CN112626081B

CN112626081B - Longan flowering regulation gene DlWRKY25, and regulation protein and application thereof

Info

Publication number: CN112626081B
Application number: CN202011537418.8A
Authority: CN
Inventors: 决登伟; 桑雪莲; 石胜友; 唐建民
Original assignee: Chongqing University of Arts and Sciences
Current assignee: Inner Mongolia Chifeng Baishan Pharmaceutical Co ltd
Priority date: 2020-12-23
Filing date: 2020-12-23
Publication date: 2021-10-01
Anticipated expiration: 2040-12-23
Also published as: CN112626081A

Abstract

A longan flowering regulation gene DlWRKY25 has a nucleotide sequence shown in SEQ ID No. 1. The whole length of an Open Reading Frame (ORF) of the longan DlWRKY25 gene is 1038bp, 345 amino acids are coded, a typical WRKY structural domain and a zinc finger structure are contained, and the longan DlWRKY25 gene belongs to III-type WRKY protein. The results of transgenic Arabidopsis show that longan is a typical transcription factorDlWRKY25Positively regulating and controlling the plant to flower; over-expressionDlWRKY25The transgenic Arabidopsis plants can show early-flowering phenotypes of different degrees compared with wild type plants, the wild type plants bloom in about 26 days, and the transgenic plants bloom in 16-18 days.

Description

Longan flowering regulation gene DlWRKY25, and regulation protein and application thereof

Technical Field

The invention relates to the technical field of molecular biology, in particular to a longan flowering regulation gene and application thereof.

Background

Longan (Dimocarpus longana Lour.) is one of the important economic fruit trees in the hot zone of China, and has more than 2000 years of cultivation history in China. The longan cultivar has seasonal flower bud differentiation and needs low-temperature induction, and in the main production area of China, such as Guangdong Yuexi area and Hainan, the KClO is often used because the low temperature required by longan flower formation is not available₃The flower forcing is realized, but the method has great limitations, such as the restriction of factors such as varieties, environment and the like. The research on the longan flower forming mechanism is to solve the problemThe fundamental way of the problem is to look up related documents, the study of longan flowering is mostly at physiological and biochemical level, and the molecular mechanism study reports are few.

As an important transcription regulation factor in higher plants, the gene family is named because of containing a highly conserved WRKY structural domain, and the structural domains are an N-terminal core sequence WRKYGQK and a C-terminal zinc finger structure respectively. WRKY proteins are generally divided into 3 major classes based on the number of domains and the characteristics of zinc finger sequences. The current research shows that most members of the WRKY gene family participate in the growth and development of plants and different adversity stress response processes. It can specifically bind with cis-acting element in downstream gene promoter region to affect plant growth, stress response and hormone signal transduction. Research on the WRKY gene mainly focuses on the response to various adversity stresses, and less research is related to flower formation. Genetic regulation of flowering time and identification and clarification of genes related to flowering have important significance for shortening childhood period, improving fruit tree yield and the like. Although research has shown that WRKY plays a role in plant flowering, for example, FvWRKY71 can promote flowering and increase branching. Under long-term illumination, over-expression of WRKY25 resulted in early flowering of Arabidopsis. MdWRKY35 may participate in different flower development processes by responding to different adversity stresses. However, how WRKY affects the flower-forming process is not completely clear at present.

Disclosure of Invention

The invention aims to provide a longan flowering regulation gene, and expressed protein and application thereof.

The purpose of the invention is realized according to the following technical scheme:

a longan flowering regulation gene DlWRKY25 has a nucleotide sequence shown in SEQ ID No. 1.

A longan flowering regulation protein has an amino acid sequence shown as SEQ ID No. 2.

The invention also provides a vector containing the coding gene.

The invention also provides an engineering bacterium containing the carrier.

The invention further provides application of the gene in promoting plant flowering.

Further, the engineering bacteria are infected into plants to obtain transgenic plants capable of promoting flowering.

The invention has the following beneficial effects:

the gene DlWRKY25 is subjected to cloning, subcellular localization analysis and transgenic function analysis, and the expression rule of the gene DlWRKY25 in the development process of different organs and floral organs is researched by qRT-PCR, so that the function of the gene DlWRKY25 is analyzed; the function of the strain in the flower forming process of the longan is clarified, and a foundation is laid for accelerating the cultivation of new varieties of early and late maturing longans by utilizing molecular assistance.

The whole length of an Open Reading Frame (ORF) of the longan DlWRKY25 gene is 1038bp, 345 amino acids are coded, a typical WRKY structural domain and a zinc finger structure are contained, and the longan DlWRKY25 gene belongs to III-type WRKY protein. The qRT-PCR result shows that the gene has tissue expression specificity, relatively high expression in stem, and inferior to pericarp and young fruit organs.

Transient expression results of tobacco epidermal cells show that the fluorescence signals are mainly concentrated in cell nuclei, and the protein encoded by the DlWRKY25 is localized in the cell nuclei.

The transgenic arabidopsis results show that longan DlWRKY25 positively regulates plant flowering as a typical transcription factor; the Arabidopsis thaliana plant over-expressing the DlWRKY25 gene can show early-flowering phenotype of different degrees compared with the wild type, the wild type plant flowers in about 26 days, and the transgenic plant lines flower in 16-18 days.

DlWRKY25 shows down-regulated expression in early stage of inducing flower formation of longan in 'season honey', the T1 period is only 5 times of the T2 period, and the expression level of DlWRKY52 does not show significant differential expression in the inducing process of flower formation of longan in 'Shixia'.

The invention lays a good foundation for deeply researching the molecular biological function of the longan DlWRKY25 gene in the growth and development process.

Drawings

FIG. 1 shows PCR amplification of longan DlWRKY25 gene.

FIG. 2 is a diagram showing the sequence alignment of WRKY protein between different species (pistachio, Pistacia vera, XP-031278909.1; rubber, Hevea brasiliensis, XP-021670644.1). The left box part represents the WRKYGQK amino acid sequence and the right box part represents the C2H2 zinc finger motif.

FIG. 3 is the phylogenetic tree analysis chart of similar sequences in longan DlWRKY25 and GenBank.

FIG. 4 is a schematic diagram of the relative expression level of DlWRKY25 in different tissues of longan. Different letter targets indicate that the difference reaches a significant level.

FIG. 5 is an expression pattern diagram of DlWRKY25 in the process of inducing flower formation of 2 longan varieties. Different letter targets indicate that the difference reaches a significant level. T1 represents the resting stage, T2 represents the emergence stage of the floral meristem ("red dots"), T3 represents the formation stage of the floral organ.

FIG. 6 is a subcellular localization of DlWRKY25 protein in tobacco. GFP: green fluorescent protein; chloroplast: chloroplast autofluorescence; bright: bright field; merged: fusing 2 kinds of fluorescence and bright field; the scale bar is 10 μm.

FIG. 7 is a phenotype map of flowers of DlWRKY25 transgenic Arabidopsis thaliana.

Detailed Description

The following examples are intended to illustrate the invention but are not intended to limit the scope of the invention.

EXAMPLE 1 cloning of the Gene of interest

1 materials and methods

1.1 sources of plant Material

The four season honey longan with continuous flower formation habit and the normal flower formation main variety Shixia longan with consistent growth vigor and tree age (9 ages) in 3 groups of longan germplasm gardens of southern subtropical crop research institute of tropical agricultural academy of China.

1.2 sampling purposes and sites

Flower formation induction expression analysis sampling site: three terminal buds in the flowering induction period are respectively in a T1 dormant period (the terminal buds do not sprout, the water content is low, and the hardness is high); t2 "red spot" period (the terminal bud begins to sprout and develop into inflorescence, the axis of bud is elongated, the axillary bud at the base part is obviously enlarged and turns red, commonly called "red spot"); t3 stage 1 inflorescence, (top bud fully developed into inflorescence, not flowering).

Tissue expression analysis sampling site: collecting organs such as flower, flower bud, leaf, pericarp, pulp, root, seed, stem and young fruit (whole fruit 60 days after flower) of the four-season honey longan.

All the test devices are repeated for 3 times, and after sampling, the samples are immediately put into liquid nitrogen for quick freezing and are transferred into a refrigerator with the temperature of minus 80 ℃ for storage for later use.

1.3 cloning and bioinformatics analysis of DlWRKY25 Gene sequence

The nucleotide Sequence and amino acid Sequence information of the gene DlWRKY25 (Dlo-011410.1) were obtained from the longan genome database (NCBI Sequence Read Archive, SRA 315202). Primers W25-S and W25-A (Table 1) were designed from the ORF sequence of the DlWRKY25 gene using Primer Premier 5.0, and were synthesized by Tianyihuiyuan Biotechnology Ltd (Guangzhou). The RNA of 'season honey' longan leaves is extracted by using a plant RNA extraction kit of Beijing Huayue biology company, a PrimeScript RT-PCR kit of Takara company is adopted, the specific operation steps refer to an instruction book, and cDNA is reversely transcribed as a template to perform PCR amplification cloning of a DlWRKY25 gene. The amplification conditions were: pre-denaturation at 94 ℃ for 5 min; denaturation at 94 ℃ for 30s, annealing at 60 ℃ for 30s, and extension at 72 ℃ for 60s for 35 cycles (denaturation-extension); extending for 10min at 72 ℃, and storing at 4 ℃. And (3) carrying out gel cutting recovery and purification on the amplification product, connecting the amplification product to a pMD18-T vector, transforming DH5 alpha competent cells, carrying out PCR (polymerase chain reaction) screening on positive clones, and selecting positive monoclonals to send to Yihui-Chiyuan biological technology limited company (Guangzhou) for sequencing.

The protein domains were predicted using the online software SMART program (http:// SMART. embedded elberg. de /), and the isoelectric points and molecular weights of the proteins were analyzed using ExPASY (http:// expay. org/tools /). According to the cDNA sequence obtained by cloning, the amino acid sequence is subjected to homology comparison by using BLASTp, and meanwhile, the MEGA 5 software is used for amino acid sequence homology analysis and phylogenetic analysis to construct a Neighbor-Joining evolutionary tree, wherein the number of times of repetition is 1000, and the others are all default settings.

1.4 expression analysis

qRT-PCR primers qW25-S and qW25-A (Table 1) were designed based on the cloned DlWRKY25 gene sequence and tested in NCBI using BLASTn to ensure primer specificity. The specific primer sequences are shown in Table 1 by taking an Actin gene (Dlo-028674) of longan as an internal reference gene.

Primer information used in Table 1

The apparatus used for the qRT-PCR reaction was LightCycler 480 from Roche and the PCR reaction enzyme was SYBR Green Master Mix from Takara. The reaction system was 20mL, in which 40ng of template cDNA, 250nM of each of the upstream and downstream primers, 10. mu.L of SYBR Green Master Mix, and ddH for the remainder₂And (4) supplementing and finishing. Reaction procedure: pre-denaturation at 94 ℃ for 5 min; melting curves were made after 40 cycles at 94 ℃ for 10s, 59 ℃ for 20s, 72 ℃ for 30s (95 → 65 ℃, 0.1 ℃/s). By use of 2^-ΔΔCtCalculating the relative expression quantity of the DlWRKY52 gene. All samples were run in 3 replicates and negative controls were set. Mean statistics were performed using Excel software, one-way anova with SPSS software was performed to analyze the significance of differences in changes of the gene of interest in different tissues and materials (P < 0.05), and plotted using SigmaPlot 12.5 software.

Example 2 subcellular localization analysis

Primers (terminator removed) were designed based on the cloned gene sequence of DlWRKY25 (table 1), and the full length ORF of DlWRKY25 was amplified and PCR reaction was performed as described above. The PCR product is detected by 1% agarose gel electrophoresis, purified and then connected to a pMD18-T vector to transform DH5 alpha. Single colonies were picked and subjected to PCR detection and sequencing of the upgraded particles. Then the target gene with correct sequence is connected with an expression vector pBWA (V) HS-osgfp. Transferring the plasmid after enzyme connection into escherichia coli DH5 alpha, selecting correct strains for sequencing after positive detection, and then extracting to obtain pBWA (V) HS-DlWRKY25-osgfp plasmid. Then, the successfully constructed plasmid is transferred into agrobacterium LB4404 by an electrical transformation method, after being cultured for 2d at 28 ℃, the agrobacterium is scraped off and inoculated in YEB culture medium, the medium is centrifuged for 4min at 1h at 170r/min and 4000r/min, supernatant is removed, and bacteria liquid is collected and 10nmol/L MgCl is used₂Resuspending the cells in the suspension and adjusting D₆₀₀About 0.6. Selecting tobacco plants with good growth condition, and using a 1mL syringe with a needleSucking the prepared bacterial liquid, injecting from the back of the tobacco leaves, carrying out 2d low-light culture on the marked tobacco leaves, shearing the injected tobacco leaves after the culture is finished, preparing into a glass slide, and observing and photographing under a laser confocal microscope.

Example 3 construction of overexpression vector and functional verification of transgenic Arabidopsis

Using specific PCR primers OEW25-S/OEW25-A (Table 1), PCR amplification was performed using longan cDNA as a template. The 5 'end of the primer is respectively added with a BamH I restriction enzyme site, and the 5' end of the reverse primer is respectively added with a Sac I restriction enzyme site. The obtained PCR product was ligated with pMD19-T vector and sequenced. Finally, extracting a plasmid with correct sequencing, carrying out double digestion on pBI121 and the plasmid with correct sequencing by using BamHI and SacI respectively, constructing a plant expression vector containing a DlWRKY25 target gene by using T4 DNA ligase, and naming the plant expression vector as pBI121-DlWRKY 25. The constructed over-expression vector pBI121-DlWRKY25 was transformed into Agrobacterium strain GV3101 by freeze-thawing method in liquid nitrogen (Clough, Steven J, Bent, et al. floral dip: a amplified method for Agrobacterium-mediated transformation of Arabidopsis thaliana [ J ]. Plant Journal, 1998(16), 735-. Positive arabidopsis thaliana was screened on MS solid medium containing 30ug/ml, and positive transgenic arabidopsis thaliana seedlings were detected with pBI121 plasmid specific primers. Transgenic plants of the T3 generation were grown separately in the same environment as the wild type and compared for their flowering phenotype.

Example 4 results and analysis

1. Cloning and bioinformatics analysis of DlWRKY25 Gene

Using longan cDNA as a template, a fragment of about 1000bp was amplified using primers W25-S/W25-A (Table 1) (FIG. 1). And (5) displaying a sequencing result. The fragment is completely consistent with a target sequence (Dlo-001658.1) in a longan ('red nucleus') genome database, has the size of 1038bp, encodes 345 amino acids, and has the molecular weight of 38.65kDa and the theoretical isoelectric point of 5.93. According to the positioning information of longan WRKY family genome, is named as DlWRKY 25. Amino acid sequence analysis shows that DlWRKY25 contains 1WRKY structural domain and C₂CH-type zinc finger structure (C-X)₇-C-X₂₃-HXC), belonging to Group III in WRKY family (fig. 2).

A homology search was performed on the amino acid sequence of D1WRKY25 using BLASTp, and then a phylogenetic tree was constructed using MEGA 6.0 software (FIG. 3). The results indicate a distant WRKY relationship with monocots, such as ZmWRKY70(ACG28802.1) of maize (Zea mays).

2. Tissue expression characteristic analysis of DlWRKY25 gene

The qRT-PCR result shows that the gene DlWRKY25 is expressed in 9 longan tissues tested, wherein the expression is highest in stems and is second to pericarp and young fruit (figure 4).

3. Expression mode of DlWRKY25 gene in floral organ development process

Using qRT-PCR technology, we analyzed the expression of DlWRKY25 in 3 flowering stages of 'season honey' and 'Shixia' longan. The result shows that DlWRKY25 is down-regulated in early induction period of 'season honey' longan flowering, and the T1 period is only 1/5 of the T2 period. While the expression level of DlWRKY25 in the induction process of the longan flower formation has not been significantly differentially expressed (FIG. 5).

4. Subcellular localization analysis of DIWRKY25 gene

In order to detect the location of the DlWRKY25 protein in cells, the invention constructs a fusion protein expression vector (35S: D1WRKY25-GFP) containing enhanced Green Fluorescent Protein (GFP), transforms tobacco to perform homeotropic expression, and observes by using a laser confocal microscope. As shown in fig. 6, under excitation at 480nm wavelength, 35S: d1WRKY25-GFP only observed fluorescence signal in nucleus, no GFP signal in cytoplasm and cell membrane, and 35S: the GFP control group showed no clear localization, and GFP signals were observed throughout the cells. This result indicates that the DlWRKY25 protein is localized on the nucleus.

5. Arabidopsis thaliana phenotype analysis of DIWRKY25 gene transfer

The result shows that the Arabidopsis thaliana plant over-expressing the DlWRKY25 gene can show early-flowering phenotype in different degrees compared with the wild type, the wild type plant flowers in about 26 days, and the transgenic plant flowers in 16-18 days (figure 7), which shows that the over-expression of the DlWRKY25 gene can obviously promote the flowering of the plant.

Sequence listing

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Claims

1. Longan flowering regulation geneDlWRKY25The nucleotide cDNA sequence is shown in SEQ ID No. 1.

2. The longan flowering regulatory gene expression regulatory protein of claim 1, wherein the amino acid sequence of the protein is shown in SEQ ID No. 2.

3. A vector comprising the flowering-controlling gene of longan according to claim 1.

4. An engineered bacterium comprising the vector of claim 3.

5. The use of the longan flowering regulating gene of claim 1 for promoting plant flowering.

6. The use of claim 5, wherein: infecting the plant with the engineering bacterium of claim 4 to obtain transgenic plant capable of promoting flowering.