CN116082481A - Transcription factor PbZIP 4 and protein affecting sensitivity of plants to anthracnose and application thereof - Google Patents

Abstract

The invention provides a transcription factor PbbZIP4 and a protein for influencing the sensitivity of plants to anthracnose and application thereof, belonging to the technical field of functional genes. The transcription factor PbZIP 4 for influencing the sensitivity of plants to anthracnose provided by the invention has an amino acid sequence shown in SEQ ID NO: 1. The invention can improve the sensitivity of plants to anthracnose by over-expressing the transcription factor PbbZIP4, and can effectively improve the resistance of plants to anthracnose stress when the expression of the transcription factor PbZIP 4 is reduced by gene silencing. Therefore, the application of the transcription factor PbbZIP4 as a target point in reducing or improving the sensitivity of plants to anthracnose is provided.

Description

Transcription factor PbZIP 4 and protein affecting sensitivity of plants to anthracnose and application thereof

Technical Field

The invention belongs to the technical field of functional genes, and particularly relates to a transcription factor PbbZIP4 and a protein for influencing the sensitivity of plants to anthracnose and application thereof.

Background

Pears are widely planted in the world, and the third fruits which are more inferior to apples and oranges in China are very wide in planting area, so that a three-region four-point production area layout mode with planting from northeast to Guangxi and from Yunnan to Shandong is formed. Although the pear planting area is wide and most of pear garden layout planning is reasonable, the problems of different areas, influence of various natural disasters, long-term unreasonable development and utilization of human beings and the like are caused, so that the pear garden layout planning has a great threat to agricultural production, grain safety and planting of various fruits, vegetables and landscape plants, and the development of the pear industry is also caused to face the influence of saline-alkali, drought, freeze injury and flooding. Stress environments are one of the major factors limiting plant growth, stress being classified into non-anthracnose and anthracnose. In anthrax, fungal infection is a very damaging stress and fungal hyphae can infect plant roots, stems, leaves and fruits, causing a great economic loss. For pear crops, pear anthracnose caused by bacillus mucilaginosus is one of the most extensive fungal diseases, and causes great harm to pear yield. Therefore, there is an urgent need to obtain drought-resistant and disease-resistant pear varieties by breeding means.

Unlike animals, plants cannot actively avoid the stress of the adverse environment, and thus plants have evolved a series of systemic response mechanisms to cope with the stress of various adverse environments. The system response mechanism is that the gene regulation network can rapidly induce the expression of the in-vivo stress related genes when being subjected to external stress signals, so as to cope with various stress environments.

bZIP transcription factors are a conserved bZIP domain consisting of 40-80 amino acids with 2 structural features. The basic region of DNA binding (N-X7-R/K-X9) is used for binding to specific DNA sequences, while a plurality of heptamer repeat sequences consisting of leucine or other hydrophobic amino acids (e.g., ile, val, phe or Met) constitute bZIP motifs for dimer specific binding. Several studies have shown that bZIP transcription factors function in a variety of ways, including biological processes, seed maturation, senescence and adaptive biological and non-anthracnose responses. However, there is no report on bZIP transcription factors having the function of regulating the plant to show different degrees of sensitivity to anthracnose.

Disclosure of Invention

Therefore, the invention aims to provide a transcription factor PbbZIP4, whether the transcription factor PbZIP 4 is expressed or not can directly influence the sensitivity of plants to anthracnose, and provides a new idea for cultivating new varieties of plants resistant to anthracnose.

The invention provides a transcription factor PbZIP 4 for influencing the sensitivity of plants to anthracnose, wherein the amino acid sequence of the transcription factor PbZIP 4 is shown in SEQ ID NO: 1.

The invention provides a coding gene of the transcription factor PbbZIP4, and the nucleotide sequence is shown in SEQ ID NO: 2.

The invention provides a primer pair for amplifying the coding gene, which comprises the nucleotide sequence shown in SEQ ID NO:3 and the nucleotide sequence of the forward primer is shown as SEQ ID NO: 4.

The invention provides application of the transcription factor PbbZIP4 affecting the sensitivity of plants to anthracnose or the coding gene serving as a target point in reducing or improving the sensitivity of plants to anthracnose.

The invention provides an application of an agent for inhibiting the function of the transcription factor PbbZIP4 or an agent for knocking out or silencing the expression of the coding gene in cultivating plant varieties resistant to anthrax virus.

Preferably, the agent that inhibits the function of the transcription factor PbbZIP4 comprises a transcription factor PbbZIP4 inhibitor.

Preferably, the agent that knocks out or silences expression of the encoding gene comprises the recombinant viral silencing vector pTRV2-PbbZIP4.

Preferably, the plant comprises a dicot.

Preferably, the dicotyledonous plant comprises pear.

The invention provides a method for cultivating anthracnose-resistant plant varieties, wherein the knockout or inhibition of the expression of a transcription factor PbZIP 4 in plants is realized, and the amino acid sequence of the transcription factor PbZIP 4 is shown as SEQ ID NO. 1.

The invention provides a transcription factor PbZIP 4 for influencing the sensitivity of plants to anthracnose, wherein the amino acid sequence of the transcription factor PbZIP 4 is shown in SEQ ID NO: i is shown. The test result shows that the transcription level of PbbZIP4 gradually decreases after the plant is treated by anthracnose, and the invention provides a transcription factor PbZIP 4 sensitive to anthracnose expression to play an important role in the anthracnose resistance of the plant. The transcription factor PbbZIP4 is transferred into pear callus, the obtained over-expression transgenic plant shows anthrax sensitivity, and the obtained transgenic plant can be used as an indication plant of anthrax indexes to early warn fungal infection of the plant; meanwhile, experiments for knocking out the PbbZIP4 gene in the plant or silencing and expressing the PbbZIP4 gene are also carried out, and the results show that the transformed plant shows the anthracnose infection resistance. The transcription factor PbbZIP4 provided by the invention has important significance for researching the anthracnose tolerance of breeding new anthracnose-tolerant varieties.

Drawings

FIG. 1 is a schematic diagram of a technical process of the present invention;

FIG. 2 shows the expression pattern of PbbZIP4 in anthrax treatment;

FIG. 3 shows subcellular localization of PbZIP 4 coding gene, wherein A is imaging of GFP gene under GFP field, bright field, fusion field; FIG. B is an image of the PbZIP 4-GFP encoding gene in GFP fields, bright fields, DAPI fields, and fusion fields;

FIG. 4 is an identification of PbZIP 4 overexpressing calli;

FIG. 5 is phenotypic and physiological data of PbZIP 4 overexpressing callus anthracnose treatment;

FIG. 6 is an identification of PbZIP 4-silenced plants;

FIG. 7 is phenotypic and physiological data of anthrax treatment of PbZIP 4-silenced plants.

Detailed Description

The invention provides a transcription factor PbZIP 4 for influencing the sensitivity of plants to anthracnose, wherein the amino acid sequence of the transcription factor PbZIP 4 is shown in SEQ ID NO:1 (MSVPIRAGDGEAKNPMLSISSSSSSLEQLQQVQQPSGSSSLRPPHPSLLLHSNTKNSNKLDVPWFWSLDDDDDDGDNVPEESDEDMFTVPDVEALPPSNNNINNAASTIANATSNNNNPDAQSGFPAKRRRGRNPVDKEYRRLKRLLRNRVSAQQARERKKVYVNDLESRAKELDDRNSKLEEKISTLVNENTMLRKVLMNTRPKVDESIEQKQGSVK).

The invention provides a coding gene of the transcription factor PbbZIP4, and the nucleotide sequence is shown in SEQ ID NO:2 (ATGTCAGTCCCAATCAGAGCAGGAGATGGTGAAGCCAAAAACCCCATGTTATCCATCTCCTCCTCCTCCTCCAGTTTGGAGCAACTACAGCAAGTACAGCAGCCATCTGGTTCTTCTTCCTTGAGGCCTCCTCATCCTTCTCTTCTTCTTCATAGTAACACCAAGAACAGTAACAAACTAGACGTTCCTTGGTTTTGGTCATTGGATGATGATGATGATGATGGTGATAATGTTCCAGAAGAGAGCGATGAAGATATGTTCACGGTTCCGGACGTGGAGGCGTTGCCCCCTTCTAATAATAATATTAATAATGCAGCCTCTACCATCGCCAATGCCACCAGTAACAACAACAATCCAGATGCCCAGTCTGGCTTTCCGGCCAAGCGCCGCCGAGGCCGAAATCCGGTCGATAAGGAGTACAGGCGACTGAAGAGATTGCTAAGGAACAGGGTGTCTGCTCAACAAGCCCGGGAGAGGAAAAAGGTTTACGTCAACGATCTGGAATCAAGAGCCAAAGAATTGGATGATAGGAATTCAAAGTTGGAAGAAAAGATCTCTACGCTTGTCAATGAAAACACCATGCTTCGAAAGGTTCTTATGAACACAAGGCCAAAAGTGGACGAAAGTATTGAGCAAAAGCAAGGATCAGTTAAGTAA).

The invention provides a primer pair for amplifying the coding gene, which comprises the nucleotide sequence shown in SEQ ID NO:3 and the nucleotide sequence of the forward primer is shown as SEQ ID NO: 4. The amplification reaction procedure of the primer pair is preferably: pre-denaturation at 94℃for 3min; denaturation at 94℃for 30s, annealing at 58℃for 90s, extension at 72℃for 90s,35 cycles, and extension at 72℃for 10min after the cycle is completed. The primer pair has good specificity for amplifying PbZIP 4.

The invention provides application of the transcription factor PbbZIP4 affecting the sensitivity of plants to anthracnose or the coding gene serving as a target point in reducing or improving the sensitivity of plants to anthracnose.

In the embodiment of the invention, the transcription level of the transcription factor PbZIP 4 gradually decreases after the plant is treated by anthracnose, which indicates that the transcription factor PbZIP 4 responds to the stress of anthracnose. Meanwhile, the transcription factor PbbZIP4 is transferred into pear callus, the transcription factor PbZIP 4 in the obtained transgenic plant is overexpressed, and compared with wild callus, the obtained transgenic callus can effectively weaken the anthracnose resistance of the transgenic plant and has larger cell damage. Meanwhile, the transcription factor PbbZIP4 is subjected to gene silencing treatment, and compared with a wild plant, the obtained transgenic plant can effectively enhance the anthracnose resistance and has less cell damage.

In the invention, a recombinant vector for over-expressing a transcription factor PbbZIP4 coding gene is also provided, and the recombinant vector can be transferred into plants under the mediation of agrobacterium to improve the sensitivity of the plants to anthracnose and can be used for indicating plants for environmental anthracnose. The recombinant vector is preferably p1300-PbbZIP4. The construction method of the recombinant vector is preferably to amplify a transcription factor PbZIP 4 coding gene, clone the gene to XbaI and BamHI sites in a p1300 vector, and obtain the positive recombinant vector through screening and identification. The primer pair for amplifying the transcription factor PbZIP 4 coding gene comprises a nucleotide sequence shown in SEQ ID NO:9 (acgggggactctagaATGTCAGTCCCAATCAGAGCAGG) and the forward primer and nucleotide sequence are set forth in SEQ ID NO:10 The reverse primer of (aaggttaccgaattctctagaCCACCAGTAACAACAACAATCCA). The annealing temperature of the amplification is preferably 58 ℃. The plant material preferably comprises callus tissue. The method for transferring the plant is not particularly limited, and transformation methods well known in the art may be employed.

In the invention, a recombinant vector for silencing expression of a transcription factor PbZIP 4 coding gene is also provided, the recombinant vector is transformed into a plant under the mediation of agrobacterium, the expression of the transcription factor PbZIP 4 in the obtained transgenic plant is reduced, and meanwhile, the resistance of the plant to anthracnose is improved. Therefore, the transgenic plant has good anthracnose stress resistance and can be used for cultivating new anthracnose-resistant plant varieties. The recombinant vector is preferably a recombinant viral silencing vector pTRV2-PbbZIP4. The construction method of the recombinant virus silencing vector pTRV2-PbbZIP4 is preferably to amplify the DNA sequence of the PbZIP 4, clone the DNA sequence into XbaI and SmaI of pTRV2, and obtain the positive recombinant virus silencing vector pTRV 2-PbZIP 4 through screening and identification. The primer for amplifying the DNA sequence of PbZIP 4 preferably comprises a nucleotide sequence shown in SEQ ID NO:11 and the nucleotide sequence of the forward primer is shown as SEQ ID NO: 12. The annealing temperature of the amplified DNA sequence of PbZIP 4 is preferably 58 ℃.

The invention also provides an application of the agent for inhibiting the function of the transcription factor PbbZIP4 or the agent for knocking out or silencing the expression of the coding gene in cultivating plant varieties resistant to anthrax virus.

In the present invention, the agent that inhibits the function of the transcription factor PbbZIP4 includes a transcription factor PbbZIP4 inhibitor. The plant preferably comprises a dicotyledonous plant. The dicotyledonous plant preferably comprises pear.

The invention provides a method for cultivating anthracnose-resistant plant varieties, wherein expression of a transcription factor PbZIP 4 in plants is knocked out or inhibited, and the amino acid sequence of the transcription factor PbZIP 4 is shown as SEQ ID NO. 1.

In the present invention, the method of knocking out or inhibiting expression of the factor PbbZIP4 in plants is the same as that described above and will not be described in detail herein.

The following examples are provided to illustrate in detail one transcription factor PbbZIP4, protein and its use which affect plant susceptibility to anthrax, but are not to be construed as limiting the scope of the invention.

Example 1

Cloning of full-length cDNA of birch-leaf pear transcription factor gene PbZIP 4 gene

A transcription factor gene PbZIP 4 is selected from the pyrus pyrifolia, primers are designed by adopting a primer premier 5.0 according to the sequence of the transcription factor gene PbZIP 4, and the full length of the transcription factor gene PbZIP 4 is amplified from the pyrus pyrifolia by using an RT-PCR method. The detailed steps are as follows: the synthesis of the first strand of cDNA was performed with reference to the protocol of the TIANGEN reverse transcription kit. The first strand cDNA thus obtained was used for amplification of the transcription factor gene PbZIP 4 gene. The total volume of the PCR reaction was 50. Mu.l, wherein 1. Mu.l of the Dunaliella cDNA, 2.5. Mu.l of each of the upstream and downstream primers (SEQ ID NO3: ATGTCAGTCCCAATCAGAGCAGG and SEQ ID NO4: CTTAACTGATCCTTGCTTTTGCTCA), 1. Mu.l of the enzyme, 25. Mu.l of Buffer, and 18. Mu.l of sterilized dd water were used. PCR was performed as follows: pre-denaturation at 94℃for 3min; denaturation at 94℃for 30s, annealing at 58℃for 90s, extension at 72℃for 90s,35 cycles, and extension at 72℃for 10min after the cycle is completed. After amplification, a single-band PCR product is generated, and after electrophoresis by 1% agarose gel, a target band is cut off, and a specific target band is recovered according to the using instruction steps of the gel recovery kit.

The recovered and purified product was ligated to pEASY-Bluntzero vector at a gene to vector molar ratio of 3:1 in the ligation system. The total reaction volume was 5. Mu.l, 4.5. Mu.l of the PCR-purified product, 0.5. Mu.1 vector, and ligated at 25℃for 30min, transformed into E.coli competent DH 5. Alpha. Using the heat shock method, PCR-verified with the target gene sequence primer and sequenced (by Shanghai Biotechnology Co., ltd.). Sequencing results are shown in SEQ ID NO: 2.

Example 2

qRT-PCR analysis of transcription factor PbbZIP4 under biological stress condition treatment for classificationResponse mode of PbZIP 4 gene in the Lepidotized birch-leaf pear to anthrax treatment, and analysis of the expression mode of the PbZIP 4 gene is carried out by using Real-time PCR technology. RNA was extracted by using Chengdu Fuji Biotechnology Co., ltd Plant Total RNA Isolation Kit Plus, and the synthesis of the first strand of DNA was performed by referring to the manual of TANGEN reverse transcription kit. In a 10. Mu.l reaction system are: mu.l of 2X SYBR Premix ExTaq, 0.1. Mu.l of cDNA, 0.4. Mu.l of primer (SEQ ID NO: 5)，TATGTTCACGGTTCCGGACGAnd SEQ ID NO:6，ATTGGCGATGGTAGAGGCTG) 4.5 μl of water. Tubulin is an internal reference gene, and the corresponding primer is SEQ ID NO:7(TGGGCTTTGCTCCTCTTAC)And SEQ ID NO:8(CCTTCGTGCTCATCTTACC). The procedure for real-time quantitative PCR is shown in table 1:

TABLE 1 real-time quantitative PCR procedure

The results are shown in FIG. 2. Sampling the seedlings of the pyrus ussuriensis at corresponding time points under the treatment of anthracnose spore suspension, and analyzing the relative expression quantity of the coding genes by adopting real-time quantitative PCR. It can be seen that PbbZIP4 has a very strong response to anthrax and that the expression level is in a down-regulating trend.

Example 3

Subcellular localization of the gene encoding PbbZIP4

Xba I and BamH I cleavage sites were added before and after the gene sequence according to the nucleotide sequence of the encoding gene of PbbZIP4 and the pJIT166-GFP vector map, respectively. The plasmid extracted from the target gene with correct sequencing result is used as a template, and amplified by a primer (SEQ ID NO:9 and SEQ ID NO: 10) added with enzyme cutting sites, and the PCR procedure is as follows: pre-denaturation at 94℃for 3min; denaturation at 94℃for 30s, annealing at 58℃for 60s, extension at 72℃for 90s,35 cycles; extending at 72℃for 10min. The stop codon TAG was removed 3' from the gene in order to allow fusion of the gene with GFP. After electrophoresis of the PCR product by 1% agarose gel, the target band was recovered by using a gel kit. The pJIT166-GFP vector plasmid was digested with Xba I and BamH I restriction enzymes, which were digested at 37℃for 4 hours, and then purified. The digested pJIT166-GFP vector was ligated with the gel-recovered PbZIP 4 fragment by recombinant ligase at 37℃for 30min, and transformed into E.coli competent DH5 a. And (3) detecting the converted bacterial liquid by using PCR, sending the bacterial liquid with positive PCR identification to a company for sequencing, extracting plasmids of bacterial liquid with correct sequencing results, and naming the obtained recombinant vector as GFP-PbZIP 4.

Agrobacterium-mediated transient transformation of tobacco cells: recombinant GFP-PbbZIP4 vector plasmid is transformed into a competent GV3101 of agrobacterium, and the activated agrobacterium containing recombinant plasmid is propagated in LB liquid medium containing 505mg/mL of Cana and 50mg/mL of rifampicin in a constant temperature shaker at 250rpm and 28 ℃. Centrifuge at 6000rpm for 10min and use the aggressive dye liquor (100mL:10mL 100mMMgCl) ₂ ，10mL 100mM MES，75μL200mM AS，ddH ₂ O80 mL) to OD ₆₀₀ 0.8. And (3) after incubation for 3-4 hours at room temperature, infecting tobacco leaf cells. The infected tobacco was incubated for 24h in the dark, then stained with DAPI for labeling nuclei, and pelleted. Photographs were taken using an inverted laser scanning confocal microscope (Zeiss LSM 780).

The results are shown in FIG. 3. FIG. 3 shows subcellular localization of the gene encoding PbZIP 4, wherein the localization of PbZIP 4 to the nucleus can be obtained according to the cell localization map.

Example 4

Genetic transformation of callus

1. Plant transformation vector construction

According to the multiple cloning site of the PCMBIA1300 vector and the coding region sequence of the PbbZIP4 gene, enzyme cutting sites XbaI and BamHI are added, and the primer 5.0 software is used for designing the upstream and downstream PCR primers (SEQ ID NO:9 and SEQ ID NO: 10) according to the general principle of primer design. PCR amplification was performed using the clone of the PbbZIP4 gene as a template. The annealing temperature of PCR amplification is 58 ℃, and the PCR reaction system and the amplification program are the same as those of PbZIP 4 gene cloning. And (5) performing gel purification and recovery after amplification. The PCMBIA1300 vector double cleavage reaction volume was 40. Mu.l, wherein: the vector plasmid containing PCMBIA1300 was 10. Mu.l, 10 XM buffer solution was 4. Mu.l, xbaI and BamHI were each 1. Mu.l, and double distilled water was added thereto at 24. Mu.l. And placing the mixture at 37 ℃ for enzyme digestion for 3-4 hours, and purifying and recycling the mixture. The molar ratio of the PbZIP 4 gene to the carrier PCMBIA1300 was 2:1 and the total reaction volume was 10. Mu.l. The method comprises the following steps: 10 Xbuffer 1. Mu.l, DNA recombinase 1. Mu.l, double digestion recovered PbZIP 4 gene 4ul, double digestion recovered PCMBIA1300 vector 2. Mu.l, double distilled water 2. Mu.l, and ligation product was obtained by reaction at 37℃for 30 min. The ligation product was transformed into E.coli DH 5. Alpha. And cultured in LB solid plates containing 50mg/L kanamycin for 16h. And (3) carrying out shaking after spotting on the screened positive clone, extracting plasmids, carrying out PCR identification, sequencing to determine that no coding frame mutation exists, obtaining recombinant clone containing the inserted target fragment, naming the recombinant clone as a p1300-PbbZIP4 recombinant vector, and introducing the recombinant vector p 1300-PbZIP 4 into agrobacterium GV3101 by using a freeze thawing method.

2. Agrobacterium-mediated callus genetic transformation was as follows:

(1) Culturing agrobacterium: streaking on a LB plate added with 50mg/L kanamycin and 50mg/L rifampicin, placing in a 28 ℃ incubator for culturing for 36-48 hours, scraping streak bacterial plaque, adding into a liquid MS (2.37 g/LMS+50g/L sucrose+0.1 mg/L IBA, pH=5.8) culture medium, transferring at 28 ℃ for 30min, shaking and culturing, and adding a surfactant sweet77 200 mu L/L for dip dyeing when the bacterial liquid concentration reaches OD=0.8-1.0.

(2) Dip dyeing: taking wild callus with good state, removing all seed pods, soaking in agrobacterium tumefaciens bacterial liquid for 1h, and culturing for 24h in dark place.

3. Screening of transgenic positive seedlings

Transferring the PbZIP 4 gene transferred callus obtained by the method to an MS selective medium containing 50mg/L hygromycin and 50mg/L timentin, culturing at 22 ℃ in dark, selecting callus with fast growth after one month of growth, and transplanting to a new medium for subculture.

3.1 transgenic callus DNA extraction

The method is used for obtaining the PbZIP 4 gene transferred callus, extracting DNA from the callus, designing primer gene inner primer, and carrying out PCR amplification to identify positive callus.

(1) Appropriate amount of callus was ground to powder with liquid nitrogen, then 500. Mu.l of CTAB preheated at 65℃ (100 mmol/L Tris-HCl pH8.0,1.5mmol/L NaCl,50mmol/L EDTA pH=8.0, 2% w/v CTAB, fully dissolved in 65℃water bath for use) and 10. Mu.l of beta-mercaptoethanol were added and mixed well.

(2) Heating in 65 deg.c water bath for 30min, taking out every 10min, and mixing; 10000g of the mixture is centrifuged for 10min at normal temperature; taking the supernatant, adding 500 mu l of chloroform isoamyl alcohol (the volume ratio of chloroform to isoamyl alcohol is 24:1), and mixing the mixture upside down;

(3) 10000g is centrifuged for 10min, 450 μl of supernatant is taken to a new 1.5ml centrifuge tube, 450 μl of isopropanol is added, and the mixture is inverted and mixed well.

(4) 10000g, centrifuging for 10min, discarding supernatant, rinsing with 1mL75% ethanol for 2 times, 10000g, centrifuging for 10min, thoroughly removing ethanol, and air drying on a super clean bench until DNA is colorless and transparent

(5) Adding 50 μl of ultrapure water, placing in a 65 ℃ incubator, dissolving for 40min, and performing gel detection.

3.2 Positive transgenic callus detection

PCR amplification was performed using primer gene specific primers. The reaction procedure and system are shown in Table 3 and Table 4, respectively. PCR was performed using the upstream and downstream primers of the gene (SEQ ID NO:3, ATGTCAGTCCCAAAGAGCAGG and SEQ ID NO:4, CTTAACTGATCTTGCTTTTGCTCA) and fragments of the expected size could be amplified in the selected transgenic lines, indicating that they were positive transgenic lines.

TABLE 3 PCR reaction procedure

Step (a)	94℃	58℃	72℃	4℃	Cycle number
						Step 1	3min				1
Step 2	30s	30s	55s		35
						Step 3			90s		1
Step 4			10min
						Step
5				30min

TABLE 4 PCR reaction System

As shown in FIG. 4, none of the transgenic lines OE1, OE2, OE3, OE4 were able to run out of the band of interest, whereas none of the WT indicated that OE1, OE2, OE3, OE4 were transgenic positive calli.

Example 5

Identification of resistance of PbbZIP4 transgenic plants

To identify whether PbbZIP4 transgenic calli are associated with anti-anthracnose stress, control and transgenic lines were subjected to anthracnose stress treatment. The results show that the pear callus which overexpresses PbZIP 4 is more sensitive to anthracnose than wild type callus, the growth speed of mycelium on the pear callus which overexpresses PbZIP 4 is obviously faster than that of wild type, catalase CAT, chitinase CHI, phenylalanine ammonia lyase PAL and polyphenol oxidase PPO are all physiological indexes which can measure the capability of resisting pathogenic bacteria of plants, after being infected by anthracnose, the CAT, CHI, PAL, PPO activity of the PbZIP 4 overexpresses callus is obviously lower than that of a control, and the activity of the PbZIP 4 overexpresses the callus is not different before being infected, so that the pear callus which overexpresses PbZIP 4 is more susceptible to anthracnose than the control (figure 5).

Example 6

Transient transformation of pear seedlings

1. Construction of virus-induced gene silencing vector

Viral silencing vectors were constructed as described in example 4. The double enzyme cutting sites of the virus silencing vector pTRV2 are XbaI and SmaI, the upstream and downstream primers (SEQ ID NO:11 and SEQ ID NO: 12) are designed by using primer 5.0 software according to the general principle of primer design, the PbZIP 4 gene is amplified (the reaction program is the same as that of Table 3) and inserted into the middle of the two enzyme cutting sites on the vector, so as to obtain a recombinant vector pTRV 2-PbZIP 4, and the recombinant vector pTRV 2-PbZIP 4 is transferred into the competence of agrobacterium GV 3101.

2. Virus-induced gene silencing of pear seedlings

(1) Culturing agrobacterium: the agrobacterium tumefaciens bacteria liquid stored in the ultralow temperature refrigerator is taken and cultured for 12 hours at 28 ℃ and 220rpm in LB liquid medium added with 50mg/L kanamycin and 50mg/L rifampicin. The cultured bacterial liquid was centrifuged at 6000g for 10min to collect bacterial cells, and the bacterial cells were inoculated with an infection liquid (10 mM MgCl) ₂ 10mM MES, 200mM acetosyringone, pH 5.6) to reach od=0.8-1.0;

(2) Bacterial liquid induction: placing the bacterial liquid with the adjusted OD value under a dark condition, and inducing for 4 hours at normal temperature at 100 rpm;

(3) Pear seedling injection: pTRV1 and pTRV2 bacterial solutions are mixed according to the ratio of 1:1 to be used as a control group, pTRV1 and pTRV 2-PbZIP 4 bacterial solutions are mixed according to the ratio of 1:1 to be used as an experimental group, and seedlings of the pyrus pyrifolia are respectively injected for 45 days, and the seedlings have consistent growth conditions and good health conditions.

3. Identification of positive seedlings for virus-induced gene silencing inhibition

And (3) carrying out dark treatment on the pear seedlings after injection at normal temperature for 12 hours, then recovering normal culture for 5 days, independently sampling each strain, extracting Miao Yangpin RNA of the control and experimental groups, detecting the structural integrity of the pear seedlings by running glue, measuring the concentration of the pear seedlings by using Nanodrop (the concentration is 200-1000 ng/. Mu.l), carrying out reverse transcription to obtain cDNA after adjusting the total amount of the RNA to 3 mu g, and carrying out amplification by using Tubulin of the pear as an internal reference. The Tubulin primer nucleotide sequence is:

tubulin forward primer: 5'-TGGGCTTTGCTCCTCTTAC-3' (SEQ ID NO: 7)

Tubulin reverse primer: 5'-CCTTCGTGCTCATCTTACC-3' (SEQ ID NO: 8)

As shown in FIG. 6, the amplified bands with Tubulin have consistent brightness, which indicates that the concentration of the reverse transcribed cDNA is the same, and then qRT-PCR detection is performed by using a PbZIP 4 specific primer, the expression level of the strain to be detected is analyzed, and three plants with lower expression level are selected as virus silencing positive strains according to the expression level of the PbZIP 4 gene.

The results are shown in FIG. 6, and the gene expression level of the gene silencing positive plants is detected by qRT-PCR using the gene specific primer and the internal reference primer Tubulin. This suggests that the PbZIP 4 gene of the virus-silenced Duke seedling positive line is silenced.

Example 7

Identification of PbZIP 4 virus-silenced plant resistance

To investigate the function of PbbZIP4 in coping with anthrax, pbbZIP 4-silenced plants and control ex vivo leaves were treated with sterile needle puncture followed by spraying spore suspension of anthrax and leaves were incubated in darkness at 25 ℃ for 4 days.

The results show that the leaf spot diameter of PbbZIP4 silenced plants is significantly smaller than the control and the leaf pore opening is smaller. The CAT, CHI, PAL, PPO content in the leaves of PbbZIP4 silenced plants after infection by anthracnose was significantly higher than that of the control, while only the PPO content was different before infection, but the difference was more significant after treatment. Taken together, the pears that silence PbbZIP4 by VIGS are more susceptible to anthrax infection than the control.

Analysis of the results shows that the PbbZIP4 gene is closely related to anthracnose resistance of plants, and the over-expressed PbZIP 4 gene can effectively enhance the active oxygen scavenging capacity of transgenic plants, maintain the ion balance and osmotic potential steady state in cells, thereby improving the anthracnose resistance of plants.

The foregoing is merely a preferred embodiment of the present invention and it should be noted that modifications and adaptations to those skilled in the art may be made without departing from the principles of the present invention, which are intended to be comprehended within the scope of the present invention.

Claims (10)

1. A transcription factor PbbZIP4 affecting plant susceptibility to anthracnose, wherein the transcription factor PbbZIP4 has an amino acid sequence as set forth in SEQ ID NO: 1.

2. The coding gene of the transcription factor PbZIP 4 as claimed in claim 1, wherein the nucleotide sequence is shown in SEQ ID NO: 2.

3. A primer pair for amplifying the coding gene of claim 2, comprising a nucleotide sequence set forth in SEQ ID NO:3 and the nucleotide sequence of the forward primer is shown as SEQ ID NO: 4.

4. Use of the transcription factor PbbZIP4 according to claim 1 affecting the susceptibility of plants to anthracnose or the coding gene according to claim 2 as a target for reducing or increasing the susceptibility of plants to anthracnose.

5. Use of an agent that inhibits the function of the transcription factor PbbZIP4 of claim 1 or an agent that knocks out or silences the expression of the coding gene of claim 2 for breeding plant varieties that are resistant to anthrax virus.

6. The use of claim 5, wherein the agent that inhibits the function of the transcription factor PbbZIP4 comprises a transcription factor PbbZIP4 inhibitor.

7. The use of claim 5, wherein the agent that knocks out or silences expression of the coding gene comprises recombinant viral silencing vector pTRV2-PbbZIP4.

8. The use according to any one of claims 4 to 7, wherein the plant comprises a dicotyledonous plant.

9. The use according to claim 8, wherein the dicotyledonous plant comprises pear.

10. A method for breeding anthracnose-resistant plant varieties, which is characterized in that the expression of a transcription factor PbZIP 4 in plants is knocked out or inhibited, wherein the amino acid sequence of the transcription factor PbZIP 4 is shown as SEQ ID NO. 1.

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