CN119082123A

CN119082123A - Application of a tree peony PsPYL4 gene in regulating root development

Info

Publication number: CN119082123A
Application number: CN202411244532.XA
Authority: CN
Inventors: 文书生; 崔慧颖; 苗大鹏; 杨玉洁; 齐兆凤; 李爱民; 李胜皓; 顾亚男
Original assignee: Nanjing Forestry University
Current assignee: Nanjing Forestry University
Priority date: 2024-09-06
Filing date: 2024-09-06
Publication date: 2024-12-06

Abstract

The invention discloses a peony PsPYL gene and application thereof in regulating root system development, wherein the nucleotide sequence of the PsPYL gene is shown as SEQ ID NO.1, and the amino acid sequence of an expressed protein is shown as SEQ ID NO. 2. The gene sequence of PsPYL4 is cloned from peony cultivar noon, expression analysis is carried out in peony, then the peony cultivar is constructed into an over-expression vector to be introduced into target plants to verify the functions, and compared with wild plant seedlings, the arabidopsis seedlings over-expressing PsPYL genes are found that the growth speed of PsPYL over-expression lines is faster, rosette leaves are larger, the length of main roots is obviously shortened, the number of lateral roots is obviously increased, the content of endogenous hormone IAA, ABA, ZR, GAs in the leaves is increased, the average number of isolated leaves is obviously increased on an MS culture medium containing IAA, the average root length is obviously increased on an MS culture medium containing IBA, so that the gene has wide application in cultivation and genetic improvement of peony and other horticultural plants.

Description

Application of peony PsPYL gene in regulation and control of root system development

Technical Field

The invention belongs to the technical field of plant genetic engineering, and particularly relates to application of peony PsPYL4 genes in regulating root system development.

Background

Peony (Paeoniasect. Moutan) is a peony (Paeonia) fallen leaf sub-shrub of Paeoniaceae (Paeoniaceae) Paeonia, which has rich flower color and graceful flower shape, and is a unique rare plant of China integrating ornamental, medicinal, edible and oil application. The peony is mainly planted, separated and grafted in the traditional way, but has the defects of low propagation coefficient, long period and the like. In recent years, the micropropagation technology can be used for mass propagation of the peony in a short period, but the problems of difficult occurrence of adventitious roots of test tube seedlings, low rooting quality and the like still exist. Therefore, research on molecular mechanisms of genes acting on plant root system development has important significance for breeding, production and application of peony.

Previous studies have found that endogenous abscisic acid (ABA) has an inhibitory effect on adventitious root formation, and that endogenous auxin (IAA/ABA) values can be used as a measure of plant rooting capacity. The abscisic acid receptor protein PYL, which is a core member in the ABA signal transduction pathway, is a major component of ABA signal perception. There are 14 ABA receptors in Arabidopsis, including PYR1, PYL1-13, and all PYR/PYL (except PYL 13) are capable of activating the expression of the ABA response gene, where PYL8 plays a role in auxin-mediated lateral root growth by interacting with MYB77, MYB44 and MYB73, the lateral root growth of the seedling of the PYL8 mutant is more sensitive to ABA, the ability of the PYL8-1PYL9 double mutant to inhibit primary root growth and lateral root formation by exogenous ABA is reduced by four times compared to PYL8, and the inhibition of lateral root growth by ABA on PYL1/2/4 mutant is reduced by four times, whereas PYL9 controls the expression of the auxin response gene in the lateral root primordia by directly regulating the transcriptional activity of MYB77 and MYB44, independent of ABA signaling. The above studies indicate that PYL not only regulates adventitious root formation through an abscisic acid signal transduction pathway, but also participates in important transcription factors regulating auxin pathways to affect adventitious root formation.

In conclusion, PYL can regulate the growth and development of root systems and plant morphogenesis in other plant species, but the gene has not been reported in peony. Therefore, by utilizing the genetic engineering technology, psPYL gene cloned from peony is transferred into a model plant, which has important significance for researching the functions of the plant and has great application prospect.

Disclosure of Invention

Aiming at the defects in the prior art, the invention aims to provide a peony PsPYL gene. Another object of the present invention is to provide the application of peony PsPYL gene in plant breeding.

In order to achieve the above object, the present invention adopts the following technical scheme:

a peony PsPYL gene has a nucleotide sequence shown in SEQ ID NO. 1.

The amino acid sequence of the expression protein of the peony PsPYL gene is shown as SEQ ID NO. 2.

The peony PsPYL gene is applied to plant growth and development.

Namely, the application of peony PsPYL4 in changing the growth of leaves of Arabidopsis thaliana 'Columbia' seedlings, the application in promoting the vertical rooting of Arabidopsis thaliana 'Columbia' seedlings and the application in changing the endogenous hormone content of Arabidopsis thaliana 'Columbia' leaves.

And (3) connecting the peony PsPYL gene to a vector, carrying out agrobacterium-mediated transformation to wild arabidopsis thaliana 'Columbia', screening, and culturing to obtain a transgenic plant.

Compared with the prior art, the invention has the beneficial effects that through cloning and identification of the peony PsPYL4 gene, expression analysis and genetic transformation of the gene, the function of the gene is verified, the arabidopsis seedling over-expressing the PsPYL4 gene is found to have the advantages that compared with wild type plant seedlings, the growth speed of the PsPYL over-expressed plant line is higher, the rosette leaves are larger, the length of main roots is obviously shortened, the number of lateral roots is obviously increased, the content of endogenous hormone IAA, ABA, ZR, GAs in the leaves is increased, the average number of in vitro leaves is obviously increased on an MS culture medium containing IAA, the average root length is obviously increased on an MS culture medium containing IBA, and the gene can be widely applied to generation and propagation breeding of adventitious roots of peony.

Drawings

FIG. 1A is a bacterial detection electrophoresis chart of peony PsPYL4 gene clone, wherein M is DL2000 Marker, the length of a target strip is 642bp, and B is a PsPYL4 over-expression vector double enzyme digestion verification electrophoresis chart;

FIG. 2 is PsPYL showing the expression of PsPYL4 during the rooting phase of peony;

FIG. 3 is a schematic diagram of the structure of an overexpression vector cloned and constructed by peony PsPYL gene;

FIG. 4A is a graph showing the PCR results of transgenic Arabidopsis plants, wherein M represents DL2000 Marker, WT uses wild DNA, (-) uses ddH ₂ 0 as a template and 1-10 uses the DNA template of the transgenic plant as a negative control, B is the relative expression level of the transgenic Arabidopsis plant transgenic lines, and note that WT is wild Arabidopsis and OE is the transgenic line;

FIG. 5 is a comparison of an overexpressed PsPYL4 gene plant with a wild type Arabidopsis plant, wherein A is a seedling comparison, B is a plant stem extraction comparison, C is a plant maturity comparison, if not specifically labeled, scale is 1cm, WT is wild type Arabidopsis, OE is a transgenic strain;

FIG. 6 is a comparison of root growth of an over-expressed PsPYL gene plant with a wild type Arabidopsis plant, and a statistical plot of main root and lateral root, where WT is wild type Arabidopsis and OE is a transgenic line;

FIG. 7 is a comparison of endogenous hormone levels in leaves of an overexpressed PsPYL gene plant versus a wild type Arabidopsis plant, where WT is the wild type Arabidopsis;

FIG. 8 is a rooting index comparison of plants overexpressing PsPYL gene under IAA treatment with wild type Arabidopsis plants;

FIG. 9 is a rooting index comparison of plants overexpressing PsPYL gene under IBA treatment with wild type Arabidopsis plants.

Detailed Description

The invention will be further illustrated with reference to specific examples.

Example 1

The material used in this example is peony 'noon' rhizome tissue, which is quick frozen in liquid nitrogen after harvesting, and stored in an ultra-low temperature refrigerator (-80 ℃).

1) Extraction of total RNA of all tissues of peony

The method is carried out according to the instruction of an OMEGA plant RNA small extraction kit, and comprises the following specific operations:

The ultralow temperature frozen peony tissues are rapidly transferred into a mortar precooled by liquid nitrogen, the tissues are ground by a pestle, liquid nitrogen is continuously added until the tissues are respectively ground into powder, the ground powder samples are respectively added into 1.5 mL sterilized tube containing 5000 μl RB Buffer, and the powder samples are uniformly mixed by shaking until no obvious precipitate exists in the lysate and then transferred into a 2mL collecting tube containing GDNA FILTER Column, and the lysate is centrifuged for 5 minutes at 14,000 rpm and 4 ℃. The filtrate was carefully pipetted into a fresh 1.5 mL sterilized Tube, the volume was measured, 1/2 volume absolute ethanol was added, the Vortex was shaken for 20 minutes and transferred to 2ml Collection Tube,12,000 rpm,4 ℃containing Hibind RNA Mini Column for centrifugation for 1 minute, 400. Mu.L of RWF Buffer was added to Hibind RNA Mini Column, centrifuged at 10,000 rpm for 30 seconds, the filtrate and Collection Tube were discarded, hibind RNA Mini Column was placed in fresh 2mL Collection Tube, 500. Mu.L of RNA WashBufferII was added to Hibind RNA Mini Column, centrifuged at 10,000 rpm for 30 seconds, the filtrate was discarded, 500. Mu.L of RNA WashBuffer II was added to Hibind RNA Mini Column, centrifuged at 10,000 rpm for 30 seconds, the filtrate was discarded, hibind RNA Mini Column was relocated to 2mL Collection Tube, centrifuged at 2 minutes at maximum speed, the filtrate and Collection Tube were discarded, hibind RNA Mini Column was placed on RNASE FREE Collecttube of 1.5 mL, 65℃and 50. Mu.L of DEPC WATER were added at the center of Hibind RNA Mini Column membrane, and left standing for 5 minutes at room temperature, and RNA was eluted at maximum speed for 1 minute. The obtained RNA is stored in a refrigerator at-80 ℃ for standby after concentration and purity detection.

The result of sucking 2 mu L of RNA and detecting by using 1% agarose gel electrophoresis shows that 28S and 18S bands are clear, the brightness of the 28S band is about twice of that of the 18S band, and the quality of the RNA is good. RNA purity is detected by a trace nucleic acid protein tester, and OD ₂₆₀/OD₂₈₀ and OD ₂₆₀/OD₂₃₀ are both 1.8-2.1, so that the integrity is good, and the RNA can be used for reverse transcription.

2) Synthesis of first strand cDNA

And (3) performing reverse transcription according to a root reverse transcription kit by using the obtained total RNA as a template. The specific operation is as follows:

Template RNA and 5X Fastking-RT Supermix were thawed on ice, and RNase-Free ddH ₂ O was quickly placed on ice after thawing at room temperature. The reagents were vortexed and centrifuged briefly before use. A Total of 20. Mu.l of the reaction system, including 5X Fastking-RT Supermix 4. Mu.l, total RNA 800ng, was prepared in a centrifuge tube, the remaining volume was made up with RNase-Free ddH ₂ O and the whole process was performed on ice. After centrifuging the centrifuge tube, putting the centrifuge tube into a PCR instrument, removing genome at 42 ℃ for 15min, performing reverse transcription reaction, inactivating enzyme at 95 ℃ for 3min, and placing on ice to obtain cDNA solution.

3) Design and cloning of target gene primer

According to the existing peony transcriptome sequencing result, designing peony PsPYL gene amplification primers by using CE Design, wherein the primer sequences are as follows:

PsPYL4-F:

5’-acgggggactctagaggatccATGTTTTCAAATCCTCCAAAATCA-3’

PsPYL4-R:

5’-ataagggactgaccacccgggTCATGAGTTGTTGCGGTTTCG-3’

cloning of peony PsPYL gene was performed using PRIMERSTAR MAX high-fidelity enzyme of Takara using cDNA as a template. 50 μl of PCR reaction solution was prepared, including PRIMERSTAR MAX μl, forward Primer 2.5 μl, REVERSE PRIMER 2.5.5 μl, templete 2.5 μl, the remainder was made up with ddH ₂ O, and centrifuged well. The PCR reaction conditions were 94℃pre-denaturation for 3min,98℃denaturation for 10s,60℃annealing for 10s,72℃extension for 30s,32 cycles, 72℃total extension for 5min,16℃incubation.

After the PCR reaction is completed, all PCR products are taken and detected by 1.8% agarose gel electrophoresis, the correct bands are cut, and the PCR target amplification products are purified and recovered. The DNA gel recovery kit of TransGen is adopted to purify and recover the target fragment, and the specific operation is as follows:

Cutting the correct strips from agarose gel, weighing in a clean centrifuge tube, adding 3 times volume of solution GSB (if the gel weight is 0.1g, the volume can be regarded as 100 mu L, 300 mu L of GSB solution is added), turning the centrifuge tube up and down every 2-3 minutes during the water bath at 55 ℃, until the gel is completely melted, adding 1 time volume of isopropanol when the gel solution is cooled to room temperature, uniformly mixing, adding the mixed solution into a centrifuge column, standing for 1min, centrifuging at 12000rpm for 1min, discarding waste liquid, adding 650 mu L of solution WB, centrifuging at 12000rpm for 1min, discarding waste liquid, centrifuging at 12000rpm for 2min, removing residual WB as far as possible, placing the adsorption column in a cover for 5min at room temperature, drying, placing the centrifuge column in a clean centrifuge tube, suspending and dropwise adding 30 mu L of ddH ₂ O at 60-70 ℃ above the adsorption film, standing for 2min at room temperature, centrifuging at 12000rpm for 2min, and eluting DNA. Taking 2 mu L of the recovered and purified product, performing gel electrophoresis detection by using 1.5% agarose, and placing the rest in a refrigerator at-20 ℃ for subsequent connection with a pBI121 vector to construct an overexpression vector.

4) Extraction of plasmids:

extracting plasmids according to the specification of a small and medium-amount kit of the Tiangen plasmids, wherein the specific steps are as follows:

Adding 20mL of the shaken bacterial solution into a centrifuge tube, centrifuging for 10min at 8000 rpm, and pouring out the supernatant; adding 500 mu L P1 solution (containing RNase A) into a centrifuge tube with bacterial sediment, fully sucking and mixing uniformly, transferring to a new 1.5mL centrifuge tube, adding 500 mu L P solution into the centrifuge tube, gently turning up and down for 6-8 times to enable bacterial sediment to be fully cracked, continuously adding 700 mu L P solution into the centrifuge tube, immediately turning up and down for 6-8 times, fully mixing uniformly, wherein white flocculent sediment appears, centrifuging at 12000rpm for 10min, adding 500 mu L balance liquid BL into an adsorption column CP4, centrifuging at 12000rpm for 1min, discarding waste liquid, transferring supernatant collected in the previous step into a filter column CS, centrifuging at 12000rpm for 2min, carefully adding the solution in the collection tube into the adsorption column CP4 in batches, centrifuging at 12000rpm for 1min, discarding waste liquid, placing the adsorption column CP4 into the collection tube, adding 500 mu L deproteinized liquid PD into the adsorption column CP4, immediately turning up and down for 1min, centrifuging at 12000rpm, placing the adsorption column CP4 into the collection tube again, rinsing at 12000rpm for 1min, removing the residual liquid in the centrifuge tube, transferring to the filter column CP to 12000 min, removing the residual liquid from the filter column, transferring to the filter column CP to the filter column, and transferring the solution to the filter column after the filter column is placed at 12000rpm for 1min, and the time when the filter column CP is not filled with water, and the filter column is left with 1min, and the filter column is left for 1min, and the filter column is placed, and the filter column is left for 1min, and the filter is left for 1, and the filter. The plasmid concentration was determined and prepared for the next experiment.

5) Double enzyme digestion reaction

The extracted pBI121 plasmid is digested with BamHI and SmaI at 37deg.C for 30min, and subjected to gel electrophoresis detection, and the linear vector is recovered by purification, and stored at-20deg.C for use. The double cleavage reaction system was 50. Mu.L of pBI121 plasmid 20. Mu.L, 5 Xbuffer 5. Mu.L, bamHI 1. Mu.L, smaI 1. Mu.L, ddH ₂ O23. Mu.L.

6) Recombination reactions

The agarose gel electrophoresis detects the purity and concentration of the target gene and the vector pBI121 which are recovered by purification after enzyme digestion. The ligation reaction was performed using 3. Mu.L of the insert, 7. Mu.L of the linearized pBI121 vector, 4. Mu.L of 5 XCE II buffer, exnase II. Mu.L, and ddH ₂ O Up to 20. Mu.L. The reaction was carried out at 37℃for 30min, left at normal temperature (without immediate cooling), and transformed to E.coli competence after 10 min.

7) Transfer of ligation products into E.coli

Competent cell Trans5α strain (stored at-80 ℃) was thawed on ice. Adding 10 μl of recombinant product into 100 μl of competent cells, placing the centrifuge tube on ice for 10min, 42 ℃ metal bath, heat shock 90 s, immediately placing on ice for 2 min, adding 500 μl of antibiotic-free LB liquid medium in a super clean bench, placing on a 37 ℃ and 200 rpm shaker for 25min for resuscitation, centrifuging 6000 rpm for 1 min, sucking 350 μl of supernatant, suspending the precipitated strain, coating on LB solid medium plate (Kan concentration is 50 mg/L), and culturing at 37deg.C overnight.

8) Identification of recombinants

Single colonies on the plates were picked up and backed up to LB liquid medium containing antibiotics (Kan) and shake cultured overnight at 37℃with 200 rpm ℃. Bacterial liquid PCR is performed by using the full-length primers of the target genes to screen positive clones, and the bacterial detection result is shown in figure 1A. The positive clones after screening were sent to Nanjing qingke company for sequencing. Positive clones with correct sequencing results are amplified, plasmids are extracted by using a root plasmid extraction kit and subjected to double enzyme digestion verification, whether the sizes of fragments after enzyme digestion are consistent is judged, and the enzyme digestion results are shown in figure 1B.

According to analysis of a sequencing result, finally determining and cloning to obtain 1 peony PsPYL4 coding genes, namely PsPYL genes, wherein the nucleotide sequence of the coding genes is shown as SEQ ID NO.1, the coding length of the PsPYL genes is 1642bp, the coding genes contain an ATG start codon and a TAA stop codon, and 213 amino acids are coded, and the amino acid sequence is shown as SEQ ID NO. 2.

Example 2

Designing a fluorescent quantitative primer by taking the cloned peony PsPYL4 gene as a reference, wherein the primer sequence is as follows:

qPsPYL4-F:5’-ACGCCCCTCCCCTATAACAT-3’

qPsPYL4-R:5’-AGGCTTGAGGGTTGTCGAAG-3’

meanwhile, UBIQUITIN is used as an internal reference gene, and the primer sequence is as follows:

UBIQUITIN-F:5’-GACCTATACCAAGCCGAAG-3’

UBIQUITIN-R:5’-CGTTCCAGCACCACAATC-3’

The preparation of the reaction solution was carried out using instructions of TB Green ^® Premix Ex TaqTM kit (TAKARA company) and run on a Applied Biosystems real-time fluorescence quantitative analyzer with PCR procedures of 95℃5min, 95℃10s,60℃30s, 40 cycles, 95℃15s,60℃1min,95℃15s. After the reaction is finished, an amplification curve is obtained, data is derived through StepOne Software v 2.3.3, excel is used for data analysis, a relative expression quantity is calculated according to a CT value by a relative quantification method of 2 ^-ΔΔCt, and the data analysis result is shown in figure 2.

The research results of the embodiment show that PsPYL gene is expressed in the process of adventitious root generation of peony, the expression quantity is firstly down-regulated and then up-regulated in the dedifferentiation period, the expression quantity is firstly down-regulated in the induction period, then the expression quantity reaches the highest value in 25d after the continuous down-regulation in the differentiation period, and then the expression is down-regulated to the lowest value in the end 30d of root induction. Demonstrating the important role of this gene in adventitious root development (FIG. 2).

Example 3

1) Preparation and transformation of Agrobacterium competent cells

The infection experiment of arabidopsis thaliana is carried out by utilizing the competence of agrobacterium GV3101, wherein recombinant agrobacterium GV3101 is transformed by ice bath, agrobacterium competent cells are melted, 1000ng of recovered and purified plasmids are added into 100 mu L of agrobacterium competence, the mixture is gently mixed, ice bath 5min, liquid nitrogen is quickly frozen for 5min, heat shock 5min is carried out in 37 ℃ metal bath, the mixture is quickly placed in ice bath 5min, 800 mu L of LB culture medium without any antibiotics is added, 28 ℃ is carried out, 200 rpm is resuscitated for 2h, 4000 rpm is centrifuged for 3 min, 350 mu L of supernatant is absorbed, residual bacterial liquid is fully absorbed and uniformly mixed, and then the mixture is coated on LB solid culture medium added with 50 mg/L kanamycin and 200 mg/L of rifampicin, and the mixture is subjected to inversion culture at 28 ℃ for 30-48 h.

The identification of agrobacterium recombinants includes picking single colony from plate culture medium, inoculating to liquid culture medium containing corresponding antibiotic, culturing at 28 deg.c and 200 rpm deg.c overnight, PCR, detecting the PCR product with 1.5% agarose gel electrophoresis, identifying whether to contain target segment, adding proper amount of sterile 50% glycerin into the positive clone, and storing at-80 deg.c for use.

2) Agrobacterium-mediated transformation of Arabidopsis thaliana

The inflorescence infection method is adopted to transfer the target gene into the arabidopsis thaliana, and the specific operation method comprises the steps of keeping the healthy growth state of the arabidopsis thaliana (Columbia type) until the arabidopsis thaliana flowers, and activating the agrobacterium GV3101 strain carrying the target gene. Picking single colony, inoculating in 50mL LB culture solution containing kanamycin and rifampicin, shaking at 28deg.C and 200 rpm until the bacterial solution just becomes turbid, about 8-10 h, sucking 1mL bacterial solution, inoculating in a triangular flask (50 mL) shaking 24 h until OD value is about 0.8-1.0, centrifuging the bacterial solution 6000 rpm at room temperature for 10 min, removing supernatant, suspending with 3% sucrose solution with pH=5.8, adding Silwet L-77 with concentration of 0.03% (300 μl/L) before soaking, mixing thoroughly, soaking aerial parts of Arabidopsis in agrobacterium suspension solution for 1min, slightly shaking, sealing the soaked Arabidopsis with tinfoil paper, standing for 24 h in dark place, uncovering tinfoil paper, and culturing under normal condition.

The 3% sucrose solution heavy suspension comprises MS culture medium, sucrose 30g/L, silwet-77. Mu.l/L. (note: pH was adjusted to 5.8 after preparation, bacterial liquid was centrifuged and resuspended, silwet L-77 was added, and the conversion relationship between the resuspension and bacterial liquid was such that the amount of resuspension was calculated as bacterial liquid OD. Bacterial liquid volume=0.8. Resuspension).

3) Screening of transgenic plants

The collected T0 generation transgenic arabidopsis seeds are sterilized by alcohol and sodium hypochlorite, and the steps are that a proper amount of obtained transgenic seeds are placed in a 1.5mL centrifuge tube, soaked in 10% NaClO and alcohol mixed solution (the proportion is 1:1 in terms of volume ratio) for 5 minutes, sterilized by 75% alcohol for 5-6 times, each time is 2 min, washed by sterile water for 3-4 times, and suspended by sterile water.

Sterilized transgenic Arabidopsis seeds were sown on 1/2MS solid medium containing antibiotics (kanamycin 50 mg/L), wrapped with tinfoil and placed in a 4℃refrigerator for vernalization. After 2 days, the medium was taken out of the refrigerator and placed at 22℃for light cultivation. After about one week, the arabidopsis thaliana which can grow normally on the culture medium is transplanted into soil for continuous growth.

4) DNA detection of transgenic plants

A proper amount of T1 generation Arabidopsis and tender leaves of transgenic plants are detected by adopting a plant DNA kit of AG Ai Kerui biological company, and the specific operation steps are that 0.1mg of plant sample is weighed and ground by liquid nitrogen, then 500 uL Buffer LS-3 and 10 uL 50 xDTT Buffer are rapidly added into the ground sample powder, 10 uL RNase A is added into the mixture, the mixture is fully and uniformly mixed by shaking, a centrifuge tube is placed in a 56 ℃ water bath kettle, heating is carried out for 10min (the period can be reversed and uniformly mixed), and 1/8 volume Buffer PA of lysate is added into the mixture and fully and uniformly mixed. Placing on ice for 5min at 12,000 rpm, centrifuging at room temperature for 5min, adding Buffer BS-2 with the same volume as the supernatant, mixing thoroughly, transferring the above solution to PLANT DNA MINI Column (the volume of the solution passing through the Column is usually not more than 750 uL twice, each time), standing at room temperature for 1min, centrifuging at 12,000 rpm for 1min, discarding the filtrate, adding Buffer WA 500 uL at 12,000 rpm for 1min, discarding the filtrate, adding Buffer WB 750 uL (63 ml of absolute ethyl alcohol has been added in advance) to the Mini Column for 1min, discarding the filtrate, repeating the operation once, centrifuging at 12,000 rpm for 2min on new 2 ml Collection Tube, placing Mini Column on new 1.5 ml, adding 50 uL O at the center of Mini Column membrane for 65 ° CddH ₂ min, standing at room temperature for rpm, centrifuging at room temperature for 53000 min, eluting DNA. PCR was performed using the above DNA, and the results are shown in FIG. 4A.

5) Fluorescent quantitative PCR detection of transgenic plants

Total RNA was extracted from young stem and leaf of the above-mentioned over-expressed peony PsPYL4 gene Arabidopsis, and the reverse transcription and fluorescence quantitative primers, method and procedure were the same as in example 2, and the final data analysis results are shown in FIG. 4B.

6) Obtaining of transgenic homozygous lines

Sterilizing the harvested transgenic T1 generation seeds, screening and culturing, transplanting the transgenic T1 generation seeds into nutrient soil, culturing at 22 ℃ in 16 h light/8 h in the dark, reserving the initially confirmed transgenic plants after detection, harvesting the T1 generation seeds after maturation, numbering to obtain the T2 generation seeds, sterilizing the T2 generation seeds as the T1 generation seeds, coating the T2 generation seeds on a screening culture medium containing antibiotics, placing the T2 generation seeds at 22 ℃ for continuous light, culturing the T2 generation seeds with different numbers in the dark according to 22 ℃ and 16 h light/8 h in the nutrient soil after about 10 days, selecting plants with the survival proportion of 75% for transplanting, carrying out positive detection on leaves, continuing numbering the positive T2 generation plants, collecting the seeds to obtain the T3 generation seeds, screening the T2 generation seeds with the screening culture medium after sterilization, continuously culturing the T2 generation seeds under light, observing the T3 generation plants with different numbers for about 10 days, and completely surviving and not appearing separated into T3 generation homozygous plants.

7) Phenotypic observation

And the transgenic plant with obvious phenotype is selected for observation, and compared with wild arabidopsis, the transgenic arabidopsis plant has the advantages of higher growth speed, larger rosette leaves, obviously shortened main root length and obviously increased lateral root number.

Sowing T3 generation transgenes and wild arabidopsis thaliana on a 1/2MS culture medium containing kan, transferring positive seedlings to the 1/2MS culture medium for vertical culture after 5d of culture, observing 7d and recording root growth change, and finding that the length of main roots of the transgenosis plant is obviously shortened and the number of lateral roots is obviously increased compared with the wild type.

The endogenous hormone content of the arabidopsis leaves is measured by an enzyme-linked immunosorbent assay (ELISA). The sample was triturated in 10mL of an 80% (v/v) methanol extraction medium containing 1mM Butyl Hydroxy Toluene (BHT) as an antioxidant. The extract was incubated for 4 h at 4℃and centrifuged at 4000 rpm for 15 min. The supernatant was passed through a C-18 column and washed successively with 80% (v/v) methanol, 100% (w/v) ether, and 100% (w/v) methanol. The hormone fractions were then dried under N2 and dissolved in Phosphate Buffered Saline (PBS) containing 0.1% (v/v) Tween 20 and 0.1% (w/v) gelatin for analysis. Monoclonal antigens and antibodies against IAA, ABA, GAs (GA 1+ GA 3), meJA and BR in ELISA kits were produced by the plant hormone institute of the university of agriculture, china. Enzyme-linked immunosorbent assay was performed using 96-well microtiter plates. Each well was coated with 100. Mu.L of coating buffer (1.5 g/L Na ₂CO₃、2.93 g/L NaHCO₃、0.02 g/L NaN₃) containing 0.25. Mu.g/mL of anti-hormone antigen, and incubated at 37℃for 30min. After washing 4 times with PBS containing 0.1% (v/v) Tween 20, each well was filled with 50. Mu.L of sample extract and 50. Mu.L of 20. Mu.g/mL antibody, incubated and washed as described above. 100. Mu.L of a color developing solution containing 1.5 mg/mL of 0-phenylenediamine and 0.008% (v/v) H ₂O₂ was added to each well. Each well 12 mol/L H ₂SO₄ stopped the reaction. ELISA (model EL310, bio-TEK, winioski, VT) 490 nm was used for color development. Hormone content was calculated according to Weiler et al (1981). 3 biological replicates were set for each hormone. As a result, the endogenous hormones IAA, ABA and ZR in the leaves of the transgenic plants are found to increase in the content of GAs.

In this example, the recombinant plasmid of the overexpressed peony PsPYL gene was transferred into the model plant Arabidopsis thaliana, and phenotypic observation and analysis were performed. From the results, it can be seen that the arabidopsis seedling over-expressing PsPYL4 gene has a higher growth rate of PsPYL over-expressed strain, a larger rosette leaf, a significantly shortened main root length, a significantly increased lateral root number, a significantly increased endogenous hormone IAA, ABA, ZR, GAs content in the leaf, a significantly increased average root number of the isolated leaf on an IAA-containing MS medium, and a significantly increased average root length on an IBA-containing MS medium, and the gene is seen to have an effect on plant growth, adventitious root formation and hormonal level change.

Claims

1. Application of peony PsPYL4 in changing the leaves of Arabidopsis thaliana 'Columbia' seedlings, the nucleotide sequence of which is shown in SEQ ID NO.1; comprising: connecting the peony PsPYL4 gene to a vector, transforming it into wild-type Arabidopsis thaliana 'Columbia' through Agrobacterium-mediated transformation, screening, culturing, and obtaining transgenic plants.

2. Application of peony PsPYL4 in changing the endogenous hormone content of Arabidopsis thaliana 'Columbia' leaves, the nucleotide sequence of which is shown in SEQ ID NO. 1; comprising: connecting the peony PsPYL4 gene to a vector, transforming it into wild-type Arabidopsis thaliana 'Columbia' through Agrobacterium-mediated transformation, screening, culturing, and obtaining transgenic plants.

3. Application of peony PsPYL4 in promoting vertical rooting of Arabidopsis thaliana 'Columbia', whose nucleotide sequence is shown in SEQ ID NO. 1; comprising: connecting the peony PsPYL4 gene to a vector, transforming it into wild-type Arabidopsis thaliana 'Columbia' through Agrobacterium-mediated transformation, screening, culturing, and obtaining transgenic plants.