CN113963745A

CN113963745A - Method for constructing plant development molecule regulation network and application thereof

Info

Publication number: CN113963745A
Application number: CN202111486035.7A
Authority: CN
Inventors: 李英; 高志民
Original assignee: International Center for Bamboo and Rattan
Current assignee: International Center for Bamboo and Rattan
Priority date: 2021-12-07
Filing date: 2021-12-07
Publication date: 2022-01-21

Abstract

The invention relates to a method for constructing a plant development molecule regulation and control network and application thereof, belonging to the technical field of molecular biology. The invention aims at a batch of miRNA-target gene pairs identified by omics, verifies the interaction relation of candidate miRNA and target genes thereof by using a dual-luciferase report detection technology and a fluorescence in situ hybridization technology in sequence, positions the candidate miRNA and the target genes in tissues and cells, accurately realizes the positioning of node genes and key switch miRNA in developing tissues, can accurately reflect the characteristics of molecular regulation and control in the plant tissue development process with space-time specificity, and has very high guiding significance and application value for molecular design and breeding.

Description

Method for constructing plant development molecule regulation network and application thereof

Technical Field

The invention relates to the technical field of molecular biology, in particular to a method for constructing a plant development molecular regulation network and application thereof.

Background

mirnas are a class of small non-coding RNAs of about 21nt in length, and are key regulators of almost all biological processes in eukaryotes. In plants, mirnas can bind to complementary sequences of target mRNA molecules to degrade the mRNA or inhibit translation of the mRNA, and regulate expression of target genes at the post-transcriptional level, and have been a research hotspot in a variety of research fields, such as genetics, development, and stem cells. For example, miR165/miR166 regulates SAM termination, organ polarity change, and fiber development defect between vascular tissues and bundles, etc. by affecting the expression of HD-ZIP III transcription factor genes. Meanwhile, Argonautes (AGOs) regulates and controls the development of stem apical meristems and the formation of leaf polarity of Arabidopsis thaliana by competitively binding miRNA 165/166. These results all indicate that miRNA can combine with upstream regulatory genes and downstream target genes, and play an important role in key developmental processes such as embryonic development, meristem and organogenesis of plants.

In addition, miRNAs influence plant development and morphogenesis by participating in regulation of biological pathways related to plant substance and energy metabolism, plant hormone signaling, and stress response. Such as microRNA165/166, can be used in conjunction with plant hormone signaling pathways to maintain stability of arabidopsis root vascular bundle configuration (Muraro et al, 2014). Previous research results have shown that almost all miRNAs are regulated by multiple transcription factors, and that miRNAs also act on numerous target genes including transcription factor genes. Moreover, the transcription factor specifically binds to a DNA cis-acting element of a downstream gene promoter region, and plays a role in activating or inhibiting the transcription of genes (including miRNA), so that a more complex transcription regulation network consisting of miRNA-transcription factor-target genes (miRNA) is formed and participates in a plurality of biological pathways including plant growth and development. Namely, miRNA, an upstream regulatory gene and a downstream target gene form a complex regulatory network, and participate in multiple biological processes of substance and energy metabolism, hormone signal transduction, adversity stress response and the like of plants, and the growth and development of the plants are regulated and controlled at the transcription level.

There are also some records related to the study of plant growth and Development using miRNA, for example, the study reports "Integrated Analysis of Small RNA, transcription and Degradome Sequencing of Providers New instructions in flow Development and Abscission in Yellow Lupin (Lupinus luteus L.)" and "Integrated transcription, Small RNA and degraded sequences of proteins degradation in coding in chip" all find candidate miRNA, detect differentially expressed miRNA, although the degradative group detection finds partial miRNA degradation target gene sites, it is difficult to determine whether the miRNA-target gene pairs are from the same growth and Development period after the sample mixing, i.e. there is a positive interaction in the sequence, and there is a false interaction. Although qRT-PCR is used for detecting that the expression quantities of the two genes have a negative correlation relationship, whether a candidate miRNA-target gene pair can interact in a cell or not is not verified, the positions of the miRNA and the target gene in plant tissues are not determined, and high false positive probability exists. A regulation and control network is constructed by utilizing the found candidate miRNA and the target gene thereof, but the key node miRNA and the target gene thereof have high false positive probability, the plant development process cannot be truly reflected, and the value of guiding breeding is not great.

Disclosure of Invention

The invention aims to provide a method for constructing a plant development molecule regulation and control network and application thereof, which can accurately realize the positioning of node genes and miRNA in development tissues, thereby accurately reflecting the characteristics of the molecular regulation and control in the plant tissue development process with space-time specificity.

In order to solve the technical problems, the invention provides the following technical scheme:

the invention provides a method for constructing a plant development molecule regulation network, which comprises the following steps:

sequencing a plant tissue gene library to obtain miRNAs-target gene pairs;

and verifying the miRNAs-target gene pairs by using a dual-luciferase report detection technology, and positioning the action positions of the miRNAs-target gene pairs in plant tissue cells by using a fluorescence in-situ hybridization technology to obtain the plant development molecule regulation and control network.

Preferably, the sequencing comprises transcriptome sequencing, small RNA sequencing and degradome sequencing.

Preferably, the transcriptome sequencing comprises:

and sequencing, analyzing data and screening the plant tissue gene library to obtain the genes with different plant tissue development periods and differential expression.

Preferably, the small RNA sequencing comprises:

and sequencing, analyzing data and screening the plant tissue gene library to obtain miRNAs which are differentially expressed in adjacent development periods of the plant, and predicting target genes of the miRNAs.

Preferably, the sequencing by degrader comprises:

sequencing a plant tissue gene library, comparing the cluster Tags obtained by sequencing with an Rfam database to obtain an unannotated sequence, analyzing the unannotated sequence to obtain a degradation site, and analyzing and screening to obtain the miRNAs-target gene pair.

Preferably, the plant tissue gene library is a cDNA library constructed from plant tissue RNA.

Preferably, the verification comprises the following steps:

and detecting the miRNAs-target gene pair by using dual luciferase to obtain the miRNA-target gene pair with a positive detection result.

Preferably, the positioning comprises the following steps:

and carrying out fluorescence in situ hybridization on the miRNA-target gene pair with the positive detection result, and observing whether the miRNA and the target gene can be positioned in the same tissue of the sample in pairs.

Preferably, the visualized plant development molecule regulation and control network can be obtained by drawing with cytoscape software according to the positioning positions of the miRNA and the target gene.

The invention also provides the application of the method or the molecular regulation network constructed by the method in guiding molecular breeding.

The invention provides a method for constructing a plant development molecule regulation and control network, which aims at batch miRNA-target gene pairs identified by omics, successively verifies the interaction relation of candidate miRNAs and target genes thereof by using a dual-luciferase report detection technology and a fluorescence in situ hybridization technology, and positions the candidate miRNAs and the target genes thereof in tissues and cells, accurately realizes the positioning of node genes and key switch miRNAs in development tissues, can accurately reflect the characteristics of molecular regulation and control in the plant tissue development process with space-time specificity, and has very high guiding significance and application value for molecular design and breeding.

Drawings

FIG. 1 is a flow chart of sequencing and data analysis of small RNAs.

FIG. 2 is a flow chart of the degradation group experiment.

Fig. 3 is a flowchart of the data analysis of the degradation group.

FIG. 4 shows that ped-miR160a-5p can play a role in the primary layer (arrow 1) of the early shoot bud development in a fluorescence in situ hybridization Fish experiment.

FIG. 5 shows molecular control network formed by main action factors in early and middle development stages of moso bamboo shoot bud development.

FIG. 6 shows that ped-miR166a-3p can play a role in apical meristem (arrow 1) in early shoot bud development stage through fluorescence in situ hybridization Fish experiments.

FIG. 7 shows that the main action factors of moso bamboo shoot in the sprouting stage and the early development stage form a molecular regulation network.

FIG. 8 shows that ped-miR166a-3p can play a role in vascular tissues (arrow 1) in the middle stage of shoot bud development in a fluorescence in situ hybridization Fish experiment.

FIG. 9 shows that the main action factors in the middle of the shoot development of moso bamboo shoot and the shoot development of thick bamboo shoot form a molecular regulation network.

Detailed Description

sequencing a plant tissue gene library to obtain miRNAs-target gene pairs;

In the present invention, the sequencing includes transcriptome sequencing, small RNA sequencing and degradome sequencing. The transcriptome sequencing comprises: and sequencing, analyzing data and screening the plant tissue gene library to obtain the genes with different plant tissue development periods and differential expression. In the present invention, the platform for sequencing the transcriptome is preferably the Illumina platform. The data analysis preferably comprises the steps of: filtering Data obtained by sequencing to obtain Clean Data, performing sequence comparison with a reference genome of a researched species, and performing library quality evaluation such as insert length inspection, randomness inspection and the like on the obtained Mapped Data; performing structure level analysis such as alternative splicing analysis, new gene discovery, gene structure optimization and the like; and carrying out differential expression analysis according to the expression quantity of the genes in the sample groups of different development stages of the plant tissues. In the present invention, the purpose of the transcriptome sequencing is to obtain differentially expressed genes in adjacent developmental stages of plant tissues.

In the present invention, the sequencing of the small RNA comprises: and sequencing, analyzing data and screening the plant tissue gene library to obtain miRNAs which are differentially expressed in adjacent development periods of the plant, and predicting target genes of the miRNAs. The platform for sequencing the small RNA is preferably an Illumina platform. The data analysis after the sequencing of the small RNA preferably comprises the following steps: and performing quality control on original sequencing data obtained by sequencing the small RNA to obtain sRNA, performing classification annotation on the sRNA to obtain miRNAs, and screening the miRNAs with differential expression after performing identification analysis and expression quantity analysis on the miRNAs. In the invention, when the miRNAs with differential expression are screened, proper differential expression analysis software is preferably selected according to actual conditions. In a specific embodiment of the present invention, the DESeq2 software is preferably used for differential expression analysis between sample groups to obtain a differentially expressed miRNA set between two biological conditions. In the invention, after differential expression miRNAs are obtained by screening, target gene prediction is carried out by using TargetFinder software according to known miRNA, newly predicted miRNA and gene sequence information of corresponding species.

In the present invention, the sequencing of the degradation group comprises: sequencing a plant tissue gene library, comparing the cluster Tags obtained by sequencing with an Rfam database to obtain an unannotated sequence, analyzing the unannotated sequence to obtain a degradation site, and analyzing and screening to obtain the miRNAs-target gene pair. The platform for sequencing the degraded set is preferably Illumina Hiseq 2500. The cluster Tags are preferably the original Tags obtained by sequencing by de-ligating and filtering the low quality. In the present invention, the degradation site is preferably detected by using the Cleaveland3 software. In the present invention, the purpose of sequencing the degraded set is to screen for miRNAs-target gene pairs.

In the present invention, the plant tissue gene library is a cDNA library constructed from plant tissue RNA. The plant tissue according to the invention is preferably a certain and/or certain tissue or cell of the plant. In the present invention, the construction of the cDNA library preferably comprises the following steps: (1) enriching mRNA of plant tissues by using magnetic beads with oligo (dT); (2) adding Fragmentation Buffer to randomly break mRNA; (3) using mRNA as a template, synthesizing a first cDNA chain by using hexabasic random primers (random hexamers) consisting of bases A, T, C and G, then adding buffer solution, dNTPs, RNase H and DNA polymerase I to synthesize a second cDNA chain, and purifying cDNA by using AMPure XP beads; (4) carrying out end repair on the purified double-stranded cDNA, adding A tail and connecting a sequencing joint, and then carrying out fragment size selection by using AMPure XPbeads; (5) and finally, obtaining a cDNA library through PCR enrichment. In the present invention, the extraction of the plant tissue RNA is preferably performed by using a kit (purchased from Tiangen Biochemical technology, Ltd.).

In the invention, the verification comprises the following steps: and detecting the miRNAs-target gene pair by using dual luciferase to obtain the miRNA-target gene pair with a positive detection result. In the present invention, the purpose of the validation is to validate whether the miRNA is able to bind to the target gene. In the invention, the dual-luciferase assay is preferably a dual-luciferase reporter assay kit.

In the invention, the positioning comprises the following steps: and carrying out fluorescence in situ hybridization on the miRNA-target gene pair with the positive detection result, and observing whether the miRNA and the target gene can be positioned in the same tissue of the sample in pairs. The fluorescence in situ hybridization method is not particularly limited, and a FISH in situ hybridization kit is preferably used in the embodiment of the invention.

According to the positioning positions of the miRNA and the target gene, the visualized plant development molecule regulation and control network can be obtained by drawing with cytoscape software. In the invention, the plant development molecule regulation and control network can regulate and control genes and miRNA for plant development and can analyze the interaction relationship between the genes and miRNA.

The invention also provides the application of the method or the molecular regulation network constructed by the method in guiding molecular breeding. The molecular breeding is not particularly limited in the present invention, and preferably includes genetic engineering breeding.

In the invention, the raw materials, reagents and equipment are known products, and conventional commercial products can be adopted.

In the present invention, the experimental methods used are, unless otherwise specified, all the technical means which are conventional in the art.

In order to further illustrate the present invention, the following embodiments are described in detail, but they should not be construed as limiting the scope of the present invention.

Example 1

Firstly, collecting tissues or organs of plants at different development stages, and setting three repetitions in samples at each stage.

Secondly, RNA extraction: the RNA of the tissues or organs at different development stages is extracted by using a kit purchased from Tiangen Biochemical technology (Beijing) Co., Ltd, and the specific operation is carried out according to the kit instruction.

Third, library construction

1. The purity, concentration and integrity of the RNA samples were tested using an Agilent Bioanalyzer 2100System (Agilent Technologies, Calif., USA) instrument.

2. After the sample is detected to be qualified, constructing a library according to the following flow:

(1) enriching eukaryotic mRNA by magnetic beads with oligo (dT);

(2) adding Fragmentation Buffer to randomly break mRNA;

(3) synthesizing a first cDNA chain by using mRNA as a template and hexabasic random primers (random hexamers), then adding buffer solution, dNTPs, RNase H and DNA polymerase I to synthesize a second cDNA chain, and purifying cDNA by using AMPure XP beads;

(4) carrying out end repair on the purified double-stranded cDNA, adding A tail and connecting a sequencing joint, and then carrying out fragment size selection by using AMPure XP beads;

(5) and finally, obtaining a cDNA library through PCR enrichment.

3. After the library is constructed, the effective concentration of the library (the effective concentration of the library is more than 2nM) is accurately quantified by using a Q-PCR method so as to ensure the quality of the library. The qualified library was saved for subsequent experiments.

Fourthly, transcriptome sequencing and analyzing the library

1. Sequencing the qualified gene library by using an Illumina platform, and performing bioinformatics analysis on the obtained data.

2. Bioinformatics analysis

2.1 bioinformatics analysis procedure summarisation

Filtering off-machine Data to obtain Clean Data, performing sequence comparison with the reference genome of the species to obtain Mapped Data, and performing library quality evaluation such as insert length inspection, randomness inspection and the like; performing structure level analysis such as alternative splicing analysis, new gene discovery, gene structure optimization and the like; and performing expression level analysis such as differential expression analysis, differential expression gene function annotation, function enrichment and the like according to the expression quantity of the gene in different samples or different sample groups.

2.2 sequencing data and quality control thereof

Based on Sequencing By Synthesis (SBS) technology, the Illumina high-throughput Sequencing platform sequenced cDNA libraries to produce a large amount of high quality Data, called Raw Data (Raw Data), with most of the base quality scores reaching or exceeding Q30.

2.2.1 sequencing of base quality values

The Base Quality value (Quality Score or Q-Score) is an integer mapping of the probability that Base recognition (Base Calling) is erroneous. The commonly used formula for the base quality value of Phred is:

Q＝-10×log 10PQ＝-10×log 10P

wherein P is the probability of base recognition error.

2.2.2 sequencing of base content distribution

The base type distribution check is used for detecting whether AT and GC separation phenomena exist, the sequence detected by the RNA-Seq is a cDNA fragment which is randomly interrupted, and the contents of G, C, A and T are theoretically equal on each sequencing cycle due to the principles of random interruption and base complementary pairing, and the whole sequencing process is stable and constant and is in a horizontal line.

2.2.3 sequencing quality control

Before data analysis can be performed, it is first necessary to ensure that these Reads are of sufficiently high quality to ensure that subsequent analysis is accurate. The data were subjected to strict quality control, with the following filtering regime: (1) removing the Reads containing the linker; (2) low quality Reads were removed (including Reads with a greater than 10% N removed; Reads with a number of bases with a quality Q ≦ 10 that is greater than 50% of the entire Read).

The high quality clear Data obtained after the above series of quality control is provided in FASTQ format.

2.3 alignment of transcriptome data with reference genomic sequences

Sequence alignment and subsequent analysis were performed using HISAT2 using the genome as a reference.

The reads on the alignment were assembled and quantified using StringTie.

2.4 transcriptome library quality assessment

Qualified transcriptome libraries are a prerequisite for transcriptome sequencing, and to ensure the quality of the libraries, the transcriptome sequencing libraries were evaluated for quality from 3 different perspectives: (1) evaluating the randomness of mRNA fragmentation and mRNA degradation by testing the distribution of the inserted fragments on the gene; (2) evaluating the degree of dispersion of the lengths of the inserts according to the length distribution of the inserts; (3) by plotting the saturation map, the library capacity and the sufficiency of Mapped Data were evaluated.

2.5 analysis of novel genes

2.5.1 novel Gene discovery

Based on the selected reference genome sequence, mapping Reads are spliced by using StringTie software, compared with the original genome annotation information, the original unannotated transcription region is searched, and a new transcript and a new gene of the species are discovered, so that the original genome annotation information is supplemented and perfected. Novel genes were discovered by filtering out sequences that encode peptide chains that are too short (less than 50 amino acid residues) or that contain only a single exon.

2.5.2 novel Gene functional Annotation

And (3) carrying out sequence alignment on the discovered new gene with NR, Swiss-Prot, GO, COG, KOG, Pfam and KEGG databases by using BLAST software, obtaining a KEGG ontology result of the new gene by using KOBAS2.0, and comparing the amino acid sequence of the new gene with the Pfam databases by using HMMER software after the amino acid sequence of the new gene is predicted to obtain annotation information of the new gene.

2.6 analysis of Gene expression level

2.6.1 Gene expression quantification

The number of fragments extracted from a transcript is related to the amount of sequencing Data (or Mapped Data), the length of the transcript, and the expression level of the transcript, and in order for the number of fragments to actually reflect the expression level of the transcript, the number of Mapped Reads in the sample and the length of the transcript need to be normalized. StringTie uses the maximum flow algorithm and FPKM (fragments Per. Kilost of transcript Per Million fragments mapped) as an index for measuring the expression level of transcripts or genes, and the calculation formula of FPKM is as follows:

wherein, the cDNA Fragments represent the number of Fragments aligned to a certain transcript, namely the number of double-ended Reads; mappled Fragments (Millons) represent the total number of Fragments aligned to the transcript, in units of 10^ 6; transcript Length (kb): transcript length, in units of 10^3 bases.

2.7 differential expression analysis

Gene expression is temporally and spatially specific, and genes or transcripts whose expression levels differ significantly under two different conditions are referred to as Differentially Expressed Genes (DEG).

The set of genes obtained by differential expression analysis is called differential expression gene set and named by means of "A _ vs _ B". Differentially expressed genes can be classified into Up-regulated genes (Up-regulated genes) and Down-regulated genes (Down-regulated genes) based on the relative high or low expression levels between the two samples (groups). The expression level of the up-regulated gene in sample (group) B is higher than that in sample (group) A; otherwise, the down-regulation of the gene is realized. The up and down adjustments are relative, as determined by the order of a and B given.

2.7.1 differential expression screening

For samples with biological duplication, DESeq is suitable for performing differential expression analysis between groups of samples to obtain a differential expression gene set between two biological conditions; for samples without biological replicates, differential analysis was performed using EBseq.

In the process of detecting the differential expression gene, the Fold Change is more than or equal to 2 and the FDR is less than 0.01 as a screening standard. Fold difference (Fold Change) represents the ratio of the expression levels between the two samples (groups). The False Discovery Rate (FDR) is obtained by correcting the difference significance p-value (p-value). Because differential expression analysis of transcriptome sequencing is to carry out independent statistical hypothesis test on a large number of gene expression values, and a false positive problem exists, in the differential expression analysis process, a well-known Benjamini-Hochberg correction method is adopted to correct a significance p value (p-value) obtained by the original hypothesis test, and FDR is finally adopted as a key index for screening the differential expression genes.

Fifthly, sequencing and analyzing the small RNA of the gene library

The experimental flow is shown in fig. 1, and specifically as follows:

1.1 converting an original image Data file obtained by utilizing the Illumina platform to an original sequencing sequence (Raw Data or Raw Reads) through Base recognition (Base Calling), and storing the result in a FASTQ file format, wherein the FASTQ file format comprises sequence information of the sequencing sequence and corresponding sequencing quality information.

1.1.1 sequencing of base quality values

Q＝-10×log10PQ＝-10×log10P

where P is the probability of base recognition error.

1.1.2 sequencing data yield statistics

The original sequence obtained by sequencing contains a linker sequence or a low-quality sequence, and in order to ensure the accuracy of information analysis, the quality control of the original data is required to obtain a high-quality sequence (namely Clean Reads), and the standard of the quality control of the original sequence is as follows:

(1) for each sample, sequences with low quality values are discarded.

(2) Removing Reads with unknown base N (N is an unidentifiable base) content of more than or equal to 10%;

(3) removing reads without 3' linker sequence;

(4) shearing off the 3' linker sequence;

(5) removing sequences shorter than 18 or longer than 30 nucleotides;

1.2sRNA Classification Annotation

1.2.1ncRNA and repeat sequence annotation

Utilizing Bowtie software to respectively carry out sequence alignment on Clean Reads with a Silva database, a GtRNAdb database, a Rfam database and a Repbase database, and filtering ncRNAs such as ribosomal RNA (rRNA), transfer RNA (tRNA), small nuclear RNA (snRNA) and repeated sequences to obtain Unnnated Reads containing miRNA.

1.2.2 alignment with reference genome

And (3) carrying out sequence comparison on Unnnatated Reads and the reference genome of the species by using Bowtie software to obtain position information on the reference genome, namely Mapped Reads.

1.3MiRNA analysis

1.3.1 identification of miRNA

In the aspect of identification of known miRNA, reads aligned to a reference genome are aligned with the mature sequence of miRNA of the species (if not temporarily included, the related species thereof can be selected) included in the database of miRBase (v22), and the range of 2nt upstream and 5nt downstream of the mature sequence, at most one mismatch is allowed, so that the identified reads are considered as the identified known miRNA.

The miRNA transcription initiation site is mostly positioned on a gene spacer region, an intron and a reverse complementary sequence of a coding sequence, the precursor of the miRNA transcription initiation site has a marker hairpin structure, and the formation of a mature body is realized by the shearing of Dicer/DCL enzyme. For the biological characteristics of mirnas, prediction of new mirnas was performed using miRDeep2 software for sequences for which no known miRNA was identified.

By utilizing a miRDeep2 software package, position information on a genome is compared through reads to obtain a possible precursor sequence, and prediction of a new miRNA is finally realized by scoring by adopting a Bayesian model based on distribution information (based on miRNA generation characteristics, match, star and loop) of the reads on the precursor sequence and precursor structure energy information (RNAfold randfold).

1.3.2miRNA base bias analysis

The Dicer enzyme and the DCL enzyme have strong bias in recognizing and cutting precursor miRNA, and the first base pair U at the 5' end. Typical miRNA base ratios were obtained by base bias analysis of mirnas.

1.3.3miRNA base editing analysis

The miRNA has post-transcriptional base editing that results in changes in the seed sequence (seed sequence) and, in turn, changes in the target gene of action. miRNA undergoing base editing was detected using isomori id software, which first called bowtie and precursor sequences for a first round (r0) of alignment, with the fully aligned sequences as reference templates for the next round of alignment. In the second round (r1), one mismatch was allowed, one mismatch at the 3 'end was designated M3, one mismatch at the 5' end was designated M5, and one mismatch in the middle was designated MM.

1.3.4miRNA family analysis

miRNA has high conservation among species, and miRNA family analysis is carried out on the detected known miRNA and new miRNA based on sequence similarity, so as to research the conservation of the miRNA in evolution.

1.4 analysis of miRNA expression level

1.4.1 quantitation of miRNA

And (3) carrying out statistics on the expression quantity of miRNA in each sample, and carrying out normalization processing on the expression quantity by using a TPM (trusted platform Module) algorithm [5 ]. The TPM normalization processing formula is as follows:

in the formula, readcount represents the number of reads compared to a certain miRNA; mapped Reads indicates the number of Reads aligned to all miRNAs.

1.5 differential expression analysis of miRNA

1.5.1 differential expression miRNA screening

The DESeq2 is used for detecting the differential expression miRNA, DESeq2 is suitable for experiments with biological repetition, and differential expression analysis between sample groups can be carried out to obtain a differential expression miRNA set between two biological conditions. In the differential expression miRNA detection process, the | log2(FC) | is more than or equal to 1.00; FDR P value is less than or equal to 0.05 and is used as a screening standard.

The set of genes found by differential expression analysis is called the differentially expressed gene set (DEG).

1.5.2 clustering analysis of differentially expressed miRNAs

And performing hierarchical clustering analysis on the screened differentially expressed miRNAs, and clustering the miRNAs with the same or similar expression behaviors.

1.6 prediction of miRNA target genes

And (3) performing target gene prediction by using TargetFinder software according to the known miRNA and the gene sequence information of the newly predicted miRNA and the corresponding species.

Sixthly, performing degradation group sequencing and analysis on the sample

The experimental flow is shown in fig. 2, and the specific steps are as follows:

1. sample detection and sequencing

(1) Capturing plant sample mRNA through magnetic beads, and connecting 5' adaptor; (2) carrying out mixed reverse transcription on a random primer and mRNA; (3) PCR amplification, and after the whole library preparation work is completed, sequencing the constructed library by using Illumina Hiseq 2500.

2. Information analysis process

The original Tags were subjected to de-piecing and low quality filtering to obtain clean Tags and cluster Tags (clustered data of clean Tags). The cluster Tags and Rfam databases were aligned and the non-coding RNAs were annotated away, and the unannotated sequences were used for degradation site analysis. The degradation group analysis flow is shown in FIG. 3.

3. Biological information analysis result

3.1 basic analysis

3.1.1 sequencing results statistics

The original Tags was of low quality via de-heading and filtration.

3.1.2 non-coding RNA Annotation

Comparing the cluster Tags with genome and Rfam libraries to obtain statistic results and non-coding RNA annotation information compared with the genome, and using the sequence which is not annotated for subsequent analysis.

3.2 degradation site analysis

And (3) carrying out degradation site detection by using known miRNA in an miRNA library and an miRNA predicted in small RNA project analysis and a gene transcript sequence information file of a corresponding species through Cleaveland3 software. The condition P-value <0.05 is set.

Seven, two luciferase reporter assays

1. The main experimental reagents are shown in the following table 1

TABLE 1 Main Experimental reagents and sources

2. Experimental procedure

2.1 Gene Synthesis and vector construction

2.1.1 primer design and Synthesis

And (3) designing primers by using Primer5 software according to the target gene sequence in the miRNA-target gene pair obtained in the step 3.2, synthesizing the sequences, and performing gene amplification.

Gene amplification

1) The PCR amplification reaction system is shown in Table 2 below

TABLE 2PCR amplification reaction System

2) The PCR amplification reaction conditions are shown in Table 3 below

TABLE 3PCR amplification reaction conditions

Electrophoresis detection of amplified fragments reference plasmid extraction kit (purchased from Axygen, manufactured by Lot 12123); the recovery procedure was performed according to the instructions of a DNA recovery kit (purchased from Tiangen Biochemical technology Ltd., production batch No. DP 214-03).

2.1.2 enzyme digestion of vector and target Gene

The reagents used are as in Table 4 below

TABLE 4 digestion reagents and systems

Enzyme digestion is carried out for 1-2h at the temperature of 37 ℃; carrying out electrophoresis detection on the enzyme digestion product; the recovery procedure was performed according to the instructions of a DNA recovery kit (purchased from Tiangen Biochemical technology Ltd., production batch No. DP 214-03).

2.1.3 ligation of vector to Gene of interest

Linking reagents are shown in Table 5 below

TABLE 5 linking reagents and systems

Reacting for 0.5-1h at 16 ℃ to ensure the full connection of the vector and the target gene.

2.1.4 transformation

1) The DNA fragment to be transformed was added to a tube containing TOP10 competent cells (25 ng of DNA was required for 50. mu.L of competent cells) in a volume not exceeding 5% of the competent cells, and the contents were mixed by gentle rotation several times and ice-cooled for 30 min.

2) The tube mixture was placed in circulating water warmed to 42 ℃ and heat-shocked for 90s without shaking the tubes.

3) The tube was quickly transferred to an ice bath to cool the cells for 1-2 min.

4) 200 μ LSOC liquid medium was added to each tube, the medium was warmed to 37 ℃ with a water bath, and then the tubes were transferred to a shaker set at 37 ℃ and cultured at 220rpm for 45min to resuscitate the cells and express the plasmid-encoded resistance marker gene.

5) Appropriate volumes (up to 200. mu.L per 90mm plate) of transformed competent cells were transferred to LB medium containing the corresponding antibiotic.

6) And (4) inverting the plate, and culturing at 37 ℃ until bacterial plaque appears after 12-16 hours.

2.1.5 colony PCR validation

And randomly selecting a plurality of colonies after the colonies grow on the plate, carrying out colony PCR verification, and detecting transformants.

2.1.6 sequencing validation

The positive clones are sent to a sequencing platform of the company for sequencing verification.

2.2 plasmid transfection

(1) Inoculating cells into a 24-well culture plate one day before transfection, wherein the cell density is 70-80% during transfection, and the culture medium is DMEM + 10% FBS;

(2) dissolving miRNA with DEPC water to a concentration of 20 μ M, adding miRNA (final concentration of transfected cells is 50nM) and 2 μ g plasmid into 50 μ L serum-free DMEM medium, incubating at room temperature for 5min, adding 2 μ L lipo2000 transfection reagent into 50 μ L serum-free DMEM medium, mixing the two, incubating at room temperature for 15min, and supplementing serum-free DMEM medium to 500 μ L;

(3) removing the culture medium in the dish, adding 500 mu L of the transfection compound prepared in the previous step, and culturing for 4-6h at 37 ℃;

(4) removing the culture medium by suction, adding 0.5mL of complete culture medium, and culturing at 37 ℃ for 48 h;

(5) the transfection was repeated 5 times;

wherein:

amount of DNA: lipofectamine 2000 ═ 1:1

b. Cell density of approximately 80% of the plates, transfection

2.3 Dual luciferase assay (Dual luciferase reporter assay kit, RG027)

The operation of the part is carried out by adopting the operation instruction of the dual-luciferase reporter gene detection kit, and the corresponding reagent in the kit. The packaging list is as follows:

RG 027-1: reporter gene cell lysate

RG 027-2: luciferase detection reagent

RG 027-3: renilla luciferase detection buffer solution

RF 027-4: substrate for Renilla luciferase assay (100X)

The specific experimental method is as follows:

(1) cell lysis: adding 200 μ L cell lysate (RG027-1) into each well of 24-well plate, incubating at room temperature for 10min, and fully lysing cells;

(2) after full cracking, collecting the cracking solution, centrifuging at 10000rpm for 5min, and taking the supernatant as a solution to be detected;

(3) firefly luciferase assay reagent (RG027-2) and Renilla luciferase assay buffer (RG027-3) were thawed and brought to room temperature. Renilla luciferase assay substrate (100X) was placed in an ice bath for use.

(4) Starting a chemiluminescence apparatus, taking another 96-hole plate, adding 100 mu L of cell lysate supernatant into the 96-hole luminescent plate, adding 100 mu L of firefly luciferase detection working solution (RG027-2), blowing, sucking and uniformly mixing;

(5) measuring a luminous value on a computer, and integrating for 1 s;

(6) after the step of measuring the firefly luciferase is completed, adding 100 mu L of renilla luciferase detection working solution (RG027-3), blowing, sucking and mixing uniformly, measuring a luminescence value on a machine, and integrating for 1 s;

(7) in the case of Renilla luciferase as an internal control, the RLU value obtained by firefly luciferase assay was divided by the RLU value obtained by Renilla luciferase assay, and the degree of activation of the target reporter gene was compared between different samples based on the obtained ratio.

Eight, fluorescent in situ hybridization

And (3) continuously carrying out fluorescence in situ hybridization detection on the miRNA-gene pairs with positive detection results, and observing whether the candidate miRNA and the gene can be positioned in the same tissue of the sample in pairs.

Wherein, all the test instruments in the experimental process are shown in Table 6

TABLE 6 test apparatus

Product name	Brand	Goods number
			Polylysines	COOlaber	CP8681
Paraformaldehyde	Aladdin	P395744
			Water bath tub	Leica	HI1220
Upright fluorescence microscope	Leica	DM3000
			FISH in situ hybridization kit	Yongfan organism	C007

The experimental procedure was as follows:

(1) slicing and paving: and (3) using an ophthalmological forceps to pick up the plant tissue slices, gently spreading the plant tissue slices on the water surface at the temperature of 40-45 ℃, and naturally flattening the slightly-wrinkled slices by virtue of the tension of water and the temperature of the water.

(2) Pasting and baking: after the section to be cut is fully flattened on the constant temperature water surface, the wax sheet is fished to the middle section of the glass slide, the residual water on the glass slide is poured out, the section to be cut is placed in a constant temperature box at the temperature of 60-65 ℃ or a drying oven of a section rinsing and drying temperature controller for drying for 15-30 minutes, and the paraffin wax for dissolving the tissue gap is removed.

(3) Paraffin section dewaxing to water: placing the slices in xylene I10 min-xylene II 10 min-absolute ethanol I5 min-absolute ethanol II 5 min-95% ethanol 5 min-90% ethanol 5 min-80% ethanol 5 min-70% ethanol 5 min-distilled water washing.

(4) Manufacturing a wet box: the resulting mixture was placed in a wet box using 5 XSSC (pH7.5) (35mL) + formamide (35 mL).

(5)30％H₂O₂1 part and 9 parts of pure methanol mixed solution are treated for 10min at room temperature. Washing with DEPC for 3 times, each for 1 min;

(6) placing the slices in a wet box, dripping 0.25% hydrochloric acid on the tissue, washing with DEPC water for 2 times at room temperature for 15min, each time for 1min (hydrochloric acid can neutralize charged basic protein and reduce background)

(7) Proteinase K covers the tissue and the temperature is 37 ℃ for 20min in a molecular hybridization instrument. Proteinase K can digest and expose the masked target nucleic acid, increasing the binding efficiency of the probe.

(8) Washing with 0.2% or 0.1mol/L glycine wash solution for 1min (now prepared), and terminating proteinase K.

(9) PBS was washed twice for 1min each.

(10) The tissue was fixed with 4% PFA paraformaldehyde for 10 min.

(11) PBS was washed three times for 1min each.

(12) Wash with acetic anhydride, ph 8.0 (acetylation, lower background) for 5min at room temperature, 2 times. (acetic anhydride solution preparation: 126ul of acetic anhydride is added into each 50mL of triethanolamine aqueous solution, the existing preparation is prepared, the preparation is prepared according to the proportion, and the amount of the acetic anhydride is used). Wash 5 times with PBS for 1min each time.

(13) Wash twice with 5 XSSSCph7.5 for 1min each time.

(14) The sections were placed in a wet box, the tissue covered with prehybridization solution and prehybridized for 1h at 65 ℃.

(15)100uM of probe mother liquor can be diluted by 1:500-1000, a section is covered by a FITC-labeled probe, the section is subjected to dark reaction at 62-70 ℃ for 24-72 h in a hybridization instrument, the reaction temperature and the reaction time depend on different tissues and probes, and the temperature of 65 ℃ is generally recommended to be 48 h.

(16) Wash 1 time 1min with 2 × SSC ph7.5 at room temperature.

(17) Washing with formamide plus a 1:1 mixture of 4 XSSC (ph 4.5) at 60 ℃ or 65 ℃ for three times, each for 20 min.

(18) Washed 5 times with PBS, each for 1min, at room temperature.

(19) DAPI was diluted 1000-fold with DEPC treated water and stained for 5 min.

(20) Wash 3 times with PBS for 5min each.

(21) Dripping an anti-quenching agent, covering a cover glass, sealing a piece with nail polish, and observing by a fluorescence microscope; the position of the probe is the action position of the target gene or miRNA.

The positive miRNA-gene pair is detected to have the condition that one miRNA corresponds to a plurality of target genes or a plurality of miRNAs correspond to one target gene, so that a complex regulation and control network is formed, and a visualized regulation and control network is drawn by using cytoscape software.

Example 2

The method for constructing the plant molecular regulation network described in example 1 was used to construct the molecular regulation network of the primary layer of bamboo in the early development stage.

137 miRNAs are identified in a bamboo shoot bud sample in the early development stage of the moso bamboo, wherein the miRNAs comprise 120 known miRNAs and 17 new miRNAs; 38652 genes. 130 miRNAs are identified from the shoot sample in the middle development stage, including 115 known miRNAs and 15 new miRNAs; 39584 genes. The expression analysis identifies 30 miRNAs which are differentially expressed in the two groups of samples and 61 genes which are differentially expressed; the 101 set of miRNA-target gene pairs were identified by degraded set sequencing analysis. Dual-luciferase reporter assays verify that 65 sets of miRNA-target gene pairs are capable of interacting intracellularly. Fluorescence in situ hybridization Fish experiments position that ped-miR160a-5p and the like can play a role in the primary layer of the shoot bud development middle stage, the results are shown in figure 4, and 1 primary layer development molecular control network in the moso bamboo development early stage is constructed, as shown in figure 5.

Example 3

The method for constructing the plant molecular regulation network described in example 1 is used for constructing the molecular regulation network of the shoot apical meristem in the early development stage of moso bamboos.

Wherein 129 miRNAs were identified in moso bamboo in the germinating samples, including 117 known and 12 new miRNAs; 38462 genes. While early developmental samples identified 137 miRNAs, including 120 known and 17 new miRNAs; 38652 genes. The expression analysis identifies 26 miRNAs which are differentially expressed in two groups of samples and 75 genes which are differentially expressed; degradation group sequencing analysis identified 98 groups of miRNA-target gene pairs. The dual-luciferase reporter assay verifies that the 56 sets of miRNA-target gene pairs are capable of interacting intracellularly. The fluorescence in situ hybridization Fish experiment positions that ped-miR166a-3p and the like can play a role in the apical meristem at the early stage of the shoot bud development, the result is shown in figure 6, and 1 molecular control network of the apical meristem at the early stage of the shoot bud development is constructed, as shown in figure 7.

Example 4

The method for constructing the plant molecule regulation network described in example 1 is used for constructing the molecular regulation network of the vascular tube in the middle of the moso bamboo development.

Wherein, moso bamboo and bamboo shoot buds in the middle development stage are collected, and 130 miRNAs including 115 known miRNAs and 15 new miRNAs are identified in a bamboo shoot bud sample in the middle development stage of the moso bamboo through small RNA sequencing, transcriptome sequencing and analysis; 39584 genes. 146 miRNAs were identified in the shoot bud sample in the middle of the thick bamboo development, including 110 known and 36 new miRNAs, 38627 genes. The 86 miRNA-target gene pairs were identified by the degraded set sequencing analysis. The dual-luciferase reporter assay verifies that the 67 set of miRNA-target gene pairs are capable of intracellular interaction. Fluorescence in situ hybridization Fish experiment positioning ped-miR166a-3p and the like can play a role in the vascular tissues of moso bamboo and bamboo shoot buds at the period, the result is shown in figure 8, and 1 molecular regulation network of vascular at the middle development stage of moso bamboo shoot buds is constructed, as shown in figure 9.

The above description is only an embodiment of the present invention, and not intended to limit the scope of the present invention, and all modifications of equivalent structures and equivalent processes, which are made by the present specification, or directly or indirectly applied to other related technical fields, are included in the scope of the present invention.

Claims

1. A method of constructing a plant development molecule regulatory network, said method comprising the steps of:

sequencing a plant tissue gene library to obtain miRNAs-target gene pairs;

2. The method of claim 1, wherein the sequencing comprises transcriptome sequencing, small RNA sequencing, and degradome sequencing.

3. The method of claim 2, wherein the transcriptome sequencing comprises:

4. The method of claim 2, wherein the small RNA sequencing comprises:

5. The method of claim 2, wherein the degraded set sequencing comprises:

6. The method of claim 1 or any one of claims 2 to 5, wherein the plant tissue gene library is a cDNA library constructed from plant tissue RNA.

7. The method according to claim 1, wherein said verifying comprises the steps of:

8. The method of claim 1, wherein said positioning comprises the steps of:

9. The method of claim 8, wherein the plant developmental molecule control network is visualized by mapping with cytoscape software according to the location of the miRNA and the target gene.

10. Use of the method of any one of claims 1 to 9 or a molecular regulatory network constructed by the method for directing molecular breeding.