CN118879707A - Cabbage flower development related gene module N-miR172-BraNPY and application thereof - Google Patents

Abstract

The invention discloses a cabbage flower development related gene module N-miR172-BraNPY and application thereof, wherein a precursor sequence of the cabbage N-miR172 is shown as SEQ ID No.1, and a target gene BraNPY5 sequence is shown as SEQ ID No. 2. The miRNA over-expression vector and the STTM inhibition expression vector are transformed into Col-type Arabidopsis through an agrobacterium flower dipping transformation method to obtain a heterologous expression Arabidopsis strain, and the result shows that the inhibition expression of the N-miR172 can cause the delay of the development of young leaves of Arabidopsis, the delay of flowering and the decline of pollen vigor, and the 3-time over-expression of the N-miR172 can cause the development failure, the dwarf plants and the decline of seed germination vigor of Arabidopsis. The result shows that the cabbage N-miR172 plays an important role in the development of flowers and organs and the transformation of vegetative growth into reproductive growth, and the gene module can be applied to breeding of cabbages and other horticultural plants.

Description

Cabbage flower development related gene module N-miR172-BraNPY and application thereof

Technical Field

The invention belongs to the technical field of plant genetic engineering, in particular to the technical field of application of cabbage N-miR172, a target gene BraNPY thereof and a coding protein thereof in a plant breeding process, and particularly relates to a cabbage flower development related gene module N-miR172-BraNPY5 and application thereof.

Background

Cabbage (Brassica rapal. Syn. B. Campestris l.) belongs to Brassica (Brassica) plants, is a worldwide vegetable which originates in China and is widely cultivated in various countries worldwide, has various edible parts including leaf balls, rosette leaves, young leaves, flower stems, young leaves and the like, can be fried, boiled and dehydrated, has unique flavor and high nutritive value, and is popular with people. Chinese cabbage hybrid vigor is obvious, and a cytoplasmic male sterile line (CMS) is generally adopted for seed production, but in actual production, the problems of poor flowering habit, malformation of flower buds, inadequacy of flower organs and the like often occur in the cytoplasmic male sterile line, and the seed production yield, quality and efficiency are obviously affected.

MiRNA is an endogenous non-coding small RNA with the length of 20-24 nt in organisms, cannot be translated into protein to perform functions, but can be combined with an mRNA, and can negatively regulate the expression of genes at the post-transcriptional level through cleavage and inhibition of translation. Many studies indicate that mirnas are widely involved in the proliferation, differentiation and apoptosis processes of cells, and play an important role in various biological processes such as growth and development of plants, metabolism, responses to biotic and abiotic stress, and the like. In recent years, a great deal of research shows that a plurality of miRNAs regulate gene pathways related to flower development through different ways and participate in the flower development process. miR156 plays an important role in the process of changing the vegetative growth of plants into reproductive growth, and overexpression of miR156 can lead to prolonged vegetative growth time and delayed flowering of Arabidopsis, tobacco and poplar. The miR319 family plays an important role in the development process of plant flowers, and mainly regulates the development of plant flower organs. Research shows that miR319a can control the size and shape of flower organs, and petals and stamens can be shortened when miR319a is mutated. Therefore, the further elucidation of the molecular regulatory mechanism of miRNA on the development of white cabbage flowers has important theoretical and practical significance.

Disclosure of Invention

The invention aims to provide a cabbage flower development related gene module N-miR172-BraNPY and application thereof, aiming at the problem of insufficient breeding resources in the prior art.

The aim of the invention is realized by the following technical scheme: the invention provides a cabbage flower development related gene module N-miR172-BraNPY, wherein the N-miR172 and a target gene BraNPY thereof are provided with:

(1) Nucleotide sequences shown as SEQ ID No.1 and SEQ ID No. 2; or (b)

(2) The nucleotide sequences shown in SEQ ID No.1 and SEQ ID No.2 are substituted, deleted and/or added with one or more nucleotides; or (b)

(3) A nucleotide sequence which hybridizes under stringent conditions to the DNA sequence defined in (1).

The invention provides a biological material containing the cabbage flower development related gene module N-miR172-BraNPY, wherein the biological material is an expression vector, an expression cassette, a host cell or engineering bacteria.

The invention provides verification of the targeting relationship of the cabbage N-miR172 and a target gene BraNPY thereof in plants.

The invention provides application of the cabbage N-miR172 and a target gene BraNPY thereof or corresponding biological materials thereof in regulating and controlling plant flower development functions.

The invention provides application of the cabbage N-miR172 and a target gene BraNPY thereof or corresponding biological materials thereof in the process of regulating and controlling plant vegetative growth to reproductive growth.

The invention provides application of the cabbage N-miR172 and a target gene BraNPY thereof or corresponding biological materials thereof in preparation of transgenic plants.

The invention provides application of the cabbage N-miR172 and a target gene BraNPY thereof or corresponding biological materials thereof in plant germplasm resource improvement.

Further, the application is specifically:

Plant growth is retarded by up-regulating the expression of the N-miR172 of the Chinese cabbage, the plant is dwarf, and the flowering of the plant is delayed and the pollen activity is reduced by inhibiting the expression of the N-miR172 of the Chinese cabbage.

The N-miR172 precursor sequence of the Chinese cabbage is shown as SEQ ID No.1, the sequence of a target gene BraNPY of the Chinese cabbage is shown as SEQ ID No.2, and the targeted combination of N-miR172 and BraNPY in plants is verified through a tobacco transient expression experiment. The method comprises the steps of respectively constructing an over-expression vector of N-miR172 and an STTM inhibition expression vector, transferring the over-expression vector into arabidopsis thaliana through an agrobacterium-mediated method, obtaining a transgenic arabidopsis thaliana strain of the over-expression and the inhibition expression of the N-miR172 of the cabbage, finding that the transgenic arabidopsis thaliana of which the growth is slow and the development is blocked by 3 times and inhibiting the development of young leaves of the plant of which the N-miR172 is expressed from abnormal development, and obviously reducing the flowering delay and the pollen vigor, so that the N-miR172 of the cabbage possibly participates in the development process of a flower organ of the cabbage, has important influence on the reproductive growth of the plant, and has good application prospect.

The invention has the beneficial effects that the function and expression analysis of the cabbage flower development related gene module N-miR172-BraNPY are provided, the breeding of white cabbage flowers is facilitated, the cabbage N-miR172 plays an important role in the aspect of the transformation of flower organ development and nutrition growth into reproductive growth, and the gene module can be applied to the breeding of cabbage vegetables and other horticultural plants and has good application prospects.

Drawings

FIG. 1 is an electrophoretogram of an N-miR172 precursor fragment and BraNPY CDS fragment amplification; wherein (A) in FIG. 1 is an electropherogram of an N-miR172 precursor fragment, and (B) in FIG. 1 is an electropherogram of a BraNPY CDS fragment;

FIG. 2 is a graph of the analysis of binding sites, fluorescent proteins, and relative fluorescent content of N-miR 172-targeted BraNPY (scale 100 μm) verified by transient expression of tobacco; wherein (A) in FIG. 2 is a schematic diagram of 3 vectors SK-N-miR172, SK and pFGC-BraNPY-target and a binding site diagram of miRNA and a target gene, (B) in FIG. 2 is a fluorescent protein diagram after SK-N-miR172 and no-load are respectively co-transfected with pFGC-BraNPY5-target bacterial solutions, and (C) in FIG. 2 is a relative fluorescence content analysis diagram of N-miR172 targeting BraNPY 5;

FIG. 3 is a diagram showing analysis of relative expression amounts of N-miR172 and target gene BraNPY in various organs of Chinese cabbage;

FIG. 4 is a diagram of the PCR detection results of transgenic plants; wherein, (a) in fig. 4 is a diagram of the detection result of the inhibition expression strain PCR, and (B) in fig. 4 is a diagram of the detection result of the overexpression strain PCR;

FIG. 5 is a graph showing the results of qRT-PCR analysis of transgenic Arabidopsis; wherein (A) in FIG. 5 is a graph of the relative expression level of N-miR172 and AtNPY of 6 over-expression positive seedlings, and (B) in FIG. 5 is a graph of the relative expression level of N-miR172 and AtNPY5 of 5 under-expression positive seedlings;

FIG. 6 is a schematic representation of the phenotype of 10-day seedlings of transgenic Arabidopsis thaliana (scale 2 mm);

FIG. 7 is a schematic diagram of bolting situation of transgenic Arabidopsis grown for 24 days (scale 2 mm);

FIG. 8 is a schematic diagram of the viability of over-expressed and inhibited expression of N-miR172 Arabidopsis pollen;

FIG. 9 is an N-miR172 Arabidopsis plant over-expressed and inhibited from expression; wherein (A) in FIG. 9 is the phenotype of an Arabidopsis plant (scale is 2 cm), and (B) in FIG. 9 is the relative expression level of OE-N-miR172-6 strain N-miR 172;

FIG. 10 is an Arabidopsis seedling (scale 1 cm) sown for 7 days; wherein (A) in FIG. 10 is a Col-0 Arabidopsis thaliana map, (B) in FIG. 10 is an OE-N-miR172-5 strain map, (C) in FIG. 10 is an STTM-N-172-1 strain map, and (D) in FIG. 10 is an OE-N-miR172-6 strain map;

FIG. 11 is a graph of BraNPY subcellular localization results (scale 100 μm);

FIG. 12 is a graph showing the results of histochemical staining of a BraNPY promoter-GUS fusion expression vector transformed Arabidopsis positive plants; wherein, (a) in fig. 12 is a chemical dyeing result chart of sepals, (B) in fig. 12 is a chemical dyeing result chart of petals, (C) in fig. 12 is a chemical dyeing result chart of stamens, (D) in fig. 12 is a chemical dyeing result chart of pistils, (E) in fig. 12 is a chemical dyeing result chart of open flowers, (F) in fig. 12 is a chemical dyeing result chart of roots, (G) in fig. 12 is a chemical dyeing result chart of stems, and (H) in fig. 12 is a chemical dyeing result chart of leaves, and (I) in fig. 12 is a chemical dyeing result chart of horns;

FIG. 13 is a schematic diagram of the construction of a functional complementation experimental vector; wherein, (a) in fig. 13 is a pFGC1008 overexpression vector, (B) in fig. 13 is a STTM suppression expression vector, (C) in fig. 13 is a promoter-GUS fusion expression vector, (D) in fig. 13 is a BraNPY-eGFP fusion expression vector, and (E) in fig. 13 is an SK-miR172 expression vector.

Detailed Description

The present invention is illustrated by the following specific examples, in which technical means not described in detail are conventional techniques well known to those skilled in the art. The examples are intended to be illustrative of the present invention and not to be construed as limiting the scope of the invention, as other examples based on the examples are intended to be within the scope of the invention by any person skilled in the art without undue burden.

The invention provides a Chinese cabbage flower development related miRNA and a target gene thereof, wherein the miRNA and the target gene are cloned from Chinese cabbage 219-06, namely a Chinese cabbage flower development related gene module N-miR172-BraNPY, wherein the N-miR172 and the target gene BraNPY thereof have sequences shown as SEQ ID No.1 and SEQ ID No. 2.

The embodiment of the invention also provides application of the N-miR172 and the target gene BraNPY thereof in aspects of regulating and controlling leaves, flowering time and pollen viability, and the application is specifically described below.

Example 1: verification of interaction between N-miR172 and target gene BraNPY5

1. Extracting total RNA of plants and synthesizing cDNA:

Extracting total RNA of cabbage tissues by adopting a Trizol method, and obtaining cDNA of the gene through reverse transcription, wherein specific steps refer to TOYOBO reverse transcription kit instruction book. The length of miRNA itself is shorter, usually 18-23 nt, so that a stem-loop needs to be added to the miRNA, and under the action of reverse transcriptase, a stem-loop reverse transcription primer is firstly combined with the 3' end of the miRNA, so that the artificial lengthened miRNA first strand cDNA is obtained, and the specific steps are referred to a Vazyme miRNA special stem-loop reverse transcription kit.

2. Obtaining target genes and linearization vectors:

Amplifying miRNA precursor fragments and target gene CDS fragments by using Chinese cabbage DNA and cDNA as templates respectively, designing amplification primers with homology arms according to the sequence of a carrier enzyme cleavage site, wherein the amplification system is as shown in table 1: KOD One Master Mix. Mu.L of each of the upstream and downstream primers, 500 ng of DNA/cDNA, and a total of 50. Mu.L of ddH ₂ O (double distilled water); the amplification procedure was: denaturation to extension at 98℃for 3min, 98℃for 10 s,53℃for 5 s,68℃for 2 min cycles, separation by 1.5% agarose gel electrophoresis after amplification, photographing with gel imager JS-680D (Shanghai Peqing, china) to obtain the corresponding electropherogram, as shown in FIG. 1, and further determining the fragment of interest, as shown in FIG. 1 (A), lane 2 is the N-miR172 precursor fragment, as shown in FIG. 1 (B), and lane 1 is the BraNPY CDS fragment. And (3) recovering the target fragment by using a gel recovery kit, and placing the target fragment at-20 ℃ for standby after measuring the concentration.

Tobacco transient expression experiments prove that two vectors are needed in total for the interaction of miRNA and target genes, pGreen II-0029 60-SK (SK) MCS is connected with miRNA precursor fragments after being cut by Bam H I and Xba I, and pFGC5941-eGFP is connected with CDS fragments of the target genes after being cut by Bam H I and Hind III. The enzyme digestion system is as follows: plasmid 1000 ng, 1. Mu.L each of the two endonucleases, 10 Xmix 5. Mu.L each, ddH ₂ O up to 50. Mu.L each, were incubated at 37℃for 30min and recovered using a PCR product recovery kit to give a linearized vector.

Table 1: primer for constructing tobacco transient expression experimental vector

3. Constructing a vector by a homologous recombination method:

Amplifying the inserted fragment by using a homologous arm primer after double enzyme cutting of the vector, and adding homologous arms at two ends of the fragment; and then the homologous recombination enzyme is used for connection, and the reaction system is as follows: 5 XCE I Buffer 4 μL, exnase I2 μL, fragment and carrier are added according to actual concentration, specifically: determining the amount added into a reaction system according to the base pair numbers of the linearization vector and the insert, wherein the addition amount (ng) of the linearization vector is base pair number multiplied by 0.02, the addition amount (ng) of the insert is base pair number multiplied by 0.04, the proper addition amount of the vector is 50-200 ng, the proper addition amount of the insert is 10-200 ng, and the insert is added in the highest or lowest addition amount when the insert is actually added beyond or insufficient; and finally, supplementing the recombinant vector to 20 mu L by using ddH ₂ O, placing the recombinant vector at 37 ℃ for incubation for 30min, and rapidly transferring the recombinant vector to ice for 5min after the incubation is completed.

4. E.coli transformed by freeze thawing method:

Thawing 50 mu L of escherichia coli competent cells on ice, adding 5 mu L of a connecting product, flicking, uniformly mixing, carrying out ice bath for 25 min, carrying out heat shock 90 s at 42 ℃, and immediately transferring to ice for 3-5 min; 700 mu L of non-resistance LB liquid medium which is balanced to room temperature is added into an ultra-clean workbench, and the temperature is 37 ℃, and the recovery is 1 h after 200 rpm; 5000 Centrifuging at rpm 1 min for bacterial recovery, discarding most of the supernatant on an ultra-clean workbench, and reserving about 100 mu L for re-suspending bacterial cells; transferring the bacterial liquid to LB solid culture medium (Kan. Antibiotic concentration is 50 mg.L ^-1, cmr. Antibiotic concentration is 34 mg.L ^-1) containing corresponding antibiotics (Kan. Or Cmr.), spreading uniformly with a spreader, drying, and culturing in a 37 ℃ incubator under inversion for 12 h.

The monoclonal propagation is selected, bacterial liquid is shaken for 2h at 37 ℃ and 200 rpm, and then the bacterial liquid can be used for Polymerase Chain Reaction (PCR) detection, and the detection primers are shown in table 2. The upstream detection primers of the miRNA vector and the eGFP vector of the over-expression vector are 35S-F and pFGC5941-F respectively, the downstream primer is a downstream amplification primer of the inserted fragment, and the detection system is as follows: 2X RAPID TAQ MASTER Mix 12.5. Mu.L, 1. Mu.L of each of the upstream and downstream primers, 1. Mu.L of the bacterial solution, and 9.5. Mu.L of ddH ₂ O. The PCR detection procedure was 98℃for 3 min,98℃for 10 s,56℃for 10 s,72℃for 15 s,72℃for 2 min cycles of denaturation to extension. The PCR product is separated by 1.5% agarose gel electrophoresis, the length of the target fragment is compared, and the bacterial liquid with correct strip is sent to a test. Extracting plasmid after sequencing verification, and preserving at-20 ℃.

Table 2: primer for PCR detection of tobacco transient expression experimental vector

5. Tobacco transient expression experiments verify that N-miR172 interacts with target gene BraNPY:

Soaking seeds of Nicotiana benthamiana with water overnight, sowing in seedling substrate (vermiculite is high), covering with fresh-keeping film, punching holes on the surface for ventilation, and accelerating germination at 25deg.C. After the tobacco bud, growing for 2-3 days (d), transplanting after cotyledons are opened, wherein the daily illumination is set to 4000 lux, and the ambient temperature is 24 ℃/16 h,22 ℃/8 h. About 4 weeks after seedling transplantation, tobacco can be used for transient expression experiments.

Adding 50 mu L of target bacterial liquid subjected to two-time activation into 15-20 mL of LB liquid culture medium containing Rif antibiotics (50 mg.L ^-1) and Kan antibiotics (50 mg.L ^-1), culturing for 16-20 h at the temperature of 28 ℃ 200 rpm, centrifuging and collecting bacteria by using a large table-type low-temperature centrifuge 5810R (Eppendorf, germany) at 5000 rpm for 15 min, and re-suspending the bacteria by using a heavy suspension, wherein the heavy suspension is a mixed liquid of 10 mmol.L ^-1 MgCl₂、10 mmol·L^-1MES、100 μmol·L^-1 acetosyringone; and then measuring the OD value of the heavy suspension by using an ultraviolet spectrophotometer UV-6100 (Shanghai Ling Ji Zhong, china), adjusting OD ₆₀₀ = 0.8-1.0, and injecting the tobacco after standing for 2-3 hours in a dark place.

Marking the leaves suitable for injection, respectively injecting negative control and experimental group bacterial liquid at the left side and the right side, and uniformly mixing the bacterial liquid containing the miRNA precursor and the bacterial liquid containing the target gene binding site carrier according to the proportion of 4:1 in advance during injection, wherein 6 leaves are injected in each group of experiments, and each leaf is injected for 2-3 times. After darkening 48 h, a small round hole was punched at the injection site with a small punch with the back face facing upwards, and the fluorescent signal intensity of the control group and the experimental group was observed with a axio zoom.v16 fluorescent microscope (carzeiss, germany), whose fluorescent protein pattern is shown in (B) of fig. 2, from which it was observed that the fluorescent signal of the tobacco leaf injected with SK-N-miR172 was reduced, relative fluorescent values were analyzed using ImageJ software as shown in (C) of fig. 2, and the result showed that the fluorescent value of tobacco co-transformed with miR172 over-expression vector was significantly reduced, indicating that N-miR172 was able to bind to BraNPY5 in plants in a targeted manner and regulated its expression, wherein 3 vectors SK-N-miR172, SK and pFGC-BraNPY5-target and the binding site of miRNA to the target gene are shown in (a) of fig. 2, and SK-miR172 expression vector is shown in (E) of fig. 13.

6. Analysis of relative expression quantity of N-miR172 and target gene BraNPY in various organs of Chinese cabbage:

The root, stem, leaf, normal flower, nested flower and fruit of the sequenced sample were taken, and the relative expression levels of N-miR172 and its target gene were detected by means of real-time fluorescent quantitative reverse transcription polymerase chain reaction (qRT-PCR), and the primers used for the detection were as shown in Table 3. The results of quantitative PCR are shown in FIG. 3, which shows the relative expression levels of N-miR172 and target gene BraNPY in various organs of Chinese cabbage, and as can be seen from FIG. 3, N-miR172 is expressed highest in leaves, then flowers are nested, and the expression levels in stems, fruits and roots are lower than those in normal flowers. Correspondingly, the target gene and miRNA show a more obvious opposite expression mode, braNPY reaches more than 20 times in flowers, the expression quantity in leaves and stems is lower, and the analysis result further supports the targeting relationship of N-miR172 and BraNPY 5.

Table 3: primer for qRT-PCR detection of nested flower sample

Example 2: construction of N-miR172 over-expression and inhibition expression vector

1. Construction of the over-expression vector:

the operations of the amplification of the precursor fragment of the N-miR172 and the construction of the vector are shown in example 1, the overexpression vector is pFGC1008, enzyme cutting sites are SalI I and Kpn I as shown in (A) in fig. 13, a target fragment is obtained by adding a homology arm primer, PCR detection is carried out after the target fragment is recombined to obtain the vector, an upstream primer is a vector primer, and a downstream primer is Pre-N-miR172-R as shown in Table 4.

Table 4: primer for N-miR172 over-expression vector

2. Construction of an inhibition expression vector:

The mature sequence of the N-miR172 and the complementary sequence thereof on the stem loop are searched in the sequencing result, a segment of STTM sequence is designed based on the mature sequence, the middle segment is a 48 nt fixed sequence, and base sequences incompletely complementary with the N-miR172 are arranged on two sides of the sequence, so that the miRNA can be combined and can not be cut by RISC. After the completion of the design, the STTM fragment was synthesized by Wohan far biotechnology Co., ltd, and ligated to pBWA (V) HS vector as shown in FIG. 13 (B), an inhibition expression vector of miR172 was constructed, designated STTM-N-172, and the construction primers and detection primers were as shown in Table 5.

Table 5: primers for STTM-N-172 inhibition expression vector

Example 3: screening of transformation Arabidopsis thaliana and positive plants by flower dipping method

1. Transformation of Arabidopsis thaliana by the floral dip method:

The pods and flowers that have been opened of the wild type Arabidopsis thaliana were removed one day in advance, and watered through. 200 mu L of target bacterial liquid subjected to twice activation is added into an LB liquid culture medium containing Rif (50 mg.L ^-1) and Cmr (34 mg.L ^-1) of 100 mL, the bacterial strain is cultured at the temperature of 28 ℃ 200 rpm for 16~20 h,5000 rpm 15 min centrifugal collection, 5% sucrose solution is used for resuspension until OD ₆₀₀ =0.8, 20 mu L of Silwet L-77 surfactant is added, after uniform mixing, the arabidopsis flower buds are poured into the bacterial liquid for 1 min, the bacterial liquid is dried after being taken out, the bacterial liquid is placed in a tray in a flat mode, and the bacterial strain is recovered to normal culture until the seeds are collected after dark culture for 24h. To increase the conversion efficiency, the conversion was repeated once again after one week.

2. Screening positive plants:

The pFGC1008 over-expression vector has hygromycin resistance marker gene, and the plant is transformed into the plant to have hygromycin resistance. The harvested T ₀ generation seeds are required to be sowed on a culture medium for screening after being disinfected for a plurality of times, the disinfection and sowing are completed on an ultra-clean workbench, and the specific screening steps are as follows:

2.1, sub-packaging T ₀ generation seeds into 2 ml centrifuge tubes, and soaking in ddH ₂ O for 1 min. After centrifugation, the upper layer of water and some seeds of poor quality were aspirated with a pipette.

2.2, Disinfecting 1 time with 75% ethanol, washing the seeds 3 times with ddH ₂ O, repeating this step 1 min times, thoroughly disinfecting the seeds and washing off residual alcohol on the seed surface.

2.3, Uniformly sowing seeds on a 1/2 MS sowing culture medium containing 90 mg.L ^-1 hygromycin after sterilization, and culturing at 25 ℃.

2.4, Observing screening conditions after culturing for one week, wherein the arabidopsis with hygromycin resistance can normally grow vertically in a culture medium, and the arabidopsis without the resistance can die 3-4 d after sprouting.

2.5, Transplanting positive seedlings growing well in the culture medium into the matrix, covering the film for two days until the seedlings adapt to the environment, and uncovering the film.

2.6, After 10 d seedlings were transplanted, DNA was extracted from the leaves grown normally, and PCR was performed, and the result was shown in FIG. 4, and the identification of the target band was positive seedlings. FIG. 4 (A) shows the result of PCR detection of an expression-inhibiting strain, wherein lanes 1-5 are STTM-N-172-1-5, lane 6 is a positive control, lane 7 is a wild type, and lane 8 is H ₂ O. In FIG. 4, (B) shows the result of PCR detection of the over-expressed strain, wherein lanes 1 to 6 are OE-N-miR172-1 to 6, respectively, lane 7 is a positive control, lane 8 is a wild type, and lane 9 is H ₂ O.

3. QRT-PCR analysis of transgenic Arabidopsis thaliana:

the study has verified that N-miR172 and target gene BraNPY5 exist BraNPY homologous gene AtNPY5 in Arabidopsis thaliana, and through a plurality of software predictions of TARGETFINDER, PSROBOT and PSRNATARGET, atNPY5 is also the target gene of N-miR172, so that N-miR172 and AtNPY relative expression amount analysis is simultaneously carried out on transgenic plants identified as positive, and the result is shown in FIG. 5. As shown in (a) of fig. 5, in 6 over-expressed lines, the N-miR172 expression levels of OE-N-miR172-4, 5, 6 plants were significantly up-regulated compared to the wild type, and the other three plants were not significantly different. Meanwhile, the observation also shows that the predicted target gene AtNPY expression quantity is down-regulated in three up-regulated over-expression lines, and the down-regulation amplitude of AtNPY expression quantity of the two other lines is obvious or extremely obvious except OE-N-miR 172-4. As shown in FIG. 5 (B), the relative N-miR172 expression levels of STTM-N-172-1, 2 and 5 plants were significantly reduced in the inhibition-expression lines, and significant upregulation of the target gene AtNPY in these three positive plants was also detected.

Example 4: phenotype observation of N-miR172 transgenic positive plant

1. And (3) observing the growth condition of young leaves:

And (3) collecting the T ₁ -generation positive Arabidopsis single plant, selecting a strain with larger difference according to the expression quantity analysis of N-miR172 of the T ₁ -generation transgenic Arabidopsis, continuously screening positive seedlings by using a hygromycin-containing 1/2 MS culture medium, and breeding the T ₂ -generation observation phenotype. It was observed that the true leaf growth of the expression-suppressing strain corresponding to the expression-suppressing vector STTM-N-172 was slow compared to the wild-type WT, but the over-expression strain corresponding to the over-expression vector OE-N-miR172 was not significantly different from the wild-type, as shown in FIG. 6. After 10 d of the plants are sown, 2 true leaves of the wild arabidopsis are grown, the whole leaves form a cross shape, but the true leaves of the expression-inhibiting strain are smaller and do not grow completely, the whole plant presents a straight shape, and the over-expression strain arabidopsis is not different from the wild type.

2. Flowering time statistics:

The T ₂ generation transgenic arabidopsis is continuously cultured and observed, when the transgenic arabidopsis grows to 21 d, the wild type starts bolting first, and at 23d, half of the wild type arabidopsis starts bolting, however, the first bolting plant does not appear in the STTM-N-172 transgenic strain until 24 d, and as shown in fig. 7, the bolting time of more than half plants is delayed by 3-4 d compared with the wild type. The bolting time of the wild type, over-expressed and inhibited expression N-miR172 strain was counted, and the results show that the STTM-N-172 transgenic strain has obvious flowering delay, as shown in Table 6, wherein the values are expressed as mean value + -standard deviation, 24 strains are counted in each sample, and multiple comparison test (p < 0.05) is adopted in each column.

Table 6: flowering time statistics of wild-type and transgenic arabidopsis thaliana

3. Pollen viability detection:

And sucking 30 mu L of Alexander dye liquor onto a glass slide, clamping the vigorous flower of Arabidopsis thaliana by using forceps, repeatedly dipping the anther on the dye liquor for several times, and directly putting the anther into the dye liquor after picking up the anther and stirring by using forceps in order to ensure that enough pollen grains are observed. To ensure the reliability of the statistical result, at least 3 plants are observed for each plant, and the total number of the counted pollen grains is not less than 1000 grains. The viable pollen grains are in a purple full round shape in the staining solution, while the pollen grains with poor vitality are shrunken and shrunken, and are stained blue by the staining solution.

As shown in FIG. 8, the wild type and the Arabidopsis thaliana pollen which is over-expressed with the N-miR172 have better vigor, a large number of purple full pollen grains are observed in a microscope field of view, but the vigor of the Arabidopsis thaliana plant pollen which is inhibited to express the N-miR172 is obviously reduced, and a large number of blue non-vigor pollen grains exist in anthers. The proportion of viable pollen grains was photographed and counted, and the result showed that the proportion of normal pollen of the wild type was 98.72%, the pollen viability of the N-miR 172-overexpressed Arabidopsis was 97.81%, and the proportion of normal pollen of Arabidopsis that inhibited the expression of N-miR172 was only 46.06%, as shown in Table 7.

Table 7: overexpression and inhibition of expression of N-miR172 Arabidopsis normal pollen proportion (%)

4. Phenotype observation of high expression N-miR172 strain:

When T ₂ generation over-expressed transgenic seedlings are observed, 6 positive seedlings of the OE-N-miR172-6 strain are subjected to bad growth, small and many leaves, short plants and other phenotypes, as shown in (A) in fig. 9, 6 positive seedlings of the T ₂ generation OE-N-miR172-6 strain are mixed and sampled, RNA is extracted, the relative expression quantity of the N-miR172 is measured, qRT-PCR results show that compared with a wild type, the content of the N-miR172 in the T ₂ generation OE-N-miR172-6 strain is obviously improved to 3 times that of the wild type, and the content of the N-miR172 in the T ₂ generation OE-N-miR172-6 strain is shown in (B) in fig. 9. Culturing T ₂ generation transgenic plants until the seeds are harvested, wherein the quantity of seeds of OE-N-miR172-6 strain is small, and the germination rate is obviously reduced, as shown in a graph in FIG. 10, wherein (A) in FIG. 10 is a Col-0 Arabidopsis thaliana map, (B) in FIG. 10 is an OE-N-miR172-5 strain map, (C) in FIG. 10 is an STTM-N-172-1 strain map, and (D) in FIG. 10 is an OE-N-miR172-6 strain map. From the results shown in fig. 10, it was found that the 3-fold higher expression of N-miR172 to wild type resulted in the plant dysplasia of arabidopsis thaliana, dwarfing of the plant and a decrease in seed vigor.

Example 5: analysis of the spatiotemporal expression Pattern of target Gene BraNPY5

1. Subcellular localization analysis:

The vector used for subcellular localization was pFGC5941-eGFP, and the BraNPY protein subcellular localization vector was constructed in example 1, and tobacco and bacterial liquid were prepared in the same manner as in example 1. Selecting tobacco leaves with good growth vigor and flatness, pricking holes on the back of the leaves at positions far away from veins by using needles, uniformly mixing a marker with no-load and corresponding genetic bacterial liquid in a ratio of 1:1 in advance, sucking a proper amount of mixed bacterial liquid by using a 1mL injector, injecting the mixed bacterial liquid into tobacco from wounds, and culturing in a dark environment of 48: 48 h. Blades of about 1 cm ² a size near the pinhole were cut with scissors with the back facing up and the fluorescence signal distribution was observed with a axio zoom.v16 fluorescence microscope, which showed BraNPY to be positioned on the membrane as shown in fig. 11.

2. Promoter-GUS fusion expression vector construction:

The promoter and GUS fusion expression vector is a pCAMBIA1300-GUS vector stored in a laboratory, restriction enzymes used upstream and downstream are Hind I and XbaI respectively, and as shown in (C) in FIG. 13, the purified linearization vector is obtained by enzyme digestion of 1h and then use of a PCR product recovery kit. Amplification of the promoter Using cabbage DNA as a template, the gene sequence was queried from NCBI (https:// www.ncbi.nlm.nih.gov /), the sequence about 2000 bp upstream of the BraNPY gene was selected as the promoter for the gene of interest, and the activity of the selected promoter sequence was examined by means of the PLANTCARE database (http:// bioinformation. Psu. Be/webtools/plantcare/html /). The amplification primers were designed based on the promoter and vector cleavage site sequences, and the homologous set was followed by using the vector upstream primer and the fragment downstream primer as PCR detection primers as shown in Table 8, and sequencing was performed to obtain pBraNPY-GUS plasmid as shown in (D) of FIG. 13.

Table 8: primer for promoter-GUS fusion expression vector

3. Histochemical staining and observation:

after pBraNPY-GUS plasmid is transformed into Arabidopsis thaliana, positive seedlings are obtained through screening, roots, stems, leaves, flowers and fruits of Arabidopsis thaliana positive plants are picked up by forceps and placed into a 2mL centrifuge tube, fresh GUS staining solution is added to completely submerge the Arabidopsis thaliana positive seedlings, the Arabidopsis thaliana positive seedlings are stained at 37 ℃ for 12H in a dark place, the Arabidopsis thaliana positive seedlings are decolorized by 75% ethanol for 1-3H, 75% ethanol is replaced every hour, after the decolorization is completed, the staining conditions of the Arabidopsis thaliana positive seedlings are observed by a DVM6 super depth fluorescence microscope (Leica, germany) microscope and a BX53 microscope (OLYMPUS, japan), the staining results are shown in FIG. 12, the staining results of sepals are shown in FIG. 12 (A), the staining results of petals are shown in FIG. 12 (B), the staining results of stamen are shown in FIG. 12 (C), the staining results of pistils are shown in FIG. 12 (D), the staining results of the open flowers are shown in FIG. 12 (E), the staining results of the roots are shown in FIG. 12 (F), the staining results of the map 12 (G) are shown in FIG. 12 (H) and the staining results of the map (G) are shown in the results of the map 12 (I) are shown. As is clear from the color results shown in FIG. 12, the BraNPY gene promoter can promote the downstream GUS gene expression in the root, stem, leaf, flower and fruit of Arabidopsis thaliana, wherein the root has the strongest promoting activity, and each part of the root and root hair have stronger GUS expression. In addition, the expression activity in leaves, petals, sepals and stamens is high, the expression activity in stems is low, and only a small amount of GUS signals exist at the basal part. There is a strong starter activity in the stigma, middle and basal part of the pistil, but only the top and basal parts have starter activity when it develops into a horn.

The foregoing description of the preferred embodiments of the present invention is provided to enable those skilled in the art to best explain the principles of the invention and its practical application, and to thereby enable others skilled in the art to best understand and utilize the invention. It will be apparent to one skilled in the art that these embodiments do not address all of the details in detail, and that certain improvements or modifications may be made thereto. Accordingly, all such modifications and improvements made upon the present invention are intended to be within the scope of the invention as claimed.

Claims (6)

1. A cabbage flower development related gene module N-miR172-BraNPY, wherein said N-miR172 and target gene BraNPY thereof have:

(1) Nucleotide sequences shown as SEQ ID No.1 and SEQ ID No. 2; or (b)

(2) The nucleotide sequences shown in SEQ ID No.1 and SEQ ID No.2 are substituted, deleted and/or added with one or more nucleotides; or (b)

(3) A nucleotide sequence which hybridizes under stringent conditions to the DNA sequence defined in (1).

2. A biological material containing the cabbage flower development related gene module N-miR172-BraNPY of claim 1, characterized in that the biological material is an expression vector, an expression cassette, a host cell or an engineering bacterium.

3. An application of the cabbage N-miR172 of claim 1 and a target gene BraNPY thereof or the biological material of claim 2 in regulating and controlling plant flower development function.

4. An application of the cabbage N-miR172 as claimed in claim 1 and target gene BraNPY thereof or the biological material as claimed in claim 2 in regulating the transformation process from vegetative growth to reproductive growth of plants.

5. Use of the cabbage N-miR172 of claim 1 and its target gene BraNPY or the biological material of claim 2 in the preparation of transgenic plants.

6. An application of the cabbage N-miR172 of claim 1 and target gene BraNPY thereof or the biological material of claim 2 in plant germplasm resource improvement.