CN116769797B

CN116769797B - Application of methyl jasmonate and PpyMYC2 gene in germination

Info

Publication number: CN116769797B
Application number: CN202311014167.9A
Authority: CN
Inventors: 滕元文; 王旭旭; 白松龄; 王培辉; 史宝靖
Original assignee: Hainan Research Institute Of Zhejiang University
Current assignee: Hainan Research Institute Of Zhejiang University
Priority date: 2023-08-14
Filing date: 2023-08-14
Publication date: 2023-10-24
Anticipated expiration: 2043-08-14
Also published as: CN116769797A

Abstract

The invention discloses methyl jasmonatePpyMYC2The application of the gene in germination can promote the dormancy of the dormant buds of the crisp pears to be released in advance when the cold energy is insufficient by methyl jasmonate (MeJA) treatment.PpyMYC2The gene is a gene responding to methyl jasmonate in crisp pear, cloned by a facultative primer, and transferred into arabidopsis thaliana by using a transgenic technology based on a strong promoter driving principle to obtain transgenic arabidopsis thaliana, and the Overexpression (OE) is foundPpyMYC2The gene can improve the germination rate of the arabidopsis seeds. And the virus-mediated transgenic technology is utilized to transfer the over-expression vector of the gene into pear buds to find out the over-expressionPpyMYC2The gene can promote the germination of dormant pear buds when the cold quantity is insufficient, and promote gibberellin biosynthesis in pear budsGA20OXsThe expression of the gene has important significance for the cultivation of pear trees in high-temperature areas in the south and the rapid breeding of the south propagation.

Description

Application of methyl jasmonate and PpyMYC2 gene in germination

Technical Field

The invention belongs to the technical field of biology, and particularly relates to application of methyl jasmonate and PpyMYC2 genes in germination.

Background

The pear belongs to a temperate deciduous fruit tree, and is used for forming winter with excessive cold sleep buds. Dormancy can improve the survival rate of deciduous fruit trees under harsh environment, and the low temperature is the main factor that induces the pear to get into dormancy, and dormancy is relieved and is needed certain low temperature accumulation, and the low temperature accumulation is not enough can influence the bud in fruit tree spring and then influence the flowering phase of fruit tree, so can normally remove dormancy and will directly influence the body productivity of fruit tree and the nutrition growth of fruit in the second year. The "warm winter effect" caused by global warming nowadays has started to affect the release of dormancy due to insufficient winter accumulation of deciduous fruit trees in the shadow areas.

Several studies have shown that Jasmonic Acid (JA) is widely involved in plant resistance to various biotic and abiotic stresses. The jasmonic acid treatment can promote germination of dormant apple seeds, which indicates that jasmonic acid can also participate in the regulation of dormancy. MYC2 is a bHLH-type transcription factor that responds to jasmonic acid signals, mediating crosstalk between the jasmonic acid, abscisic acid (ABA) and Gibberellin (GA) signaling pathways. Gibberellin is intimately involved in the release of shoot dormancy. Recently, MYC2 has been found to mediate jasmonic acid/gibberellin crosstalk by regulating tomato steroid alkaloid production and gibberellin catabolic genes.

Disclosure of Invention

The invention aims at overcoming the defects of the prior art and provides an application of methyl jasmonate and PpyMYC2 genes in promoting pear bud dormancy release.

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

an application of methyl jasmonate in promoting dormancy of pear buds is provided.

Further, specifically:

spraying methyl jasmonate solution on the surface of pear bud to promote the release of pear bud dormancy.

The application of PpyMYC2 gene in promoting pear bud dormancy release is that the nucleotide sequence of the PpyMYC2 gene is shown as SEQ ID NO.1, and the amino acid sequence is shown as SEQ ID NO. 2; the PpyMYC2 gene can respond to methyl jasmonate MeJA treatment, so that the dormancy of pear buds is promoted.

Further, specifically: in pear buds, virus-mediated transient transformation is utilized to carry out overexpression on PpyMYC2 genes, so that pear bud dormancy is promoted to be relieved.

Further, the overexpression of the PpyMYC2 gene in pear buds by using virus-mediated transient transformation is specifically as follows: and injecting the infection liquid containing the PpyMYC2 gene overexpression plasmid into branches containing pear dormant buds to enable the PpyMYC2 gene to be overexpressed.

Further, the vector of the PpyMYC2 gene over-expression plasmid is an IL-60-BS virus vector.

The application of PpyMYC2 gene in promoting the germination and growth of Arabidopsis thaliana seeds is provided, and the nucleotide sequence of the PpyMYC2 gene is shown as SEQ ID NO. 1.

Further, in arabidopsis, the overexpression vector of the PpyMYC2 gene is transferred into arabidopsis by using a transgenic technology based on the driving principle of a strong promoter cauliflower mosaic virus promoter (35 s), so that a PpyMYC2 arabidopsis transgenic plant is obtained. Experiments prove that the germination rate of the transgenic arabidopsis seeds is higher under the condition of no low-temperature stratification, the transgenic arabidopsis seedlings have longer hypocotyls and roots, and meanwhile, the activity of GA20OX enzyme in the transgenic arabidopsis seedlings is higher than that of a control. The method comprises the following steps:

and immersing the arabidopsis inflorescence into an invasion solution containing the PpyMYC2 gene over-expression plasmid for transfection, continuously screening the transfected arabidopsis into a successful generation to obtain generation homozygote, and collecting and obtaining transgenic arabidopsis seeds.

The invention provides application of methyl jasmonate in promoting release of dormancy of pear buds, and simultaneously clones a complete coding section of a methyl jasmonate response PpyMYC2 gene from pear buds through a plant genetic engineering technology, verifies the function of the gene, and after the fact that the PpyMYC2 gene is over-expressed, the germination rate of arabidopsis seeds is increased without low-temperature lamination, and the germination rate of pear buds in the dormancy stage is obviously increased when the cooling capacity is insufficient. Overexpression of PpyMYC2 in Arabidopsis thaliana promotes germination of Arabidopsis thaliana seeds that have not been stratified at low temperature, and increases GA20OX enzyme activity in Arabidopsis thaliana seedlings. Transient overexpression of PpyMYC2 in pear buds can promote expression of gibberellin biosynthesis enzyme genes (GA 20 OXs), so that pear buds are released from dormancy when low-temperature accumulation is insufficient. The research and exploration of the effect of the PpyMYC2 gene in pear dormancy removal are applied to pear tree cultivation in a high-temperature region in the south and pear south propagation breeding, so that a method for promoting pear buds to be able to normally remove dormancy under the condition of insufficient cold required is more concise and efficient, and the method has practical significance in the field of dormancy research.

Drawings

FIG. 1 is a graph showing the results of the germination rate of dormant pear sprouts and the expression level of PpyMYC2 gene in pear sprouts in a clear water control group, a positive control group and an experimental group, wherein A in FIG. 1 is clear water control group (Mock), a positive control group (GA ₄₊₇ (100 [ mu ] M)) and an experimental group (MeJA (5 [ mu ] M)) are used for processing germination rate statistical graphs of dormant pear buds, and B in FIG. 1 is a PpyMYC2 gene expression quantity statistical graph of the dormant pear buds processed by a clear water control group, a positive control group and the experimental group;

FIG. 2 is an electropherogram of PCR amplified products of pear buds;

FIG. 3 is a graph of gene expression and germination rate results for Arabidopsis seeds OE#4 and OE#5 ectopically expressing PpyMYC2 and a control blank; wherein A in FIG. 3 is a bar graph of the expression level of the PpyMYC2 gene detected by qRT-PCR, B in FIG. 3 is a line graph of the germination rate over time, and WT in the graph represents a blank control, which is an empty vector;

FIG. 4 is a graph showing the effect of ectopic expression of the PpyMYC2 gene on the hypocotyl and root length of Arabidopsis thaliana; wherein A in FIG. 4 is a photograph of Arabidopsis seeds OE#4 and OE#5 ectopically expressing PpyMYC2 and a blank control, B in FIG. 4 is a statistical plot of hypocotyl lengths of Arabidopsis seeds OE#4 and OE#5 ectopically expressing PpyMYC2 and a blank control, and C in FIG. 4 is a statistical plot of root lengths of Arabidopsis seeds OE#4 and OE#5 ectopically expressing PpyMYC2 and a blank control;

FIG. 5 is a statistical graph of GA20OX enzyme activity of Arabidopsis seeds OE#4 and OE#5 ectopically expressing PpyMYC2 and a blank;

FIG. 6 is a graph showing the results of gene expression and germination rates of transgenic PpyMYC2-OE and control IL60-2 pear buds; wherein A in FIG. 6 is a bar chart of the expression level of the PpyMYC2 gene detected by qRT-PCR, and B in FIG. 6 is a bar chart of the germination rate of pear buds;

FIG. 7 shows PpyGA20OXs Gene expression and GA in transgenic PpyMYC2-OE and control IL60-2 pear bud ₃ Content statistics wherein A in FIG. 6 is a histogram of the expression level of PpyGA20OX1L1 gene, B in FIG. 6 is a histogram of the expression level of PpyGA20OX2L2 gene, and C in FIG. 6 is GA ₃ Content bar graph.

Detailed Description

The following description of the embodiments of the present invention will be made clearly and completely, and it is apparent that the described embodiments are only some embodiments of the present invention, but not all embodiments. All other embodiments, which can be made by those skilled in the art based on the embodiments of the invention without making any inventive effort, are intended to be within the scope of the invention.

Example 1: treatment of pear bud with methyl jasmonate (MeJA)

1. MeJA treatment affects pear bud germination

And before dormancy in pear buds is released in 12 months and 15 days, branches of Dangshan pear with full annual flower buds are taken from a Dangshan pear plantation and are divided into three groups for treatment. The agents and concentrations used for each treatment group were: clear water control group: clear water; positive control group: 100. mu M GA ₄₊₇ And experimental group: 5. micro M MeJA; each group of treated shoots was divided into three replicates and their bases were immersed in the above agents for 3 days, after which the germination rate was counted after incubation for 21 days at 25 ℃ (16 h day)/21 ℃ (8 h black night) with a relative humidity of 70±5%. As can be seen from A in FIG. 1, the germination rate of the MeJA treated branches is higher, and the threshold for relieving dormancy of pear buds is reached in advance, namely the germination rate>50%。

2. Pear bud RNA extraction and reverse transcription

1. The total RNA of plant tissues is extracted by adopting an RNAprepPure polysaccharide polyphenol plant total RNA extraction kit (tengen), and the steps comprise:

(1) Homogenizing: respectively taking three groups of 100 mg 'Dangshan pear' pear buds, rapidly grinding into powder in liquid nitrogen, adding 500 mu L SL (containing 5% beta-mercaptoethanol), immediately vortex shaking and vigorously mixing;

(2) Centrifuging the solution obtained after the uniform mixing in the step (1) at 12000 rpm for 2 min;

(3) Transferring the supernatant after centrifugation in the step (2) to a CS filter column provided with a collecting pipe, centrifuging at 12000 rpm for 2 min, transferring the supernatant in the collecting pipe to a new RNase-Free centrifuge tube, and avoiding suction of a suction head into sediment and cell fragments in the transferring process as much as possible;

(4) Continuously adding absolute ethyl alcohol with the volume which is 0.4 times that of the supernatant into an RNase-Free centrifuge tube slowly, uniformly mixing, transferring the obtained solution and the sediment after centrifugation in the step (3) into a CR3 adsorption column, centrifuging at 12000 rpm for 15 s, discarding waste liquid in a collecting tube, and placing the CR3 adsorption column into the collecting tube;

(5) Adding 350 mu L of RW1 deproteinized liquid into the CR3 adsorption column, centrifuging at 12000 rpm for 15 s, discarding the waste liquid in the collecting pipe, and placing the CR3 adsorption column back into the collecting pipe;

(6) Preparing DNaseI working solution: taking 10 mu L of DNaseI storage solution, putting into a new RNase-Free centrifuge tube, adding 70 mu L of RDD solution, and slowly and uniformly mixing;

(7) Adding 80 mu L of DNaseI working solution into the center of the CR3 adsorption column, and standing at room temperature for 15 min;

(8) Adding 1350 mu L of deproteinized liquid RW into the CR3 adsorption column, centrifuging at 12000 rpm for 15 s, discarding the waste liquid in the collecting pipe, and placing the CR3 adsorption column back into the collecting pipe;

(9) Adding 500 mu L of rinsing liquid RW containing ethanol into the CR3 adsorption column, centrifuging at 12000 rpm for 15 s, discarding the waste liquid in the collecting pipe, and placing the CR3 adsorption column back into the collecting pipe;

(10) Repeating step (9);

(11) Centrifuging at 12000 rpm for 2 min, collecting new RNase-Free centrifuge tube, placing CR3 adsorption column therein, suspending, and dripping 30-50 μl ddH ₂ O (RNase-Free) to the middle of the adsorption film, standing at room temperature for 2 min, centrifuging at 12000 rpm for 1 min, and centrifuging to obtain the extracted RNA solution.

2. Synthesis of first strand of reverse transcribed cDNA

The reverse transcription was performed using PrimeScript ™ II 1st Strand cDNA Synthesis Kit of Takara doctor materials Co., ltd (Beijing, takara, china) as follows:

(1) Preparation of RNA denaturation systems as shown in Table 1

TABLE 1 RNA denaturation system

(2) Gently mixing the RNA denaturation system, incubating at 65 ℃ for 5 min, and then rapidly carrying out ice bath;

(3) Preparing reverse transcription reaction liquid shown in table 2 in a Microtube, wherein the total amount of the reverse transcription reaction liquid is 20 mu L;

TABLE 2 reverse transcription reaction solution

(4) Slowly and uniformly mixing the reverse transcription reaction liquid obtained in the step (3), and carrying out reverse transcription reaction under the following conditions: 42. 30 min at C, then heating at 95deg.C for 5 min, and immediately ice-bathing after inactivating the enzyme.

3. MeJA treatment promotes PpyMYC2 gene expression

qRT-PCR (primer synthesized by Zhejiang Shangya Biotechnology Co., ltd.) was applied to the MeJA-treated pear buds to detect the expression level of PpyMYC2 gene in three groups of treated pear buds, and the result is shown as B in FIG. 1, and the gene expression level of PpyMYC2 was significantly increased in the MeJA-treated pear buds compared with the control, indicating that the PpyMYC2 gene can be induced to be expressed by MeJA by the jasmonic acid signal transduction transcription factor.

The expression level of the gene was detected using a Bio-Rad CFX96 fluorescent quantitative PCR instrument with the Baozhen TB Green ™ Fast qPCR Mix dye, and the primers were found in the sequence Listing. The reaction system and experimental steps are as follows:

the PCR reaction solutions shown in Table 3 were prepared on ice:

TABLE 3 PCR reaction solution

The PCR reaction was performed in a two-step method on a CFX96 quantitative PCR apparatus under the following reaction conditions: pre-denaturing at 95 ℃ 30 s, then denaturing at 95 ℃ 5s, annealing at 60 ℃ 30 s for 40 cycles, and finally drawing a dissolution curve;

calculation of the expression level of the Gene: by 2 ^-ΔΔCT The method.

Example 2: cloning of the Pyrus pyriMYC 2 Gene

1. Full-length cDNA sequence acquisition

PCR amplification of PpyMYC2 gene was performed using Phusion DNA polymerase from Simer Feishmania technology (China), the primer sequences are as follows:

PpyMYC2-F：actcttgaccatggtagatctATGACGGACTACCGGATACCG，SEQ ID NO.5；

PpyMYC2-R：aagttcttctcctttactagtAGTCTGGCGGTTTTCCCCG，SEQ ID NO.6；

the PCR amplification reaction system and the steps are as follows:

(1) The reaction solution (ice bath) shown in table 4 was prepared:

TABLE 4 reaction solution

(2) PCR reaction, the reaction conditions are: pre-denaturation at 98 ℃ 30 s; denaturation at 98℃of 10 s; annealing at 60 ℃ for 30 s; extending at 72 ℃ for 1 min; denaturation, annealing and extension were performed for 34 cycles altogether; finally, final extension is carried out for 10 min at 72 ℃;

(3) The PCR results were detected by gel electrophoresis using a 1wt% agarose gel, and the electrophoresis results are shown in FIG. 2, whereby a PpyMYC2 gene fragment having the size of 2031 and bp was obtained.

(4) And (3) recovering agarose gel electrophoresis products, wherein agarose gel DNA recovery kits of Tiangen biochemical technology (Beijing) limited company are adopted for recovering the agarose gel electrophoresis products, and the operation steps are carried out according to instructions.

Example 3: ectopic overexpression of the Pyrus Pyriculatus PpyMYC2 Gene in Arabidopsis thaliana

1. Construction of PpyMYC2 Gene expression vector

To investigate the function of the PpyMYC2 gene, the fragment of total 2031 bp, which contains the coding region of the PpyMYC2 gene, obtained by cloning in example 2, was correctly inserted into the pCambia-1302 expression vector. Specifically, first, pCambia-1302 expression vector plasmids were digested with FastDiget endonuclease Bgl II and Spe I of Sesameiser technologies (China), and the digestion reaction system is shown in Table 5.

TABLE 5 cleavage reaction System

And (3) enzyme cutting for 20min at 37 ℃, detecting the enzyme cutting result by agarose gel electrophoresis, and recovering the cut gel after observation.

Then, the gel recovered product was ligated with the digested expression vector at 37℃for 30 min using a ClonExpress II one-step cloning kit (ClonExpress II One Step Cloning Kit available from Vazyme Co.) and the ligation reaction system was as shown in Table 6.

TABLE 6 ligation reaction System

Then transforming the connection product into DH5 alpha, screening positive monoclonal colonies by colony PCR, wherein the PCR reaction conditions are as follows: 98. pre-denaturation at 30 ℃ s; 98. denaturation at 10 ℃ s; 60. annealing at 30 deg.c s; 72. extending at the temperature of 1 min; denaturation, annealing and extension were performed for 34 cycles altogether; finally, the mixture is finally extended for 10 min at 72 ℃; sequencing in Qingdao Optimachia biotechnology limited company, amplifying and culturing positive monoclonal colony with correct sequence, and extracting plasmid to obtain PpyMYC2 gene expression vector (PpyMYC 2-OE); the PpyMYC2 gene expression vector and pCambia-1302 empty vector (WT) are transferred into agrobacterium GV3101 and stored at-80 ℃ for standby.

2. Obtaining of PpyMYC2 over-expressed Arabidopsis thaliana

1. The wild arabidopsis seeds are respectively sterilized with 70vol% alcohol for 3 min,4wt% sodium hypochlorite for 8-10 min (shaking for multiple times), then washed with sterilized water for 5 times, the seeds are directly paved on the surface of a seed germination culture medium for sowing after being sucked dry by sterile filter paper, then are cultivated at 25-28 ℃ for 16 h long-day or 8 h short-day for 10 days until seedlings grow out, and are transplanted to a substrate for cultivation until flowering.

2. The monoclonal colony of the agrobacterium in the first step is inoculated in 10 mL containing 50 mg.L ^-1 Shake culturing in hygromycin liquid medium at 28deg.C and 200 rpm until OD600 is 0.6-0.8 (about 48 h); adding 1. 1 mL bacterial liquid into 20 mL YEP liquid culture medium, shake culturing at 28deg.C and 200 rpm until OD600 is 0.6-0.8 (about 5 h), centrifuging to collect bacterial cells, and culturing with culture medium containing 0.05 g mL ^-1 Sucrose and 0.03-0.05% by volume of Silweet suspension are diluted 20 times to obtain an infection liquid containing plasmids.

3. Immersing Arabidopsis inflorescence in an invasion solution containing plasmid for 15-20 s, collecting pods and 25 mg mL ^-1 Hygromycin resistance screening and qRT-PCR detection to obtain positive transgenic Arabidopsis plants, as shown in figure 2. And (3) carrying out continuous generation screening on the positive transgenic plants for 3 generations to obtain generation homozygotes, collecting seeds, and carrying out phenotype analysis. The statistics show that the germination rate of the PpyMYC2 gene expression quantity of the Arabidopsis seeds with ectopic expression of PpyMYC2 laminated at a low temperature without 4 ℃ is significantly higher than that of a blank control, as shown in figure 3.

4. Healthy control Arabidopsis thaliana and PpyMYC2 ectopic expression Arabidopsis thaliana seedlings grown for 5 days are selected, placed under a split mirror, and the axis length and root length of the lower embryo of the Arabidopsis thaliana are observed, measured and counted. As shown in FIG. 4, it was found that the ectopic expression of PpyMYC2 in Arabidopsis seedlings possessed longer hypocotyls and roots than the control, indicating that overexpression of PpyMYC2 may increase the enzymatic activity of Arabidopsis GA20 OX.

5. A healthy 5-day-growth control Arabidopsis thaliana and PpyMYC2 ectopic-expression Arabidopsis thaliana seedling were selected, and 10-fold volumes of phosphate buffer (containing 136.89 mM NaCl,2.67 mM KCl,8.1 mM Na) were sampled and added ₂ HPO ₄ And 1.76 mM KH ₂ PO ₄ ) Grinding the materials. The enzyme activity of GA20OX in Arabidopsis was measured using a plant gibberellin-20-oxidase (GA 20 OX) enzyme-linked immunosorbent assay kit from Shanghai Co., ltd. As shown in FIG. 5, it was found that GA20OX enzyme activity in P pyMYC2 ectopic expression Arabidopsis thaliana seedlings was higher than that in p-pairArabidopsis thaliana was irradiated.

Example 3: transient overexpression of pear PpyMYC2 Gene in pear bud

1. Construction of viral expression vector for PpyMYC2 Gene

To investigate the function of the PpyMYC2 gene, the fragment of total 2031 bp, which contains the coding region of the PpyMYC2 gene, obtained by cloning in example 2, was correctly inserted into an IL-60-BS viral vector. IL-60-BS vector is cut by using endonuclease Sal І and Nhe I, and the sequence of the gene amplification primer is shown as SEQ ID NO.7 and SEQ ID NO. 8.

After gene amplification, vector enzyme digestion, one-step connection, transformation of escherichia coli DH5 alpha and positive clone screening, sequencing is carried out on Hangzhou Shangya biotechnology Co-Ltd, and the correctly sequenced positive monoclonal colony and IL-60-BS empty vector (control group) are respectively subjected to amplification culture, and plasmids are extracted and stored at the temperature of minus 20 ℃ for standby.

2. Obtaining of Pyrus bud overexpressed by PpyMYC2

The branches of Dangshan pear with full flower buds are taken from a plant garden without entering into a dormant Dangshan pear for 1 month at 10 months, and the branches are accumulated at a low temperature of 720 and h at a temperature of 10 ℃ to enable certain low-temperature accumulation amount to exist. The stand-by plasmid was diluted to a final concentration of 5 ng. Mu.l using the infection solution ^-1 . The plasmid is injected into branches of the flowers and buds to be infected 'Dangshan pear' by using a 1 ml injector, and the injected branches are planted on the flower mud fully absorbed with water, and are dark-cultured for 3 days at 15 ℃. After 3 days of infection, 30 buds were taken for RNA extraction and detection. The IL-60-BS empty vector plasmid is transferred into a control group IL60-2, the positive monoclonal colony plasmid is transferred into a transgene group PpyMYC2-OE, each group of branches is 8, each group of branches is not less than 30 buds, three repetitions are carried out, and the germination rate is counted after continuous culture for 20 days at 25 ℃ (16 h daytime)/21 ℃ (8 h black night) with the relative humidity of 70+/-5%.

The total RNA of empty vector and PpyMYC2-OE transgenic pear buds is extracted, a Bio-Rad CFX96 fluorescence quantitative PCR instrument is used, the expression level of PpyMYC2 genes in the empty vector and the transgenic pear buds is detected by using a Baotor TB Green ™ Fast qPCR Mix dye, qPCR-PpyMYC2 primers are shown as SEQ ID NO.3 and SEQ ID NO.4, the result is shown as A in figure 6, and compared with a control group IL60-2, the transcription level of PpyMYC2 is obviously increased in the transgenic pear buds, which indicates that the PpyMYC2 is successfully and transiently over expressed in the pear buds.

3. Analysis of germination rate of transient overexpressed pear buds

The germination rate of transiently expressed pear buds was counted, and as shown in B in FIG. 6, the germination rate of the transgenic group PpyMYC2-OE pear buds was found to be significantly higher than that of the control group IL60-2 after transiently infecting pear buds. And as shown in FIG. 7, GA20OXs gene expression and GA in pear buds of PpyMYC2 were overexpressed ₃ The content is obviously higher than that of a control group, which shows that the PpyMYC2 gene positively regulates and controls the dormancy release of pear buds by regulating gibberellin biosynthesis.

In conclusion, meJA can promote the release of dormancy of pear buds with insufficient low-temperature accumulation, and a PpyMYC2 gene responding to MeJA is separated from pears, and through the verification analysis of the transfer function of pear buds, ppyMYC2 has remarkable effects in promoting arabidopsis thaliana, pear bud germination and release of pear bud dormancy, reduces the limitation of cold required to pear bud germination, and has important significance in low-temperature mediated release of pear dormancy and creation of pear south breeding conditions.

In the present specification, each embodiment is described in a progressive manner, and each embodiment is mainly described in a different point from other embodiments, and identical and similar parts between the embodiments are all enough to refer to each other.

The previous description of the disclosed embodiments is provided to enable any person skilled in the art to make or use the present invention. Various modifications to the embodiments described above will be readily apparent to those skilled in the art, and the generic principles defined herein may be applied to other embodiments without departing from the spirit or scope of the invention. Thus, the present invention is not intended to be limited to the embodiments shown herein but is to be accorded the widest scope consistent with the principles and novel features disclosed herein.

Claims

1. The method comprises the following steps ofPpyMYC2The use of a gene for promoting the release of dormancy of pear buds, characterized in that in pear buds, the gene is used for the followingPpyMYC2Overexpression of genes, saidPpyMYC2Nucleotide sequences of genes, e.g. SEQ ID No. 1.

2. Use according to claim 1, characterized in that it is in particular: in pear buds, the virus-mediated transient transformation is used for the preparation of the strainPpyMYC2The gene is over-expressed to promote the dormancy of pear buds to be released.

3. The use according to claim 1, characterized in that in pear buds the virus-mediated transient transformation is used for the saidPpyMYC2The gene is subjected to overexpression specifically comprises the following steps: will contain the saidPpyMYC2Injecting the infection liquid of the gene overexpression plasmid into branches containing pear dormant buds to enable the branches to be subjected to the following steps ofPpyMYC2And (5) gene overexpression.

4. The use according to claim 3, wherein thePpyMYC2The vector of the gene overexpression plasmid is an IL-60-BS virus vector.

5. The method comprises the following steps ofPpyMYC2The application of the gene in promoting the germination and growth of Arabidopsis seeds is characterized in thatPpyMYC2The nucleotide sequence of the gene is shown as SEQ ID NO. 1.

6. The use according to claim 5, characterized in that it is in particular:

soaking the arabidopsis inflorescence into the solutionPpyMYC2And (3) carrying out transfection in an invasion solution of the gene overexpression plasmid, continuously screening transfected functional Arabidopsis thaliana to obtain generation homozygotes, and collecting and obtaining transgenic Arabidopsis thaliana seeds, wherein the germination rate of the transgenic Arabidopsis thaliana seeds is high, and the growth speed is high.