CN111718940A - Sequence of a carrot-related DcWRKY69 gene in response to exogenous hormones and its application - Google Patents

Abstract

WRKY transcription factors can regulate plant growth, development and material metabolism by participating in hormone signaling pathways. In the present invention, the carrot variety 'Heitian Wucun' is used as the test material, and the DcWRKY69 gene is cloned from the cDNA of its leaves by RT-PCR method. The results of sequence analysis showed that the DcWRKY69 gene contains an open reading frame (ORF) of 783 bp in length, encoding 260 amino acids, with a highly conserved WRKY domain; DcWRKY69 protein contains a total of 34 phosphorylation sites, belonging to a hydrophilic protein , its secondary structure consists of 21.92% α-helix, 4.23% β-sheet, 11.92% extended backbone and 61.92% random coil. RT‑qPCR results showed that DcWRKY69 gene responded to MeJA, SA, GA ₃ and ABA treatments, and its expression level was most significantly up-regulated at 8, 12, 1 and 4 h after treatment, and there were significant differences compared with the control. The invention clones the DcWRKY69 gene from carrot, and the gene can participate in the response process of carrot to exogenous hormone.

Description

Sequence of a carrot response to exogenous hormone-related DcWRKY69 gene and its application

technical field

The invention belongs to the field of plant genetic engineering, and relates to a WRKY gene related to carrot response to exogenous hormones and its application. In the present invention, a gene DcWRKY69 related to responding to exogenous hormones is cloned and obtained from carrots, and the gene can be applied to the research on the response of carrots to exogenous hormones

Background technique

Carrot (Daucus carota L.) is a biennial herb of the genus Daucus in the family Umbelliferae, and is one of the top ten vegetable crops in the world with a very wide cultivation area (Ou Chenggang et al., Chinese Vegetables, 2009(4): 1-6). The fleshy root of carrot contains protein, vitamins and carotenoids and other nutrients, and is the main edible part (Yan Yihong, Food Research and Development, 2003, 24(6): 120-122). The growth and development of carrots, such as material accumulation, structural changes, and gene regulation, are directly or indirectly regulated by plant hormones (Wang Guanglong, Nanjing: Nanjing Agricultural University, 2016: 4-15).

Plant hormones are widely involved in a series of physiological activities of plants, such as biotic and abiotic stress responses, carbohydrate synthesis, plant senescence and organ development by regulating the expression of related genes (Xiong Guosheng et al., Science Bulletin, 2009, 54(18) : 2718-2733). The rational use of plant hormones can play a positive role in regulating carrot quality (Huang Minghui et al., Northern Horticulture, 2015, 39(12): 178-182).

WRKY transcription factors (WRKY transcription factors) are a class of zinc-finger transcriptional regulators that mainly exist in plants (Gao Guoqing et al., Bulletin of Botany, 2005, 22(1): 11-18). In terms of plant hormone signal transduction, WRKY transcription factors are mainly involved in the pathways of abscisic acid (ABA, abscisic acid), salicylic acid (SA, salicylic acid), and methyl jasmonate (MeJA, methyl jasmonate), thereby regulating plant growth. The process of growth and development, material metabolism, disease resistance and stress resistance (Li Ran et al., Chinese Journal of Ecology, 2011, 31(11): 3223-3231), and can also regulate plant senescence by participating in the gibberellin (GA, gibberellin) pathway Progress (Chen et al., Molecular Plant, 2017, 10(9): 1174-1189).

SUMMARY OF THE INVENTION

The invention provides a preparation method and application of DcWRKY69 gene related to carrot response to exogenous hormones. The obtained DcWRKY69 gene of carrot is beneficial to deeply understand the response mechanism of carrot to exogenous hormones.

Description of drawings

Figure 1. Conserved domain prediction of carrot DcWRKY69 transcription factor

Figure 2. Phylogenetic tree of carrot DcWRKY69 protein and other plant WRKY proteins

Figure 3. Hydrophobicity and hydrophilicity analysis of the amino acid sequence of carrot DcWRKY69 protein

Figure 4. Prediction of phosphorylation sites of carrot DcWRKY69 protein

Figure 5. Secondary structure prediction of carrot DcWRKY69 protein

Figure 6. Expression levels of carrot DcWRKY69 gene under different exogenous hormone treatments

Detailed ways

1. Plant material and treatment: The carrot material 'Heitian Wucun' was planted in the artificial climate room of the State Key Laboratory of Crop Genetics and Germplasm Innovation of Nanjing Agricultural University in September 2018. The culture conditions were as follows: light time of 16h·d ^-1 , day temperature of 25℃, night temperature of 18℃, and light intensity of 300μmol·m ^-2 ·s ^-1 . When it grows to 60 days, the leaves of plants with good growth are selected and treated with 0.1mmol·L ^-1 methyl jasmonate (MeJA), 1mmol·L ^-1 salicylic acid (SA), 0.1mmol·L ^-1 gibberellin respectively. (GA ₃ ) and 0.1 mmol·L ^-1 abscisic acid (ABA) treatment. The four hormones were all dissolved in absolute ethanol, and after adding 5 μl of Tween-20 surfactant, the leaves of the plants were sprayed. After 0, 1, 2, 4, 8, and 12 h of treatment, the leaves of the plants under the four hormone treatments were collected respectively.

2. Extraction of carrot total RNA and synthesis of cDNA: using a plant total RNA extraction kit (Beijing Tiangen Biochemical Technology Co., Ltd.), the total RNA of carrot leaf material was extracted, and its concentration was measured using a nano-ultraviolet detector Nano-Drop, The extracted RNA samples were reverse transcribed into cDNA using the HiScript II Q RT SuperMix for qPCR (+gDNA wiper) (Nanjing Nova Biotechnology Co., Ltd.) kit.

3. Cloning of carrot DcWRKY69 gene: Based on the carrot genome database in our laboratory (Xu et al., Database, 2014. DOI: 10.1093/database/bau096), the sequence of carrot DcWRKY69 gene was retrieved and a pair of cloning primers were designed. The forward primer was DcWRKY69-F1: 5'-TCTCTCTAGCCAGCCAATAGC-3', and the reverse primer was DcWRKY69-R1: 5'-CAACAAAAAGCAGTTTT ATTT-3'. PCR amplification was performed using carrot leaf cDNA as a template. The total volume of the amplification system was 30 μL, including 15 μL of PrimerStar Max Premix (2X) high-fidelity enzyme, 10 μL of ddH ₂ O, 2 μL of cDNA template, and 1.5 μL of forward and reverse primers. The amplification program was as follows: pre-denaturation at 98°C for 10s; denaturation at 98°C for 10s, annealing at 60°C for 10s, extension at 72°C for 30s, a total of 35 cycles; extension at 72°C for 10s. Take 5 μL of the amplified product and use 1.2% agarose gel for electrophoresis detection. After obtaining the correct target product, the remaining 25 μL PCR product is entrusted to General Biosystems Co., Ltd. for sequencing.

3. Sequence analysis: On the NCBI website (http://blast.ncbi.nlm.nih.gov/Blast.cgi), the nucleotide and amino acid sequences of the obtained target gene fragments were subjected to Blast comparison and conservative domain prediction; DNAMAN6.0 software was used for protein hydrophilicity/hydrophobicity analysis; the construction of phylogenetic tree and the generation of graphic report were completed by MEGA7.0 software; the prediction analysis of phosphorylation sites was performed in NetPhos 3.1 Server (http://www.cbs. dtu.dk/services/NetPhos/); on the SOPMA website (http://pbil.ibcp.fr/) for prediction of the secondary structure of proteins.

4. Real-time quantitative PCR reaction: select Hieff qPCR SYBR Green Master Mix (Shanghai Yisheng Biotechnology Co., Ltd.), and perform real-time quantitative PCR operation according to the operation instructions. A pair of fluorescent quantitative detection primers were designed with the help of Primer Premier 6.0 software. The forward primer sequence was DcWRKY69-F2: 5'-GGAAGAAGAGCAGGAAGAAGAAG-3', and the reverse primer sequence was DcWRKY69-R2: 5'-AGCCACTCAACGGAGAATGTA-3'. The carrot Actin gene was used as the internal reference gene (Tian et al., PLoS One, 2015, 10(2): e0117569), and the primer sequences were: forward Actin-F: 5'-CGGTATTGTGTTGGACTCTGGTGAT-3'; reverse Actin-R: 5'-CAGCAAGGTCAAGACGGAGTATGG-3'. The total qRT-PCR reaction system was 20 μL, including 10 μL of SYBR Premix Ex Taq enzyme, 2 μL of cDNA template, 7.2 μL of ddH ₂ O, and 0.4 μL of forward and reverse primers. The reaction program was: pre-denaturation at 95 °C for 5 min; denaturation at 95 °C for 10 s, and annealing at 60 °C for 30 s, a total of 40 cycles. Fluorescence was continuously measured during the stepwise temperature rise from 65°C to 95°C, and a melting curve was drawn. The relative expression of genes was calculated by the 2- ^ΔΔCt method, where ΔΔCt=(C _{t, target gene} -C _{t, Actin} ) _treatment- (C _{t, target gene} -C _{t, Actin} ) _control . Three biological replicates were set up for each treatment sample, and the obtained values were analyzed at the significant level of difference using IBM SPSS Statistic 20 and WPS Excel 2019.

5. Experimental results: 1). Predict the conserved domain of the amino acid sequence encoded by the carrot DcWRKY69 gene cloned in the present invention. It is found that the carrot DcWRKY69 protein has a conserved WRKY domain and belongs to the WRKY superfamily (Fig. 1). 2). A phylogenetic tree was constructed using the amino acid sequences of carrot DcWRKY69 and WRKY proteins of 13 other plants. The results showed that the evolution of Umbelliferae carrot and Euphorbiaceae rubber tree and cassava, Jugaceae walnut and Malvaceae plant cotton The relationship is relatively close (Figure 2). 3). The analysis of the hydrophilicity/hydrophobicity of the amino acid sequence of carrot DcWRKY69 protein showed that the glutamic acid (Glu) at positions 123, 125 and 126 and the lysine (Lys) at position 124 had the strongest hydrophilicity . The most hydrophobic site is leucine (Leu) at position 99. Overall, most of the amino acids in the carrot DcWRKY69 protein were hydrophilic amino acids, so it was speculated that the protein was a hydrophilic protein (Figure 3). 4). The prediction results of the phosphorylation sites of carrot DcWRKY69 protein show that carrot DcWRKY69 protein contains a total of 34 phosphorylation sites, including 23 serine (Ser) phosphorylation sites and 9 threonine (Thr) phosphorylation sites. site and 2 tyrosine (Tyr) phosphorylation sites (Figure 4). 5). The secondary structure of the carrot DcWRKY69 protein cloned by the present invention is predicted and analyzed, and it is found that the carrot DcWRKY69 protein is mainly composed of 21.92% α-helix (Alpha helix), 4.23% β-sheet (Beta turn), 11.92% The Extended strand consisted of 61.92% random coil (Fig. 5). 6). The results of real-time quantitative PCR showed that the expression of DcWRKY69 gene in carrots was induced under the treatment of methyl jasmonate (MeJA), salicylic acid (SA), gibberellin (GA3) and abscisic acid (ABA), but the relative expression There was a certain difference in the amount of the two groups, and their expression levels were most significantly up-regulated at 8, 12, 1, and 4 h after treatment, and there were significant differences compared with the control (Figure 6).

Claims (5)

1. A response to exogenous hormone-related gene DcWRKY69 obtained from carrot. 2. The amino acid sequence of the DcWRKY69 protein encoded by the DcWRKY69 gene of claim 1. 3. a method for preparing the described DcWRKY69 gene of claim 1, comprising the following steps: 1) Based on the carrot genome database of our laboratory, retrieve the nucleotide sequence of the carrot DcWRKY69 gene; 2) Design cloning primers, forward: 5'-TCTCTCTAGCCAGCCAATAGC-3', reverse 5'-CAACAAAAAGCAGTTTTATTT-3', clone the DcWRKY69 gene from carrot variety 'Heitian Wucun' by PCR method. 4. The research on the function of the carrot DcWRKY69 gene according to claim 1: using the fluorescent quantitative PCR method, complete the carrot DcWRKY69 gene in methyl jasmonate (MeJA), salicylic acid (SA), gibberellin (GA ₃ ) and abscisic acid. (ABA) expression analysis under treatment. 5. The application of the carrot DcWRKY69 gene of claim 1 under the treatment of exogenous hormones.

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