, transformed into Agrobacterium, and then dipped in Arabidopsis inflorescence to screen the transgenic plants.5.根据权利要求2-4任一项所述方法获得的转基因植物在培育抗干旱、抗盐、耐低温的植物中的应用；5. the application of the transgenic plant obtained according to the method of any one of claims 2-4 in cultivating drought-resistant, salt-resistant, low-temperature-resistant plants;所述植物为拟南芥。The plant is Arabidopsis.6.根据权利要求5所述的应用，其特征在于，育种方法包括转基因、杂交、回交、自交或无性繁殖。6. The use according to claim 5, characterized in that the breeding method comprises transgenic, hybridization, backcrossing, self-crossing or asexual reproduction.

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