CN116622655A

CN116622655A - Cold-resistant gene PtrP5CS1 of hovenia dulcis thunb and application thereof in plant cold-resistant genetic improvement

Info

Publication number: CN116622655A
Application number: CN202310690936.0A
Authority: CN
Inventors: 刘继红; 肖玮; 李春龙
Original assignee: Huazhong Agricultural University
Current assignee: Huazhong Agricultural University
Priority date: 2023-06-12
Filing date: 2023-06-12
Publication date: 2023-08-22

Abstract

The invention belongs to the field of plant genetic engineering and discloses a cold-resistant gene of hovenia dulcis thunbPtrP5CS1And the application thereof in the genetic improvement of plant cold resistance,PtrP5CS1the gene is a synthetase gene separated from the orange material with extreme cold resistance, and the sequence of the synthetase gene is shown as SEQ ID NO. 1. The gene is constructed into an over-expression and interference vector, and is introduced into the hovenia dulcis thunb through agrobacterium-mediated genetic transformation, and the obtained transgenic plant is verified by biological functions, which shows that the invention is clonedPtrP5CS1The gene has the function of improving the cold resistance of plants. The gene was found to beThe plant stress-resistant molecular design breeding provides new genetic resources, provides new genetic resources for green agriculture and water-saving agriculture, and is beneficial to reducing the agricultural production cost and realizing environmental friendliness.

Description

Cold-resistant gene PtrP5CS1 of hovenia dulcis thunb and application thereof in plant cold-resistant genetic improvement

Technical Field

The invention belongs to the field of plant genetic engineering, and particularly relates to a pyrroline-5-carboxylic acid synthetase coding gene PtrP5CS1 obtained by separating and cloning from hovenia dulcis (Poncirust rifoliata), and application of the gene in plant cold resistance genetic improvement, wherein the gene is interfered in plants, and the cold resistance of the obtained transgenic plants is obviously reduced.

Background

In the process of long evolution, plants have evolved into complex mechanisms to alleviate cold-induced injury, maintain growth and development under adverse conditions, and improve survival levels. After a period of time under non-lethal, non-frozen low temperature conditions, the process of obtaining low temperature resistance by activating metabolism in the body in advance is called cold acclimation (Steponkus 1984, monroy and Dhindsa 1995,Shelp et al 2012,Liu et al 2020). In this process, plants act by altering the expression of metabolic pathway genes and accumulating a large amount of protective substances to combat low temperature injury, which act by stabilizing cell osmotic pressure, maintaining cell structure at low temperature, maintaining cell membrane fluidity, and promoting ROS clearance (Kaplan and Guy 2004,Chinnusamy et al 2007,Baier et al 2019,Chai et al 2019), mainly including permeabilizing substances: such as soluble sugars (Yuan et al 2015,Zhao et al 2019,Liang et al 2021,Sun et al 2021,Huang et al 2022), proline (Browse and Xin 2001), betaine (Ming et al 2021), polyamines (Kou et al 2018,Li et al 2022) and unsaturated fatty acids (Liu et al 2022). These substances act to maintain osmotic balance, protect cellular structures, and in addition, part of the osmoregulating substances can act as oxidative scavengers, or as carbon/nitrogen sources to maintain energy metabolism under adverse conditions (Ghosh 2021).

Low temperature is an important environmental factor limiting crop growth and yield, and low temperature stress can cause plant chlorosis, stunted growth and development, and even death of plants. At the microscopic level, it appears that low temperature causes accumulation of ROS in plants, oxidative damage, inactivation and denaturation of proteins, and thus metabolic disorders and inhibition of photosynthesis (Pearce 2001,Foyer et al 2002). The low temperature below zero for a long period of time causes weakening of cell membrane fluidity, intracellular ice crystal formation, disruption of cell structure, and outflow of cell contents, which eventually leads to cell death (Steponkus 1984,Steponkus et al 1998).

Proline is a class of protein amino acids that are synthesized in the cytoplasm by the glutamate pathway (Szoke et al 1992), and degradation of proline occurs in mitochondria, which is reduced to glutamate under the action of ProDH and P5CDH (Deuschle et al 2004). Among them, P5CS (pyrroline-5-carboxylate synthetase, pyrroline-5-carboxylic acid synthase) is a rate-limiting enzyme gene for proline synthesis, and in most higher plants, P5CS is encoded by two homologous genes (Strizhov and Ja 2008, jimez-lopez et al 2010,Brocker et al 2013). Proline is considered an important osmoprotectant substance that protects subcellular structures and macromolecular substances under osmotic stress (savure 2009, ghosh 2021). In addition, proline can enhance the activity of different enzymes by protecting the integrity of the protein as a chaperone (Funck et al 2012); proline also relieves stress-induced injury by scavenging free radicals (Kavi Kishor et al 2005,Miller et al 2010). Under extreme stress conditions, proline is able to supply energy as an important nitrogen source (Kohl et al 1988,Mattioli et al 2012,Liang et al 2013). External application of proline or substances affecting the proline content was found to affect stress resistance of plants. In bermuda grass, proline treatment relieves the damage to plants by drought stress (Jiang et al 2023). Mutation of the proline-synthesizing gene OsOAT results in extremely low temperature sensitivity of rice (Yan et al 2023). Accumulation of proline in the cytoplasm is extremely important for plants to resist low temperature stress (Heyer et al 2021). Although the synthetic pathway of proline is already quite clear, accumulation of proline is also considered as an important protective adaptation mechanism of plants under adverse conditions, functional verification of proline in low temperature stress still requires more exploration.

Disclosure of Invention

The invention aims to provide a hovenia dulcis thunb cold-resistant gene which is a pyrroline-5-carboxylic acid synthetase gene separated from hovenia dulcis thunb (Poncirus trifoliata) and cloned into a very cold-resistant gene, and the applicant names the gene as PtrP5CS1, the nucleotide sequence of the gene is shown as SEQ ID NO.1, and the coded protein is shown as SEQ ID NO. 2.

The invention also aims at providing an application of the cold-resistant gene PtrP5CS1 in controlling the cold-resistant property of plants. The gene is silenced and expressed in plants to obtain plants with reduced cold resistance.

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

The applicant is based on a transcription factor isolated and cloned from the extreme cold-resistant hovenia dulcis (Poncirus trifoliata) by a plant gene cloning technology, and names the transcription factor as PtrP5CS1, the sequence of which is shown as SEQ ID NO.1, and the corresponding amino acid sequence of which is shown as SEQ ID NO. 2; open Reading Frame (ORF) prediction shows that the gene is protein containing one ORF and having a length of 2148bp and encoding 715 amino acids, the molecular weight of the protein is 77.29kDa, and the isoelectric point is 6.20.

The expression of the protein shown in SEQ ID NO.2, which is an expression cassette, recombinant vector or recombinant microorganism comprising the polynucleotide encoding SEQ ID NO.2, is also within the scope of the present invention.

The protection scope of the invention also comprises:

application of the protein shown as SEQ ID NO.2, polynucleotide for encoding the protein shown as SEQ ID NO.2 or substance for expressing the protein shown as SEQ ID NO.2 in controlling cold resistance of plants;

in the above application, preferably, the plant is trifoliate orange;

in the above application, preferably, the control is to knock out, inhibit or silence the expression level of the coding gene of the protein shown in SEQ ID No.2 in Zhi so as to weaken the cold resistance of Zhi.

In the above application, it is preferable that the silencing is performed by constructing an interference vector using a polynucleotide encoding a protein shown in SEQ ID NO.2, and interfering it in Zhi through Agrobacterium-mediated genetic transformation.

Compared with the prior art, the invention has the following advantages:

the discovery and identification of PtrP5CS1 gene provides new gene resources for plant stress-tolerant molecular design breeding, and provides new genetic resources for green agriculture and water-saving agriculture, and the development and utilization of the genetic resources are beneficial to reducing the agricultural production cost and realizing environmental friendliness.

Drawings

Fig. 1 is a technical flow chart of the present invention.

FIG. 2 is a schematic diagram showing the expression pattern of PtrP5CS1 of the present invention in response to low temperature stress treatment;

wherein: a is the relative expression level of PtrP5CS1 gene under low temperature (4 ℃) treatment; b is a GUS fusion vector schematic diagram; c, GUS staining of PtrP5CS1 promoter transient transformation callus; and D, quantitatively analyzing.

FIG. 3 is a schematic diagram showing the expression pattern of PtrP5CS1 of the present invention in response to various treatments.

Wherein: a is ethylene treatment; b is dehydration treatment; c is salt treatment.

FIG. 4 is a schematic representation of positive identification and relative expression analysis of the VIGS silencing material of the present invention;

wherein: a is PtrP5CS1 gene and TRV1 specific primer identification positive plants (TRV 2-PtrP5CS 1) of the invention;

b is the identification of a positive TRV2 empty-load transformed plant by the TRV2 and TRV1 specific primer;

c is an analysis of PtrP5CS1 expression levels in PtrP5CS1 interference material.

Wherein, "M" represents a marker, "P" represents a plasmid, "W" represents water, "wt" represents wild-type, and "TRV2" represents an empty control;

FIG. 5 is a graph showing the measurement of proline content before and after the treatment of interfering PtrP5CS1 gene plants (TRV 2-PtrP5CS1 for short);

FIG. 6 is a schematic diagram showing cold resistance analysis of Zhi PtrP5CS1 gene silencing;

wherein: a is the phenotype of empty load TRV2 and interference plant TRV2-PtrP5CS1 before and after low temperature treatment; b is a chlorophyll fluorescence phenotype chart before and after low-temperature treatment of the hovenia dulcis thunb; c is Fv/Fm value before and after low temperature treatment of the interference hovenia dulcis thunb; d is the relative conductivity before and after the low-temperature treatment of the interference hovenia dulcis thunb; e is MDA content before and after low temperature treatment of the interference hovenia dulcis thunb.

FIG. 7 is a graph showing measurement of physiological indexes related to active oxygen before and after treatment of interfering PtrP5CS1 gene;

wherein: a is the empty load TRV2 before and after the low temperature treatment and the interference plant TRV2-PtrP5CS1 before and after the low temperature treatment ₂ O ₂ The content is as follows; b is O before and after low-temperature treatment of the interference hovenia dulcis thunb ₂ ^·- The content is as follows; c (C)Is a DAB dyeing chart after interference and low-temperature treatment; d is an NBT staining chart after interference and low-temperature treatment.

Detailed Description

The present invention will be described in detail with reference to specific examples. From the following description and examples, one skilled in the art can ascertain the essential characteristics of this invention, and without departing from the spirit and scope thereof, can make various changes and modifications of the invention to adapt it to various usages and conditions.

Example 1:

cloning of full-length cDNA of Hovenia dulcis PtrP5CS1 Gene

Using the hovenia dulcis thunb cDNA as a template, and adopting high-fidelity enzyme for amplification, wherein an amplification system is shown in a table 1, an amplification program is shown in a table 2, and an amplification primer sequence is as follows: forward primers:5'-ATGGACTCAACGGACAGTTCCA-3' and Reward primers 5'-CTAGGATTGCACGGCAAGATTC-3'.

Purifying and recovering amplified product by AxyPrep-96 DNA gel recovery kit (Axygene, USA), connecting the purified product with pEASY-Blunt vector (full gold, china), incubating at room temperature for 5min, and transforming E.coli competent DH5 alpha. Coating a plate, selecting a monoclonal, then carrying out PCR positive identification (GenStar, china), wherein a positive identification system is shown in Table 4, obtaining positive clone, and then carrying out sequencing by the Wuhantian Yihua gene technology Co., ltd, and obtaining the full-length sequence of the PtrP5CS1 gene according to the sequencing result.

TABLE 1 Gene amplification System

TABLE 2 Gene amplification PCR procedure

TABLE 3pEASY-Blunt vector ligation System

TABLE 4 reaction system for positive identification of colonies

Sequencing results show that the CDS length of the gene is 2148bp, 715 amino acids are encoded, the molecular weight of the protein is 77.29kDa, the isoelectric point is 6.20, the gene is named PtrP5CS1, the polynucleotide sequence is shown as SEQ ID NO.1, and the amino acid sequence is shown as SEQ ID NO. 2.

Example 2: analysis of PtrP5CS1 Gene expression under different stress Condition treatments

The expression pattern of PtrP5CS1 gene was analyzed by real-time fluorescent quantitative PCR (qRT-PCR) using AceQ qPCR SYBR Green Master Mix (Norpran, china) with reference to the specification. The reaction system is shown in Table 5, and the prepared reaction system is reacted by using a Quantum studio 7Flex system (Applied Biosystems, USA) fluorescence quantitative analyzer, and the reaction procedure is shown in Table 6. The relative expression level of the genes is 2 ^-ΔΔCT The method is used for calculation, wherein citrus Actin is selected as an internal reference gene (Forward primer: 5'-CCGACCGTATGAGCAAGGAAA-3'; reverse primer: 5'-TTCCTGTGGACAATGGATGGA-3'), ptrP5CS1 real-time quantitative primer (Forward primers:5'-TTGTTATCCCCAGAGGCAGC-3'; forward primers: 5'-CCGCCAAATAAACCGACACC-3').

TABLE 5 quantitative PCR reaction System

TABLE 6 qPCR reaction procedure

The results of this experiment showed that the expression level of PtrP5CS1 gene was continuously induced at low temperature, and the expression level was highest at 72 hours, increased by about 10-fold as compared with that before the treatment, and then decreased slowly (A in FIG. 2). Meanwhile, the expression level of PtrP5CS1 gene was up-regulated by ethylene induction, and the expression level was highest at 72 hours, increased about 6-fold compared with that before the treatment, and then decreased slowly (A in FIG. 3). Meanwhile, ptrP5CS1 is also expressed under dehydration stress, and the highest induction multiple is 14 times (B in FIG. 3). When treated with salt, the gene was up-regulated 15-fold at 12h (C in FIG. 3). In combination, ptrP5CS1 gene is subject to low temperature, dehydration, salt stress and ethylene induced expression, and may play an important role in plant abiotic stress.

Example 3: GUS staining analysis of PtrP5CS1 Gene promoter transient transformation calli

1. Vector construction

The experiment is based on PtrP5CS1 gene promoter sequence in the fructus Aurantii whole gene database, specific primers are designed, high-fidelity enzyme (Norvezan, china) is used for amplification by taking the hovenia gDNA as a template, an amplification reaction system and a program refer to Table 1 and Table 2, and the sequence is determined after a pEASY intermediate vector is connected. The amplification primer sequences were as follows:

proPtrP5CS1-F：5’-ATATAAACCAGGTGGCCAGTCACAG-3’

proPtrP5CS1-R：5’-TTGACGGAAGAAGAAAGCGAGTCA-3’

purifying and recovering amplified product by AxyPrep-96 DNA gel recovery kit (Axygene, USA), connecting the purified product with pEASY-Blunt vector (full gold, china), incubating at room temperature for 5min, and transforming E.coli competent DH5 alpha. And (3) plating, selecting a monoclonal, carrying out PCR positive identification, wherein a positive identification system is shown in Table 4, obtaining positive clones, and then sending the positive clones to Wuhan Tianhua gene technology limited company for sequencing, and obtaining PtrP5CS1 gene promoter sequences according to sequencing results.

The plasmid with correct sequence is used as a template, a specific primer is designed to amplify, and 15-20bp sequences at the tail ends of linearization vectors are respectively added at the 5' ends to serve as homologous sequences, and the primer pair is used to amplify to obtain an insert with the homologous sequences. The one-step method was used to insert between PstI and BamHI cleavage sites ligated to DX2181G vector using One Step Cloning Kit (Norwegian, china), the specific method is shown in Table 7, and the constructed vector was transferred to GV3101 competent after sequencing correctly. The primers for constructing the vector were as follows:

proPtrP5CS1-DX-F：

5’-CTACAGCGCTAAGCTTGGCTGCAGATATAAACCAGGTGGCCAGTCACAG-3’

proPtrP5CS1-DX-R：

5’-AAGGGACTGACCACCCGGGATCCTTGACGGAAGAAGAAAGCGAGTCA-3’

TABLE 7 one-step ligase reaction System

2. Instant transformation of sweet orange callus

1) Suspension callus: transferring the well-grown sweet orange callus to 50mL MT liquid culture medium for culturing, shaking and scattering on a room temperature darkroom shaking table (25 ℃ C., 120 r/min) for about 5d;

2) Activating agrobacterium: streaking agrobacterium preserved at-80deg.C in LB solid medium (containing carrier-resistant antibiotic) with sterile inoculating loop, and culturing in 28 deg.C incubator for 2d to obtain monoclonal;

and (3) performing expansion culture: selecting the first streaked monoclonal, inoculating to 10mL of LB liquid culture medium (containing carrier-resistant antibiotics) for small shaking activation, and then sucking 1mL of small shaking bacteria liquid to 50mL of fresh culture medium for large shaking culture at 28 ℃ over night;

3) Preparing an aggressive dyeing liquid: 8000r/min,5min, centrifuging to collect bacteria, cleaning with 10mL MT suspension culture medium, centrifuging to remove supernatant, and suspending in MT suspension cultureIn the base, the OD is regulated ₆₀₀ The value is 0.6-0.8, to about 50mL of suspension culture medium (MT+0.5 g/L maltose powder+1.5 g/L L-glutamine) containing 50mg/LAS (Acetosyringone), and the infection is carried out for 20min by a shaking table at 28 ℃ and 200 r/min;

4) Infection and cultivation: standing the shaken-up callus for 45min, gently pouring out the upper liquid, transferring the callus by using a sterile spoon or forceps, spreading the callus in a glass dish paved with sterile filter paper, drying the callus (standing for about 40min at low wind speed in an ultra-clean workbench or until water is absorbed), pouring the callus into an agrobacterium infection solution, carrying out table infection for 10min at 28 ℃, standing for 30min, drying the callus by using the same method, transferring the callus to a co-culture medium (MT solid medium +50mg/L AS) paved with sterile filter paper, and co-culturing for 3d in a room temperature and darkroom.

GUS staining analysis

GUS staining was performed on sweet orange calli using GUS staining kit (coolaber, SL7160, china) and the GUS staining results were quantified by Image J software.

The experimental result shows that the callus of the pPtrP5CS1 subjected to no low-temperature treatment is light blue, and the callus of the pPtrP5CS1 subjected to GUS transformation is obviously deepened in blue after low temperature, and the control group of DX2181G no-load transformation is not stained with color before and after low temperature (figures 2C-D), which shows that the low temperature can enhance PtrP5CS1 promoter activity, and further proves that PtrP5CS1 is subjected to low-temperature strong induction expression.

Example 4: plant transformation vector construction

1. Vector construction

The specific primer is designed to amplify 427bp fragment of 3' -end non-conserved region of PtrP5CS1 gene by using the hovenia dulcis-cDNA as a template, one Step Cloning Kit (Noruzan, china) is adopted, one-step method is inserted and connected between two enzyme cutting sites of BamHI and SmaI on pTRV2 vector, the specific method is shown in Table 7, the constructed vector is transferred to GV3101 competence after being sequenced correctly, pTRV2-PtrP5CS1 agrobacterium is constructed, and pTRV1 and pTRV2 are respectively transferred to GV3101 competence.

The primers for constructing the vector were as follows:

pTRV2-PtrP5CS1-F(BamHI)：

5’-AGAAGGCCTCCATGGGGATCCTTTGGCGGACCAAGAGCAAG-3’；

pTRV2-PtrP5CS1-R(SmaI)：

5’-TGTCTTCGGGACATGCCCGGGAGGATTGCACGGCAAGATTC-3’。

example 5: VIGS interference hovenia dulcis and identification of positive seedlings

Vigs infestation

1) Preparing agrobacterium infection liquid:

agrobacteria such as pTRV1, pTRV2 and pTRV2-PtrP5CS1 are respectively streaked on LB solid medium (50 mg/L rifampicin, 50mg/L kanamycin) and cultured for 2 days at 28 ℃, and then the obtained product is selected and monoclonal in 5mL LB liquid medium containing the same antibiotics at 28 ℃ and 220r/min, so that the bacterial cells are fully activated (24-48 h). The activated agrobacterium liquid is inoculated into fresh LB culture medium containing antibiotics according to the proportion of 1:100, and cultured overnight at 28 ℃ and 220 r/min. Centrifuging at 4000r/min, collecting thallus, adding MES buffer (10 mmol/L MES,10mmol/L MgCl) ₂ 200. Mu. Mol/L AS, pH=5.6-5.7) suspended cells, and OD was adjusted ₆₀₀ To 2.0. Mixing pTRV1 and pTRV2 or pTRV2-PtrP5CS1 bacterial heavy suspension according to a ratio of 1:1, uniformly mixing, placing in a 28 ℃ incubator, and standing for 2-3h to obtain the final product.

2) Infection:

fresh semen Hoveniae is taken out from fruits, pectin is removed by soaking in 1mol/L NaOH solution for 15min, then the fruits are washed clean by sterile water, spread on wet clean gauze, placed in an incubator (28 ℃ in darkness) for germination, and used for VIGS infection after the buds of the seeds germinate to 1-2cm long. Slightly pricking small holes on germinated buds by using a syringe needle, completely soaking in prepared agrobacterium tumefaciens dip, vacuumizing for 10min, rapidly deflating to enable the agrobacterium tumefaciens to be immersed in germinated seeds, and repeating for 3 times. Standing for 15min, taking out the infected seeds, airing on dry filter paper, standing for 2-3min, spreading in a large dish of filter paper soaked with sterile water, and standing for 2-3d dark culture in a room temperature culture room in a shading manner; the seeds were removed, rinsed with clear water to clean residual bacterial liquid, sown in the matrix (soil: vermiculite=3:1), grown in incubator for 25d and positive identification was performed.

2. Identification of Positive Material

When the resistant buds root and 2-3 leaves grow, taking a small number of leaves for DNA extraction, wherein the DNA extraction steps are as follows:

1) A small amount of leaves are taken and placed into a 1.5mL centrifuge tube, liquid nitrogen is ground into powder, 600 mu L of CATB extracting solution is added, and the preparation method of the CTAB extracting solution is shown in Table 8;

2) Fully and uniformly mixing, and then placing the mixture into a 65 ℃ water bath pot for water bath for 90min, and reversing and uniformly mixing every 30 min;

3) After completion of the water bath, 700 μl of 24:1 (chloroform: isoamyl alcohol) is mixed with the extract, the mixture is vigorously mixed for 10min, the mixture is centrifuged for 15min at the normal temperature of 12000r/min, and the supernatant (about 500 mu L) is sucked and transferred into a new 1.5mL centrifuge tube;

4) Adding pre-cooled isopropanol with the same volume as the supernatant, mixing the mixture upside down, and placing the mixture in a refrigerator at the temperature of minus 20 ℃ for precipitation (the precipitation time can be prolonged);

5) Taking out after precipitation is completed, and centrifuging for 10min at 12000 r/min. Pouring out the supernatant, adding 1mL of pre-cooled 75% ethanol, cleaning for 2-3 times, discarding the ethanol, and air-drying in a fume hood;

6) Add 20-30. Mu.L ddH to each tube ₂ O dissolves DNA, and the dissolved DNA is stored in a refrigerator at-20 ℃.

Concentration measurement, 1. Mu.L of each sample was taken and measured on a NanoDrop2000 ultra-micro spectrophotometer (Thermo, USA), OD thereof ₂₆₀ /OD ₂₈₀ When the ratio is in the range of 1.8-2.0, the purity of DNA is high. And also detected by gel electrophoresis.

The identification of the plant positive primer sequences is as follows:

TRV1-F：5’-ATTGAGGCGAAGTACGATGG-3’

TRV1-R：5’-CCATCCACAATTATTTTCCGC-3’

TRV2-F：5’-ATTCACTGGGAGATGATACGCT-3’

TRV2-R：5’-AGTCGGCCAAACGCCGATCTCA-3’

TABLE 8CTAB extract formulation

Identifying the hovenia dulcis positive plants with the VIGS silence, and taking the extracted hovenia dulcis DNA as a template, and adopting a TRV1 forward primer, a TRV2 forward primer and a reverse primer positive plant constructed by a TRV2-PtrP5CS1 vector. Wherein, the negative control distilled water (W) and wild type hovenia dulcis (wt) DNA are used as template amplification products without bands, and the interference plant DNA is amplified with bands which are consistent with the size of the positive control TRV2-PtrP5CS1 plasmid (P). And the TRV1 forward and reverse primers were used, and the positive control TRV1 plasmid and the interference line both amplified bands of uniform size, indicating successful transformation of Zhi (A in FIG. 4). Similarly, when the empty plants of the transformed TRV2 were identified, the TRV1 forward and reverse primers and the TRV2 forward and reverse primers amplified bands consistent with the TRV1 or TRV2 plasmid size, while the negative control distilled water (W) and wild-type (wt) DNA were used as templates, the amplified products were not band (B in FIG. 4). The expression level of PtrP5CS1 gene in the positive plants is also detected by real-time fluorescence quantification, the expression level of PtrP5CS1 in the empty load control plants (TRV 2) is 1, and the result shows that compared with the empty load control plants, the expression level of PtrP5CS1 in the interference plants is suppressed to a degree between 47 and 94 percent, the expression of the target gene is obviously lower than that of the empty load control plants, and the plants with higher suppression degree are randomly selected for subsequent cold resistance analysis (C in the description).

Example 6: identification of interference PtrP5CS1 cold resistance

Plants with the best interference effect (# 3, #6, #13, #28, #38 and #42 in example 5) were selected for low temperature resistance identification, wherein no-load control plants (TRV 2) were used as negative controls, "TRV2-PtrP5CS1" indicated by PtrP5CS1 interference line plants, and "TRV2-PtrP5CS1+pro" indicated by PtrP5CS1 interference line plants water-cultured for 2d with 1mM proline (distilled water solution). Empty control and interference plants were treated at-4 ℃ and the proline content of PtrP5CS1 interference plants before and after low temperature treatment was significantly lower than that of the control group (fig. 5). After the low temperature treatment, the leaf injury degree of the PtrP5CS1 interference plant is highest, the leaf injury degree is represented by serious wilting and curling, the empty control only has a small part of leaf curling, and the interference material with proline is not generated after the low temperature treatment (A in fig. 6). The results of chlorophyll fluorescence imaging and maximum photosynthetic efficiency (Fv/Fm) also indicate that the maximum photosynthetic efficiency Fv/Fm of PtrP5CS1 interfering plants is significantly lower than that of the TRV2 control and proline-exotic interfering materials (B in FIG. 6, C in FIG. 6). Meanwhile, after low temperature treatment, the electrolyte leakage rate and MDA content of the interference plants of PtrP5CS1 were significantly higher than those of the control group and the proline-treated group (TRV 2-PtrP5CS1 +pro) (D in FIG. 6, E in FIG. 6). These results demonstrate that interfering with the PtrP5CS1 gene reduces proline synthesis in plants, destroys the active oxygen scavenging system in plants, inhibits the scavenging of active oxygen in plants under low temperature treatment, and further makes plants more vulnerable to low temperature stress. Taken together, ptrP5CS1 is a positive regulator of plant resistance to low temperature stress.

Claims

1. From the hovenia dulcis thunb @ to achievePoncirus trifoliata) The sequence of the protein separated from the above is shown as SEQ ID NO. 2.

2. A polynucleotide encoding the protein of claim 1.

3. The sequence according to claim 2, which is set forth in SEQ ID NO. 1.

4. A substance expressing the protein shown in SEQ ID NO.2, wherein the substance is an expression cassette, a recombinant vector or a recombinant microorganism containing a polynucleotide encoding SEQ ID NO. 2.

5. Use of a protein according to claim 1, a polynucleotide according to claim 2 or a substance according to claim 4 for controlling cold resistance in plants.

6. The use according to claim 5, wherein the plant is trifoliate orange.

7. The use according to claim 6, wherein the control is to knock out, inhibit or silence the expression level of the gene encoding the protein represented by SEQ ID NO.2 in Zhi to attenuate cold resistance.

8. The use according to claim 7, wherein the silencing is by constructing an intervention vector using the polynucleotide of claim 2, which is interfered in trifoliate orange by agrobacterium-mediated genetic transformation.