A gene module N-miR172-BraNPY5 related to cabbage flower development and its application

Abstract

The present invention discloses a gene module N-miR172- BraNPY5 and its application, the precursor sequence of cabbage N-miR172 is shown in SEQ ID No.1, and its target gene BraNPY5 The sequence is shown in SEQ ID No. 2. The miRNA overexpression vector and the STTM inhibition expression vector were transformed into Col-type Arabidopsis by Agrobacterium floral transformation to obtain heterologous expression Arabidopsis strains. The results showed that the inhibition of N-miR172 expression led to delayed development of young leaves, delayed flowering, and decreased pollen vitality in Arabidopsis, while 3-fold overexpression of N-miR172 led to poor development of Arabidopsis, short plants, and decreased seed germination vitality. This shows that cabbage N-miR172 plays an important role in the development of floral organs and the transition from vegetative growth to reproductive growth, and this gene module can be applied to the breeding of cabbage and other horticultural plants.

Description

Technical Field

[0001] The present invention belongs to the technical field of plant genetic engineering, and specifically relates to cabbage N-miR172 and its target gene BraNPY5 The technical field of application of its encoding protein in plant breeding, in particular, relates to a gene module N-miR172- BraNPY5 and its applications. Background Art

[0002] Chinese cabbage( Brassica rapa L. syn. B. campestris L.) belongs to the genus Brassica in the family Cruciferae. Brassica ) plant, is a world vegetable that originated in my country and is widely cultivated in countries around the world. It has a variety of edible parts, including leaf heads, rosette leaves, young leaves, flower stems, stalk leaves, etc. It can be fried, cooked into soup, and dehydrated. It has a unique flavor and high nutritional value and is loved by people. Cabbage has obvious hybrid vigor, and cytoplasmic male sterile lines (CMS) are usually used for seed production. However, in actual production, cytoplasmic male sterile plants often have problems such as poor flowering habits, deformed buds, and incomplete development of flower organs, which significantly affect seed production yield, quality, and efficiency.

[0003] miRNA is a type of endogenous non-coding small RNA in organisms. It is 20-24 nt in length and cannot be translated into proteins to perform functions. However, it can bind to a type of mRNA and negatively regulate gene expression at the post-transcriptional level by cutting and inhibiting translation. Many studies have shown that miRNA is widely involved in cell proliferation, differentiation and apoptosis, and plays an important role in various biological processes such as plant growth and development, metabolism, and response to biotic and abiotic stresses. In recent years, a large number of studies have shown that many miRNAs regulate flower development-related gene pathways through different pathways and participate in the flower development process. miR156 plays an important role in the process of plants switching from vegetative growth to reproductive growth. Overexpression of miR156 will lead to prolonged vegetative growth time and delayed flowering in Arabidopsis, tobacco and poplar. The miR319 family also plays an important role in the flower development of plants, mainly regulating the development of plant flower organs. Studies have shown that miR319a can control the size and shape of flower organs. When miR319a mutates, it will cause petals and stamens to become shorter. Therefore, further clarifying the molecular regulatory mechanism of miRNA on cabbage flower development has important theoretical and practical significance. Summary of the invention

[0004] The purpose of the present invention is to provide a gene module N-miR172- BraNPY5 and its applications.

[0005] The purpose of the present invention is to achieve the following technical solutions: The present invention provides a gene module N-miR172- BraNPY5 , the N-miR172 and its target genes BraNPY5 have:

[0006] (1) the nucleotide sequences shown in SEQ ID No.1 and SEQ ID No.2; or

[0007] (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

[0008] (3) A nucleotide sequence that hybridizes to the DNA sequence defined in (1) under stringent conditions.

[0009] The present invention provides a method comprising the above-mentioned cabbage flower development-related gene module N-miR172- BraNPY5 The biological material is an expression vector, an expression cassette, a host cell or an engineered bacterium.

[0010] The present invention provides the above-mentioned Chinese cabbage N-miR172 and its target gene BraNPY5 Validation of targeting relationships in planta.

[0011] The present invention provides the above-mentioned Chinese cabbage N-miR172 and its target gene BraNPY5 Or the application of its corresponding biological materials in regulating plant flower development function.

[0012] The present invention provides the above-mentioned Chinese cabbage N-miR172 and its target gene BraNPY5 Or the application of its corresponding biological materials in regulating the transition from plant vegetative growth to reproductive growth.

[0013] The present invention provides the above-mentioned Chinese cabbage N-miR172 and its target gene BraNPY5 Or the use of corresponding biological materials in preparing transgenic plants.

[0014] The present invention provides the above-mentioned Chinese cabbage N-miR172 and its target gene BraNPY5 Or the application of its corresponding biological materials in the improvement of plant germplasm resources.

[0015] Furthermore, the application is specifically:

[0016] Up-regulating the expression of N-miR172 in Chinese cabbage delayed plant development and made the plants short, while inhibiting the expression of N-miR172 in Chinese cabbage delayed flowering and reduced pollen viability.

[0017] The cabbage N-miR172 precursor sequence provided by the present invention is shown in SEQ ID No.1, and its target gene BraNPY5 The sequence is shown in SEQ ID No. 2. The transient expression experiment of tobacco was used to verify the expression of N-miR172 and BraNPY5 Targeted binding in plants. An N-miR172 overexpression vector and an STTM inhibition expression vector were constructed, respectively, and transferred into Arabidopsis thaliana by Agrobacterium-mediated method to obtain transgenic Arabidopsis thaliana lines with overexpression and inhibition of N-miR172 in cabbage. The results showed that transgenic Arabidopsis thaliana with 3-fold expression of N-miR172 had slow growth and developmental stunting, and plants with suppressed expression of N-miR172 had abnormal young leaf development, delayed flowering, and significantly reduced pollen vitality, which indicated that cabbage N-miR172 may be involved in regulating the development of cabbage floral organs, have an important impact on the reproductive growth of plants, and have good application prospects.

[0018] The beneficial effect of the present invention is that the present invention provides a gene module N-miR172- BraNPY5 Functional and expression analysis are beneficial to the breeding of cabbage flowers. Cabbage N-miR172 plays an important role in the development of floral organs and the transition from vegetative growth to reproductive growth. This gene module can be applied to the breeding of cabbage vegetables and other horticultural plants, and has good application prospects. BRIEF DESCRIPTION OF THE DRAWINGS

[0019] Figure 1 N-miR172 precursor fragment and BraNPY5 Electrophoresis diagram of CDS fragment amplification; among them, Figure 1 (A) is the electrophoresis diagram of N-miR172 precursor fragment. Figure 1 (B) in BraNPY5 Electropherogram of CDS fragment;

[0020] Figure 2 Validation of N-miR172 targeting for transient expression in tobacco BraNPY5 (Scale bar is 100 μm) of binding sites, fluorescent proteins and relative fluorescence content analysis; Figure 2 (A) is a schematic diagram of the three vectors SK-N-miR172, SK and pFGC-BraNPY5-target and a map of the binding sites of miRNA and target genes. Figure 2 (B) shows the fluorescence protein image of tobacco after SK-N-miR172 and empty vector were co-transfected with pFGC-BraNPY5-target bacterial solution. Figure 2 (C) is the target of N-miR172 BraNPY5 Relative fluorescence content analysis diagram;

[0021] Figure 3 N-miR172 and its target genes in various organs of Chinese cabbage BraNPY5 Schematic diagram of relative expression analysis;

[0022] Figure 4 This is the result of PCR detection of transgenic plants; Figure 4 (A) is the result of PCR detection of the expression inhibition strain. Figure 4 (B) is the PCR test result of the overexpression strain;

[0023] Figure 5 The figure shows the results of qRT-PCR analysis of transgenic Arabidopsis thaliana; Figure 5 (A) shows the expression of N-miR172 and AtNPY5 Relative expression results. Figure 5 (B) shows the expression of N-miR172 in 5 seedlings with positive inhibition. AtNPY5 Relative expression results graph;

[0024] Figure 6 Schematic diagram of the phenotype of 10-day-old transgenic Arabidopsis seedlings (scale bar is 2 mm);

[0025] Figure 7 This is a schematic diagram of the bolting of transgenic Arabidopsis thaliana after 24 days of growth (the scale bar is 2 mm);

[0026] Figure 8 Schematic diagram of Arabidopsis pollen viability in overexpressed and suppressed N-miR172;

[0027] Fig. 9 Arabidopsis plants that overexpress and suppress N-miR172 expression; Fig. 9 (A) shows the phenotype of Arabidopsis plants (bar is 2 cm). Fig. 9 (B) in the figure shows the relative expression level of N-miR172 in the OE-N-miR172-6 strain;

[0028] Fig.10 7-day-old Arabidopsis seedlings (scale is 1 cm); Fig.10 (A) is the Col-0 Arabidopsis thaliana image. Fig.10 (B) is the OE-N-miR172-5 strain map. Fig.10 (C) is the STTM-N-172-1 strain map. Fig.10 (D) in the middle is the map of the OE-N-miR172-6 strain;

[0029] Fig.11 This is the result of BraNPY5 subcellular localization (scale bar is 100 μm);

[0030] Fig.12 for BraNPY5 The histochemical staining results of Arabidopsis positive plants transformed with promoter and GUS fusion expression vector; Fig.12 (A) is the result of chemical staining of sepals. Fig.12 (B) is the result of chemical staining of petals. Fig.12 (C) is the result of chemical staining of stamens. Fig.12 (D) is the result of chemical staining of the pistil. Fig.12 (E) is the result of chemical staining of an open flower. Fig.12 (F) in the figure is the chemical staining result of the root. Fig.12 (G) in the figure is the result of chemical staining of the stem. Fig.12 (H) in the figure is the chemical staining result of leaves. Fig.12 (I) in the figure is the result of chemical staining of silique;

[0031] Fig.13 This is a schematic diagram of constructing a functional complementation experimental vector; Fig.13 (A) is the pFGC1008 overexpression vector. Fig.13 (B) is the STTM inhibition expression vector. Fig.13 (C) in the figure is a promoter-GUS fusion expression vector. Fig.13 (D) in the figure is the BraNPY5-eGFP fusion expression vector. Fig.13 (E) in the figure is the SK-miR172 expression vector. DETAILED DESCRIPTION

[0032] The present invention is described below by specific examples, and the technical means not described in detail in the examples belong to conventional techniques well known to professionals in the field. The examples are only used to illustrate the present invention, but do not limit the scope of the present invention. Any other embodiments obtained by any technician in the field without creative work based on the examples of the present invention belong to the protection scope of the present invention.

[0033] The present invention provides a cauliflower flower development-related miRNA and a target gene thereof. The miRNA and the target gene are cloned from cauliflower "219-06", namely, the cauliflower flower development-related gene module N-miR172- BraNPY5 , among which N-miR172 and its target genes BraNPY5 The sequences are shown as SEQ ID No.1 and SEQ ID No.2.

[0034] The present invention also provides the above-mentioned N-miR172 and its target gene BraNPY5The application in regulating leaves, flowering time and pollen vitality is described in detail below.

[0035] Example 1: N-miR172 and target genes BraNPY5 Mutual Authentication

[0036] 1. Plant total RNA extraction and cDNA synthesis:

[0037] The total RNA of cabbage tissue was extracted by Trizol method, and the cDNA of the gene was obtained by reverse transcription. The specific steps refer to the instruction manual of TOYOBO reverse transcription kit. The miRNA itself is short, usually 18-23 nt, so it is necessary to add a stem-loop to the miRNA. Under the action of reverse transcriptase, the stem-loop reverse transcription primer first binds to the 3' end of the miRNA to obtain the artificially lengthened first-chain cDNA of the miRNA. The specific steps refer to the Vazyme miRNA special stem-loop reverse transcription kit.

[0038] 2. Obtaining the target gene and linearized vector:

[0039] The miRNA precursor fragment and the target gene CDS fragment were amplified using cabbage DNA and cDNA as templates, respectively. Amplification primers with homology arms were designed according to the vector restriction site sequence, as shown in Table 1. The amplification system was: KOD One Master Mix 25 μL, upstream and downstream primers 1.5 μL each, DNA / cDNA 500 ng, ddH2O (double distilled water) to 50 μL; the amplification program was: 98℃ 3 min, 98℃ 10 s, 53℃ 5 s, 68℃ 5 s, 68℃ 2 min, denaturation to extension for a total of 35 cycles. After amplification, the fragments were separated by 1.5% agarose gel electrophoresis and photographed with a gel imager JS-680D (Shanghai Peiqing, China), and the corresponding electrophoresis patterns were obtained, as shown in Table 1. Figure 1 As shown, the target fragment is then determined, such as Figure 1 As shown in (A), lane 2 is the N-miR172 precursor fragment, such as Figure 1 As shown in (B), lane 1 is BraNPY5 CDS fragment: Use gel recovery kit to recover the target fragment, determine the concentration and store at -20℃ for later use.

[0040] Tobacco transient expression experiments verify the interaction between miRNA and target genes. Two vectors are needed: pGreen II-002960-SK (SK) MCS Bam HI and XB I digested and connected miRNA precursor fragments, pFGC5941-eGFP was used Bam HI and HinThe CDS fragment of the target gene was connected after d III restriction digestion. The restriction digestion system was: 1000 ng of plasmid, 1 μL of each of the two endonucleases, 5 μL of 10×mix, ddH2O was added to 50 μL, incubated at 37°C for 30 min, and then recovered with a PCR product recovery kit to obtain a linearized vector.

[0041] Table 1: Primers used for the construction of tobacco transient expression vectors

[0042]

[0043] 3. Construction of vector by homologous recombination method:

[0044] After double digestion of the vector, the insert fragment was amplified with homology arm primers, and homology arms were added to both ends of the fragment; then the homologous recombinase was used for ligation, and the reaction system was: 5×CEΙΙ Buffer 4 μL, ExnaseΙΙ 2 μL, and the fragment and vector were added according to the actual concentration, specifically: the amount added to the reaction system was determined according to the base pair number of the linearized vector and the insert fragment, where the amount of linearized vector added (ng) was the base pair number × 0.02, and the amount of insert fragment added (ng) was the base pair number × 0.04, the appropriate amount of vector added was 50~200 ng, and the appropriate amount of insert fragment added was 10~200 ng. If it exceeded or was insufficient during actual addition, the highest or lowest amount could be added; finally, ddH2O was added to 20 μL, and the mixture was incubated at 37℃ for 30 min. After incubation, it was quickly transferred to ice for 5 min to obtain the recombinant vector.

[0045] 4. Freeze-thaw method to transform E. coli:

[0046] Thaw 50 μL of competent E. coli cells on ice, add 5 μL of ligation product, flick to mix, place on ice for 25 min, heat shock at 42°C for 90 s, and immediately transfer to ice for 3-5 min; add 700 μL of resistance-free LB liquid medium equilibrated to room temperature on a clean bench, and resuscitate at 37°C, 200 rpm for 1 h; centrifuge at 5000 rpm for 1 min to collect bacteria, discard most of the supernatant on the clean bench, and keep about 100 μL for resuspending the bacteria; transfer the bacterial solution to LB solid medium containing the corresponding antibiotic (Kan. or Cmr.) (Kan. antibiotic concentration is 50 mg·L -1 , Cmr. antibiotic concentration was 34 mg·L -1 ) and apply it evenly with an applicator. After it dries, place it upside down in a 37℃ incubator for 12 h.

[0047] Single clones were selected for propagation, and the bacterial solution was shaken at 37℃ and 200 rpm for 2 h before polymerase chain reaction (PCR) detection. The detection primers are shown in Table 2. The upstream test primers of the overexpression vector miRNA vector and eGFP vector were 35S-F and pFGC5941-F, respectively, and the downstream primers were downstream amplification primers of the insert fragment itself. The detection system was: 2×Rapid TaqMaster Mix 12.5 μL, 1 μL of upstream and downstream primers, 1 μL of bacterial solution, and 9.5 μL of ddH2O. The PCR detection program was 98℃ for 3 min, 98℃ for 10 s, 56℃ for 10 s, 72℃ for 15 s, and 72℃ for 2 min, with a total of 35 cycles from denaturation to extension. The PCR products were separated by 1.5% agarose gel electrophoresis, and the length of the target fragment was compared. The bacterial solution with the correct band was sent for verification. After sequencing verification, the plasmid was extracted and stored at -20℃.

[0048] Table 2: Primers used for PCR detection of tobacco transient expression vectors

[0049]

[0050] 5. Tobacco transient expression experiment verifies N-miR172 and target genes BraNPY5 Mutual verification:

[0051] Soak the seeds of Nicotiana benthamiana in water overnight, sow them in the seedling medium (with a high proportion of vermiculite) the next day, cover with plastic wrap and poke holes on the surface for ventilation, and place them in a 25°C environment for germination. After the tobacco sprouts, grow for 2 to 3 days (d), and transplant the seedlings after the cotyledons open. The daily light setting is 4000 lux, and the ambient temperature is 24°C / 16 h, 22°C / 8 h. About 4 weeks after transplanting, the tobacco can be used for transient expression experiments.

[0052] In 15~20 mL of Rif. antibiotics (50 mg·L -1 ) and Kan. Antibiotics (50 mg·L -1 50 μL of the twice activated target bacterial solution was added to the LB liquid medium, cultured at 28°C and 200 rpm for 16-20 h, and centrifuged at 5000 rpm for 15 min in a large desktop low-temperature centrifuge 5810R (Eppendorf, Germany) to collect the bacteria, and resuspended them in a resuspension solution with a concentration of 10 mmol·L -1 MgCl2, 10 mmol·L -1 MES, 100 μmol·L -1 The OD value of the resuspension was measured by UV-6100 spectrophotometer (Shanghai Lingxi, China) and the OD was adjusted. 600=0.8~1.0, keep away from light for 2~3h and then inject tobacco.

[0053] The leaves suitable for injection were marked, and the negative control and experimental group bacterial solutions were injected on the left and right sides respectively. The bacterial solution containing the miRNA precursor and the bacterial solution containing the target gene binding site vector were mixed in a ratio of 4:1 in advance. Six leaves were injected in each group of experiments, and each leaf was injected 2 to 3 times. After 48 hours of dark culture, a small hole was punched at the injection site with an even hole, with the back facing up. The fluorescence signal strength of the control group and the experimental group was observed with an axio Zoom.V16 fluorescence microscope (Carl Zeiss, Germany). The fluorescence protein image is shown in the figure. Figure 2 As shown in (B), it can be observed that the fluorescence signal of tobacco leaves injected with SK-N-miR172 is weakened, and the relative fluorescence value is analyzed using ImageJ software. Figure 2 As shown in (C), the results showed that the fluorescence value of tobacco co-transfected with miR172 overexpression vector was significantly reduced, indicating that N-miR172 can target and bind to BraNPY5 in plants and regulate its expression. The three vectors SK-N-miR172, SK and pFGC-BraNPY5-target and the binding sites of miRNA and target genes are shown in Figure 2 As shown in (A), the SK-miR172 expression vector is as follows Fig.13 As shown in (E) in the figure.

[0054] 6. N-miR172 and target genes in various organs of Chinese cabbage BraNPY5 Relative expression analysis:

[0055] The roots, stems, leaves, normal flowers, nested flowers and fruits of the sequencing samples were respectively taken, and the relative expression levels of N-miR172 and its target genes were detected by real-time fluorescence quantitative reverse transcription polymerase chain reaction (qRT-PCR). The primers used for the detection are shown in Table 3. The results of quantitative PCR are shown in Figure 3 As shown, the relationship between N-miR172 and target genes in various organs of Chinese cabbage is shown. BraNPY5 The relative expression level was Figure 3 It can be seen that N-miR172 and are expressed most highly in leaves, followed by flowers, and are higher in nested flowers than in normal flowers, while less expressed in stems, fruits, and roots. Correspondingly, the target gene and miRNA showed a relatively obvious opposite expression pattern. BraNPY 5 reached more than 20 times in flowers, while the expression level in leaves and stems was lower. This analysis result further supports the relationship between N-miR172 and BraNPY5 targeted relationship.

[0056] Table 3: Primers used for qRT-PCR detection of nested flower samples

[0057]

[0058] Example 2: Construction of N-miR172 overexpression and inhibition expression vector

[0059] 1. Construction of overexpression vector:

[0060] The precursor fragment amplification and vector construction of N-miR172 are shown in Example 1. The overexpression vector is pFGC1008, such as Fig.13 As shown in (A), the restriction site is Sal Ι and Kpn Ι. After adding homology arm primers, the target fragment was amplified and the vector was recombined and then PCR detection was performed. The upstream primer was the vector primer and the downstream primer was Pre-N-miR172-R, as shown in Table 4.

[0061] Table 4: Primers used for N-miR172 overexpression vector

[0062]

[0063] 2. Construction of inhibition expression vector:

[0064] The mature sequence of N-miR172 and its complementary sequence on the stem loop were searched in the sequencing results. Based on this, a STTM sequence was designed. The middle section was a 48 nt fixed sequence, and the two sides had base sequences that were not completely complementary to N-miR172, which could bind to miRNA but would not be cut by RISC. After the design was completed, it was synthesized by Wuhan Boyuan Biotechnology Co., Ltd. and connected to the pBWA(V)HS vector. Fig.13 As shown in (B) in the figure, an inhibitory expression vector of miR172 was constructed and named STTM-N-172. The construction primers and detection primers are shown in Table 5.

[0065] Table 5: Primers used in STTM-N-172 inhibition expression vector

[0066]

[0067] Example 3: Transformation of Arabidopsis thaliana by floral dipping method and screening of positive plants

[0068] 1. Transformation of Arabidopsis thaliana by floral dipping method:

[0069] Remove the fruit pods and opened flowers of wild-type Arabidopsis thaliana one day in advance and water thoroughly. -1 ) and Cmr. (34 mg·L -1200 μL of the twice activated target bacterial solution was added to the LB liquid medium, cultured at 28°C and 200 rpm for 16-20 h, centrifuged at 5000 rpm for 15 min, and resuspended in 5% sucrose solution to OD 600 =0.8, add 20 μL of Silwet L-77 surfactant, mix well, and then put the Arabidopsis buds upside down into the bacterial solution for 1 min. Take them out and wipe them dry, place them flat on the tray, and culture them in the dark for 24 h before returning to normal culture until harvest. To improve the transformation efficiency, repeat the transformation once more after one week.

[0070] 2. Screening of positive plants:

[0071] The pFGC1008 overexpression vector has a hygromycin resistance marker gene, which will make the plant hygromycin resistant when transferred into the plant. The harvested T0 generation seeds need to be sterilized several times before being sown on the culture medium for screening. Sterilization and sowing are completed on a clean bench. The specific screening steps are as follows:

[0072] 2.1. Place T0 generation seeds in 2 ml centrifuge tubes and soak them in ddH2O for 1 min. After centrifugation, use a pipette to remove the water on the upper layer and some seeds of poor quality.

[0073] 2.2. Disinfect the seeds with 75% ethanol once, wash the seeds with ddH2O three times, 1 min each time, and repeat this step three times to fully disinfect the seeds and wash away the alcohol remaining on the surface of the seeds.

[0074] 2.3. After disinfection, spread the seeds evenly on a soil containing 90 mg·L -1 Culture on 1 / 2 MS seeding medium containing hygromycin at 25°C.

[0075] 2.4. Observe the screening results after one week of culture. Arabidopsis thaliana with hygromycin resistance can grow normally upright in the culture medium, while Arabidopsis thaliana without resistance will die 3 to 4 days after germination.

[0076] 2.5. Transplant the positive seedlings that grow well in the culture medium into the substrate and cover them with film for two days until the seedlings adapt to the environment and then remove the film.

[0077] 2.6. 10 days after transplanting, take leaves with normal growth to extract DNA and perform PCR test. The results are as follows: Figure 4 As shown, the seedlings with the target band were identified as positive seedlings. Figure 4 (A) is the PCR detection result of the expression inhibition strain, where lanes 1-5 are STTM-N-172-1~5, lane 6 is the positive control, lane 7 is the wild type, and lane 8 is H2O. Figure 4(B) is the PCR detection result of the overexpression strain, where lanes 1-6 are OE-N-miR172-1~6, lane 7 is the positive control, lane 8 is the wild type, and lane 9 is H2O.

[0078] 3. qRT-PCR analysis of transgenic Arabidopsis:

[0079] This study has investigated the relationship between N-miR172 and target genes BraNPY5 It has been verified that there is BraNPY5 Homologous genes AtNPY5 , predicted by TargetFinder, psRobot and psRNATarget, AtNPY5 is also a target gene of N-miR172. Therefore, the transgenic plants identified as positive were simultaneously subjected to N-miR172 and AtNPY5 Relative expression analysis, the results are as follows Figure 5 As shown. Figure 5 As shown in (A), among the six overexpression lines, the expression of N-miR172 in OE-N-miR172-4, 5, and 6 plants was significantly upregulated compared with the wild type, while there was no significant difference in the other three plants. At the same time, it was also observed that in the three upregulated overexpression lines, the predicted target genes AtNPY5 The expression levels of miR172-4 and OE-N-miR172-4 were down-regulated in the other two strains. AtNPY5 The downregulation of expression is significant or extremely significant. Figure 5 As shown in (B), in the suppressed expression lines, the relative expression of N-miR172 in STTM-N-172-1, 2, and 5 plants was significantly downregulated. Similarly, the target gene was also detected in these three positive plants. AtNPY5 Significant increase in .

[0080] Example 4: Phenotypic observation of N-miR172 transgenic positive plants

[0081] 1. Observation of young leaf growth:

[0082] The positive Arabidopsis plants of the T1 generation were harvested, and the expression level of N-miR172 in the transgenic Arabidopsis of the T1 generation was analyzed. The lines with greater differences were selected to continue screening positive seedlings with 1 / 2 MS medium containing hygromycin, and the T2 generation was propagated to observe the phenotype. It was found that compared with the wild-type WT, the true leaf growth of the suppression expression line corresponding to the suppression expression vector STTM-N-172 was slow, but the overexpression line corresponding to the overexpression vector OE-N-miR172 had no significant difference with the wild type, such as Figure 6As shown in the figure. 10 days after sowing, the wild-type Arabidopsis has grown two true leaves, forming a "cross" shape as a whole, but the true leaves of the suppressed expression strain are smaller and not fully grown, and the plant is in the shape of a "single" shape as a whole. There is no difference between the overexpression strain and the wild type.

[0083] 2. Flowering time statistics:

[0084] The T2 transgenic Arabidopsis thaliana were continuously cultured and observed. When the wild type plants grew to 21 days, they began to bolt first. At 23 days, half of the wild type Arabidopsis plants began to bolt. However, the first bolting plant of the STTM-N-172 transgenic line did not appear until 24 days. Figure 7 As shown in Table 6, the bolting time of more than half of the plants was delayed by 3-4 days compared with the wild type. The bolting time of the wild type, overexpression and inhibition of N-miR172 expression was counted separately. The results showed that the STTM-N-172 transgenic line had a significant flowering delay, as shown in Table 6. The values ​​are expressed as mean ± standard deviation. Each sample counted 24 plants, and each column used a multiple comparison test (p < 0.05).

[0085] Table 6: Flowering time statistics of wild type and transgenic Arabidopsis

[0086]

[0087] 3. Pollen vitality detection:

[0088] Pipette 30 μL of Alexander stain onto a glass slide, pick up a vigorously growing flower of Arabidopsis with tweezers, and dip the anther in the stain several times. To ensure that enough pollen grains are observed, the anther can be removed and placed directly into the stain, and stirred with tweezers. To ensure the reliability of the statistical results, at least 3 strains were observed for each line, and the total number of pollen grains counted was not less than 1000. Vigorous pollen grains appear purple and round in the stain, while pollen grains with poor vitality are shriveled and stained blue by the stain.

[0089] The staining results are as follows Figure 8 As shown in Table 7, the pollen vitality of the wild type and Arabidopsis overexpressing N-miR172 was good, and a large number of purple plump pollen grains were observed in the microscope field, but the pollen vitality of Arabidopsis plants with suppressed expression of N-miR172 was significantly reduced, and a large number of blue and inactive pollen grains were found in the anthers. The proportion of viable pollen grains was photographed and counted, and the results showed that the normal pollen proportion of the wild type was 98.72%, the pollen vitality of Arabidopsis overexpressing N-miR172 was 97.81%, and the normal pollen proportion of Arabidopsis with suppressed expression of N-miR172 was only 46.06%, as shown in Table 7.

[0090] Table 7: Ratio of normal pollen in Arabidopsis thaliana with overexpression and suppression of N-miR172 (%)

[0091]

[0092] 4. Phenotypic observation of strains with high expression of N-miR172:

[0093] When observing the T2 generation of overexpressing transgenic seedlings, it was found that the six positive seedlings of the OE-N-miR172-6 line all showed poor growth, small and numerous leaves, and dwarf plants. Fig. 9 As shown in (A), six positive seedlings of the T2 generation OE-N-miR172-6 strain were mixed and sampled, RNA was extracted and the relative expression level of N-miR172 was determined. The qRT-PCR results showed that compared with the wild type, the N-miR172 content in the T2 generation OE-N-miR172-6 strain was significantly increased, reaching 3 times that of the wild type, as shown in Figure 2. Fig. 9 As shown in (B). When the T2 transgenic plants were cultured until harvest, the number of seeds of the OE-N-miR172-6 line was small and the germination rate was significantly reduced, as shown in Fig.10 As shown, Fig.10 (A) is the Col-0 Arabidopsis thaliana image. Fig.10 (B) is the OE-N-miR172-5 strain map. Fig.10 (C) is the STTM-N-172-1 strain map. Fig.10 (D) is the OE-N-miR172-6 strain map. Fig.10 As shown in the results, overexpression of N-miR172 to 3 times of the wild type resulted in poor development of Arabidopsis plants, dwarfing of plants and decreased seed vigor.

[0094] Example 5: Target Gene BraNPY5 Analysis of spatiotemporal expression patterns

[0095] 1. Subcellular localization analysis:

[0096] The vector used for subcellular localization was pFGC5941-eGFP, which has been constructed in Example 1. BraNPY5 The subcellular localization vector of the protein, tobacco and bacterial solution were prepared in the same way as in Example 1. Select tobacco leaves that are growing well and are relatively flat, and use a needle to pierce holes on the back of the leaves away from the veins. Mix the marker with the empty vector and the corresponding gene bacterial solution in a ratio of 1:1 in advance, and use a 1 mL syringe to draw an appropriate amount of the mixed bacterial solution, inject it into the tobacco from the wound, and culture it in a dark environment for 48 h. Use scissors to cut off a 1 cm thick section of the needle hole. 2 The leaves of different sizes were placed with the back facing upwards, and the distribution of fluorescence signals was observed using an Axio Zoom.V16 fluorescence microscope. The results showed that BraNPY5 Positioned on the membrane, such as Fig.11 shown.

[0097] 2. Construction of promoter-GUS fusion expression vector:

[0098] The promoter and GUS fusion expression vector used was the pCAMBIA1300-GUS vector stored in the laboratory, and the restriction endonucleases used in the upstream and downstream were Hin d ΙΙΙ and XB I, such as Fig.13 As shown in (C), the linearized vector was purified using a PCR product recovery kit after 1 h of restriction digestion. The promoter was amplified using cabbage DNA as a template, and the gene sequence was queried from NCBI (https: / / www.ncbi.nlm.nih.gov / ) and selected BraNPY5 The sequence of about 2000 bp upstream of the gene was used as the promoter of the target gene, and the activity of the selected promoter sequence was detected by the PlantCare database (http: / / bioinformatics.psb.ugent.be / webtools / plantcare / html / ). Amplification primers were designed according to the promoter and vector restriction site sequences. After homology and grouping, the vector upstream primer and the fragment downstream primer were used as PCR detection primers as shown in Table 8. After sequencing verification, the pBraNPY5-GUS plasmid was obtained, as shown in Table 8. Fig.13 As shown in (D) in the figure.

[0099] Table 8: Primers used for promoter-GUS fusion expression vector

[0100]

[0101] 3. Histochemical staining and observation:

[0102] The pBraNPY5-GUS plasmid was transformed into Arabidopsis. After the positive seedlings were screened, the roots, stems, leaves, flowers, and fruits of the positive Arabidopsis plants were picked with tweezers and placed in 2 mL centrifuge tubes. Freshly prepared GUS staining solution was added until the materials were completely immersed. The materials were stained at 37°C in the dark for 12 h, and decolorized with 75% ethanol for 1-3 h. The 75% ethanol was replaced every hour. After complete decolorization, the staining of each part was observed with a DVM6 ultra-depth fluorescence microscope (Leica, Germany) and a BX53 microscope (OLYMPUS, Japan). The staining results are shown in Figure 2. Fig.12 As shown, the staining results of sepals are as follows Fig.12 As shown in (A) in the figure, the dyeing result of the petals is as follows Fig.12 As shown in (B), the staining results of the stamens are as follows Fig.12 As shown in (C), the staining results of the pistil are as follows Fig.12 As shown in (D), the staining results of the open flowers are as follows Fig.12 As shown in (E), the root staining results are as follows Fig.12 As shown in (F), the staining results of the stem are as follows Fig.12 As shown in (G), the staining results of the leaves are as follows Fig.12 As shown in (H) in the figure, the staining results of the silique are as follows Fig.12 As shown in (I) in . Fig.12 The color results shown show that BraNPY5 The promoter of the gene can activate downstream GUS Gene expression, among which the root has the strongest activation activity, and all parts of the root including the root hairs have strong GUS In addition, the expression activity in leaves, petals, sepals, and stamens is high, while the expression activity in stems is very low, with only a small amount at the base. GUS There is strong activation activity in the stigma, middle and base of the pistil, but when it develops into a silique, only the top and base have activation activity.

[0103] The above are preferred specific embodiments of the present invention. These examples are intended to better explain the principles and practical applications of the present invention so that those skilled in the art can better understand and utilize the present invention. However, these embodiments do not describe all the details in detail, and some improvements or modifications can be made on the basis of them, which is obvious to any person skilled in the art. Therefore, these modifications and improvements made on the basis of the present invention all belong to the scope of protection claimed by the present invention.

Claims

1. A precursor gene N-miR172 of a gene related to cauliflower development, characterized in that: The nucleotide sequence of the precursor gene N-miR172 is shown in SEQ ID No.

1.

2. A biological material containing the precursor gene N-miR172 of the cabbage flower development-related gene according to claim 1, characterized in that: The biological material is an expression vector, an expression box or an engineered bacterium.

3. A use of the precursor gene N-miR172 according to claim 1 in regulating the developmental function of Arabidopsis thaliana after expression or inhibition of expression, characterized in that: After transformation with the overexpression vector of the precursor gene N-miR172, Arabidopsis thaliana grows slowly and its development is blocked; or After transformation with the inhibitory expression vector of the precursor gene N-miR172, the young leaves of the plant developed abnormally, flowering was delayed, and pollen vitality was reduced.

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