CN111718941A - Gene sequence of a transcription factor AgbZIP16 related to abiotic stress in celery and its application - Google Patents

Abstract

The bZIP transcription factor family belongs to a large number of transcription factor families, which are very conserved and involved in plant growth and development, metabolic regulation, and biotic and abiotic stress responses. This patent clones a new bZIP-like transcription factor gene AgbZIP16 from two celery varieties 'Liuhe Huangxinqin' and 'Ventura'. The gene contains an open reading frame with a length of 1221 bp, encoding 406 amino acids, and contains a BRLZ conserved domain typical of the bZIP family. The protein encoded by AgbZIP16 is a hydrophilic protein, and has the closest evolutionary relationship with the carrot bZIP transcription factor protein, which belongs to the same Umbelliferae, and is highly conserved. Fluorescence quantitative expression analysis showed that the relative expression levels of AgbZIP16 in 'Liuhe Huangxinqin' and 'Ventura' were the highest in roots and the lowest in petioles, showing significant tissue specificity. At the same time, high temperature, low temperature, drought and salt stress can induce the expression of AgbZIP16 gene to change, and its expression response in different celery varieties is significantly different. The invention utilizes polymerase amplification technology to clone and obtain the AgbZIP16 transcription factor gene from celery, which confirms that the gene is involved in the regulation of celery's stress resistance.

Description

Gene sequence of a transcription factor AgbZIP16 related to abiotic stress in celery and its application

technical field

The invention belongs to the field of plant genetic engineering, and relates to a bZIP transcription factor of plants and its application. The invention obtains a gene AgbZIP16 responding to abiotic stress from celery varieties 'Liuhe Huangxinqin' and 'Ventura', and the gene can be applied to the research and application of celery's stress resistance mechanism.

Background technique

Celery (Apium graveolens L.) is a one- or two-year herbaceous plant belonging to the genus Apiaceae. It is rich in vitamins, carotene, cellulose and volatile aromatic components, and has both edible and medicinal value. Celery is one of the most important leafy vegetable crops in the world. It has strong cold resistance and has a long history of cultivation and a wide range of cultivation in my country.

Plant growth and development are affected by abiotic stress factors, such as high temperature, low temperature, drought, salt stress, etc., which will damage plant cells, thereby reducing yield and quality (Sun Lijun et al., Genetics, 2012, 34(8): 993- 1002.). Transcription factors play an important role in regulating the expression of corresponding genes by binding to downstream gene promoters during plant growth and development (Du H et al., BMC Plant Biology, 2012, 12(106): 1-22 .).) The basic leucine zipper transcription factor family (Basic region/leucine zipper, bZIP) belongs to a large number of transcription factor families and is very conserved, and is found in humans, animals, plants and microorganisms. Its conserved domain consists of about 60-80 amino acid residues, including two conserved regions: basic amino acid region and leucine zipper region (Talanian RV et al., Science, 1990, 249 (4970): 769-771 .).) The results show that bZIP transcription factors are involved in plant growth and development, metabolic regulation, and biotic and abiotic stress responses (Toh S et al., Plant Signal & Behavior, 2012, 7(5): 556-558; Wang J et al., Journal of Integrative Plant Biology, 2011, 53.212-231.). It binds to response elements such as abscisic acid (ABA) and cytokinin (CTK), and regulates the transcription of these response genes, thereby improving the stress resistance of plants (Li Dongyue et al., Zhejiang Agricultural Journal, 2017, 29 (1 ): 168-175.).

SUMMARY OF THE INVENTION

The invention provides a preparation method and application of celery transcription factor AgbZIP16 gene. The obtained AgbZIP16 gene is beneficial to deeply understand the response mechanism of celery under stress.

Description of drawings

Figure 1. Prediction of conserved domains of celery AgbZIP16 amino acid sequence

Figure 2. Phylogenetic tree of AgbZIP16 and bZIP protein sequences from other species

Figure 3. Hydrophobicity and hydrophilicity analysis of celery AgbZIP16 amino acid sequence

Figure 4. Expression of AgbZIP16 in different tissue parts of celery

Figure 5. Expression of AgbZIP16 under different abiotic stresses in celery

Detailed ways

1. Extraction of total RNA from celery and synthesis of cDNA: Take two celery samples 'Liuhe Huangxinqin' and 'Ventura', extract total RNA according to the instructions of RNA Simple Total Rna Kit (Tiangen, Beijing), and use Nanodrop2000 microspectroscopy The total RNA concentration was detected by a photometer (Thermo Scientific, USA), and the total RNA was reverse transcribed into cDNA according to the instructions of the Prime Script RTreagent Kit.

2. Cloning of the celery transcription factor AgbZIP16 gene: The gene sequence encoding celery AgbZIP16 was retrieved from the celery transcriptome database (JiaXL et al., Scientific Reports, 2015, 5:8259.) determined by this research group, and according to the A pair of specific primers were designed with Primer Premier 6.0. The sequence of the forward primer was: 5'-ATGGGCAGTAGTGAAATGGAAA-3'; the sequence of the reverse primer was: 5'-TCATGCAGCCTCTGTATGACCG-3'. The target gene fragment was amplified by PCR using the cDNA obtained by reverse transcription as a template. The PCR reaction system was 20 μl in total, including 1 μL of cDNA (2.5ng.μL ⁻¹ ), 1 μL of forward and reverse primers, 7 μL of ddH ₂ O, and 10 μL of PrimeSTAR Max Premix (TaKaRa, Dalian). The reaction program was: pre-denaturation at 94 °C for 5 min; denaturation at 94 °C for 30 s, annealing at 54 °C for 30 s, extension at 72 °C for 1 min, a total of 35 cycles; extension at 72 °C for 10 min. The PCR amplification products were detected by electrophoresis using 1.5% agarose gel, and the recovered reaction products were sequenced by Nanjing GenScript Biotechnology Co., Ltd.

3. Sequence analysis: use BioXM 2.6 software to analyze the nucleotide and amino acid sequence of the cloned AgbZIP16 gene fragment; use the BLAST tool in the NCBI database to obtain the conservative domain prediction and homology analysis of the target gene; use DNAMAN 6.0 software The hydrophilicity and hydrophobicity of the amino acid sequence encoded by the target gene were analyzed; the phylogenetic tree was drawn by MEGA5.2 software.

et al.，Nucleic Acids Res，2001，29(14)：2994-3005)，对两个芹菜品种中AgbZIP16基因在不同组织和不同逆境条件下的相对表达量进行分析。4. Real-time quantitative PCR reaction: According to the celery AgbZIP16 gene sequencing results, Primer Premier 6.0 was used to design fluorescent quantitative primers, the forward primer was AgbZIP16-qF: 5'-AACCCATCAGGAGCAGCCACTAATG-3', and the reverse primer was AgbZIP16-qR: 5'- CCGCACTAACAGTTCCTCCAGTAAT-3'. The celery actin gene was used as an internal reference gene (Jiang Q et al., Plos One, 2014, 9(3): e92262.). The expression of this gene in different tissues and under different abiotic stresses was analyzed using a real-time PCR detection system. The reaction system was 20 μL, including 0.4 μL of forward and reverse primers, 10 μL of SYBRGreen I mix, 7.2 μL of ddH ₂ O and 2 μL of 15-fold diluted cDNA. The reaction program was: pre-denaturation at 95 °C for 30 s, denaturation at 95 °C for 5 s, and annealing and extension at 60 °C for 30 s, a total of 40 cycles. Using Excel software, the method of 2- ^ΔΔCt (

et al., Nucleic Acids Res, 2001, 29(14): 2994-3005), analyzed the relative expression levels of AgbZIP16 gene in two celery varieties in different tissues and under different stress conditions.

5. Experimental results: 1). The amino acid sequence encoded by the celery AgbZIP16 gene cloned in the present invention is predicted to be a conservative domain. The AgbZIP16 transcription factor gene contains a typical BRLZ conserved domain of the bZIP family, which confirms that the cloned AgbZIP16 gene belongs to the bZIP transcription factor family members (Figure 1). 2). The amino acid sequence encoded by the celery AgbZIP16 gene cloned by the present invention was compared with the bZIP protein amino acid sequence of other species, and it was found that AgbZIP16 has the closest evolutionary relationship with the carrot bZIP transcription factor belonging to the same Umbelliferae (Fig. 2). 3). The amino acid sequence encoded by the celery AgbZIP16 gene cloned by the present invention is subjected to hydrophobicity analysis, and the result shows that the celery AgbZIP16 amino acid is a hydrophilic protein, and the position with the strongest hydrophilicity is the 312-position glutamine (Glu), The most hydrophobic position is isoleucine (Ile) at position 239 (Figure 3). 4). Fluorescence quantitative PCR results showed that the relative expression of AgbZIP16 gene in 'Liuhe Huangxinqin' and 'Ventura' was the highest in the root and the lowest in the petiole; the expression of AgbZIP16 in the root of'Liuhe Huangxinqin' was detected. 2.64-fold and 1.05-fold higher than those in petioles and leaves, respectively, and 2.89-fold and 1.17-fold higher in 'Ventura' roots than those in petioles and leaves, respectively (Fig. 4). 5). The results of real-time PCR showed that the AgbZIP16 gene in celery all responded to high temperature, low temperature, drought and salt treatment. The expression level of AgbZIP16 in 'Liuhe Huangxinqin' was down-regulated after stress treatment; the expression of AgbZIP16 in 'Ventura' The level of high temperature treatment was significantly higher than that of the control at 4 h; the expression level of low temperature, drought and salt treatment showed an overall trend of first increase and then decrease, and the expression level was significantly increased at 24 h of salt treatment and 8 h of drought treatment (Fig. 5).

Claims (5)

1. The bZIP transcription factor gene AgbZIP16 is obtained from two celery varieties of 'Liuhe yellow celery' and 'wentula'.

2. The amino acid sequence of the celery bZIP transcription factor AgbZIP16 protein.

3. A method for preparing the AgbZIP 16-derived gene of claim 1, comprising the steps of:

1) carrying out retrieval analysis based on celery transcriptome sequencing information to obtain a gene sequence of celery AgbZIP 16;

2) designing a primer, and carrying out forward: 5'-ATGGGCAGTAGTGAAATGGAAA-3', reverse 5 ' -TCATGCAGCCTCTGTATGACCG-3, and cloning AgbZIP16 transcription factor gene from celery, celery hexaply yellow celery and Wen Tura by PCR method.

4. The celery AgbZIP16 gene function research of claim 1.

1) And (3) adopting a fluorescent quantitative PCR method to complete the expression quantity analysis of the AgbZIP16 gene of celery in roots, leaves and petioles of the Liuhe yellow celery and the Wendiula.

2) And (3) completing the expression analysis of the celery AgbZIP16 gene under the abiotic stress (high temperature, low temperature, drought and high salt) by adopting a fluorescent quantitative PCR method.

5. The use of the celery bZIP transcription factor AgbZIP16 gene as claimed in claim 1.

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