CN111718941A

CN111718941A - Celery abiotic stress related transcription factor AgbZIP16 gene sequence and application thereof

Info

Publication number: CN111718941A
Application number: CN201910229877.0A
Authority: CN
Inventors: 熊爱生; 沈迪; 徐志胜; 刘洁霞
Original assignee: Nanjing Agricultural University
Current assignee: Nanjing Agricultural University
Priority date: 2019-03-22
Filing date: 2019-03-22
Publication date: 2020-09-29

Abstract

The bZIP transcription factor family belongs to a large number of transcription factor families, is well conserved, and is involved in plant growth and development, metabolic regulation and biotic and abiotic stress response. The patent clones a new bZIP transcription factor gene AgbZIP16 from two celery varieties of 'Liuhe yellow celery' and 'wentula'. The gene comprises an open reading frame with the length of 1221bp, encodes 406 amino acids and contains a BRLZ conserved domain typical of a bZIP family. The protein coded by AgbZIP16 belongs to hydrophilic protein, has a high conservative property in the recent evolutionary relationship with carrot bZIP transcription factor protein of the same genus and the family Umbelliferae. Fluorescence quantitative expression analysis shows that the relative expression of AgbZIP16 in the celery containing six ingredients and the asparagus containing 'wentula' is the highest in roots and the lowest in petioles, and the AgbZIP16 shows obvious tissue specificity. Meanwhile, the AgbZIP16 gene expression can be induced to change under the conditions of high temperature, low temperature, drought and salt stress, and the expression response of the AgbZIP16 gene in different celery varieties has obvious difference. The invention utilizes polymerase amplification technology to clone AgbZIP16 transcription factor gene from celery, and proves that the gene participates in stress resistance regulation of celery.

Description

Celery abiotic stress related transcription factor AgbZIP16 gene sequence and application thereof

Technical Field

The invention belongs to the field of plant genetic engineering, and relates to a bZIP transcription factor of a plant and application thereof. The gene AgbZIP16 for responding to abiotic stress is obtained from celery varieties 'Liuhe yellow celery' and 'wentula', and can be applied to the research and the application of a stress-resistant mechanism of celery.

Background

Celery (Apium graveolens L.) is a first-and second-year-old herb of Apium of Umbelliferae, contains abundant vitamins, carotene, cellulose and volatile aromatic components, and has edible and medicinal values. Celery is one of important leaf vegetable crops all over the world, has strong cold resistance, and has a long cultivation history and a wide planting range in China.

Plant growth and development are affected by abiotic stress factors, such as high temperature, low temperature, drought, salt stress and the like, which can damage plant cells, and further reduce yield and quality (Sunliemilitary et al, genetics, 2012, 34 (8): 993-. The transcription factor can control the expression of corresponding genes through the combination with downstream gene promoters during the growth and development of plants, and plays an important role (Du H et al, BMC Plant Biology, 2012, 12 (106): 1-22.). The Basic region/leucoine zipper (bZIP) family belongs to one of the transcription factor families with a large number, is well conserved and is found in human bodies, animals, plants and microorganisms. The conserved domain of the polypeptide consists of about 60-80 amino acid residues and comprises two conserved regions: a basic amino acid region and a leucine zipper region (Talanian R V et al, Science, 1990, 249 (4970): 769-771.). The results of the studies indicate that bZIP transcription factors are involved in Plant growth and development, metabolic regulation, and response to biotic and abiotic stress (Toh S et al, Plant Signal & Behavior, 2012, 7 (5): 556-. It is combined with abscisic acid (ABA), Cytokinin (CTK) and other response elements, and regulates the transcription of these response genes, thereby improving the stress resistance of plants (plum winter month, et al, Zhejiang agriculture bulletin, 2017, 29 (1): 168-.

Disclosure of Invention

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

Drawings

FIG. 1 prediction of conserved domain of amino acid sequence of celery AgbZIP16

FIG. 2 phylogenetic tree of AgbZIP16 with bZIP protein sequences from other species

FIG. 3 analysis of the hydrophobicity and hydrophilicity of the amino acid sequence of celery AgbZIP16

FIG. 4 expression of AgbZIP16 in different tissue parts of celery

FIG. 5 expression of AgbZIP16 under different abiotic stresses in celery

Detailed Description

1. Extraction of celery total RNA and synthesis of cDNA: taking two celery samples of 'Liuhe yellow celery' and 'Wen Tura', extracting Total RNA respectively according to the instruction of an RNA Simple Total Rna Kit (Beijing Tiangen), detecting the concentration of the Total RNA by using a Nanodrop2000 micro-spectrophotometer (Thermo Scientific in America), and then carrying out reverse transcription on the Total RNA into cDNA according to the instruction of a Prime Script RTreagen Kit.

2. Cloning of celery transcription factor AgbZIP16 gene: the gene sequence encoding the celery AgbZIP16 was retrieved from the celery transcriptome database (JiaX L et al, Scientific Reports, 2015, 5: 8259.) determined in this subject group, and based on this sequence a pair of specific primers was designed using Primer Premier 6.0, the forward Primer sequence being: 5'-ATGGGCAGTAGTGAAATGGAAA-3', respectively; the reverse primer sequence is as follows: 5'-TCATGCAGCCTCTGTATGACCG-3' are provided. And carrying out PCR amplification on the target gene segment by using cDNA obtained by reverse transcription as a template. A total of 20. mu.l of PCR reaction was included with cDNA (2.5 ng.. mu.L)^-1)1 μ L of forward and reverse primers, 1 μ L of each, ddH₂O7. mu.L, PrimeSTAR Max Premix (TaKaRa Co., Ltd.) 10. mu.L. The reaction procedure is as follows: pre-denaturation at 94 ℃ for 5 min; denaturation at 94 ℃ for 30s, annealing at 54 ℃ for 30s, and extension at 72 ℃ for 1min for 35 cycles; extension at 72 ℃ for 10 min. The PCR amplification product is detected by electrophoresis using 1.5% agar gel, and the recovered reaction product is obtained by the Biotech of Nanjing JinsleiLimited sequencing.

3. Sequence analysis: analyzing the nucleotide and amino acid sequence of the AgbZIP16 gene fragment obtained by cloning by using BioXM 2.6 software; obtaining conservative domain prediction and homology analysis of a target gene by using a BLAST tool in an NCBI database; performing hydrophilic and hydrophobic analysis on the amino acid sequence coded by the target gene by using DNAMAN 6.0 software; the phylogenetic tree was drawn using MEGA5.2 software.

4. Real-time quantitative PCR reaction: according to the celery AgbZIP16 gene sequencing result, a Primer Premier 6.0 is adopted to design a fluorescent quantitative Primer, and a forward Primer is AgbZIP 16-qF: 5'-AACCATCAGGAGCAGCCACTAATG-3', the reverse primer is AgbZIP 16-qR: 5'-CCGCACTAACAGTTCCTCCAGTAAT-3' are provided. The celery actin gene is used as an internal reference gene (Jiang Q et al, Plos One, 2014, 9 (3): e 92262). The expression condition of the gene under different tissues and different abiotic stresses is analyzed by using a fluorescent quantitative PCR detection system. 20 μ L of reaction system, including 0.4 μ L of forward and reverse primers, 10 μ L of SYBRGreen I mix, ddH₂O7.2. mu.L and 2. mu.L of 15-fold diluted cDNA. The reaction procedure is as follows: pre-denaturation at 95 ℃ for 30s, denaturation at 95 ℃ for 5s, and annealing and extension at 60 ℃ for 30s, and the cycle is 40 times. Excel software was used, with 2^-ΔΔCtMethod (1)

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

5. The experimental results are as follows: 1) the conservative domain prediction is carried out on the amino acid sequence coded by the celery AgbZIP16 gene obtained by cloning, the AgbZIP16 transcription factor gene contains a BRLZ conservative domain typical of a bZIP family, and the AgbZIP16 gene obtained by cloning is proved to belong to a bZIP transcription factor family member (figure 1). 2) Comparing the amino acid sequence coded by the celery AgbZIP16 gene obtained by cloning with the amino acid sequences of bZIP proteins of other species, the evolutionary relationship between AgbZIP16 and carrot bZIP transcription factors of the same genus and the family Umbelliferae is found to be recent (figure 2). 3) The amino acid sequence coded by the celery AgbZIP16 gene cloned by the invention is subjected to hydrophilic and hydrophobic analysis, and the result shows that the celery AgbZIP16 amino acid is hydrophilic protein, wherein the position with the strongest hydrophilicity is glutamine (Glu) at position 312, and the position with the strongest hydrophobicity is isoleucine (Ile) at position 239 (figure 3). 4) The fluorescent quantitative PCR result shows that the relative expression amounts of the AgbZIP16 genes in the celery Liuhe and the Wen Tura are the highest in roots and the lowest in petioles; AgbZIP16 was detected to be expressed in the root of ` Saxaparia longifolia ` at 2.64-fold and 1.05-fold respectively in the petioles and leaves, and in ` Wen Tura ` at 2.89-fold and 1.17-fold respectively in the petioles and leaves (FIG. 4). 5) Fluorescent quantitative PCR results show that the AgbZIP16 gene of celery responds to high temperature, low temperature, drought and salt treatment, and the expression level of AgbZIP16 in the 'Liuhe yellow celery' is down-regulated after stress treatment; the expression level of AgbZIP16 in 'wentula' was significantly higher than the control at high temperature treatment for 4 h; the expression level of the gene under low temperature, drought and salt treatment is in a trend of increasing firstly and then decreasing, and the expression level is obviously increased when the salt treatment is carried out for 24 hours and the drought treatment is carried out for 8 hours (figure 5).

Claims

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.