Effective method for rapidly predicting downstream target gene of strawberry photoresponse transcription factor

Abstract

The invention provides an effective method for rapidly predicting downstream target genes of strawberry photoresponse transcription factors, and belongs to the technical field of biological information. According to the method provided by the invention, data is measured based on a DNA affinity purification sequencing method (DAP-seq), a basic sequence element and a position of a photoresponse-related promoter are detected in a whole genome, and a downstream target gene of a strawberry photoresponse key transcription factor is predicted. According to the method, sequencing data and bioinformatics tools are combined, so that the method has the advantages of low cost, rapidness and convenience, the prediction accuracy and flux are improved, and the method has important significance in research on a regulation mechanism of the photoresponse transcription factor.

Description

Technical Field

[0001] The invention relates to the field of bioinformatics, and in particular to an effective method for rapidly predicting downstream target genes of strawberry light-responsive transcription factors. Background Art

[0002] Strawberry is known as the "Queen of Fruits" and is one of the fruits and vegetables with important economic value. It is distributed all over the world. Since 2017, China has ranked first in the world in terms of both cultivated area and total output of strawberries. In recent years, due to the rise of facility cultivation and ecological park sightseeing and picking, strawberries have become one of the main cultivated plant varieties in facility cultivation because of their advantages such as short growth cycle, early listing and high efficiency. At present, research on strawberries at home and abroad is mainly reflected in cultivation technology, quality improvement methods and post-harvest preservation.

[0003] Light is one of the most important environmental factors that determine the development process of plants, and is involved in regulating physiological and biochemical processes such as seed germination, seedling growth, organ development, and flowering time. The absorption of light by different photoreceptors leads to the regulation of the core signaling network, which further coordinates specific hormone and metabolic signaling pathways to regulate plant growth and development. The perception of light by photoreceptors activates many transcription factors of different gene families that play a regulatory role, such as bZIP, bHLH, MYB, Zinc.finger, GATA, GTl, etc., which can bind to light-responsive elements such as G-BOX, Z-BOX, GTl-motif and MYB on gene promoters to regulate gene transcription levels. Among them, HY5 in the bZIP transcription factor family is the first transcription factor discovered to promote photomorphogenesis. It can respond to light signals transmitted by different photoreceptors under light, while in a dark environment, COPl will bind to HY5 and reduce its activity. Transcription factor HY5 is a molecular switch for light-regulated plant development and a positive regulator of photomorphogenesis. It regulates the expression of thousands of genes in the plant genome during light-induced gene expression. HY5 protein plays an important role in the signal transduction process of plant hormones and in photomorphogenesis. It integrates light signal transduction and plant hormone signal transduction. Therefore, it is of great significance to study the binding sites of light-responsive transcription factors on the whole genome and their downstream target gene information.

[0004] The prediction and identification methods for transcription factor binding sites include: ChIP-seq technology, CUT&Tag technology and DAP-seq technology, among which ChIP-seq technology and CUT&Tag technology must be transformed by stable plants, but the genetic transformation cycle of strawberry is long and the transformation efficiency is low. DAP-seq technology is to identify its binding sites in the whole genome by combining in vitro expression of transcription factors with high-throughput sequencing technology. DAP-seq technology has the advantages of saving time and cost, not requiring transgenic technology, and is more suitable for predicting downstream target genes of strawberry transcription factors. However, DAP-seq technology requires precise operation and high-quality samples, not only requiring sufficient genomic DNA, but also requiring high purity, and any impurities in the sample may interfere with the test results. Therefore, the present invention provides a fast and effective method for predicting downstream target genes of strawberry transcription factors. Summary of the invention

[0005] In view of this, the purpose of the present invention is to provide an effective method for quickly predicting the downstream target genes of strawberry light-responsive transcription factors. The method provided by the present invention can accurately locate and mine the binding sites of light-responsive transcription factors on the whole genome and their downstream target genes.

[0006] In order to achieve the above object, the present invention provides the following technical solutions:

[0007] An effective method for rapidly predicting downstream target genes of strawberry light-responsive transcription factors, the method comprising the following steps:

[0008] S1, extracting strawberry fruit genomic DNA, fragmenting the genomic DNA, sorting and purifying it, and then constructing a DNA library;

[0009] S2, constructing an in vitro protein expression vector of a key transcription factor of strawberry light response, obtaining a recombinant plasmid, expressing a fusion protein with a halo tag, and combining the protein with magnetic beads; the key transcription factor of strawberry light response is FvHY5 protein;

[0010] S3, mixing the protein-bound magnetic beads and the DNA library, eluting and performing PCR enrichment, and sequencing the enriched fragments;

[0011] S4. Perform DAP-seq analysis to obtain the binding sites and downstream target genes of strawberry light-responsive key transcription factors at the genome-wide level.

[0012] In the present invention, the fragmentation of genomic DNA in S1 is preferably processing the genomic DNA into fragments of 300 bp in size.

[0013] In the present invention, the sorting and purification in S2 preferably includes magnetic bead purification.

[0014] In the present invention, the volume ratio of magnetic beads to DNA fragments during the magnetic bead purification is preferably 2:1.

[0015] In the present invention, the in vitro protein expression vector used in S2 is preferably pDonor201 vector.

[0016] In the present invention, the size of the DNA fragment after PCR enrichment in S3 is preferably 250 bp to 350 bp.

[0017] In the present invention, the DAP-seq analysis in S4 preferably includes but is not limited to transcription factor binding site motif analysis, GO annotation of downstream genes regulated by transcription factors, and analysis of downstream genes regulated by transcription factors.

[0018] In the present invention, the most significantly enriched motif of the FvHY5 protein is preferably [T / C / G / A]ACGT[G / A / C][G / T / A][C / A / G / T][C / T / A][C / A / T / G].

[0019] Beneficial technical effects: The present invention provides an effective method for quickly predicting downstream target genes of strawberry light-responsive transcription factors. The method is based on the data measured by the DAP affinity purification sequencing method (DAP-seq), detects the basic sequence elements and positions of promoters related to light response in the whole genome, and predicts the downstream target bases of key strawberry light-responsive transcription factors. By combining sequencing data and bioinformatics tools, the present invention not only has the advantages of low cost, fast and convenient, but also increases the accuracy and throughput of prediction, which is of great significance to the study of the regulatory mechanism of light-responsive transcription factors. BRIEF DESCRIPTION OF THE DRAWINGS

[0020] Figure 1 DAP-seq was used to detect the binding peak distribution of FvHY5 in the whole strawberry genome;

[0021] Figure 2 The average density and heat map analysis of FvHY5-bound genes;

[0022] Figure 3 Motifs enriched for DAP-seq-specific detection of target genes;

[0023] Figure 4 GO enrichment analysis of FvHY5-binding downstream genes detected by DAP-seq. DETAILED DESCRIPTION

[0024] In order to better understand the present invention, the content of the present invention is further explained in conjunction with the following examples, but the content of the present invention is not limited to the following examples. The materials, reagents, etc. used in the examples and test examples of the present invention, unless otherwise specified, can be obtained from commercial channels; the methods used in the examples and test examples of the present invention, unless otherwise specified, are conventional methods.

[0025] Example 1

[0026] 1. Reagents and Materials

[0027] The fruits of forest strawberry (Fragaria vesca) 'Ruegen' were collected from the sunlight greenhouse of the teaching and research base of Shenyang Agricultural University. The fruits of forest strawberry 'Ruegen' at different developmental stages were collected, frozen in liquid nitrogen, mixed and ground for the construction of DNA library.

[0028] 2. Research Methods

[0029] 2.1 DNA library construction

[0030] The CTAB method was used to extract the genomic DNA of diploid forest strawberry fruit. Weigh 0.2g of powdered pulp tissue ground in liquid nitrogen, add 588μL of CTAB solution preheated at 65℃, and add 12μL of β-mercaptoethanol and mix. Then place it in a 65℃ water bath for 5min, mix it several times, and repeat 5 times. Extract it twice with chloroform. First, add an equal volume of chloroform and mix it for 5min, then centrifuge it at 12000rpm / min for 10min. Then take 450μL of supernatant and add an equal volume of chloroform for the second extraction. Finally, take 300μL of supernatant and add two times of the volume of anhydrous ethanol precooled at -20℃ and mix it, then place it at -20℃ for 30min. Then centrifuge it at 12000rpm / min in a low-temperature centrifuge for 10min, discard the supernatant, and add 800μL of 70% ethanol to wash the DNA twice. The purified DNA was air-dried and dissolved in 50 μL of TE buffer. The concentration of DNA was detected by ultra-micro UV spectrophotometer NanoDrop 2000 (Thermo Fisher Scientific, USA), and its integrity was checked by agarose gel electrophoresis.

[0031] 5 μg of the above genomic DNA was diluted with EB buffer (10 mM Tris-HCl, pH 8.5), and then the genomic DNA was ultrasonically disrupted using a non-contact ultrasonic disruptor (brand: Diagenode, model: Bioruptor Plus), and the program was set as: 80% power, 5S on, 30S off, 18 cycles, temperature 30°C. The DNA fragments were purified with AmpureXP magnetic beads (Beckman Coulter, product number A63881): DNA fragments (volume ratio) = 2:1, and the sample was finally eluted into 30 μL EB buffer.

[0032] 2.2 FvHY5 protein expression

[0033] The FvHY5 fragment (https: / / www.rosaceae.org / , ID: FvH4_2g29440) was constructed into the pDonor201 vector using the FvHY5-pDonor201-F / R primers. The successfully sequenced plasmid was named pDonor201-FvHY5. The primer sequences are as follows:

[0034] FvHY5-pDonor201-F (SEQ ID NO.1):

[0035] 5'-ACAAGTTTGTACAAAAAAGCAGGATTCATGTTGCAAGACCAAGCCACG-3';

[0036] FvHY5-pDonor201-R(SEQ ID NO.2):

[0037] 5'-ACCACTTTGTACAAGAAAGCTGGGTAAGATCCCTCAGCATTTG-3'.

[0038] Take 2 μL of pDonor201-FvHY5 plasmid and pIX-HALO plasmid, mix with 1 μL of LR recombinase, react overnight at 25℃, and store at 4℃. Transform the recombinant plasmid into E. coli competent cells and sequence. The plasmid with correct sequencing is named pIX-HALO-FvHY5.

[0039] The fusion protein was expressed using the TNT SP6 Coupled Wheat Germ Extract System (Promega), where 2 μg to 3 μg of expression vector plasmid was added to 50 μL of the system, incubated at 30°C for 2 h, and the protein expression was detected. Bead (Promega) kit instructions for purification.

[0040] 2.3 DNA-protein binding

[0041] Add 50 μL of purified magnetic beads with FvHY5 protein to 500 μg of DNA library and rotate at low speed at room temperature 25°C for 1 hour. Then use a magnet to adsorb the magnetic beads and remove the supernatant. Wash the beads four times with 200 μL PBS+NP40 (0.005%). Then resuspend in 100 μL 1X PBS, adsorb the magnetic beads with a magnet and remove the supernatant, resuspend the HaloTag magnetic beads in 30 μL EB solution, place at 98°C for 10 minutes, then place on ice for 5 minutes, adsorb the magnetic beads with a magnet and aspirate the supernatant containing the eluted DNA. Then take 30μL of the eluted sample and add 2μL of 10μM Illumina TruSeq universal primers (SEQ ID NO.3~SEQ ID NO.4), 1μL of Phusion, 5μL of dNTP, 5μL of 5×HFbuffer, 1.5μL of DMSO. The reaction procedure is: 72℃5min; 98℃3min; 98℃15s, 63℃30s, 72℃1min, 13 cycles. The negative control of this test is the binding reaction solution of the genomic library and magnetic beads without protein binding. The PCR amplification product was purified and recovered with AMPure XP beads (BECKMAN) and dissolved with 25μL~30μL enzyme-free water. The DNA concentration was detected (the sample was qualified if it was 50ng / μL) and the enriched fragments were sequenced.

[0042] SEQ ID NO.3:

[0043] 5'-CAAGCAGAAGACGGCATACGAGATNNNNNNGTGACTGGAGTTCAGACGTGTGCTCTTCCGATCT-3';

[0044] SEQ ID NO.4:

[0045] 5'-AATGATACGGCGACCACCGAGATCTACACTCTTTCCCTACACGAGCGCTCTTCCGATCT-3'.

[0046] 2.4 DAP-seq data analysis

[0047] The original sequencing data was quality controlled and analyzed. Using Q30 as the quality control standard, reads containing adapter sequences, N ratio greater than 10%, and low-quality reads were filtered. Clean data was compared with the diploid forest strawberry reference genome sequence (Fragariavesca Genome v4.0.a2). The location of each fragment in the genome was obtained, and combined with the annotation information, information such as the genes and their functions in the region where the fragments were aligned were obtained; the peaks were found according to the enrichment of the fragments; the distribution of the filtered peaks in the introns, exons, and intergenic regions of the genome was annotated; the genes whose peaks overlapped with the promoter region were screened as the target genes of the transcription factor for GO analysis, and the motifs enriched in the upstream (promoter region) of the gene were found.

[0048] 3. Results and Analysis

[0049] 3.1 In vitro expression of FvHY5 protein

[0050] Before the DAP-seq experiment, Western blot was used to verify whether the in vitro expressed protein was correctly expressed. The in vitro expressed tag fusion protein FvHY5 was used for verification, and the tag fusion protein size was about 51KD. The test results showed that the transcription factor was correctly expressed in vitro.

[0051] 3.2 Construction of DAP-library

[0052] The DNA of the fruits of forest strawberry at different developmental stages was extracted by CTAB method and fragmented. The fragments after fragmentation were purified to remove fragments that were too large or too small. The in vitro translated fusion protein was incubated with Magne HaloTag Beads, and the transcription factor was fixed by magnetic bead capture tag, and reacted with the constructed DAP library. The DNA fragments that specifically bound to the fusion protein were then eluted and subjected to PCR reaction to construct the library.

[0053] 3.3 Distribution of DNA affinity purification sequencing peaks across the genome

[0054] DAP-seq raw data was obtained by high-throughput sequencing. After quality control using Q30 as the standard, the clean data was aligned to the diploid strawberry reference genome sequence to obtain the location of each read in the genome. The average alignment rate of the DAP-seq library constructed by this method was 74.29%, and there were sufficient peaks, indicating that the data was qualified.

[0055] In order to obtain the distribution of transcription factor binding peaks in the whole genome, the forest strawberry genome sequence was divided into promoter region (promoter), exon region (Exon), intron region (Intron), intergenic region (distalintergenic), and downstream region (downstream). All transcription factor binding peaks were summarized and classified ( Figure 1 ), a total of 41.04% of the peaks were distributed in the promoter (<=1kb), 19.37% of the peaks were distributed in the promoter (1-2kb), 12.48% of the peaks were distributed in the promoter (2-3kb), 0.03% of the peaks were distributed in the first exon, 2.92% of the peaks were distributed in other exons, 0.22% of the peaks were distributed in the first intron, 1.38% of the peaks were distributed in other introns, 0.23% of the peaks were distributed in the downstream region of the gene, and 21.11% of the peaks were distributed in the intergenic region. The results show that the transcription factor binding sites are significantly enriched in the downstream gene promoter region, which is consistent with its transcriptional regulatory function.

[0056] We found that the fragments enriched in the DAP-seq library of the FvHY5 transcription factor were mainly concentrated near the transcription start site, indicating that the FvHY5 transcription factor binds to the promoter region and participates in the transcriptional regulation of the gene ( Figure 2 ).

[0057] 3.4 Analysis of motifs of FvHY5 transcription factor binding sites

[0058] The results are as follows Figure 3 As shown. The FvHY5 transcription factor binding sites are G-box (CACGTG) and ACE-box (ACGT). Interestingly, the most significantly enriched motif of FvHY5 obtained in this DAP-seq experiment is [T / C / G / A]ACGT[G / A / C][G / T / A][C / A / G / T][C / T / A][C / A / T / G], indicating that DAP-seq has a high accuracy.

[0059] 3.5 GO annotation of downstream genes regulated by transcription factors

[0060] After screening the downstream genes bound by DAP-seq, genes whose binding sites were located in the promoter region were selected for GO (Gene Ontology) enrichment analysis. The analysis results are as follows Figure 4 As shown by Figure 4 It can be seen that the downstream genes of FvHY5 are significantly enriched in categories such as secondary metabolite biosynthesis process, phenylpropanoid metabolism process, and pectinesterase activity.

[0061] The above is only a preferred embodiment of the present invention. It should be pointed out that for ordinary technicians in this technical field, several improvements and modifications can be made without departing from the principle of the present invention. These improvements and modifications should also be regarded as the scope of protection of the present invention.

Claims

1. An effective method for rapidly predicting downstream target genes of strawberry light-responsive transcription factors, characterized in that: The method comprises the following steps: S1, extracting genomic DNA from strawberry fruit, fragmenting the genomic DNA using ultrasound, sorting and purifying it, and then constructing a DNA library; S2, constructing an in vitro protein expression vector of a key transcription factor of strawberry light response, obtaining a recombinant plasmid, expressing a fusion protein with a halo tag, and combining the protein with magnetic beads; the key transcription factor of strawberry light response is FvHY5 protein; S3, mixing the protein-bound magnetic beads and the DNA library, eluting and performing PCR enrichment, and sequencing the enriched fragments; S4. Perform DAP-seq analysis to obtain the binding sites and downstream target genes of strawberry light-responsive key transcription factors at the genome-wide level.

2. The method according to claim 1, characterized in that: In the S1, the genomic DNA is fragmented to process the genomic DNA into fragments of 300 bp in size.

3. The method according to claim 1, characterized in that The sorting and purification in S2 includes magnetic bead purification.

4. The method according to claim 1 or 3, characterized in that: The in vitro protein expression vector used in S2 is the pDonor201 vector.

5. The method according to claim 1, characterized in that The size of the DNA fragment after PCR enrichment in S3 is 250 bp to 350 bp.

6. The method according to claim 1, characterized in that The DAP-seq analysis in S4 includes, but is not limited to, transcription factor binding site motif analysis, GO annotation of downstream genes regulated by transcription factors, and analysis of downstream genes regulated by transcription factors.

7. The method according to claim 3 or 6, characterized in that: The most significant motif enriched in the FvHY5 protein was [T / C / G / A]ACGT[G / A / C][G / T / A][C / A / G / T][C / T / A][C / A / T / G].

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