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CN106854238B - Plant adversity resistance related protein TabZIP14 and its encoding gene and application - Google Patents

Plant adversity resistance related protein TabZIP14 and its encoding gene and application Download PDF

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CN106854238B
CN106854238B CN201510896097.3A CN201510896097A CN106854238B CN 106854238 B CN106854238 B CN 106854238B CN 201510896097 A CN201510896097 A CN 201510896097A CN 106854238 B CN106854238 B CN 106854238B
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孔秀英
张丽娜
夏川
张立超
赵光耀
贾继增
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Institute of Crop Sciences of Chinese Academy of Agricultural Sciences
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    • C12N15/8271Phenotypically and genetically modified plants via recombinant DNA technology with agronomic (input) traits, e.g. crop yield for stress resistance, e.g. heavy metal resistance
    • C12N15/8273Phenotypically and genetically modified plants via recombinant DNA technology with agronomic (input) traits, e.g. crop yield for stress resistance, e.g. heavy metal resistance for drought, cold, salt resistance

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Abstract

本发明公开了一种植物抗逆性相关蛋白TabZIP14及其编码基因与应用。本发明所提供的TabZIP14蛋白质,具体是如下(a)或(b):(a)由序列表中序列1所示的氨基酸序列组成的蛋白质;(b)将序列1的氨基酸序列经过一个或几个氨基酸残基的取代和/或缺失和/或添加,且与植物抗逆性相关的由序列1衍生的蛋白质。本发明所提供的TaAREB3蛋白及其编码基因在提高植物抗逆性方面具有重要意义,将在培育高抗逆性如强抗冻性和强抗盐性植物品种中发挥重要作用。The invention discloses a plant stress resistance-related protein TabZIP14, its coding gene and application. The TabZIP14 protein provided by the present invention is specifically the following (a) or (b): (a) a protein composed of the amino acid sequence shown in Sequence 1 in the sequence listing; (b) the amino acid sequence of Sequence 1 undergoes one or several Substitution and/or deletion and/or addition of amino acid residues, and a protein derived from sequence 1 related to plant stress resistance. The TaAREB3 protein and its coding gene provided by the present invention are of great significance in improving stress resistance of plants, and will play an important role in cultivating high stress resistance such as strong frost resistance and strong salt resistance plant varieties.

Description

植物抗逆性相关蛋白TabZIP14及其编码基因与应用Plant stress resistance related protein TabZIP14 and its coding gene and application

技术领域technical field

本发明属于植物基因工程领域,涉及一种植物抗逆性相关蛋白TabZIP14及其编码基因与应用。The invention belongs to the field of plant genetic engineering, and relates to a plant stress resistance-related protein TabZIP14, its coding gene and application.

背景技术Background technique

非生物胁迫是影响作物生长发育和产量的主要因素之一,开发和利用抗逆品种在盐碱地和缺水地区显得尤为重要。最近几年,在提高植物抗逆性的基因方面取得一些进展。转录因子也是响应植物非生物胁迫逆境的关键调节因子,而且过表达一些转录因子基因可以提高植物的抗逆性。bZIP就是存在于植物中的一种转录因子。Abiotic stress is one of the main factors affecting crop growth and yield, and the development and utilization of stress-resistant varieties is particularly important in saline-alkali land and water-scarce areas. In recent years, some progress has been made in genes that improve plant stress resistance. Transcription factors are also key regulators in response to plant abiotic stress stress, and overexpression of some transcription factor genes can improve plant stress resistance. bZIP is a transcription factor that exists in plants.

在逆境胁迫下植物体内会产生一系列应答反应,伴随着许多生理生化及发育上的变化。明确植物对逆境的反应机制,将为抗逆基因工程研究和应用提供科学论据。目前,植物抗逆性研究已逐渐深入到细胞、分子水平,并与遗传学和遗传工程研究相结合,探索用生物技术来改进植物生长特性,其目的是提高植物对逆境的适应能力。Under adversity stress, plants will produce a series of responses, accompanied by many physiological, biochemical and developmental changes. Clarifying the response mechanism of plants to stress will provide scientific evidence for the research and application of stress-resistant genetic engineering. At present, the research on plant stress resistance has gradually penetrated into the cellular and molecular levels, combined with genetics and genetic engineering research, and explored the use of biotechnology to improve plant growth characteristics, the purpose of which is to improve the adaptability of plants to adversity.

因此,利用现代分子生物技术,挖掘抗逆关键基因,通过基因工程改良植物特别是农作物的抗逆性,对于保障国家粮食安全具有重要意义。Therefore, using modern molecular biotechnology to excavate key genes for stress resistance and improve the stress resistance of plants, especially crops, through genetic engineering is of great significance for ensuring national food security.

发明内容Contents of the invention

本发明的目的是提供一种植物抗逆性相关蛋白TabZIP14及其编码基因与应用。The object of the present invention is to provide a plant stress resistance related protein TabZIP14 and its coding gene and application.

本发明所提供的TabZIP14蛋白来源于小麦(Triticum aestivum L.)品种中国春,具体可为如下(a)或(b):The TabZIP14 protein provided by the present invention is derived from wheat (Triticum aestivum L.) variety China Spring, which can be specifically as follows (a) or (b):

(a)由序列表中序列1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 Sequence 1 in the Sequence Listing;

(b)将序列1的氨基酸序列经过一个或几个氨基酸残基的取代和/或缺失和/或添加,且与植物抗逆性相关的由序列1衍生的蛋白质。(b) Substitution and/or deletion and/or addition of one or several amino acid residues to the amino acid sequence of Sequence 1, and a protein derived from Sequence 1 that is related to plant stress resistance.

为了便于上述(a)中所示蛋白质的纯化,可在由序列表中序列1的氨基酸残基序列组成的蛋白质的氨基末端或羧基末端连接上如下表所示的标签。In order to facilitate the purification of the protein shown in (a) above, the amino-terminal or carboxy-terminal of the protein consisting of the amino acid residue sequence of Sequence 1 in the Sequence Listing can be attached with the tags shown in the table below.

表:标签的序列Table: Sequence of Labels

标签Label 残基Residues 序列sequence Poly-ArgPoly-Arg 5-6(通常为5个)5-6 (usually 5) RRRRRRRRRR Poly-HisPoly-His 2-10(通常为6个)2-10 (usually 6) HHHHHHHHHHHH FLAGFLAG 88 DYKDDDDKDYKDDDDK Strep-tag IIStrep-tag II 88 WSHPQFEKWSHPQFEK c-mycc-myc 1010 EQKLISEEDLEQKLISEEDL

上述(b)中的蛋白质可人工合成,也可先合成其编码基因,再进行生物表达得到。上述(b)中的蛋白质的编码基因可通过将序列表中序列2所示的DNA序列中缺失一个或几个氨基酸残基的密码子,和/或进行一个或几个碱基对的错义突变。The protein in (b) above can be synthesized artificially, or its coding gene can be synthesized first, and then obtained by biological expression. The protein-encoding gene in (b) above can be deleted by deleting one or several amino acid residue codons in the DNA sequence shown in Sequence 2 in the sequence listing, and/or carrying out one or several base pairs of missense mutation.

编码所述蛋白质的核酸分子也属于本发明的保护范围。Nucleic acid molecules encoding the protein also belong to the protection scope of the present invention.

所述核酸分子可以是DNA,如cDNA、基因组DNA或重组DNA;所述核酸分子也可以是RNA,如mRNA、hnRNA或tRNA等。The nucleic acid molecule can be DNA, such as cDNA, genomic DNA or recombinant DNA; the nucleic acid molecule can also be RNA, such as mRNA, hnRNA or tRNA.

在本发明的一个实施例中,所述核酸分子具体为编码所述蛋白质的基因,所述基因具体可为如下1)-4)中任一的DNA分子:In one embodiment of the present invention, the nucleic acid molecule is specifically a gene encoding the protein, and the gene can specifically be a DNA molecule in any of the following 1)-4):

1)序列表中序列2所示的DNA分子;1) The DNA molecule shown in sequence 2 in the sequence listing;

2)序列表中序列2的第181-1383位所示的DNA分子;2) the DNA molecule shown in No. 181-1383 of Sequence 2 in the sequence listing;

3)在严格条件下与1)或2)限定的DNA分子杂交且编码抗逆性相关蛋白的DNA分子;3) A DNA molecule that hybridizes to the DNA molecule defined in 1) or 2) under stringent conditions and encodes a stress resistance-related protein;

4)与1)-3)中任一限定的DNA序列具有90%以上同一性,且编码抗逆性相关蛋白的DNA分子。4) A DNA molecule that has more than 90% identity with the DNA sequence defined in any one of 1)-3) and encodes a stress resistance-related protein.

其中,序列2由1576个核苷酸组成,第181-1383位为ORF,编码序列表中序列1所示的蛋白质。Among them, sequence 2 consists of 1576 nucleotides, and the 181-1383rd position is ORF, encoding the protein shown in sequence 1 in the sequence listing.

含有上述核酸分子的重组载体、表达盒、转基因细胞系或重组微生物也属于本发明的保护范围。Recombinant vectors, expression cassettes, transgenic cell lines or recombinant microorganisms containing the above nucleic acid molecules also belong to the protection scope of the present invention.

所述重组载体可为重组表达载体,也可为重组克隆载体。The recombinant vector can be a recombinant expression vector or a recombinant cloning vector.

所述重组表达载体可用现有的植物表达载体构建。所述植物表达载体包括双元农杆菌载体和可用于植物微弹轰击的载体等,如pGreen0029、pCAMBIA3301、pCAMBIA1300、pBI121、pBin19、pCAMBIA2301、pCAMBIA1301-UbiN或其它衍生植物表达载体。所述植物表达载体还可包含外源基因的3’端非翻译区域,即包含聚腺苷酸信号和任何其它参与mRNA加工或基因表达的DNA片段。所述聚腺苷酸信号可引导聚腺苷酸加入到mRNA前体的3’端。使用所述基因构建重组表达载体时,在其转录起始核苷酸前可加上任何一种增强型、组成型、组织特异型或诱导型启动子,例如花椰菜花叶病毒(CAMV)35S启动子、泛素基因Ubiquitin启动子(pUbi)、胁迫诱导型启动子rd29A等,它们可单独使用或与其它的植物启动子结合使用;此外,使用本发明的基因构建重组表达载体时,还可使用增强子,包括翻译增强子或转录增强子,这些增强子区域可以是ATG起始密码子或邻接区域起始密码子等,但必需与编码序列的阅读框相同,以保证整个序列的正确翻译。所述翻译控制信号和起始密码子的来源是广泛的,可以是天然的,也可以是合成的。翻译起始区域可以来自转录起始区域或结构基因。为了便于对转基因植物细胞或植物进行鉴定及筛选,可对所用重组表达载体进行加工,如加入可在植物中表达的编码可产生颜色变化的酶或发光化合物的基因、具有抗性的抗生素标记物或是抗化学试剂标记基因等。也可不加任何选择性标记基因,直接以逆境筛选转化植株。The recombinant expression vector can be constructed with existing plant expression vectors. The plant expression vectors include binary Agrobacterium vectors and vectors that can be used for plant microprojectile bombardment, such as pGreen0029, pCAMBIA3301, pCAMBIA1300, pBI121, pBin19, pCAMBIA2301, pCAMBIA1301-UbiN or other derived plant expression vectors. The plant expression vector can also include the 3' untranslated region of the foreign gene, that is, the polyadenylation signal and any other DNA fragments involved in mRNA processing or gene expression. The polyA signal directs the addition of polyA to the 3' end of the pre-mRNA. When using the gene to construct a recombinant expression vector, any enhanced, constitutive, tissue-specific or inducible promoter can be added before its transcription initiation nucleotide, such as cauliflower mosaic virus (CAMV) 35S promoter promoter, ubiquitin gene Ubiquitin promoter (pUbi), stress-inducible promoter rd29A, etc., they can be used alone or in combination with other plant promoters; in addition, when using the gene of the present invention to construct a recombinant expression vector, it can also be used Enhancers include translation enhancers or transcription enhancers. These enhancer regions can be ATG start codons or adjacent region start codons, etc., but must be in the same reading frame as the coding sequence to ensure correct translation of the entire sequence. The sources of the translation control signals and initiation codons are extensive and can be natural or synthetic. The translation initiation region can be from a transcription initiation region or a structural gene. In order to facilitate the identification and screening of transgenic plant cells or plants, the recombinant expression vectors used can be processed, such as adding genes that encode enzymes that can produce color changes or luminescent compounds that can be expressed in plants, antibiotic markers with resistance Or anti-chemical reagent marker genes, etc. It is also possible to directly select transformed plants by stress without adding any selectable marker gene.

在本发明中,所述重组表达载体中启动所述基因转录的启动子具体为35S启动子。In the present invention, the promoter in the recombinant expression vector that initiates the transcription of the gene is specifically the 35S promoter.

进一步,所述重组表达载体为将pEarleyGate 100载体的attR1和attR2之间的小片段替换为所述基因后得到的重组质粒。Further, the recombinant expression vector is a recombinant plasmid obtained by replacing the small fragment between attR1 and attR2 of the pEarleyGate 100 vector with the gene.

更加具体的,所述重组表达载体是将所述基因(序列2的第181-1383位)通过BP反应重组到入门载体上,得到入门质粒;再将入门质粒与目标载体通过LR反应得到的目的重组质粒;所述入门载体具体为pDONR/Zeo(该载体具体为invitrogen公司产品,产品目录号为12535-035);所述目标载体具体为pEarleyGate 100(该载体具体为自invitrogen公司产品);所述pEarleyGate 100在文献“Zhang L,Zhang L,Xia C,Zhao G,Liu J,Jia J,KongX.A novel wheat bZIP transcription factor,TabZIP60,confers multiple abioticstress tolerances in transgenic Arabidopsis.Physiol Plant.2015,153(4):538-554.”中公开过。More specifically, the recombinant expression vector is obtained by recombining the gene (position 181-1383 of Sequence 2) into the entry vector through BP reaction to obtain the entry plasmid; Recombinant plasmid; the entry vector is specifically pDONR/Zeo (the carrier is specifically a product of Invitrogen Company, and the product catalog number is 12535-035); the target vector is specifically pEarleyGate 100 (the specific vector is a product of Invitrogen Company); the The pEarleyGate 100 described in the literature "Zhang L, Zhang L, Xia C, Zhao G, Liu J, Jia J, Kong X. A novel wheat bZIP transcription factor, TabZIP60, confers multiple abiotic stress tolerances in transgenic Arabidopsis. Physiol Plant. 2015, 153( 4):538-554." published in.

所述表达盒由能够启动所述基因表达的启动子,所述基因,以及转录终止序列组成。The expression cassette consists of a promoter capable of driving the expression of the gene, the gene, and a transcription termination sequence.

所述转基因细胞系为转入所述基因的非繁殖材料。The transgenic cell line is the non-reproductive material transferred with the gene.

所述蛋白质或所述核酸分子或所述重组载体、表达盒、转基因细胞系或重组微生物在如下任一中的应用也属于本发明的保护范围:The application of the protein or the nucleic acid molecule or the recombinant vector, expression cassette, transgenic cell line or recombinant microorganism in any of the following also belongs to the protection scope of the present invention:

(a)调控植物抗逆性;(a) regulating plant stress resistance;

(b)选育抗逆性提高的植物品种。(b) Breeding plant varieties with improved stress resistance.

在(a)中,所述调控植物的抗逆性具体体现在:在所述植物体内,若所述基因或所述蛋白的表达量越高,则所述植物的抗逆性越强;若所述基因或所述蛋白的表达量越低,则所述植物的抗逆性越弱。In (a), the regulation of the stress resistance of the plant is specifically reflected in: in the plant, if the expression level of the gene or the protein is higher, the stress resistance of the plant is stronger; if The lower the expression level of the gene or the protein, the weaker the stress resistance of the plant.

在(b)中,所述选育抗逆性增强的植物品种的方法,具体可包括将所述基因或所述蛋白表达量较高的植株作为亲本进行杂交的步骤。In (b), the method for breeding a plant variety with enhanced stress resistance may specifically include the step of crossing a plant with a higher expression level of the gene or the protein as a parent.

本发明还提供了一种培育抗逆性提高的转基因植物的方法。The invention also provides a method for cultivating transgenic plants with improved stress resistance.

本发明所提供的培育抗逆性提高的转基因植物的方法,具体可包括如下(a1)和(a2)的步骤:The method for cultivating transgenic plants with improved stress resistance provided by the present invention may specifically include the following steps (a1) and (a2):

(a1)向受体植物中导入所述蛋白质的编码基因,得到表达所述编码基因的转基因植物;(a1) introducing the gene encoding the protein into the recipient plant to obtain a transgenic plant expressing the gene encoding;

(a2)从步骤(a1)所得转基因植物中得到与所述受体植物相比,抗逆性提高的转基因植物。(a2) obtaining a transgenic plant with improved stress resistance compared with the recipient plant from the transgenic plant obtained in step (a1).

所述蛋白质在所述转基因植物中的表达量高于所述受体植物;编码所述蛋白质的基因是如下1)-4)中任一的DNA分子:The expression level of the protein in the transgenic plant is higher than that of the recipient plant; the gene encoding the protein is any DNA molecule in the following 1)-4):

1)序列表中序列2所示的DNA分子;1) The DNA molecule shown in sequence 2 in the sequence listing;

2)序列表中序列2的第181-1383位所示的DNA分子;2) the DNA molecule shown in No. 181-1383 of Sequence 2 in the sequence listing;

3)在严格条件下与1)或2)限定的DNA分子杂交且编码抗逆性相关蛋白的DNA分子;3) A DNA molecule that hybridizes to the DNA molecule defined in 1) or 2) under stringent conditions and encodes a stress resistance-related protein;

4)与1)-3)中任一限定的DNA序列具有90%以上同一性,且编码抗逆性相关蛋白的DNA分子。4) A DNA molecule that has more than 90% identity with the DNA sequence defined in any one of 1)-3) and encodes a stress resistance-related protein.

所述基因具体可通过上述任一所述重组表达载体导入所述受体植物中,得到所述转基因植物。具体可通过使用Ti质粒、Ri质粒、植物病毒载体、直接DNA转化、显微注射、电导、农杆菌介导、基因枪等常规生物学方法将所述重组表达载体转化植物细胞或组织,并将转化的植物组织培育成植株。Specifically, the gene can be introduced into the recipient plant through any of the above-mentioned recombinant expression vectors to obtain the transgenic plant. Specifically, the recombinant expression vector can be transformed into plant cells or tissues by using conventional biological methods such as Ti plasmid, Ri plasmid, plant virus vector, direct DNA transformation, microinjection, conductance, Agrobacterium-mediated, gene gun, etc., and The transformed plant tissue is grown into plants.

在本发明中,所述基因是通过重组农杆菌导入的,所述重组农杆菌是将所述重组表达载体导入农杆菌得到的;所述农杆菌具体为农杆菌GV3101-pMP 90,该菌在文献“Xu Z,Kim S,Hyeon D,Kim D,et al.The Arabidopsis thaliana NAC transcription factorfamily:structure–function relationships and determinants of ANAC019 stresssignaling.Biochem.J.2010,426:183-196.”中公开过。In the present invention, the gene is introduced by recombinant Agrobacterium, which is obtained by introducing the recombinant expression vector into Agrobacterium; the Agrobacterium is specifically Agrobacterium GV3101-pMP 90, which is Document "Xu Z, Kim S, Hyeon D, Kim D, et al. The Arabidopsis thaliana NAC transcription factor family: structure–function relationships and determinants of ANAC019 stress signaling. Biochem. J. 2010, 426: 183-196." .

在上述应用或方法中,所述抗逆性为如下中的至少一种:抗冻性、抗盐性。In the above application or method, the stress resistance is at least one of the following: frost resistance and salt resistance.

在本发明中,所述抗冻性中的“冻”具体体现为-10℃处理3h。In the present invention, the "freezing" in the frost resistance is specifically embodied as treatment at -10°C for 3 hours.

在本发明中,所述抗盐性中的“盐”具体体现为150-200mM NaCl处理。In the present invention, the "salt" in the salt resistance is embodied as 150-200mM NaCl treatment.

在上述应用或方法中,所述植物可为双子叶植物,也可为单子叶植物。如:小麦、烟草或拟南芥等。In the above application or method, the plant may be a dicotyledonous plant or a monocotyledonous plant. Such as: wheat, tobacco or Arabidopsis, etc.

在本发明中,所述植物为拟南芥,具体为拟南芥Columbia-0亚型。In the present invention, the plant is Arabidopsis thaliana, specifically Arabidopsis Columbia-0 subtype.

实验证明,将可表达序列表中序列2所示DNA分子(TabZIP14基因)的重组表达载体转化拟南芥得到的转基因植株,与相同条件下的野生型植株相比:在抗冻性实验(-10℃处理3h)中,野生型植株的存活率为16%,转TabZIP14基因的株系的存活率为79%。在抗盐实验中,150mM NaCl处理后,野生型拟南芥的根长明显受到抑制,T3代转TabZIP14基因拟南芥L1、L2和L3株系的根长分别是野生型植株根长的1.4倍、1.3倍和1.4倍,部分野生型的拟南芥叶子呈现白化现象,白化率为37%±0.5%,而T3代转TabZIP14基因拟南芥叶片全部保持绿色;200mM NaCl处理后,T3代转TabZIP14基因拟南芥L1、L2和L3株系的根长比分别是野生型植株根长的1.2倍、1.3倍和1.3倍,野生型植株的叶片几乎全部呈白化现象,白化率为98%±0.4%,而T3代转TabZIP14基因拟南芥叶片基本呈现绿色状态。以上结果说明转TabZIP14基因拟南芥的抗盐性明显提高。本发明所提供的TabZIP14蛋白及其编码基因在提高植物抗逆性方面具有重要意义,将在培育高抗逆性如强抗冻性和强抗盐性植物品种中发挥重要作用。Experiments have shown that the transgenic plants obtained by transforming the recombinant expression vector of the DNA molecule (TabZIP14 gene) shown in sequence 2 in the expressible sequence table to Arabidopsis, compared with wild-type plants under the same conditions: in the frost resistance experiment (- In the treatment at 10° C. for 3 h), the survival rate of wild-type plants was 16%, and the survival rate of the lines transfected with TabZIP14 gene was 79%. In the salt resistance experiment, after 150mM NaCl treatment, the root length of wild-type Arabidopsis was significantly inhibited, and the root lengths of T3 transgenic TabZIP14 gene Arabidopsis L1, L2 and L3 lines were 1.4 times the root length of wild-type plants. times, 1.3 times and 1.4 times, some wild-type Arabidopsis thaliana leaves showed albinism, and the albinism rate was 37%±0.5%, while the leaves of T3 generation Arabidopsis transgenic TabZIP14 gene remained green; after 200mM NaCl treatment, T3 generation The root length ratios of Arabidopsis transgenic Arabidopsis L1, L2, and L3 lines were 1.2, 1.3, and 1.3 times that of wild-type plants, and the leaves of wild-type plants were almost all albino, with an albino rate of 98%. ±0.4%, while the leaves of Arabidopsis transgenic with TabZIP14 gene in the T3 generation were basically green. The above results indicated that the salt resistance of Arabidopsis transgenic with TabZIP14 gene was significantly improved. The TabZIP14 protein and its coding gene provided by the present invention are of great significance in improving plant stress resistance, and will play an important role in cultivating high stress resistance such as strong frost resistance and strong salt resistance plant varieties.

附图说明Description of drawings

图1为PEG胁迫下小麦TabZIP14基因的相对表达量。Figure 1 shows the relative expression level of wheat TabZIP14 gene under PEG stress.

图2为盐胁迫下小麦TabZIP14基因的相对表达量。Fig. 2 is the relative expression level of wheat TabZIP14 gene under salt stress.

图3为低温胁迫下小麦TabZIP14基因的相对表达量。Fig. 3 is the relative expression level of wheat TabZIP14 gene under low temperature stress.

图4为ABA胁迫下小麦TabZIP14基因的相对表达量。Fig. 4 is the relative expression level of wheat TabZIP14 gene under ABA stress.

图5为T3代转TabZIP14基因拟南芥的RNA水平鉴定结果。Fig. 5 is the RNA level identification result of Arabidopsis transgenic with TabZIP14 gene in the T3 generation.

图6为低温胁迫下拟南芥的表型。Figure 6 shows the phenotype of Arabidopsis under low temperature stress.

图7为盐胁迫下拟南芥的表型。Figure 7 shows the phenotype of Arabidopsis under salt stress.

具体实施方式Detailed ways

下述实施例中所使用的实验方法如无特殊说明,均为常规方法。The experimental methods used in the following examples are conventional methods unless otherwise specified.

下述实施例中所用的材料、试剂等,如无特殊说明,均可从商业途径得到。The materials and reagents used in the following examples can be obtained from commercial sources unless otherwise specified.

KOD-Plus高保真酶:TOYOBO公司产品,产品目录号为KOD-201。KOD-Plus High Fidelity Enzyme: Product of TOYOBO Company, catalog number is KOD-201.

入门载体pDONR/Zeo:invitrogen公司产品,产品目录号为12535-035。Entry vector pDONR/Zeo: product of Invitrogen, catalog number 12535-035.

BP ClonaseTMⅡEnzyme Mix:invitrogen公司产品,产品目录号为11789-013。BP ClonaseTMⅡEnzyme Mix: product of Invitrogen Company, the product catalog number is 11789-013.

目标载体pEarleyGate 100:在文献“Zhang L,Zhang L,Xia C,Zhao G,Liu J,JiaJ,Kong X.A novel wheat bZIP transcription factor,TabZIP60,confers multipleabiotic stress tolerances in transgenic Arabidopsis.Physiol Plant.2015,153(4):538-554.”中公开过,公众可从中国农业科学院作物科学研究所获得。Target vector pEarleyGate 100: In the literature "Zhang L, Zhang L, Xia C, Zhao G, Liu J, JiaJ, Kong X.A novel wheat bZIP transcription factor, TabZIP60, confers multipleabiotic stress tolerances in transgenic Arabidopsis. Physiol Plant. 2015, 153( 4): 538-554.", publicly available from the Institute of Crop Science, Chinese Academy of Agricultural Sciences.

LR ClonaseTMⅡ Enzyme Mix:invitrogen公司产品,产品目录号为11791-019。LR ClonaseTMⅡ Enzyme Mix: product of Invitrogen Company, the product catalog number is 11791-019.

农杆菌GV3101-pMP90:在文献“Xu Z,Kim S,Hyeon D,Kim D,et al.TheArabidopsis thaliana NAC transcription factor family:structure–functionrelationships and determinants of ANAC019 stress signaling.Biochem.J.2010,426:183-196.”中公开过,公众可从中国农业科学院作物科学研究所获得。Agrobacterium GV3101-pMP90: In the literature "Xu Z, Kim S, Hyeon D, Kim D, et al. The Arabidopsis thaliana NAC transcription factor family: structure–function relationships and determinants of ANAC019 stress signaling. Biochem. J. 2010, 426: 183 -196.", which is publicly available from the Institute of Crop Science, Chinese Academy of Agricultural Sciences.

拟南芥(Arabidopsis thaliana)(Columbia-0亚型):在文献“He Y,Li W,Lv J,Jia Y,Wang M and Xia G.Ectopic expression of a wheat MYB transcription factorgene,TaMYB73,improves salinity stress tolerance in Arabidopsis thaliana.J ExpBot,2012,63(3):1511–1522.”中公开过,公众可从中国农业科学院作物科学研究所获得。Arabidopsis thaliana (Columbia-0 subtype): In the literature "He Y, Li W, Lv J, Jia Y, Wang M and Xia G. Ectopic expression of a wheat MYB transcription factorgene, TaMYB73, improves salinity stress tolerance in Arabidopsis thaliana.J ExpBot,2012,63(3):1511–1522.”, which is publicly available from the Institute of Crop Science, Chinese Academy of Agricultural Sciences.

中国春小麦(Chinese Spring Wheat):在文献“Kobayashia F,Maetaa E,Terashimaa A,Takum S.Positive role of a wheat HvABI5ortholog in abioticstress response of seedlings.Physiol Plant,2008,134:74–86.”中公开过,公众可从中国农业科学院作物科学研究所获得。Chinese Spring Wheat: disclosed in the literature "Kobayashia F, Maetaa E, Terashimaa A, Takum S. Positive role of a wheat HvABI5ortholog in bioticstress response of seedlings. Physiol Plant, 2008, 134:74-86." , publicly available from the Institute of Crop Science, Chinese Academy of Agricultural Sciences.

实施例1、TabZIP14蛋白及其编码基因的发现Embodiment 1, the discovery of TabZIP14 protein and its coding gene

为了研究小麦bZIP基因在植物抗逆方面的功能,通过生物信息学的方法,从发明人所在实验室已经测序的30000条非冗余的小麦全长cDNA序列中筛选出22个编码小麦bZIP蛋白的基因。通过半定量PCR的方法,对该22个小麦bZIP基因在逆境条件下的表达模式进行研究,筛选出受逆境诱导表达的TabZIP14基因。In order to study the function of wheat bZIP gene in plant stress resistance, 22 wheat bZIP protein-encoding genes were screened from 30,000 non-redundant wheat full-length cDNA sequences sequenced in the inventor's laboratory through bioinformatics methods. Gene. The expression pattern of the 22 wheat bZIP genes under stress conditions was studied by semi-quantitative PCR, and the TabZIP14 gene induced by stress was screened out.

TabZIP14基因全长cDNA为1576bp,序列如序列表中序列2所示。其开放阅读框为序列2的自5’末端起第181-1383位核苷酸,编码序列表中序列1所示的TabZIP14蛋白,序列1共由400个氨基酸残基组成。The full-length cDNA of the TabZIP14 gene is 1576bp, and its sequence is shown in sequence 2 in the sequence listing. Its open reading frame is 181-1383 nucleotides from the 5' end of Sequence 2, encoding the TabZIP14 protein shown in Sequence 1 in the Sequence Listing, and Sequence 1 consists of 400 amino acid residues in total.

实施例2、TabZIP14基因在不同逆境胁迫下的转录水平表达模式Example 2, Transcript level expression pattern of TabZIP14 gene under different stresses

一、TabZIP14基因在PEG胁迫下的表达模式1. Expression pattern of TabZIP14 gene under PEG stress

对两叶一心期的中国春小麦幼苗置于16.1g/100ml的PEG6000水溶液中进行处理,分别在处理前(0h)、处理后1、3、6、12、24和48小时提取小麦幼苗根组织的RNA,反转录成cDNA,以其cDNA为模板,以上游引物1和下游引物2为引物,进行实时定量PCR分析,得到TabZIP14基因的相对表达量,内参是小麦Tubulin基因。The Chinese spring wheat seedlings at the two-leaf one-heart stage were placed in 16.1g/100ml of PEG6000 aqueous solution for treatment, and the root tissues of wheat seedlings were extracted respectively before treatment (0h), after treatment 1, 3, 6, 12, 24 and 48 hours. The RNA was reverse-transcribed into cDNA, and the cDNA was used as a template, and the upstream primer 1 and downstream primer 2 were used as primers for real-time quantitative PCR analysis to obtain the relative expression of the TabZIP14 gene. The internal reference was the wheat Tubulin gene.

用于检测TabZIP14基因的引物如下:The primers used to detect the TabZIP14 gene are as follows:

上游引物1:5’-CGGCTTGCTGATGTTAATC-3’;Upstream primer 1: 5'-CGGCTTGCTGATGTTAATC-3';

下游引物2:5’-TCGCTTGTGGAGAAGTAAG-3’。Downstream primer 2: 5'-TCGCTTGTGGAGAAGTAAG-3'.

用于检测内参Tubulin基因的引物如下:The primers used to detect the internal reference Tubulin gene are as follows:

Tubulin-F:5’-TTAGACTTGCGAAGCCAGCA-3’;Tubulin-F: 5'-TTAGACTTGCGAAGCCAGCA-3';

Tubulin-R:5’-AAATGCCCTTGAGGTTTCCC-3’。Tubulin-R: 5'-AAATGCCCTTGAGGTTTCCC-3'.

结果如图1所示,由图可见TabZIP14基因响应PEG胁迫诱导。The results are shown in Figure 1, from which it can be seen that the TabZIP14 gene is induced in response to PEG stress.

二、TabZIP14基因在盐胁迫下的表达模式2. Expression pattern of TabZIP14 gene under salt stress

对两叶一心期的中国春小麦幼苗置于250mM NaCl水溶液中进行处理,分别在处理前(0h)、处理后1、3、6、12、24和48小时提取小麦幼苗根组织的RNA,反转录成cDNA,以其cDNA为模板,以上游引物1和下游引物2为引物,进行实时定量PCR分析,得到TabZIP14基因的相对表达量,内参是小麦Tubulin基因。具体引物序列参见步骤一。The Chinese spring wheat seedlings at the two-leaf one-heart stage were treated in 250mM NaCl aqueous solution, and the RNA of the root tissue of the wheat seedlings was extracted respectively before (0h), 1, 3, 6, 12, 24 and 48 hours after the treatment, and reversed Recorded into cDNA, using the cDNA as a template, using upstream primer 1 and downstream primer 2 as primers, real-time quantitative PCR analysis was performed to obtain the relative expression of TabZIP14 gene, and the internal reference was wheat Tubulin gene. See step 1 for specific primer sequences.

结果如图2所示,由图可见TabZIP14基因响应盐胁迫诱导。The results are shown in Figure 2, from which it can be seen that the TabZIP14 gene is induced in response to salt stress.

三、TabZIP14基因在低温胁迫下的表达模式3. Expression pattern of TabZIP14 gene under low temperature stress

对两叶一心期的中国春小麦幼苗置于4℃处理,分别在处理前(0h)、处理后1、3、6、12、24和48小时提取小麦幼苗叶子组织的RNA,反转录成cDNA,以其cDNA为模板,进行实时定量PCR分析,得到TabZIP14基因的相对表达量,内参是小麦Tubulin基因。具体引物序列参见步骤一。The Chinese spring wheat seedlings at the two-leaf one-heart stage were treated at 4°C, and RNA was extracted from the leaf tissues of wheat seedlings before (0h), 1, 3, 6, 12, 24 and 48 hours after treatment, and reverse-transcribed into cDNA , using its cDNA as a template, real-time quantitative PCR analysis was carried out to obtain the relative expression level of TabZIP14 gene, and the internal reference was wheat Tubulin gene. See step 1 for specific primer sequences.

结果如图3所示,由图可见TabZIP14基因响应低温胁迫诱导。The results are shown in Figure 3, from which it can be seen that the TabZIP14 gene is induced in response to low temperature stress.

四、TabZIP14基因在ABA胁迫下的表达模式4. Expression pattern of TabZIP14 gene under ABA stress

对两叶一心期的中国春小麦幼苗置于200μM ABA水溶液中进行处理,分别在处理前(0h)、处理后1、3、6、12、24和48小时提取小麦幼苗根组织的RNA,反转录成cDNA,以其cDNA为模板,以上游引物1和下游引物2为引物,进行实时定量PCR分析,得到TabZIP14基因的相对表达量,内参是小麦Tubulin基因。具体引物序列参见步骤一。The Chinese spring wheat seedlings at the two-leaf one-heart stage were treated in 200 μM ABA aqueous solution, and the RNA of the root tissue of the wheat seedlings was extracted before treatment (0h), 1, 3, 6, 12, 24 and 48 hours after treatment, and reversed Recorded into cDNA, using the cDNA as a template, using upstream primer 1 and downstream primer 2 as primers, real-time quantitative PCR analysis was performed to obtain the relative expression of TabZIP14 gene, and the internal reference was wheat Tubulin gene. See step 1 for specific primer sequences.

结果如图4所示,由图可见TabZIP14基因响应ABA诱导表达。The results are shown in Figure 4, from which it can be seen that the expression of the TabZIP14 gene responds to ABA induction.

实施例3、转TabZIP14基因植株的获得和耐逆性鉴定Embodiment 3, the acquisition of transgenic TabZIP14 plant and identification of stress tolerance

一、利用Gateway技术构建过表达载体1. Using Gateway technology to construct overexpression vectors

1、attB引物设计1. attB primer design

上游引物3:5’-GCATGGAGCGCGGCGTCTTC-3’Upstream primer 3: 5'-GCATGGAGCGCGGCGTCTTC-3'

下游引物4:5’-CTACGAGGTCGATCCGGATG-3’Downstream primer 4: 5'-CTACGAGGTCGATCCGGATG-3'

2、根据Gateway技术构建表达载体的需要,在上游引物3和下游引物4的5'端分别加入attB1和attB2重组位点(下划线标注部分为attB1和attB2重组位点),分别得到上游引物5和下游引物6。2. According to the needs of Gateway technology to construct expression vectors, attB1 and attB2 recombination sites were added to the 5' ends of upstream primer 3 and downstream primer 4 (the underlined parts are attB1 and attB2 recombination sites), respectively, to obtain upstream primers 5 and Downstream primer 6.

上游引物5:5’-GGGGACAAGTTTGTACAAAAAAGCAGGCTGCATGGAGCGCGGCGTCTTC-3’Upstream primer 5: 5'- GGGGACAAGTTTGTACAAAAAAAGCAGGCTGCATGGAGCGCGGCGTCTTC -3'

下游引物6:5’-GGGGACCACTTTGTACAAGAAAGCTGGGTCTACGAGGTCGATCCGGATG-3’Downstream primer 6: 5'- GGGGACCACTTTGTACAAGAAAGCTGGGTCTACGAGGTCGATCCGGATG -3'

3、以中国春小麦的cDNA为模板,用上游引物5和下游引物6进行PCR扩增,得到attBPCR产物,为保证PCR过程的保真性,使用KOD-Plus高保真酶进行PCR反应,PCR反应体系和反应程序如下:3. Using the cDNA of Chinese spring wheat as a template, PCR amplification was performed with upstream primer 5 and downstream primer 6 to obtain the attBPCR product. In order to ensure the fidelity of the PCR process, KOD-Plus high-fidelity enzyme was used for PCR reaction. The PCR reaction system and The reaction procedure is as follows:

4、按照如下体系和程序进行BP重组反应,得到BP反应产物:4. Carry out the BP recombination reaction according to the following system and procedure to obtain the BP reaction product:

5、入门质粒的获得5. Obtaining entry plasmid

将2.5μl BP反应产物加入50μl TOP10感受态细胞进行转化,冰浴30min,42℃热击90s,然后迅速置于冰上2min。加入500μl LB培养液,37℃,210rpm/min复苏50min,复苏液均匀涂布于加有Zeocin(浓度为40μg/ml)的固体LB选择培养基表面,37℃倒置培养过夜。pDONRTM/Zeo载体自身带有致死基因,不能在培养基上生存。通过步骤4的BP重组反应目标基因片段会取代致死基因,最终得到的克隆即为入门克隆(entry clone),质粒为入门质粒,将入门质粒送测序,结果正确。Add 2.5 μl BP reaction product to 50 μl TOP10 competent cells for transformation, ice bath for 30 min, heat shock at 42°C for 90 s, and then quickly place on ice for 2 min. Add 500 μl LB culture solution, recover at 37°C, 210 rpm/min for 50 minutes, spread the recovery solution evenly on the surface of solid LB selection medium added with Zeocin (concentration: 40 μg/ml), and incubate overnight at 37°C. The pDONR TM /Zeo vector itself has a lethal gene and cannot survive on the culture medium. Through the BP recombination reaction in step 4, the target gene fragment will replace the lethal gene, and the final clone obtained is the entry clone, and the plasmid is the entry plasmid. The entry plasmid is sent for sequencing, and the result is correct.

6、按照如下体系和程序进行LR重组反应,得到LR反应产物:6. Perform the LR recombination reaction according to the following system and procedure to obtain the LR reaction product:

7、目标质粒的获得7. Obtaining the target plasmid

转化过程同步骤5,只是将zeocin替换为卡那霉素(浓度为50μg/ml)。The transformation process is the same as step 5, except that zeocin is replaced by kanamycin (concentration: 50 μg/ml).

pEarleyGate 100载体同样带有致死基因,通过LR重组反应致死基因将会被目标基因片段取代,得到的克隆即为目标克隆(destination clone),质粒为目标质粒,将目标质粒命名为pEarleyGate 100-TabZIP14。pEarleyGate 100-TabZIP14的测序结果表明pEarleyGate 100-TabZIP14含有序列表中序列2的第181-1383位所示DNA片段。pEarleyGate 100-TabZIP14为TabZIP14基因表达载体,启动子是花椰菜花叶病毒35S启动子。The pEarleyGate 100 vector also carries a lethal gene. The lethal gene will be replaced by the target gene fragment through LR recombination reaction. The resulting clone is the destination clone, and the plasmid is the target plasmid. The target plasmid is named pEarleyGate 100-TabZIP14. The sequencing result of pEarleyGate 100-TabZIP14 shows that pEarleyGate 100-TabZIP14 contains the DNA fragment shown in the 181-1383 position of sequence 2 in the sequence listing. pEarleyGate 100-TabZIP14 is the expression vector of TabZIP14 gene, and the promoter is cauliflower mosaic virus 35S promoter.

二、转TabZIP14基因拟南芥的获得2. Obtaining the transgenic Arabidopsis thaliana

1、将步骤一获得的目标质粒转化农杆菌GV3101-pMP90,得到重组农杆菌。1. Transform the target plasmid obtained in step 1 into Agrobacterium GV3101-pMP90 to obtain recombinant Agrobacterium.

2、拟南芥侵染及转基因植株筛选及鉴定2. Arabidopsis infection and screening and identification of transgenic plants

(1)拟南芥的培养(1) Culture of Arabidopsis

将拟南芥(Arabidopsis thaliana)(Columbia-0亚型)种子用灭菌水(含有体积百分含量为10%次氯酸钠以及10%吐温-20的水溶液)摇动消毒15min,在超净工作台中用灭菌水清洗上述消毒的种子至少5次。将清洗完的种子均匀播种在MS培养基上。MS平板于4℃春化3d,然后置于22℃光照培养箱培养一周。待小苗长出四片真叶后移栽至营养钵中培养,保湿2-3d。拟南芥的生长对温度比较敏感,20-22℃为比较适宜的培养温度。当拟南芥植株生长至大部分花蕾处于即将开花状态时,进行农杆菌侵染。The seeds of Arabidopsis thaliana (Columbia-0 subtype) were sterilized with sterilized water (an aqueous solution containing 10% sodium hypochlorite and 10% Tween-20 by volume) for 15 minutes, and then used in an ultra-clean workbench. Wash the above sterilized seeds at least 5 times with sterilized water. Sow the washed seeds evenly on MS medium. MS plates were vernalized at 4°C for 3 days, and then placed in a light incubator at 22°C for one week. After the seedlings grow four true leaves, they are transplanted into a nutrient bowl for cultivation, and kept moist for 2-3 days. The growth of Arabidopsis thaliana is sensitive to temperature, and 20-22°C is a suitable culture temperature. Agrobacterium infection was performed when the Arabidopsis plants were grown to the point where most flower buds were in the imminent state of flowering.

(2)拟南芥的侵染(2) Infection of Arabidopsis thaliana

采用沾花法用步骤1所得的重组农杆菌对步骤(1)得到的拟南芥进行侵染,将侵染过的拟南芥平放于托盘中,黑暗保湿培养24h,然后放于正常培养条件下培养。拟南芥侵染1周后根据拟南芥的生长状态可再次侵染以提高转化效率,得到转TabZIP14基因拟南芥。Use the recombinant Agrobacterium obtained in step 1 to infect the Arabidopsis thaliana obtained in step (1) by the staining method, place the infected Arabidopsis flat on the tray, and culture in the dark for 24 hours, and then put it in the normal culture cultivated under conditions. One week after the Arabidopsis infection, the Arabidopsis can be re-infected according to the growth state of the Arabidopsis to improve the transformation efficiency, and the TabZIP14 gene transgenic Arabidopsis can be obtained.

(3)阳性转TabZIP14基因拟南芥的初步筛选(3) Preliminary screening of positive transgenic Arabidopsis thaliana

①收集转TabZIP14基因拟南芥T0代种子,于37℃烘箱中烘干(6-8d),然后4℃春化3d。① The T0 generation seeds of Arabidopsis thaliana transgenic with TabZIP14 gene were collected, dried in an oven at 37°C (6-8d), and then vernalized at 4°C for 3d.

②将种子直接撒播在营养钵中,22℃保湿培养1周。② Sow the seeds directly in the nutrient bowl, and cultivate them with moisture at 22°C for 1 week.

③待小苗长出4片真叶后,通过喷洒除草剂(basta)进行阳性转TabZIP14基因拟南芥筛选,非阳性转TabZIP14基因拟南芥在喷洒3天后开始出现萎蔫并停止生长,2周后基本死亡。为了将非阳性转TabZIP14基因拟南芥苗彻底除净,可连续喷洒2-3次,每次间隔2-3d。③After the seedlings grew 4 true leaves, positively transfected TabZIP14 gene Arabidopsis was screened by spraying herbicide (basta). The non-positive TabZIP14 gene transgenic Arabidopsis began to wilt and stop growing 3 days after spraying, and 2 weeks later Basically dead. In order to completely eliminate the non-positive TabZIP14 transgenic Arabidopsis seedlings, it can be sprayed 2-3 times continuously with an interval of 2-3 days between each time.

(4)阳性转TabZIP14基因拟南芥的鉴定(4) Identification of positive transgenic Arabidopsis thaliana

A.DNA水平鉴定A. DNA level identification

CTAB法提取步骤(3)初步筛选获得的阳性转TabZIP14基因拟南芥叶片的基因组DNA,以其为模板,以TabZIP14基因特异性的正向引物7和反向引物8进行PCR扩增,得到PCR扩增产物,将PCR扩增产物进行琼脂糖凝胶电泳。The genomic DNA of the positive TabZIP14 gene Arabidopsis leaves obtained by preliminary screening in CTAB method extraction step (3) is used as a template, and PCR amplification is carried out with the forward primer 7 and the reverse primer 8 specific to the TabZIP14 gene to obtain PCR The amplified product was subjected to agarose gel electrophoresis.

正向引物7:5’-CAGAAGATGATGATATGGAAGG-3’;Forward primer 7: 5'-CAGAAGATGATGATATGGAAGG-3';

反向引物8:5’-TCGCTTGTGGAGAAGTAAG-3’。Reverse primer 8: 5'-TCGCTTGTGGAGAAGTAAG-3'.

电泳结果表明,步骤(3)初步筛选获得的阳性转TabZIP14基因拟南芥在PCR扩增之后有约445bp的目的条带,进一步确定为转TabZIP14基因拟南芥。将野生型拟南芥进行上述鉴定实验无目的条带。The electrophoresis results showed that the positive TabZIP14 gene transgenic Arabidopsis obtained in step (3) had a target band of about 445 bp after PCR amplification, and was further identified as the TabZIP14 gene transgenic Arabidopsis. The wild-type Arabidopsis was subjected to the above identification experiment without any target bands.

用上述方法进行鉴定,直至获得T3代纯合转TabZIP14基因拟南芥(以下简称T3代转TabZIP14基因拟南芥),从所得T3代转TabZIP14基因拟南芥中随机选取3各株系,记为L1、L2和L3。Use the above method to identify until the T3 generation homozygous TabZIP14 gene transgenic Arabidopsis (hereinafter referred to as T3 transgenic TabZIP14 gene Arabidopsis), randomly select 3 lines from the obtained T3 transgenic TabZIP14 gene Arabidopsis, record for L1, L2 and L3.

B.RNA水平鉴定B. RNA level identification

对basta除草剂筛选获得的T3代纯合转基因植株,随机选取三株,提取总RNA,并进行第一链cDNA的合成,以上游引物1和下游引物2为引物,进行实时定量PCR分析,得到TabZIP14基因的相对表达量,内参是小麦Tubulin基因。具体引物序列参见实施例2步骤一。For the T3 homozygous transgenic plants screened by basta herbicide, three plants were randomly selected, total RNA was extracted, and the first-strand cDNA was synthesized. The upstream primer 1 and downstream primer 2 were used as primers for real-time quantitative PCR analysis to obtain The relative expression level of TabZIP14 gene, the internal reference is wheat Tubulin gene. For specific primer sequences, refer to Step 1 of Example 2.

结果显示:转TabZIP14基因的T3代纯合植株,利用RT-qPCR检测,结果表明其纯合株系L1,L2和L3中TabZIP14基因的表达比较相近。可以继续后续功能鉴定。转TabZIP14基因的L1、L2和L3株系的RNA水平鉴定结果具体如图5所示。The results showed that the T3 homozygous plants transfected with the TabZIP14 gene were detected by RT-qPCR, and the results showed that the expression of the TabZIP14 gene in the homozygous lines L1, L2 and L3 was similar. Subsequent functional qualification can be continued. The RNA level identification results of the L1, L2 and L3 strains transfected with the TabZIP14 gene are specifically shown in FIG. 5 .

三、转TabZIP14基因拟南芥的抗逆鉴定3. Stress resistance identification of Arabidopsis transgenic with TabZIP14 gene

1、抗冻性分析1. Analysis of frost resistance

将野生型拟南芥(WT)以及三个T3代转TabZIP14基因拟南芥(L1,L2,L3)的种子在22℃,12h光照下进行培养,得到3周幼苗,然后将各幼苗在-10℃处理3小时,再恢复培养4天。观察各组拟南芥冷处理前后的幼苗生长状态并统计其存活率。实验重复三次,每次重复每个株系12株。The seeds of wild-type Arabidopsis (WT) and three Arabidopsis (L1, L2, L3) transgenic with TabZIP14 gene in T3 generation were cultured at 22°C under 12h light to obtain 3-week-old seedlings, and then each seedling was placed in- Treat at 10°C for 3 hours, and resume culture for 4 days. The growth status of Arabidopsis thaliana seedlings in each group before and after cold treatment was observed, and the survival rate was counted. The experiment was repeated three times, with 12 strains per replicate.

冷处理前后的各幼苗的生长状态如图6所示,由图可以看出,与野生型拟南芥相比,转TabZIP14基因拟南芥的抗冻性明显提高。抗冻处理后,野生型植株的存活率为16%,转TabZIP14的株系存活率为79%(L1、L2和L3这三个株系的存活率均值)。The growth state of each seedling before and after cold treatment is shown in Figure 6, and it can be seen from the figure that compared with wild-type Arabidopsis, the frost resistance of the transgenic Arabidopsis with TabZIP14 gene is significantly improved. After the antifreeze treatment, the survival rate of wild-type plants was 16%, and the survival rate of TabZIP14-transformed lines was 79% (average of the survival rates of the three lines L1, L2 and L3).

2、抗盐性分析2. Salt resistance analysis

将野生型拟南芥(WT)以及三个T3代转TabZIP14基因拟南芥(L1,L2,L3)的种子在22℃,12h光照下进行垂直培养,得到5天幼苗,将各幼苗转移到含150和200mM NaCl的MS培养基上垂直培养,并分别以在不含NaCl的MS培养基上垂直培养得到的幼苗为对照。观察各组拟南芥幼苗生长状态并统计植株根长和植株白化率(盐胁迫处理后有白化叶片的植株株数与盐胁迫处理前植株株数之比)。实验重复三次,每次重复每个株系8株。The seeds of wild-type Arabidopsis (WT) and three T3 generations of Arabidopsis transgenic with TabZIP14 gene (L1, L2, L3) were vertically cultured at 22°C under 12h light to obtain 5-day seedlings, and each seedling was transferred to Vertically cultured on MS medium containing 150 and 200mM NaCl, and the vertical cultured seedlings on MS medium without NaCl were used as controls. The growth status of Arabidopsis thaliana seedlings in each group was observed, and the plant root length and plant albinism rate (the ratio of the number of plants with albino leaves after salt stress treatment to the number of plants before salt stress treatment) were counted. The experiment was repeated three times, with 8 strains per replicate.

各组拟南芥幼苗的状态如图7所示,由图可以看出,150mM NaCl处理后,野生型拟南芥的根长明显受到抑制,T3代转TabZIP14基因拟南芥L1、L2和L3株系的根长分别是野生型植株根长的1.4倍、1.3倍和1.4倍。部分野生型的拟南芥叶子呈现白化现象,白化率为37%±0.5%。而T3代转TabZIP14基因拟南芥叶片全部保持绿色。200mM NaCl处理后,T3代转TabZIP14基因拟南芥L1、L2和L3株系的根长比分别是野生型植株根长的1.2倍、1.3倍和1.3倍。野生型植株的叶片几乎全部呈现白化现在,白化率为98%±0.4%;而T3代转TabZIP14基因拟南芥叶片基本呈现绿色状态。以上结果说明转TabZIP14基因拟南芥的抗盐性明显提高。The state of Arabidopsis seedlings in each group is shown in Figure 7. It can be seen from the figure that after 150mM NaCl treatment, the root length of wild-type Arabidopsis was significantly inhibited, and the Arabidopsis L1, L2 and L3 of T3 transgenic TabZIP14 gene The root lengths of the lines were 1.4 times, 1.3 times and 1.4 times that of the wild-type plants, respectively. Some wild-type Arabidopsis leaves showed albinism, and the albino rate was 37%±0.5%. However, the leaves of Arabidopsis thaliana transgenic with TabZIP14 gene in the T3 generation remained green. After 200mM NaCl treatment, the root length ratios of Arabidopsis L1, L2 and L3 lines transduced with TabZIP14 gene in the T3 generation were 1.2, 1.3 and 1.3 times that of the wild-type plants, respectively. The leaves of wild-type plants are almost all albino, and the albino rate is 98%±0.4%; while the leaves of Arabidopsis transgenic with TabZIP14 gene in the T3 generation are basically in a green state. The above results indicated that the salt resistance of Arabidopsis transgenic with TabZIP14 gene was significantly improved.

Claims (13)

1. protein, the protein that the amino acid sequence shown in sequence 1 in sequence table forms.
2. encoding the nucleic acid molecules of protein described in claim 1.
3. nucleic acid molecules according to claim 2, it is characterised in that: the nucleic acid molecules are described in coding claim 1 The gene of protein, the gene are following DNA molecular 1) or 2):
1) DNA molecular shown in sequence 2 in sequence table;
2) DNA molecular shown in 181-1383 of sequence 2 in sequence table.
4. the recombinant vector containing nucleic acid molecules described in claim 2.
5. the recombinant vector containing nucleic acid molecules described in claim 3.
6. the expression cassette containing nucleic acid molecules described in Claims 2 or 3.
7. the recombinant microorganism containing nucleic acid molecules described in Claims 2 or 3.
8. recombinant vector according to claim 5, it is characterised in that: the recombinant vector is recombinant expression carrier or recombination Cloning vector;
The promoter for starting the genetic transcription in the recombinant expression carrier is 35S promoter.
9. weight described in nucleic acid molecules described in protein or Claims 2 or 3 described in claim 1 or claim 4 or 5 or 8 Group carrier or expression cassette as claimed in claim 6 or recombinant microorganism as claimed in claim 7 it is following it is any in application:
(a) regulate and control stress resistance of plant;
(b) plant variety that breeding resistance improves;
The resistance is at least one of following: frost resistance, salt-resistance.
10. application according to claim 9, it is characterised in that: the plant is dicotyledon or monocotyledon.
11. the method for cultivating the genetically modified plants that resistance improves includes the steps that following (a1) and (a2):
(a1) encoding gene that protein described in claim 1 is imported into recipient plant, obtains expressing the encoding gene Genetically modified plants;
(a2) it is obtained compared with the recipient plant from genetically modified plants obtained by step (a1), the transgenosis that resistance improves is planted Object;
The resistance is at least one of following: frost resistance, salt-resistance.
12. according to the method for claim 11, it is characterised in that: in step (a1), the encoding gene is to pass through right It is required that recombinant vector described in 4 or 5 or 8 imports the recipient plant.
13. according to the method for claim 11, it is characterised in that: the plant is dicotyledon or monocotyledon.
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