CN116732047A - Application of gene OsMADS5 in regulating plant root elongation - Google Patents

Abstract

The present invention relates to the field of plant growth and development molecular biology, and in particular to the application of gene OsMADS5 in regulating plant root elongation. The gene OsMADS5 has the nucleotide sequence shown in SEQ ID NO: 1. The present invention clones the gene OsMADS5, constructs a transgenic vector, obtains overexpression and knockout transgenic materials, measures the phenotypic traits of the transgenic materials, and obtains significant changes in the length of the root system in different materials. Compared with the wild type, the root system of the knockout transgenic materials The length increased significantly, and the root length of the overexpression material became shorter, indicating that the OsMADS5 gene plays an important regulatory function in the process of plant root elongation.

Description

Application of gene OsMADS5 in regulating plant root elongation

Technical Field

The present invention relates to the field of molecular biology of plant growth and development, and in particular to application of gene OsMADS5 in regulating plant root elongation.

Background Art

MADS is a large and widely distributed gene family. Studies have shown that the presence of this family of genes has been found in plants (monocots, dicots, gymnosperms, mosses, algae), animals, fungi (yeast) and other species, and it is an important type of transcriptional regulatory factor. The functions of the transcription factor MADS-box family of genes are mainly related to cell differentiation and its related developmental processes. Among them, the most studied is the role of this family of genes in the formation and development of floral organs. In addition, this family of genes plays an important role in root nutrient absorption, leaf development, vernalization, flowering time, meristem differentiation, fruit ripening, embryo development, etc. At present, about 75 MADS-box transcription factors have been found in rice, and only a few of them have been identified. At present, these identified genes are mainly involved in controlling and regulating development, including tillering, flower development, nitrate nitrogen utilization, root development, drought stress, etc. Therefore, this family of genes plays a very important regulatory role in the growth and development of rice. The gene functions of the MADS family in rice need to be further discovered.

Summary of the invention

Rice OsMADS5 encodes 226 amino acids with a molecular weight of 25.9933KD. After cloning the OsMADS5 gene and connecting it to the CRISPR-Cas9 vector to construct knockout materials, it was found that the length of the rice root system increased, and after connecting the overexpression vector to obtain overexpression plants, it was found that the length of the rice root system was significantly shortened, indicating that the OsMADS5 gene plays an important regulatory function in the process of rice root elongation.

The first aspect of the present invention provides the following technical solutions:

The invention discloses an application of the gene OsMADS5 in regulating the elongation of rice root system. The gene OsMADS5 has a nucleotide sequence shown in SEQ ID NO: 1.

The present invention clones the gene OsMADS5, constructs a transgenic vector, obtains overexpression and knockout transgenic materials, identifies the phenotypic traits of the transgenic materials, and obtains the changes in root length in different materials. Compared with wild rice materials, the knockout transgenic materials show an increase in root length, and the overexpression materials show a shortened root length, indicating that the OsMADS5 gene plays an important regulatory function in the growth and development of rice roots.

The gene OsMADS5 involved in the present invention has a significant regulatory effect on plant root elongation, indicating that the gene OsMADS5 is closely related to plant root elongation. Therefore, in practical applications, the purpose of changing the length of plant roots can be achieved by changing the expression level of the OsMADS5 gene.

The second aspect of the present invention provides the following technical solutions:

The method for detecting the root elongation performance of a plant is to detect the expression of the gene OsMADS5 of the sample to be detected to determine its root elongation performance.

In the present invention, the gene controlling rice root elongation and its application can make the gene OsMADS5 as a molecular marker for plant root elongation, that is, by detecting whether the plant has the expression of the gene OsMADS5, the root elongation of the plant can be relatively described, thereby providing good technical support for plant breeding.

In the present invention, the plants include monocots and dicots;

The monocotyledonous plants include rice, corn, and wheat;

The dicotyledonous plants include soybean, cotton and tobacco.

Among them, the present invention can detect whether the sample to be detected contains the gene OsMADS5 in a variety of ways, such as directly detecting whether it contains the gene OsMADS5 itself, or detecting the product produced by the gene OsMADS5, the product includes a direct product or an indirect product or a secondary product, etc. The product can be a gene, a protein, or a certain compound, etc.

The gene OsMADS5 can be directly detected by using a specific primer pair of the gene OsMADS5, or by using a probe or chip designed for the gene OsMADS5. Furthermore, the sample to be detected is detected by using a primer pair, a probe or a chip of the gene OsMADS5.

The primer pair, probe or chip for the gene OsMADS5 involved in the present invention can be designed according to conventional methods.

Furthermore, the nucleic acid sequences of the primer pair are shown in SEQ ID NO.2 and SEQ ID NO.3.

However, the method for detecting the OsMADS5 gene itself is not limited thereto, and any method that can be realized in molecular biology is within the protection scope of the present invention.

Similarly, the detection of the product produced by the gene OsMADS5 can also be carried out by various means, such as various ELISA detection kits.

Furthermore, the sample to be detected includes materials suitable for tissue culture of sexually propagated, asexually propagated or regenerative cells.

The samples to be detected are seminal roots and adventitious roots.

Adventitious roots develop from the embryo at the base of plant stems. The occurrence and elongation of adventitious roots expand the number and surface area of the plant's root system. The growth and development of adventitious roots are regulated by genetic factors and plant hormones.

These samples to be tested can be materials suitable for sexual reproduction, such as pollen, ovary, ovule, embryo sac, etc.;

Materials suitable for asexual reproduction may be selected from roots, stems, cuttings, protoplasts, etc.;

Materials suitable for tissue culture of regenerable cells may be selected from leaves, pollen, meristem cells, roots, root tips, seeds, embryos, cotyledons, hypocotyls and stems.

Specifically, the sample to be detected includes any one of the following materials: leaves, roots, stems, radicles, embryos, and seeds.

Among them, plants include monocotyledons and dicotyledons; for example, monocotyledons include rice, corn, and wheat; dicotyledons include soybeans, cotton, and tobacco.

The third aspect of the present invention also provides the application of the gene OsMADS5 in the study of genetic diversity of plant populations.

Among them, plants include monocotyledons and dicotyledons; for example, monocotyledons include rice, corn, and wheat; dicotyledons include soybeans, cotton, and tobacco.

Compared with the prior art, the beneficial effects of the present invention include at least the following aspects:

(1) The present invention constructs knockout materials and overexpression materials of the OsMADS5 gene, and studies have found that the knockout transgenic materials show an increase in root length, while overexpression of the OsMADS5 gene has a significant inhibitory effect on root length.

(2) The OsMADS5 gene provided by the present invention can be applied to plant root elongation performance, and the plants involved include rice, corn, wheat, soybean, cotton, tobacco, etc.

BRIEF DESCRIPTION OF THE DRAWINGS

In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings required to be used in the description of the embodiments or the prior art are briefly introduced below.

FIG1 is a diagram showing the molecular detection results of OsMADS5 gene overexpression materials in Example 1 of the present invention;

FIG2 is a schematic diagram of molecular detection of OsMADS5 gene knockout materials in Example 2 of the present invention;

FIG3 is a root phenotype diagram of OsMADS5 knockout materials and wild-type materials in an embodiment of the present invention;

FIG4 is a root phenotype diagram of OsMADS5 overexpression materials and wild-type materials in an embodiment of the present invention;

DETAILED DESCRIPTION

The embodiments of the present invention will be described in detail below in conjunction with the examples, but it will be appreciated by those skilled in the art that the following examples are only used to illustrate the present invention and should not be construed as limiting the scope of the present invention. If specific conditions are not specified in the examples, they are carried out under normal conditions or conditions recommended by the manufacturer. If the manufacturer is not specified for the reagents or instruments used, they are all conventional products that can be purchased commercially.

Example 1

The acquisition of transgenic plants comprises the following steps:

1) Extraction of total RNA

Rice seeds were sterilized with 30% NaClO, germinated, and cultured to two leaves and one heart. Rice plants of uniform size were selected, and the endosperm was removed and transplanted to 1/2 International Rice Research Institute IRRI nutrient solution with pH 5.5. When the seeds had four leaves and one heart, the solution was changed to the International Rice Research Institute IRRI complete nutrient solution (Mao D R. The methods of plant nutrition research. Beijing: Beijing Agricultural University Press, 1994). After one week of culture, the roots and leaves were quickly frozen in liquid nitrogen for preservation. About 0.1 g of the sample was weighed and crushed with liquid nitrogen. After grinding, it was added to a 1.5 ml centrifuge tube, 1 ml of Trizol reagent was quickly added, 0.2 ml of chloroform was added, and the supernatant was aspirated after centrifugation. 0.5 ml of isopropanol was added, and the supernatant was discarded after centrifugation. 70% ethanol was added to wash the precipitate. RNA was dissolved in DEPC water (volume ratio was 1‰), and the RNA quality was detected by agarose gel electrophoresis with a mass ratio of 1.0%, and the concentration and purity of the total RNA were detected by a spectrophotometer. Proceed to the next step after passing.

2) Total cDNA synthesis

To each RNA sample of 2 μg, 50 μmol·L ^-1 Oligo dT18 was added, and 1‰ DEPC water was added to make up to 10 μL. The mixture was incubated in a water bath at 70°C for 5 min. After being placed on ice for 5 min, 0.5 μL of RNase inhibitor and 5 μL of 5xRT buffer, 2.5 μL of 10 mM dNTPs, 1 μL of M-MLV reverse transcriptase, and 1‰ DEPC water were added to make up to 25 μL. The mixture was incubated in a water bath at 42°C for 60 min, and then incubated in a water bath at 70°C for 10 min to terminate the reaction (Oligo dT18 was synthesized by Nanjing GenScript Co., Ltd.; the reverse transcription kit was purchased from Fermentas, Canada).

3) Obtaining the full-length cDNA of OsMADS5 gene

Using the total cDNA of rice Zhonghua 11 obtained above as a template, PCR primers were designed, and the PCR product contained the complete OsMADS5 reading frame (from the start codon ATG to TAG). The primer sequences were:

OsMADS5-F:5’-ATGGGGCGAGGGAAAGTAGA-3’;

OsMADS5-R:5’-TCATTGGTTGAGGTGATC CATGT-3’

The PCR program is as follows: 95℃ pre-denaturation for 3min, 95℃ denaturation for 30s, 56℃ annealing for 45s, 72℃ renaturation extension for 2min, 35 cycles, 72℃ for 7min, the amplified PCR product was detected by 1% agarose gel electrophoresis, and its size was 678bp fragment. The target PCR product was separated by agarose electrophoresis and then cut and recovered. The recovered fragment was connected to the P-easy blunt vector. The total volume of the enzyme connection system was 5μL (including 1μL vector and 4μL PCR purified product). After adding the sample, mix evenly, centrifuge to the bottom of the tube, and place at 28℃ for 15min;

The enzyme-linked system was heat-shocked at 42°C into Escherichia coli DH5α competent cells, and 500-700 μL of LB liquid culture medium without antibiotics was added and shaken for 1 hour, then centrifuged at low speed to enrich the bacteria, which were spread on LB solid culture medium containing 100 μg·mL ^-1 of kanamycin and grown for 12h-14h, and positive colonies were picked for DNA sequencing. The accession number of the OsMADS5 gene is AK064184, and the full length of the open reading frame (ORF) of OsMADS5 is 678bp; the correctly sequenced bacterial solution was added with an equal volume of 50% glycerol and stored at -70°C for later use, and the P vector containing the open reading frame of OsMADS5 was named pOsMADS5inP.

4) Construction of overexpression vector pUbi-OsMADS5

According to the cDNA sequence of the rice auxin transport protein gene OsMADS5, PCR primers were designed, and the PCR product contained the complete OsMADS5 gene reading frame (from the start codon ATG to the stop codon TAG), and restriction endonuclease sites KpnI and SpeI were introduced into the upstream and downstream primers, respectively. The primer sequences were:

overOsMADS5-F:5’-CGGGATCCATGGGGCGAGGGAAAGTAGAG-3’BamHI

overOsMADS5-R:5’-CGGGGTACCTCATTGGTTGAGGTGATCCATGTAA-3’KpnI

Using the pOsMADS5inP plasmid obtained above as a template, the PCR program was as follows: pre-denaturation at 95°C for 3 min, denaturation at 95°C for 30 s, annealing at 56°C for 45 s, renaturation and extension at 72°C for 2 min, 35 cycles, and 72°C for 7 min. The amplified PCR product was detected by 1% agarose gel electrophoresis, and the PCR product size was approximately 678 bp. The target PCR product was separated by agarose electrophoresis and then cut and recovered by gel cutting. The recovered product was digested and recovered by restriction endonucleases BamHI and KpnI. At the same time, the plant overexpression vector pTCK303 plasmid was double-digested with BamHI and KpnI, and then the digested PCR fragments and vectors were recovered respectively. The vector was dephosphorylated and recovered again. After recovery, the linearized vector and the digested PCR fragments were connected with T4 ligase at 16°C overnight, transformed into Escherichia coli DH5a competent cells, and spread on LB solid culture medium containing kanamycin 50μg·mL ^-1. After growing for 12h, positive colonies were picked, and the plasmid was extracted. After the fragment size was verified by BamHI and KpnI digestion, the bacterial solution was subjected to DNA sequencing, and the bacterial solution containing the correctly sequenced clones was added with an equal volume of 50% glycerol and stored at -70°C. The extracted positive clone plasmid was named pUbi-OsMADS5;

Finally, the pUbi-OsMADS5 plasmid was transformed into competent cells of Agrobacterium tumefaciens EHA105 by electroporation, and then grown on YEP solid medium containing 50 μg·mL ^-1 of kanamycin and streptomycin for 48 h. Positive colonies were picked and the plasmids were extracted. After double enzyme digestion with BamHI and KpnI, the bacterial solution was added with an equal volume of 50% glycerol and stored at -70°C for future use in transgenic production.

5) Obtaining transgenic plants

In order to avoid plant cytoplasmic gene mutations caused by the transgenic process, we conducted different batches of transgenic experiments. From July to October 2020 and from December 2020 to February 2021, the Agrobacterium obtained above with the 35S-OsMADS5 plasmid was used to infect rice callus tissue and cultured for 3 days. After selective culture, differentiation, rooting and seedling hardening of resistant callus tissue, different batches of T0 transgenic plants were obtained in different years. In order to avoid the change of plant traits due to cytoplasmic chimeras caused by non-genomic insertions, we carried out two expansions on all transgenic materials, obtained the stable inherited T2 generation and performed physiological tests on the stable inherited T2 generation materials.

The specific preparation of transgenic plants is as follows:

5.1) Agrobacterium-mediated rice transformation

Inducing callus: Peeled rice seeds (14 seeds per plate) were placed in a triangular bottle, soaked in 70% ethanol for 1 min (to submerge the seeds), the 70% ethanol was discarded, and the seeds were washed 5-6 times with sterile water, soaked in 30% sodium hypochlorite for 30 min, and then washed 5-6 times with sterile water until clear. The seeds were moved onto sterilized filter paper with tweezers, and the water was absorbed. Finally, the seeds were placed on the induction medium and cultured in a 32°C light incubator for 5 days.

Preparation of Agrobacterium: Agrobacterium EHA105 strain with corresponding vector was streaked on AB medium (50 mg/L Kan) and cultured in the dark at 28°C for 3 days. Agrobacterium colonies were scraped off with a sterile spoon and suspended in AAM culture medium (containing As) with an OD600 of about 0.1.

Infection of callus and co-cultivation: Pick out rice callus from the subculture medium and place it in a centrifuge tube. The number of callus should not exceed the conical part of the 50ml centrifuge tube (select light yellow, round and tough callus). Take 1ml of the cultured bacterial solution in a 1.5ml centrifuge tube, centrifuge at 4℃, 5000rpm for 1 min, and remove the supernatant. Use 30ml of infection solution containing 200μmo1·L ^-1 acetosyringone (As) to make a suspension of the collected bacteria. Pour this suspension into the selected callus and infect for 5min. Pour out the liquid, take out the callus, and place it on a sterile culture dish containing absorbent paper to drain for 30-40min. Place the callus on the co-cultivation medium (with a layer of 9cm sterile filter paper on it) and culture it in the dark at 25℃ for 3 days.

Bacteria washing and antibiotic screening culture: Remove the callus from the co-culture medium and wash it with sterile water 5 times, shaking it for 5 minutes each time. Then soak it in sterile water containing 500mg·L ^-1 carbenicillin (car) for 40-60 minutes. Finally, place it on sterile filter paper to drain for 2 hours. The first round of screening: Transfer the dried callus to the selection medium containing 400mg·L ^-1 carbenicillin (car) and 50mg·L ^-1 hygromycin (Hyg) for the first selection, and culture it at 32°C under light for two weeks;

Second round of screening: The vigorously growing calli were transferred to differentiation medium containing 50 mg/L hygromycin B and 250 mg/L carboxybenzyl to induce differentiation, and kept in continuous light at 28°C for about two weeks.

Induced differentiation and rooting of resistant callus: Pick the bright yellow resistant callus and transfer it into the differentiation pot containing differentiation medium, put it in a constant temperature culture room, wait for differentiation into seedlings (about 30 days, the culture conditions in the tissue culture room are 24-30℃, 14h light/8h dark), and when the seedlings grow to about 5cm, put them into the rooting medium to strengthen the seedlings.

Hardening and transplanting of transgenic seedlings: Pick out the test tubes with relatively complete differentiation of seedling roots and stems and leaves (when the seedlings grow to the top of the test tube, open the lid in time), open the sealing film, add appropriate amount of sterile water (to prevent bacteria from growing in the culture medium), harden the seedlings for about 3 to 7 days, then wash off the agar and transplant them to the greenhouse for hydroponic or soil culture growth and testing.

5.2) Rapid detection of transgenic seedlings using hygromycin to obtain T0 generation plants

Cut and collect fresh green leaves of about 1 cm long from the seedlings to be tested (with cuts at both ends), place them flat on a medium containing hygromycin (80 mg·L ^-1 ), and culture them at 30°C, 16h/8h (light/dark) for 48h. If the leaves remain bright green, they are positive plants, while the leaves of negative seedlings show block necrosis (Zheng Ye. Establishment and Application of Rice Efficient Transgenic System. 2008). 60 strains of positive T0 plants were obtained by hygromycin screening. From April to November 2021, the overexpression materials were planted to obtain T0 generation seeds.

5.3) Molecular identification of OsMADS5 overexpression lines

After germination of T0 generation seeds, T1 generation transgenic seedlings were obtained. At the beginning of tillering of flower 11 in transgenic material OsMADS5-OE and wild type material, RNA was extracted from roots, and qRT-PCR was performed after reverse transcription. Quantitative PCR identification was performed. The results are shown in Figure 2, and stable genetic transgenic lines OE-2, OE-14, and OE-16 were obtained.

Example 2

Obtaining OsMADS5 knockout plants:

1) Select the target:

According to the NCBI OsMADS5 gene sequence, a target site was designed at the first exon of the gene.

Target:GGATCGAGAACAAGATAAGC

2) Construction of intermediate vector

2.1 Intermediate vector primer synthesis

Cas9-OsMADS5-F:GGCAGGATCGAGAACAAGATAAGC

Cas9-OsMADS5-R:AAACGCTTATCTTGTTCTCGATCC

Primers were denatured and annealed to obtain gRNA fragments. The PCR reaction system was as follows: 5 μL of forward and reverse primers, 40 μL of water was added to make up to 40 μL. The PCR reaction procedure was as follows: denaturation at 95°C for 10 min, annealing at 55°C for 10 min, and cooling at 14°C for 5 min.

2.2 Enzyme ligation to construct knockout vector and obtain transgenic material

The AarI-Cas9-PC1300 vector was digested with AarⅠ endonuclease at 37℃ overnight. The digestion system was 10μL of vector plasmid, 2μL of AarⅠ, 5μL of 10×Buffer, 1μL of 50×oligonucleotide (0.025mmol·L-1), and ddH ₂ O was added to 50μL. The digestion product was recovered and purified, and eluted with 50μL ddH ₂ O.

Store at -20℃ until use.

The gDNA fragment of OsMADS5 was connected to the AarI-Cas9-PC1300 vector. The connection system was T4

DNA Ligase 1μL, 5×Rapid Buffer 2μL, annealed product fragment 6μL and AarI-Cas9-PC1300 vector 1μL, incubate at 22℃ for 30min.

The ligation system was transformed into Escherichia coli DH5α competent cells, positive colonies were picked, and plasmids were extracted for DNA sequencing. The plasmids with correct sequencing were named Cas9-OsMADS5. Then Agrobacterium was transformed, and the positive Agrobacterium was used to infect rice callus to obtain the gene knockout material of OsMADS5. The transformation steps were the same as in Example 1.

3) Identification of gene knockout materials

The roots of transgenic materials and wild-type materials at the seedling stage of 11 were taken and DNA was extracted. Then forward and reverse primers were designed according to the two ends of the target site (target: GGATCGAGAACAAGATAAGC).

Cas9-OsMADS5-F:AACAAACGGAGCAAGACTGCAAGGG

Cas9-OsMADS5-R:ACACAACAAAGCAGCAGGGAAAAG

The PCR amplification products were subjected to Sanger sequencing, and the sequencing results were compared with those of the Nipponbare amplification products to determine whether the gene knockout was successful.

The test results are shown in Figure 1.

In order to avoid the possibility that changes in plant traits were caused by cytoplasmic chimeras resulting from non-genomic insertions, all T0 generation transgenic knockout plants were propagated twice to obtain the stably inherited T2 generation, and physiological tests were performed on the stably inherited T2 generation materials.

Test example

1. After sterilization, the flowers of T2 transgenic materials and wild-type materials were germinated at 28℃ for 1 day, then cultured at 28℃ for 5 days with 16h light and 8h dark, and then the root phenotypes of the seedling stage were counted using a root scanner. Each strain was replicated 8 times. The results are shown in Figures 3 and 4.

As can be seen from Figure 3, compared with the wild type (Zhonghua 11, ZH11), the root length of the T2 generation knockout materials (cas9-1, cas9-2, cas9-3) increased significantly. As can be seen from Figure 4, the root length of the T2 generation OsMADS5 gene overexpression materials (OE-2, OE-14, OE-16) was significantly shortened.

In summary, the OsMADS5 gene has a significant effect on root elongation.

In the present invention, the reagents and solutions involved are as follows:

1. Induction medium

pH 5.8, autoclave at 115℃ for 20 min.

2. Co-culture medium

pH 5.2, autoclave at 115°C for 20 min.

3. Choose the culture medium

pH 5.8, autoclave at 115℃ for 20 min.

4. Differentiation medium

pH 5.8, autoclave at 115℃ for 20 min.

5. Rooting medium

pH 5.8, autoclave at 115℃ for 20 min.

6. AAM culture medium

pH 5.2, autoclave at 115°C for 20 min.

7. AB medium

pH 7.2, autoclave at 115°C for 20 min.

8. Culture medium mother solution formula:

N ₆ macroelements (20X)

Dissolve the above reagents one by one, then make up to volume with distilled water at room temperature, label the preparer and preparation date, and store at 4℃.

N ₆ trace elements (1000X)

Dissolve the above reagents at room temperature and make up to volume with distilled water, label the preparer and date, and store at 4°C.

N ₆ organic matter (100X)

Add distilled water to make up to volume, label the preparer and date, and store at 4°C for no more than 1 month.

MS macroelements (20X)

Dissolve the above reagents one by one, then make up to volume with distilled water at room temperature, label the preparer and preparation date, and store at 4℃.

MS trace elements (1000X)

Dissolve the above reagents at room temperature and make up to volume with distilled water, label the preparer and date, and store at 4°C.

MS organic matter (100X)

Add distilled water to make up to volume, label the preparer and date, and store at 4°C for no more than 1 month.

Among them, iron salt (100X): dissolve 3.73g of disodium ethylenediaminetetraacetate (Na ₂ EDTA·2H ₂ O) and 2.78g of FeSO ₄ ·7H ₂ O separately, mix and use. Add distilled water to 1000ml, warm it at 70℃ for 2 hours, mark the preparer and preparation date after cooling, and store it at 4℃.

50mg/ml Myo-Inositol: Add 5g of inositol to 100ml of distilled water, label with the concentration, preparer and preparation date, and store at 4℃.

5mg/ml copper sulfate (CuSO ₄ ·5H ₂ O): Add 0.5g CuSO ₄ ·5H ₂ O to 100ml, label the concentration, preparer and preparation date, and store at 4℃.

5mg/ml cobalt chloride (CoCl ₂ ·6H ₂ O): Add 0.5g CoCl ₂ ·6H ₂ O to 100ml, label the concentration, preparer and preparation date, and store at 4℃.

2,4-D (1mg/ml): Place 100mg 2,4-D in a 100ml beaker, add 20ml water first, then add 3ml 1NKOH. After it is completely dissolved, add water to make up to 100ml. Mark the concentration, preparer and preparation date, and store at 4℃.

KT (1mg/ml): Place 100mg Kinetin (abbreviated as KT) in a 100ml beaker, add 20ml water first, then add 5ml 1N HCl, add water to make up to 100ml after complete dissolution, mark the concentration, preparer and preparation date, and store in aliquots at -20℃.

NAA (1mg/ml): Place 100mg NAA in a 100ml beaker, add 20ml water first, then add 3ml 1N KOH. After it is completely dissolved, add water to make up to 100ml. Mark the concentration, preparer and preparation date, and store at 4℃.

1N KOH: Dissolve 5.6g KOH in 100ml water, label the concentration, preparer and preparation date, and store at room temperature.

1N NaOH: Dissolve 4g NaOH in 100ml water, label the concentration, preparer and preparation date, and store at room temperature.

1N HCl: Add 12.5 ml of concentrated hydrochloric acid to 100 ml with water, label the concentration, preparer and preparation date, and store at room temperature.

Kan (50 mg/ml): Dissolve Kanamycin (Kan for short) in sterile water, 50 mg/ml, filter and sterilize, label with concentration, preparer and preparation date, and store at -20℃.

Rif (50 mg/ml): Rifampicin (Rif) was prepared into a 50 mg/ml stock solution with DMSO. The concentration, preparer and preparation date were marked and stored at -20°C.

Cb (500 mg/ml): Dissolve 1 g of carbenicillin in 2 ml of sterile water in a clean bench, filter and sterilize, label the concentration, preparer and preparation date, and store at -20°C.

AS (100 mM): 0.196 g AS was dissolved in 10 ml DMSO, divided into 1 ml tubes, marked with concentration, preparer and preparation date, and stored at -20°C.

The English abbreviations used in the culture medium of the present invention are as follows: Cb (Carbenicillin); NAA (Napthalene acetic acid); 2,4-D (2,4-Dichlorophenoxyacetic acid); AS (Acetosringone); CH (Casein Enzymatic Hydrolysate); L-pro (L-proline); L-Glu (L-glutamine); MES (2-(N-Morpholino)EthaneSulfonic Acid); N6 (N6 macroelement component solution); B5 (B5 trace element component solution).

Although the present invention has been illustrated and described with specific embodiments, it will be appreciated that many other changes and modifications may be made without departing from the spirit and scope of the present invention. Therefore, it is intended to include all such changes and modifications within the scope of the present invention in the appended claims.

Claims (10)

1. Application of the gene OsMADS5 in regulating plant root elongation, characterized in that: the gene OsMADS5 has the nucleotide sequence shown in SEQ ID NO: 1. 2. Application according to claim 1, characterized in that the root system is elongated to the length of the root system. 3. The application according to claim 1, characterized in that the knockout material of the gene OsMADS5 promotes rice plant elongation, while the overexpression material inhibits plant root elongation. 4. The application according to any one of claims 1 to 3, characterized in that the plants include monocots and dicots. 5. A method for detecting plant root elongation performance, which is characterized by detecting the expression of the gene OsMADS5 of the sample to be detected. 6. The method for detecting plant root elongation performance according to claim 5, characterized in that the sample to be detected is detected through a primer pair or probe or chip of the gene OsMADS5. 7. The method for detecting root elongation according to claim 6, wherein the nucleic acid sequence of the primer pair is as shown in SEQ ID NO. 2 and SEQ ID NO. 3. 8. The method for detecting plant root elongation performance according to any one of claims 5-7, characterized in that the sample to be detected includes tissue culture suitable for sexual reproduction, asexual reproduction or regenerative cells. Material. 9. The method for detecting plant root elongation performance according to claim 8, characterized in that the samples to be detected are seed roots and adventitious roots. 10. Application of gene OsMADS5 in the study of genetic diversity of plant populations.