CN111718940A

CN111718940A - Sequence of carrot exogenous hormone-responsive related DcWRKY69 gene and application thereof

Info

Publication number: CN111718940A
Application number: CN201910229876.6A
Authority: CN
Inventors: 熊爱生; 张榕蓉; 徐志胜
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 invention takes carrot variety 'black field five inch' as a test material, obtains DcWRKY69 gene from the leaf cDNA by cloning by an RT-PCR method, sequence analysis results show that the DcWRKY69 gene comprises 1 Open Reading Frame (ORF) with the length of 783bp, codes 260 amino acids, has a highly conserved WRKY structural domain, and DcWRKY69 protein contains 34 phosphorylation sites in total, belongs to hydrophilic protein, and has a secondary structure consisting of 21.92% of α -helix, 4.23% of β -fold, 11.92% of extension main chain and 61.92% of random coilFor MeJA, SA and GA₃The ABA treatment and the ABA treatment respond, and the expression levels are most obviously upregulated after 8 h, 12h, 1 h and 4h after the treatment, and are all significantly different from the control. The gene DcWRKY69 is cloned from carrot and can participate in the response process of carrot to exogenous hormone.

Description

Sequence of carrot exogenous hormone-responsive related DcWRKY69 gene and application thereof

Technical Field

The invention belongs to the field of plant genetic engineering, and relates to a WRKY gene related to response of carrot to exogenous hormone and application thereof. The gene DcWRKY69 related to response of exogenous hormone is obtained by cloning from carrot, and can be applied to research on response of carrot to exogenous hormone

Background

Carrot (Daucus carota L.) is a biennial herb plant of the genus Daucus of the family Umbelliferae, is one of ten vegetable crops worldwide, and has a very wide cultivation area (Ouchuan Steel et al, Chinese vegetables, 2009 (4): 1-6). The fleshy root of carrot contains many nutrients such as protein, vitamins and carotenoids, and is the main part of food (strictly red, research and development of food, 2003, 24 (6): 120-. The growth and development processes of carrot, such as substance accumulation, structural change and gene regulation, are directly or indirectly regulated by phytohormone (Wang Guanlong, Nanjing: Nanjing agriculture university, 2016: 4-15).

Plant hormones widely participate in a series of physiological activities of plants such as biotic and abiotic stress response, carbohydrate synthesis, plant senescence and organ development by regulating expression of related genes (bear wins, et al, science bulletin, 2009, 54 (18): 2718-2733). The reasonable use of plant hormones can play a positive role in regulating the quality of carrot (Huang Ming Hui et al, northern horticulture 2015, 39 (12): 178-.

WRKY transcription factors (WRKY transcription factors) are a class of zinc finger type transcription regulating factors mainly existing in plants (Gao national Qing et al, notice of botany 2005, 22 (1): 11-18). In the aspect of phytohormone signal transduction, WRKY transcription factors are mainly involved in the pathway of abscisic acid (ABA), Salicylic Acid (SA), and methyl jasmonate (MeJA, methyl jasmonate), so as to regulate the growth and development, substance metabolism and disease-resistant and stress-resistant processes of plants (Liran et al, ecological report, 2011, 31 (11): 3223-3231), and simultaneously regulate the Plant senescence process by participating in the pathway of gibberellin (GA, gibberllin) (Chen et al, Molecular Plant, 2017, 10 (9): 1174-1189).

Disclosure of Invention

The invention provides a preparation method and application of a carrot exogenous hormone-responsive related DcWRKY69 gene. The obtained carrot DcWRKY69 gene is beneficial to deeply knowing the response mechanism of carrot to exogenous hormone.

Drawings

FIG. 1 conserved domain prediction of carrot DcWRKY69 transcription factor

FIG. 2 phylogenetic tree of carrot DcWRKY69 protein and other plant WRKY protein

FIG. 3 analysis of hydrophobicity and hydrophilicity of amino acid sequence of carrot DcWRKY69 protein

FIG. 4 phosphorylation site prediction of carrot DcWRKY69 protein

FIG. 5 Secondary Structure prediction of carrot DcWRKY69 protein

FIG. 6 expression level of carrot DcWRKY69 gene under different exogenous hormone treatment

Detailed Description

1. Plant material and treatment: carrot material 'black field five inches' is planted in the artificial climate chamber of key laboratory of Nanjing agriculture university crop inheritance and germplasm innovation country in 2018 and 9 months. The culture conditions were: illumination time 16h d^-1Day temperature 25 ℃, night temperature 18 ℃, and illumination intensity 300 [ mu ] mol/m^-2·s^-1. When the plant grows for 60 days, selecting plant leaves with good growth vigor, and respectively carrying out 0.1 mmol.L^-1Methyl jasmonate (MeJA), 1 mmol. L^-1Salicylic Acid (SA), 0.1 mmol. L^-1Gibberellins (GA)₃) And 0.1 mmol. multidot.L^-1Abscisic acid (ABA) treatment. The 4 hormones are dissolved by absolute ethyl alcohol, 5 mul of Tween-20 surfactant is added, and then the spraying treatment is carried out on the leaf surfaces of the plants. After 0, 1, 2, 4, 8 and 12 hours of treatment, the leaves of the plants treated by the 4 hormones are respectively taken.

2. Extraction of carrot total RNA and cDNA synthesis: total RNA of carrot leaf material was extracted using a plant total RNA extraction kit (Beijing Tiangen Biotechnology Co., Ltd.), concentration thereof was measured using a micro ultraviolet detector Nano-Drop, and the extracted RNA sample was reverse-transcribed into cDNA using a HiScriptII Q RT Supermix for qPCR (+ gDNA wiper) (Nanjing Nozanza Biotechnology Co., Ltd.).

3. Cloning of carrot DcWRKY69 Gene: based on the carrot genome Database (Xu et al, Database, 2014. DOI: 10.1093/Database/bau096) in the laboratory, the sequence of the carrot DcWRKY69 gene is obtained by searching, and a pair of cloning primers is designed to forwardThe primer is DcWRKY 69-F1: 5'-TCTCTCTAGCCAGCCAATAGC-3', the reverse primer is DcWRKY 69-R1: 5'-CAACAAAAAGCAGTTTT ATTT-3' are provided. And (3) carrying out PCR amplification by using carrot leaf cDNA as a template. Amplification System Total volume 30. mu.L, including PrimerStar Max Premix (2X) Hi Fidelity enzyme 15. mu. L, ddH ₂2. mu.L of O10. mu. L, cDNA template and 1.5. mu.L of forward and reverse primers, respectively. The amplification procedure was: pre-denaturation at 98 ℃ for 10 s; denaturation at 98 ℃ for 10s, annealing at 60 ℃ for 10s, and extension at 72 ℃ for 30s for 35 cycles; extension at 72 ℃ for 10 s. 5 mul of the amplified product was detected by electrophoresis using 1.2% agarose gel to obtain the correct target product, and the remaining 25 mul of the PCR product was sequenced by general biosystems, Inc.

3. Sequence analysis: performing Blast comparison and conservative domain prediction on the nucleotide and amino acid sequences of the obtained target gene fragment on an NCBI website (http:// Blast. NCBI. nlm. nih. gov/Blast. cgi); performing protein hydrophilicity/hydrophobicity analysis by adopting DNAMAN6.0 software; the construction of the evolutionary tree and the generation of the graphic report are completed by MEGA7.0 software; predictive analysis of phosphorylation sites was performed on Netphos 3.1 Server (http:// www.cbs.dtu.dk/services/Netphos /); predictive analysis of the secondary structure of the protein was performed on the SOPMA website (http:// pbil. ibcp. fr.).

4. Real-time quantitative PCR reaction: a Hieff qPCR SYBR Green Master Mix (san Jose assist Biotech Co., Ltd.) was selected and subjected to real-time fluorescent quantitative PCR according to the instructions. Designing a pair of fluorescent quantitative detection primers by means of Primer Premier 6.0 software, wherein the sequence of a forward Primer is DcWRKY 69-F2: 5'-GGAAGAAGAGCAGGAAGAAGAAG-3', reverse primer sequence DcWRKY 69-R2: 5'-AGCCACTCAACGGAGAATGTA-3' are provided. The carrot Actin gene is used as an internal reference gene (Tian et al, PLoS One, 2015, 10 (2): e0117569), and the primer sequence is as follows: forward Actin-F: 5'-CGGTATTGTGTTGGACTCTGGTGAT-3', respectively; reverse Actin-R: 5'-CAGCAAGGTCAAGACGGAGTATGG-3' are provided. The overall qRT-PCR reaction was 20. mu.L, including SYBR Premix Ex Taq enzyme 10. mu. L, cDNA template 2. mu. L, ddH₂O7.2. mu.L and forward and reverse primers 0.4. mu.L each. The reaction procedure is as follows: pre-denaturation at 95 ℃ for 5 min; denaturation at 95 ℃ for 10s and annealing at 60 ℃ for 30s for 40 cycles. Continuously raising the temperature to 95 ℃ in the process of gradually raising the temperature to 65 DEG CFluorescence was measured and a melting curve was plotted. By using 2^-ΔΔCtThe relative expression amount of the gene was calculated by the method, wherein Δ Δ Ct ═ C_{t, target Gene}-C_t，Actin)_{Treatment of}-(C_{t, target Gene}-C_t，Actin)_Control. 3 biological replicates were set for each treatment sample and the resulting values were analyzed for differential significance levels using IBM SPSS statistical 20 and WPS Excel 2019.

5. The experimental results are as follows: 1) the amino acid sequence coded by the carrot DcWRKY69 gene obtained by cloning is subjected to conservative domain prediction, and the carrot DcWRKY69 protein is found to have 1 conservative WRKY structural domain and belongs to a WRKY super family (figure 1). 2) The amino acid sequence of WRKY protein of carrot DcWRKY69 and other 13 plants is used for constructing phylogenetic tree, and the result shows that the evolutionary relationship of carrot of Umbelliferae plant and rubber tree of Euphorbiaceae plant and cotton tree of cassava, walnut of Juglandaceae plant and Malvaceae plant is close (figure 2). 3) The analysis result of the amino acid sequence hydrophilicity/hydrophobicity of the carrot DcWRKY69 protein shows that glutamic acid (Glu) at the 123 th position, the 125 th position and the 126 th position and lysine (Lys) at the 124 th position have the highest hydrophilicity. The most hydrophobic site is leucine (Leu) at position 99. In general, most of the amino acids in the carrot DcWRKY69 protein are hydrophilic amino acids, from which it is presumed that the protein belongs to a hydrophilic protein (FIG. 3). 4) The phosphorylation site prediction results of carrot DcWRKY69 protein show that carrot DcWRKY69 protein contains 34 phosphorylation sites in total, wherein 23 serine (Ser) phosphorylation sites, 9 threonine (Thr) phosphorylation sites and 2 tyrosine (Tyr) phosphorylation sites (FIG. 4). 5) Predictive analysis of the secondary structure of the carrot DcWRKY69 protein cloned according to the invention revealed that the carrot DcWRKY69 protein consisted essentially of 21.92% Alpha-helix (Alpha helix), 4.23% Beta-sheet (Beta turn), 11.92% Extended strand, and 61.92% Random coil (Random coil) (fig. 5). 6) Fluorescent quantitative PCR results show that the carrot DcWRKY69 gene is induced to be expressed under the treatment of methyl jasmonate (MeJA), Salicylic Acid (SA), gibberellin (GA3) and abscisic acid (ABA), but relative expression amounts are different, the expression levels are adjusted most obviously at 8, 12, 1 and 4h after the treatment, and the differences are significant compared with a control (FIG. 6).

Claims

1. A gene DcWRKY69 for responding to exogenous hormone is obtained from carrot.

2. The amino acid sequence of DcWRKY69 gene encoded DcWRKY69 protein of claim 1.

3. A method of preparing a DcWRKY 69-derived gene as defined in claim 1, comprising the steps of:

1) based on the carrot genome database in the laboratory, the nucleotide sequence of the carrot DcWRKY69 gene is obtained by searching;

2) design of cloning primers, forward: 5'-TCTCTCTAGCCAGCCAATAGC-3', reverse 5'-CAACAAAAAGCAGTTTTATTT-3', the DcWRKY69 gene was cloned from carrot variety ` Wutian-Wucun `byPCR.

4. The carrot DcWRKY69 gene function study of claim 1: adopting a fluorescent quantitative PCR method to finish the gene conversion of carrot DcWRKY69 in methyl jasmonate (MeJA), Salicylic Acid (SA) and Gibberellin (GA)₃) And analysis of expression level under abscisic acid (ABA) treatment.

5. The carrot DcWRKY69 gene as claimed in claim 1, used under the treatment of exogenous hormone.