Strawberry powdery mildew resistance gene and application thereof
阅读说明：本技术 一种草莓白粉病抗性基因及其应用 (Strawberry powdery mildew resistance gene and application thereof ) 是由 张俊祥 史册 王岩 姚晋湘 张志宏 代红艳 于 2021-07-02 设计创作，主要内容包括：本发明涉及一种草莓白粉病抗性基因及其应用,属于分子生物学中的基因工程领域。本发明的发明人从森林草莓中克隆了FvMYB46基因,并通过稳定转基因技术证明降低FvMYB46基因表达能够提高草莓对白粉病的抗性,表明FvMYB46负调控草莓白粉病抗性,同时发现FvMYB46是通过负调控PALs基因的表达来增强草莓对白粉病的抗病性。(The invention relates to a strawberry powdery mildew resistance gene and application thereof, belonging to the field of genetic engineering in molecular biology. The inventor clones FvMYB46 gene from forest strawberries, and proves that the resistance of the strawberries to powdery mildew can be improved by reducing the expression of the FvMYB46 gene through a stable transgenic technology, so that the resistance of the strawberries to the powdery mildew is negatively regulated and controlled by FvMYB46, and the fact that the disease resistance of the strawberries to the powdery mildew is enhanced by negatively regulating and controlling the expression of PAL gene is found.)
1. A protein which is (a) or (b) or (c) below:
(a) a protein consisting of an amino acid sequence shown in a sequence 1 in a sequence table;
(b) a protein consisting of an amino acid sequence shown from the 1 st to the 335 th site of the N end of a sequence 1 in a sequence table;
(c) and (b) is protein which is related to the powdery mildew resistance of plants and is obtained by substituting and/or deleting and/or adding one or more amino acid residues in (a) or (b).
2. A gene encoding the protein of claim 1, wherein: the gene is a DNA molecule as described in any one of the following (1) to (4):
(1) the coding region is a DNA molecule shown as a sequence 2 in a sequence table;
(2) DNA molecules shown in 1 st to 1008 th sites from 5' end of a sequence 2 in a sequence table;
(3) a DNA molecule which hybridizes with the DNA sequence defined in (1) or (2) under strict conditions and codes a protein related to the powdery mildew resistance of plants;
(4) and (3) DNA molecules which have more than 90% of homology with the DNA sequences defined in (1) or (2) or (3) and encode proteins related to the powdery mildew resistance of plants.
3. A recombinant expression vector, expression cassette, transgenic cell line or recombinant bacterium comprising the gene of claim 2 or 3.
4. Use of a protein according to claim 1 or a gene according to claim 2 or 3 for modulating powdery mildew resistance in a plant.
5. A method for producing a transgenic plant, comprising introducing the gene of claim 2 or 3 into a plant of interest to obtain a transgenic plant; the powdery mildew resistance of the transgenic plant is higher than that of the target plant.
6. A method for improving powdery mildew resistance of a plant, which comprises increasing the activity and/or expression level of the protein of claim 1 in the plant of interest, thereby improving powdery mildew resistance of the plant.
7. Use of the protein of claim 1 or the gene of claim 2 or 3 or the method of claim 6 or 7 in plant breeding.
8. The use of claim 7, wherein: the breeding aims to breed plants with high powdery mildew resistance.
Technical Field
The invention belongs to the field of molecular biology and biotechnology, and particularly relates to a strawberry powdery mildew resistance gene and application thereof.
Background
Strawberry powdery mildew is caused by infection of the monocapsule shell of the collard, and hyphae are mainly distributed on two surfaces of leaves, petioles, flowers, pedicels and fruits. The strawberry powdery mildew has the characteristics of short time for infecting strawberry plants, short time for establishing a parasitic relationship with the plants, high frequency for infecting the plants and the like. The main factors influencing the strawberry powdery mildew occurrence process are as follows: growth status and growth environment of strawberry plants. The plants have different growth states and different infection effects of powdery mildew, and researches show that the infection efficiency on new leaves is far higher than that on old leaves. Generally, when the relative humidity in the environment reaches more than 80% and the temperature is 15-25 ℃, the strawberry plants are very easy to be infected with powdery mildew.
MYB transcription factors are one of the most abundant transcription factors in plants, and comprise a highly conserved DNA binding domain consisting of 51-52 amino acids in a repeat, and most MYB proteins comprise a MYB domain at the N-terminal and consist of a section of amino acid residues. The MYB family is divided into four major classes based on its domain number, including 1R-MYB, R2R3-MYB, 3R-MYB, and 4R-MYB proteins.
MYB transcription factors play an important role in growth, development and regulation of plants, form a large family of transcription factors, the functional research of the transcription factors is fast in the model plant Arabidopsis, and only part of the functions of the MYB transcription factor family in strawberry is analyzed. The current research shows that MYB46 mainly plays a role in regulating lignin synthesis, and is only found in Arabidopsis and wheat to participate in regulating diseases (Visente et al, 2011; Zheng et al, 2019), but the recognition degree of a strawberry MYB46 coding region sequence with Arabidopsis AtMYB16 is 45.81%, and the recognition degree with wheat TuMYB46 is 43.96%; the degree of identity of the amino acid sequence of the strawberry MYB46 with Arabidopsis AtMYB16 is 41.03%, and the degree of identity with wheat TuMYB46 is 40.78% (example 1). Therefore, compared with the coding regions and amino acid sequences of arabidopsis thaliana and wheat MYB46, the recognition degree of the strawberry MYB46 to arabidopsis thaliana and wheat MYB46 is low, and the function of strawberry is difficult to predict and analyze based on the functions of arabidopsis thaliana and wheat MYB 46. Therefore, is strawberry MYB46 involved in regulation of diseases such as powdery mildew? Whether the disease regulation mechanism of MYB46 in strawberries is the same as that of the crops is still unclear, so that the functions of the strawberry MYB46 can be accurately judged by analyzing the functions of the strawberries.
A large number of researches show that the cultivation and planting of disease-resistant varieties are the most economic, effective, safe and reliable ways for preventing and treating powdery mildew of strawberries, and the disease resistance identification, the resistance source screening and the disease-resistant new gene mining of the varieties are the basis for the research of improving the disease resistance of strawberries. The traditional crossbreeding takes long time and has large workload, and the accurate site-specific mutation is difficult to realize by mutagenesis means such as physics, chemistry, biology and the like. Therefore, the method has important significance for developing the powdery mildew resistance gene of the strawberry and analyzing the disease resistance mechanism of the strawberry and culturing a new powdery mildew resistance strawberry variety.
Disclosure of Invention
The invention provides a strawberry powdery mildew resistance gene and application thereof, wherein the coded amino acid sequence of the strawberry powdery mildew resistance gene FvMYB46 is shown as a sequence 1, and the nucleotide sequence of the coding region of the FvMYB46 gene is shown as a sequence 2.
The invention provides that silencing of the FvMYB46 gene by RNAi enhances the resistance of strawberries to powdery mildew.
The invention relates to an FvMYB46 gene which can enhance the disease resistance of strawberries to powdery mildew through negatively regulating the expression of PALs gene.
The technical problem of the invention can be solved by the following technical scheme:
(1) cloning of strawberry FvMYB46 gene, and selecting 335bp fragment for interference vector construction.
Extracting total RNA in strawberry leaves, and then reversely transcribing the RNA into cDNA;
using cDNA as a template, aiming at a conserved segment of an FvMYB46 gene, designing two pairs of specific primers MYB46-RNAi-F1 and MYB46-RNAi-R1, introducing an Xba1 enzyme digestion site on an upstream primer MYB46-RNAi-F1, and introducing a Sal1 enzyme digestion site on a downstream primer MYB 46-RNAi-R1; MYB46-RNAi-F2 and MYB46-RNAi-R2, Kpn1 enzyme cutting sites are introduced to an upstream primer MYB46-RNAi-F2, and Sac1 enzyme cutting sites are introduced to a downstream primer MYB 46-RNAi-R2; PCR amplification is carried out to obtain forward and reverse fragments of the FvMYB46 gene introduced with corresponding enzyme cutting sites;
wherein, the specific primer is:
MYB46-RNAi-F1：5’-CGCTCTAGATGGTCTCAAATTGCAGCACG-3’
MYB46-RNAi-R1：5’-CGCGTCGACGTTGTAGTACCCGCCATGCT-3’
MYB46-RNAi-F2：5’-CGCGGTACCGTTGTAGTACCCGCCATGCT-3’
MYB46-RNAi-R2：5’-CGCGAGCTCTGGTCTCAAATTGCAGCACG-3’
note: the first nine bases, namely CGCTCTAGA, CGCGTCGAC, CGCGGTACC and CGCGAGCTC, in primer sequences MYB46-RNAi-F1, MYB46-RNAi-R1, MYB46-RNAi-F2 and MYB46-RNAi-R2 are protective bases and enzyme digestion sites, are bases artificially introduced for constructing a vector, and do not belong to the gene sequence of FvMYB 46.
(2) RNAi interference vector construction:
(a) cloning a forward fragment of the FvMYB46 gene to a pRI101-RNAi vector by taking a recombinant plasmid pMD-T-FvMYB46 as a template and MYB46-RNAi-F1 and MYB46-RNAi-R1 as primers;
(b) after the sequencing is correct, the reverse fragment of the FvMYB46 gene is cloned to a pRI101-RNAi vector successfully connected with the forward fragment by taking MYB46-RNAi-F2 and MYB46-RNAi-R2 as primers, and the vector is named as RNAi-FvMYB 46.
(3) Genetic transformation and cultivation of transgenic strawberries: extracting constructed RNAi interference vector RNAi-FvMYB46 plasmid DNA, transferring agrobacterium to the RNAi interference vector by a freeze-thaw method, genetically transforming strawberry by a leaf disc method, and obtaining a positive transgenic plant by molecular detection, wherein the strawberry is diploid forest strawberry Ruegen.
(4) And (3) identifying powdery mildew: selecting strawberry powdery mildew strain naturally occurring in strawberry test base of Shenyang agricultural university, inoculating by spraying method, wherein spore suspension concentration is 1 × 106And (2) inoculating a control and RNAi-FvMYB46 transgenic strawberry leaves respectively per mL, identifying the damage condition of the leaves by adopting a trypan blue staining method, and finding that the FvMYB46 gene negatively regulates the resistance of strawberry powdery mildew.
(5) Regulation of FvPALs gene by FvMYB46 protein: the single-hybrid experiment of yeast and the luciferase experiment of firefly show that the FvMYB46 negatively regulates the expression of the PAL genes to enhance the disease resistance of strawberries to powdery mildew.
The invention has the beneficial effects that:
1. according to the invention, the function analysis reveals that the resistance of the strawberry to powdery mildew can be obviously improved by reducing the expression of the strawberry FvMYB46, and a foundation is laid for creating a new powdery mildew resistant strawberry germplasm through gene editing in the future.
2. The invention discovers that FvMYB46 regulates and controls powdery mildew resistance through negatively regulating and controlling PALs genes for the first time.
Drawings
FIG. 1 shows the result of amplification of the sequence of the coding region of the FvMYB46 gene.
FIG. 2 is a PCR identification electrophoretogram of transgenic plants;
wherein, M: DL2000 Marker; transgenic plants and control plants are labeled as shown.
FIG. 3 shows the relative expression level of FvMYB46 gene in RNAi-FvMYB46 transgenic plant
FIG. 4 shows RNAi interfering with the phenotype of the FvMYB46 transgenic plant.
FIG. 5FvMYB46 interfered with inoculation and trypan blue staining of powdery mildew of strawberry plants.
FIG. 6 analysis of the gene expression of the strawberry FvPALs, members FvPAL1.1 and FvPAL 1.2.
FIG. 7 Yeast Single-hybrid assay the regulation of FvPAL1.1 by FvMYB 46.
FIG. 8 firefly fluorescence reporter assay analyzes the regulation of FvPAL1.1 and FvPAL1.2 by FvMYB 46.
Detailed Description
In order to further illustrate the invention, but not to limit it, reference is made to the following examples. The experimental procedures described in the following examples are conventional unless otherwise specified. The kit biomaterials are commercially available without special instructions.
Example 1: cloning of strawberry FvMYB46 gene and comparative analysis of the gene and Arabidopsis thaliana and wheat MYB46 sequences
(1) Cloning of strawberry FvMYB46 Gene
Diploid forest strawberry 'Ruegen' is used as a test material, and the material is grown in a greenhouse.
RNA extraction: and (3) extracting the total RNA of the test material by using a CTAB method, wherein the whole operation process is carried out according to the RNA extraction process of the CTAB method, and then carrying out reverse transcription by using the total RNA as a template to obtain a first cDNA chain.
Cloning of the genes: the first strand of the reverse transcribed fruit cDNA is used as a template, primers FvMYB46-F and FvMYB46-R are used for PCR amplification, a PCR product is recovered, and a target fragment of 1008bp is obtained and is shown in figure 1.
FvMYB46-F：GCTCTAGAATGAGGAAGCCGGAACCCTA；
FvMYB46-R：CGGGATCCTCAACTCTGGTAGTCAAGTAAAG。
After recovering the target fragment, the gel was ligated to pMD18-T vector (purchased from TaKaRa Co.), transformed into E.coli competent cell TOP10 (purchased from Beijing Tiangen Biotechnology Co., Ltd.), screened for positive single colonies, extracted for plasmids, and sequenced as shown in sequence 2.
(2) Comparison analysis of strawberry FvMYB46 gene and Arabidopsis thaliana and wheat MYB46 sequence
The recognition degree of the sequence of the coding region of the strawberry MYB46 to Arabidopsis AtMYB16 is 45.81 percent, and the recognition degree to wheat TuMYB46 is 43.96 percent; the degree of recognition of the amino acid sequence of the strawberry MYB46 with Arabidopsis AtMYB16 is 41.03%, and the degree of recognition with wheat TuMYB46 is 40.78%.
Example 2: RNAi interference FvMYB46 transformation forest strawberry and gene function identification thereof
1. Construction of plant interference expression vector RNAi-FvMYB 46.
(1) Primer 5.0 software analysis is utilized to design primers and amplify FvMYB46 positive and negative fragments
MYB46-RNAi-F1：5’-CGCTCTAGATGGTCTCAAATTGCAGCACG-3’
MYB46-RNAi-R1：5’-CGCGTCGACGTTGTAGTACCCGCCATGCT-3’
MYB46-RNAi-F2：5’-CGCGGTACCGTTGTAGTACCCGCCATGCT-3’
MYB46-RNAi-R2：5’-CGCGAGCTCTGGTCTCAAATTGCAGCACG-3’
(2) Performing PCR reaction by taking PMD-T-FvMYB46 as a template;
the PCR reaction system is as follows: adding 0.2 muL of Ex Taq enzyme, 2 muL of 10 XEx Buffer, 1.6 muL of dNTPs (2.5 mmol. L-1) and 0.5 muL of forward and reverse primers into 1 muL of PMD-T-FvMYB46 plasmid, and finally supplementing the mixture to 20 muL with water;
PCR program 955 min; 30s at 95 ℃, 30s at 58 ℃, 90s at 72 ℃ and 35 cycles; extending for 7min at 72 ℃, and storing at 4 ℃; carrying out agarose gel electrophoresis on the product;
(3) the nucleic acid was recovered using a nucleic acid purification kit (Takara).
(4) After recovery, the recovered PCR product was cleaved with restriction enzymes XbaI and SalI, respectively, with pRI101-RNAi vector, purified and recovered again after cleavage, and then ligated with the vector.
The PCR reaction system is as follows: mu.L of the recovered PCR product, 1. mu. L T4DNA ligase, 10 XT41. mu.L of DNA ligase Buffer, T41 mu L of carrier;
PCR reaction procedure: connecting for 16h at 16 ℃;
(5) transformation of E.coli into the plasmid containing Amp (60. mu.g.mL)-1) The cultured cells were cultured on the LB medium plate at 37 ℃ for 12 to 16 hours. Individual white clones were picked and plated on new Amp-containing medium (60. mu.g.mL)-1) The obtained product was cultured at 37 ℃ for 12 to 16 hours on an LB medium plate (second rotation), and then colony PCR amplification was performed. After the strains which are verified to be correct are cultured, the strains are sent to the Jinzhi corporation of Suzhou for sequencing, and the strains are named as RNAi-FvMYB 46-positive.
(6) MYB46-RNAi-F2 and MYB46-RNAi-R2 are used as primers, PMD-T-FvMYB46 is used as a template, PCR reaction is carried out, Kpn I and SacI restriction endonucleases are used for carrying out enzyme digestion on the recovered PCR product and an RNAi-FvMYB 46-positive vector respectively, purification and recovery are carried out again after enzyme digestion, then vector connection is carried out, after the vector connection is finished, escherichia coli transformation, colony PCR verification and recombinant plasmid extraction are carried out, names are marked after extraction, the product is handed to a biological company Limited for inspection, DNAMAN software is used for analysis, and the RNAi-FvMYB46 recombinant vector is obtained after the analysis result is correct.
RNAi interference FvMYB46 transformation forest strawberry and gene function identification thereof
(1) Transformation of Agrobacterium
The RNAi-FvMYB46 plasmid is introduced into Agrobacterium GV3101, and the specific operation steps are as follows:
(a) taking out the two tubes of GV3101 Agrobacterium tumefaciens competence from the ultra-low temperature refrigerator, placing on ice, standing for 3min, opening the water bath, setting the temperature to 37 ℃, sucking 10 μ l of RNAi-FvMYB46 plasmid, adding into the Agrobacterium competence, and standing on ice for 10 min.
(b) After standing, putting into liquid nitrogen, and quickly freezing for 1 min.
(c) After the freezing, the mixture is quickly put into a water bath kettle at 37 ℃ and thermally shocked for 5 min.
(d) After completion of the heat shock, the mixture was transferred to ice and left to stand for 2 min. Adding 900 μ l YEP liquid culture medium (formula shown in appendix) into the centrifuge tube in an ultraclean workbench, sealing the opening of the centrifuge tube with a sealing film, placing in a constant-temperature shaking incubator at 28 deg.C, shaking for 5h, and setting the incubator at 180 rpm/min.
(e) And after the shaking culture is finished, centrifuging for 5min to collect thalli, and setting the rotating speed of a centrifuge to be 5000 rpm/min.
(f) Keeping 100 μ l of supernatant in a clean bench, suspending the thallus, mixing, spreading on YEP solid culture medium (formula shown in appendix), and culturing in 28 deg.C incubator for 2-3 d. And after full single colonies grow, carrying out PCR verification on the single colonies.
(2) Agrobacterium mediated strawberry genetic transformation
(a) Preparation of infection bacterial liquid
Taking a sterilized 150mL triangular flask, adding 50mL YEP liquid culture medium, 400 μ L rifampicin (25mg/L) and 25 μ L kanamycin (100mg/L), labeling the flask body, adding correctly verified thalli, placing in a constant-temperature shaking incubator at 28 ℃, and fully shaking and culturing for 8-12h until the color of the bacterial liquid becomes orange, and the bacterial liquid is turbid and free of impurities. And (3) reserving 2mL of bacterial liquid, adding 50mL of YEP liquid culture medium again, continuously placing the bacterial liquid in a constant-temperature shaking incubator at 28 ℃ for about 5h, measuring the OD600 of the bacterial liquid to be about 0.5 by using a spectrophotometer, collecting thalli at the moment, transferring the thalli into a 50mL centrifuge tube, setting the rotation speed of the centrifuge to be 5500rpm/min, centrifuging for 5min, pouring out the supernatant in a super workbench, resuspending the thalli by using sterilized MS suspension, measuring the OD600 of the thalli to be between 0.4 and 0.6 by using the spectrophotometer, and placing the suspension aside for later use.
(b) Infecting explants
Cutting leaves of diploid 'Ruegen' tissue culture strawberry seedlings with green color and good state into small blocks of 2-4mm, quickly putting the cut explants into liquid co-culture to avoid wilting, pouring the liquid co-culture after the leaves are completely cut, transferring the leaves into a sterilized 100mL small triangular flask, adding acetosyringone into the prepared suspension, turning upside down and uniformly mixing, pouring into the small triangular flask (the process needs to be quick), soaking the explants for 8min, shaking once every 2min to ensure that the bacterial liquid fully contacts wounds of the explants. After soaking, the bacterial solution was poured off, the leaves were transferred to filter paper with tweezers, the bacterial solution was blotted dry, the leaves were placed back up and evenly on solid co-culture medium (with filter paper added).
(c) Cultivation of explants
Solid co-culture: placing in dark condition, setting temperature at 22-25 deg.C, and culturing for 3 d.
And (3) delayed culture: and after solid co-culture for 3d, replacing the culture medium with a delay medium, reversing the leaves to enable the leaf back to face downwards, uniformly paving the leaf back on the delay medium, and culturing for 4d at the temperature of 22-25 ℃ under the dark condition.
Selecting and culturing: and after the culture of the delay culture medium is carried out for 4 days, the selection culture medium is changed, the leaves face downwards, the selection culture medium is uniformly paved on the selection culture medium, the selection culture medium is placed under the dark condition at the temperature of 22-25 ℃, and the explants with callus are transferred to the light at the temperature of 22-25 ℃ until the explants are observed to grow plump yellow green callus, so that adventitious buds of the explants are promoted to be extracted.
Differentiation culture: when the adventitious buds in the selective medium grow into plantlets, the names of the strains are marked, and the plantlets are transferred to a differentiation medium for differentiation culture, so that the number of the plantlets is increased rapidly.
(3) PCR identification and gene function preliminary identification:
(a) when the resistant seedlings grow to 7-8 leaves, extracting DNA (deoxyribonucleic acid) of young leaves of strawberry transgenic plants (CTAB method), taking clear water and 'Ruegen' strawberry plants as negative controls, taking RNAi-FvMYB46 plasmid as a positive control, and performing PCR amplification and agarose gel electrophoresis test, wherein the result is shown in figure 2, the transgenic plants have 315bp fragments, and the control plants do not, which indicates that the strawberry FvMYB46 silencing vector is successfully introduced into the strawberries.
(b) When the resistant seedlings grow to 7-8 leaves, extracting RNA of young leaves of strawberry transgenic plants, and detecting whether the target gene is transferred into the young leaves by RT-PCR
The upstream primer is 5'-GCCGGAACCCTATGCTGTAA-3', and the upstream primer is,
the downstream primer is 5'-GATCCAACGAAGCCTGCAAC-3', and the primer is,
the results are shown in FIG. 3, and it can be seen from FIG. 3 that the 350bp fragment appeared in the transgenic plant, whereas the control plant did not, indicating that the strawberry FvMYB46 silencing vector was successfully introduced into the strawberry.
(4) Phenotypic observations on transgenic strawberries
It was observed that the stem color of the transgenic plants was redder compared to the control plants (FIG. 5), but there was no significant change in flower, leaf size and plant height (FIG. 4).
Example 3: FvMYB46 interference on inoculation and trypan blue staining observation of powdery mildew of strawberry plants
Selecting naturally-occurring strawberry powdery mildew leaves in strawberry test base of Shenyang agricultural university, removing surface impurities, brushing powdery mildew spores into clean sterile water by using a small-size brush, and calculating the concentration of spore suspension to be 1 × 10 by using a blood counting chamber6And uniformly spraying the spore suspension on transgenic strawberry leaves of a control strain and an RNAi-FvMYB46 strain by using a handheld sprayer to ensure that the surfaces of the leaves are fully covered with the spore suspension, placing the leaves in a climatic incubator after inoculation, and keeping the temperature at night at 18 ℃, the temperature at day at about 25 ℃ and the relative humidity at 85%. The observed plant status after 10d is shown in FIG. 5. Compared with the transgenic strawberry leaves of three strains of RNAi-FvMYB46, powdery mildew spores are not obvious, and then the transgenic strawberry leaves of the three strains of the control and RNAi-FvMYB46 are observed to be damaged by a trypan blue staining methodIn the situation, compared with the transgenic strawberry of three strains of RNAi-FvMYB46, the blue color of the leaves is lighter, which indicates that the leaves are not damaged seriously compared with the control, and the results show that the FvMYB46 gene negatively regulates the resistance of strawberry powdery mildew.
Example 4: regulation and control analysis of FvMYB46 protein on FvPALs gene
1. Analysis of Gene expression of strawberry FvPALs Member FvPAL1.1 and FvPAL1.2
In order to verify the regulation and control effect of FvMYB46 on FvPAL1.1 and FvPAL1.2, the expression levels of the FvPAL1.1 and FvPAL1.2 genes in a control plant and an interference transgenic plant are measured by real-time fluorescent quantitative PCR. Compared with the control, the relative expression amount of the FvPAL1.1 and FvPAL1.2 genes is increased in the interference transgenic plants (figure 6). After inoculation with erysiphe necator, the expression levels of both fvpal1.1 and fvpal1.2 genes were elevated compared to the control (fig. 6). The above experiments demonstrate that FvMYB46 negatively regulates the expression of fvpall 1.1 and fvpall 1.2, and that fvpall 1.1 and fvpall 1.2 are induced by powdery mildew.
2. Regulation of FvPAL1.1 by Yeast Single-hybrid analysis FvMYB46
To verify whether FvMYB46 was able to bind to the promoter of fvpal1.1, a yeast single-hybrid assay was performed. pAbAi-proFvPAL1.1 is firstly introduced into yeast cells, cultured on an SD/-Ura culture medium, and screened out to have an ABA concentration of 150 ug/L. The pGAD424-FvMYB46 vector was then introduced into the yeast competence constructed from the pAbAi-pro PAL1.1 vector, and yeast transformed twice were able to grow on SD/-Leu medium at 150ug/L AbA. pGAD424 as a negative control was found to be unable to grow at no load on AbA-added SD/-Leu medium, while yeast cells introduced into pGAD424-FvMYB46 grew normally (FIG. 7), indicating that FvMYB46 itself could bind to the promoter of FvPAL1.1, thereby enhancing the resistance of the plant to powdery mildew.
3. Firefly fluorescence report assay analysis of FvMYB46 Regulation of FvPAL1.1 and FvPAL1.2
FvMYB46 can be combined on the promoter of FvPAL1.1, and in order to further verify the regulation relationship between FvMYB46 and PALs, the influence of FvMYB46 on the expression of the PALs gene is further analyzed by using a tobacco transient expression technology. pRI101-AN-FvMYB46 was used as AN effector in this experiment. Meanwhile, pGreenII0800-LUC-proFvPAL1.1 and pGreenII0800-LUC-proFvPAL1.2 are used as reporters in the test, and the results show that: the fluorescent signal of pGreenII0800-LUC + pRI101 empty vector combination as a negative control was weak, the fluorescence of the combination in which the FvMYB46 transcription factor participates in the proFvPAL1.1 promoter was weak, and the fluorescence of the combination in which no FvMYB46 transcription factor participates was strong (FIG. 8). Indicating that the FvMYB46 transcription factor negatively regulates the expression of the FvPAL1.1 gene.
Sequence listing
<110> Shenyang agriculture university
<120> strawberry powdery mildew resistance gene and application thereof
<130> 2
<141> 2021-07-02
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<170> SIPOSequenceListing 1.0
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<213> forest strawberry (Fragaria visco)
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Met Arg Lys Pro Glu Pro Tyr Ala Val Ser Asn Thr Gly Gly Lys Asn
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Asn Lys Asn Asn Ser Gly Met Ser Ser Asn Asn Lys Leu Arg Lys Gly
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Leu Trp Ser Pro Glu Glu Asp Glu Lys Leu Met Arg Tyr Met Leu Asn
35 40 45
Asn Gly Gln Gly Cys Trp Ser Asp Val Ala Arg Asn Ala Gly Leu Glu
50 55 60
Arg Cys Gly Lys Ser Cys Arg Leu Arg Trp Ile Asn Tyr Leu Arg Pro
65 70 75 80
Asp Leu Lys Arg Gly Ala Phe Ser Pro Gln Glu Glu Asp Leu Ile Ile
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His Phe His Ser Leu Leu Gly Asn Arg Trp Ser Gln Ile Ala Ala Arg
100 105 110
Leu Pro Gly Arg Thr Asp Asn Glu Ile Lys Asn Phe Trp Asn Ser Thr
115 120 125
Ile Lys Lys Arg Leu Lys Ile Leu Ser Ser Ser Ser Ser Thr Ala Ser
130 135 140
Pro Asn Ala Ser Asp Ser Ser Ser Glu Gln Pro Asn Asn Asn Asp Phe
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Phe Ala Ala Ala Gly Gly Gly Gly Gly Phe Met Asn Ile Ile Pro Pro
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Ser Met Met Pro Ile Tyr Pro Asp Ser Ser Met Gln Ala Thr Ser Leu
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Ile Asn His Met Phe Asp Pro Phe Pro Met Val Glu His Gly Gly Tyr
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Tyr Asn Asn Gly Asn Pro Cys Thr Ala Gln Ile Gly Ser Val Gly Ser
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Gly Ser Ser Thr Gly Asp Asp Cys Gly Phe Gly Gln Asn Ile Asp Val
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Phe Gly Asn Val Asn Met Arg Val Glu Glu Asp Ile Tyr Val Pro Pro
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Leu Glu Ser Val Ser Thr Ile Asp His Asp Gln Asn Asn Leu Lys Thr
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Glu Thr Ile Phe Tyr Asp His Asn Asn Tyr Tyr Ser Asn Thr Asn Asn
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Ile Ile Lys Gly Glu Asn Met Ile Gly Val Gly Asn Tyr Phe Glu Asp
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Asp His Gln Asp Gln Gly Leu Thr Met Gly Glu Trp Asp Leu Glu Asp
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aagctgatga ggtacatgct caacaatggc caaggctgct ggagcgacgt ggcgagaaat 180
gctggccttg agaggtgcgg aaagagttgc aggcttcgtt ggatcaatta cttgagacct 240
gaccttaaga gaggtgcctt ttctccccaa gaagaagacc tcatcatcca tttccattcc 300
cttcttggca acaggtggtc tcaaattgca gcacgcttgc caggacgaac tgataacgaa 360
atcaagaact tctggaattc gaccataaag aaacgtctaa aaattctgtc ctcctcctcc 420
tccactgcct caccaaacgc gagtgattct tcctcggagc agcctaacaa caatgacttc 480
ttcgccgcgg ccggaggagg aggagggttc atgaacatca ttccaccgtc aatgatgccc 540
atttaccctg attcatcaat gcaagccacc tccctgatca accacatgtt tgatcccttt 600
ccgatggtcg agcatggcgg gtactacaac aatggaaacc catgcactgc tcagattggt 660
tcggtcggca gtggtagcag tactggtgat gattgtggct ttggacaaaa tattgacgtg 720
tttgggaacg tcaatatgag agtagaagaa gacatctatg tgcctcccct agagagcgta 780
agcaccattg accacgatca aaataatctc aaaactgaaa cgatcttcta tgatcataac 840
aattactaca gtaacactaa caatatcatc aagggtgaaa acatgattgg ggttgggaat 900
tactttgaag atgatcatca ggatcaaggg ttaacaatgg gggagtggga cttggaggat 960
ctgatgaaag atgtttcctc ctttccttta cttgactacc agagttga 1008