CN116426548B - Tartary buckwheat cytochrome P450 enzyme FtCYP94C1, and coding gene and application thereof - Google Patents

Tartary buckwheat cytochrome P450 enzyme FtCYP94C1, and coding gene and application thereof Download PDF

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CN116426548B
CN116426548B CN202310164199.0A CN202310164199A CN116426548B CN 116426548 B CN116426548 B CN 116426548B CN 202310164199 A CN202310164199 A CN 202310164199A CN 116426548 B CN116426548 B CN 116426548B
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CN116426548A (en
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周美亮
关超男
卢翔
张凯旋
何毓琦
李光胜
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Sanya National Academy Of Southern Propagation Chinese Academy Of Agricultural Sciences
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Abstract

The invention provides a tartary buckwheat cytochrome P450 enzyme FtCYP94C1, and a coding gene and application thereof, and belongs to the technical field of genetic engineering. The coding gene is FtCYP94C1 gene SEQ ID NO.1, and the coded amino acid sequence is SEQ ID NO.2. According to the invention, by constructing plants over-expressing the FtCYP94C1 gene, the fact that the FtCYP94C1 gene can resist rhizoctonia solani infection is verified, a novel technical scheme is provided for solving the problem of plant rhizoctonia solani, and the method has a good practical prospect.

Description

Tartary buckwheat cytochrome P450 enzyme FtCYP94C1, and coding gene and application thereof
Technical Field
The invention belongs to the technical field of genetic engineering, and particularly relates to an FtCYP94C1 gene and application thereof in damping-off resistance.
Background
Tartary buckwheat (Fagopyrum tataricum), also known as tartary buckwheat, is a nutritionally balanced gluten-free crop rich in bioactive flavonoids and is widely planted in Asia and Europe. Rutin contained in Fagopyrum tataricum is used as an antioxidant, and can reduce cholesterol, thrombosis and hypertension, and enhance vascular toughness. Stem rot of buckwheat caused by the soil-borne plant pathogen rhizoctonia solani (Rhizoctonia solani, r.solani) is one of the most damaging diseases of buckwheat, which causes death of buckwheat seedlings, resulting in serious loss of buckwheat yield. Rhizoctonia solani is an infectious necrotic pathogen that can infect over 250 plants, including monocots and dicots. At present, the traditional method for preventing and treating the damping-off is chemical drug spraying to inhibit the growth of pathogenic bacteria, however, the long-term use can lead to the problems of reduced soil microorganism population, soil hardening, reduced fertility, increased later repair difficulty, pesticide residue, pathogenic bacteria drug resistance and the like. Screening of resistant germplasm resources and excellent gene mining are key to disease resistance breeding, and researches on resistance breeding of buckwheat in damping off disease are freshly reported.
In complex stress environments, plants develop various defense strategies to protect themselves from pathogen infection. The plant hormone Jasmonic Acid (JA) is a chemical substance that plays a key role in activating the plant's defense against pathogen attack. In order to coordinate growth and defense against pathogens or herbivores, plants synthesize jasmonic acid when they cope with these stresses, promote the synthesis of JA-Ile (a derivative of jasmonic acid and isoleucine) and induce plant defense-related gene expression. There is no disclosure in the prior art of JA-induced related damping-off resistance genes in tartary buckwheat. Cytochrome P450 (CYP 450) is a monooxygenase coded by a super gene family in plants, can participate in various catalytic reactions, and plays an important role in biological defense response. In the research, an important JA signal transduction gene FtCYP94C1 is obtained through gene cloning, encodes an enzyme responsible for oxidizing JA-Ile into 12OH-JA-Ile, systematically explores the damping-off resistance function of the enzyme, and fills the blank of research on the damping-off resistance function of cytochrome P450 enzyme in tartary buckwheat.
Disclosure of Invention
The invention aims at cloning a FtCYP94C1 gene and exploring the application thereof in resisting plant damping-off. The invention discovers that the gene FtCYP94C1 of the tartary buckwheat is homologous with the CYP94C1 of the arabidopsis thaliana, and plays a positive role in resisting R.solani. Overexpression of FtCYP94C1 enhances disease resistance of plants, and the content of flavonoid metabolites in the FtCYP94C1 Arabidopsis is higher than that of wild type. Thus, these results indicate that FtCYP94C1 is a positive regulator in the resistance of tartary buckwheat to rhizoctonia solani.
In one aspect, the invention provides a FtCYP94C1 gene.
The nucleotide sequence of the gene is SEQ ID NO.1, and the encoded amino acid sequence is SEQ ID NO.2.
The gene is used for encoding a tartary buckwheat cytochrome P450 enzyme FtCYP94C1.
The invention also provides an expression vector containing the FtCYP94C1 gene and a genetically engineered cell containing the expression vector.
In another aspect, the invention provides the use of the FtCYP94C1 gene in combating rhizoctonia solani infection.
Preferably, the application is damping off.
Preferably, the application object is a plant.
Further preferably, the application object is a buckwheat plant.
Furthermore, the application object is tartary buckwheat.
In particular, the application may be the breeding of buckwheat plants, preferably the breeding of tartary buckwheat.
In yet another aspect, the invention provides the use of the FtCYP94C1 gene or a vector expressing the FtCYP94C1 gene or a cell expressing the FtCYP94C1 gene in the construction of a transgenic plant.
Specifically, the plants overexpress the FtCYP94C1 gene.
In particular, the transgenic plants have damping off resistance.
In yet another aspect, the invention provides a method of constructing a transgenic plant.
The construction method comprises the step of over-expressing FtCYP94C1 genes in plants.
Preferably, the construction method comprises inserting FtCYP94C1 gene into an expression empty vector, and infecting plants after host cell transformation to obtain transgenic plants.
Preferably, the expression empty vector is a plant expression vector, and further preferably, the expression empty vector is pCAMBIA 1302.
Preferably, the host cell is a genetically engineered cell with an expression function, preferably agrobacterium, more preferably agrobacterium GV3101.
Preferably, the host cell is E.coli BL21, more preferably E.coli BL21 (DE 3).
Preferably, the construction method comprises the following steps:
(1) Cloning to obtain a transcription factor FtCYP94C1 gene;
(2) Operably constructing the FtCYP94C1 gene into an expression control sequence to form a plant expression vector containing the FtCYP94C1 gene;
(3) Transforming agrobacterium tumefaciens GV3101 with the plant expression vector containing the FtCYP94C1 gene obtained in the step (2) to obtain a agrobacterium strain containing the FtCYP94C1 gene for transformation;
(4) And (3) utilizing the agrobacterium strain constructed in the step (3) to genetically transform the target plant.
Preferably, the target plant is tartary buckwheat.
Preferably, the method for cloning the FtCYP94C1 gene is PCR.
Preferably, the PCR primer is 1302-FtCYP94C 1F/R, and the sequence is SEQ ID NO.5-6.
Preferably, the PCR is performed at 95 ℃ for 3min;95℃30s,58℃30s,72℃90s,31 cycles.
In some embodiments, the construction method comprises the steps of:
(1) Cloning to obtain a transcription factor FtCYP94C1 gene;
(2) Operably constructing the FtCYP94C1 gene into an expression control sequence to form a plant expression vector containing the FtCYP94C1 gene;
(3) Transforming agrobacterium tumefaciens GV3101 with the plant expression vector containing the FtCYP94C1 gene obtained in the step (2) to obtain an agrobacterium strain containing the FtCYP94C1 gene for transforming tartary buckwheat;
(4) And genetic transformation of Arabidopsis thaliana by using the Agrobacterium strain constructed in the step (3).
In some embodiments, the construction method includes the following steps (1) are: sterilizing with 1% sodium hypochlorite solution for 10min and 75% ethanol for 2min, cleaning with sterile water until the water is clear, sterilizing the seeds of Fagopyrum tataricum, and culturing with MS culture medium to obtain sterile seedling of Fagopyrum tataricum.
The aseptic seedling is characterized in that seeds are placed on sterilized filter paper to absorb water and planted on an MS culture medium; the culture condition is that the temperature is 22-25 ℃, the photoperiod is 16h/8h, the humidity is 75-80%, and the culture is carried out for 2-4 weeks.
The invention has the beneficial effects that:
the invention clones a FtCYP94C1 gene and explores the effect of the FtCYP94C1 gene in the anti-damping-off of tartary buckwheat; and the FtCYP94C1 gene is genetically transformed into the Arabidopsis thaliana by utilizing a genetic engineering means to obtain the transgenic Arabidopsis thaliana which is over-expressed so as to carry out subsequent experiments. The pathogen infection experiment shows that the FtCYP94C1 overexpressed Arabidopsis has higher disease resistance compared with the wild type. Metabolome results indicate that FtCYP94C1 over-expressed arabidopsis has a richer flavone compound than wild type, possibly being one of the reasons for its more disease resistance.
Drawings
FIG. 1 is a phylogenetic tree of Fagopyrum tataricum FtCYP94C1 protein and its homologous proteins.
FIG. 2 shows the expression level of FtCYP94C1 gene after infection by Rhizoctonia solani.
FIG. 3 shows the expression level of FtCYP94C1 gene after MeJA induction.
FIG. 4 is a map of pCAMBIA1302 vector.
FIG. 5 shows the analysis of the expression level of the FtCYP94C1 gene of the overexpression Arabidopsis thaliana.
FIG. 6 is an observation of the area of disease onset after infection of leaves by Rhizoctonia solani.
FIG. 7 is a graph showing statistics of incidence and disease index after infection of leaves by Rhizoctonia solani.
FIG. 8 is a DAB staining observation after infection of leaves by Rhizoctonia solani.
FIG. 9 is an analysis of flavonoid content in an over-expressed FtCYP94C1 Arabidopsis.
Detailed Description
The present invention will be described in further detail with reference to the following examples, which are not intended to limit the present invention, but are merely illustrative of the present invention. The experimental methods used in the following examples are not specifically described, but the experimental methods in which specific conditions are not specified in the examples are generally carried out under conventional conditions, and the materials, reagents, etc. used in the following examples are commercially available unless otherwise specified.
Example 1
(1) Cloning of FtCYP94C1 CDS and promoter
Selecting four-week-old seedlings of a tartary buckwheat 'product bitter No. 1' strain, taking 50-100mg of seedlings, adding liquid nitrogen, fully grinding, and extracting total RNA by using a Trizol method. Using the RNA as a templateIII 1st Strand cDNASynthesis Kit (+gDNAwipe) kit (Nanjinouzan Biotechnology Co., ltd.) was subjected to reverse transcription to obtain cDNA of seedlings.
The cDNA of the strain "Pinku No. 1" is used as a template, and FtCYP94C1-F is used for preparing the cDNA of the strain: SEQ ID NO.3, ftCYP94C1-R: carrying out PCR amplification by using SEQ ID NO.4 as a specific primer to obtain a CDS sequence of a target gene SEQ ID NO.1, wherein the PCR program is 3min at 95 ℃;95℃30s,58℃30s,72℃90s,31 cycles. The PCR purified product was ligated to pTOPO-Blunt Simple Blunt end cloning vector to obtain a T vector plasmid containing SEQ ID NO. 1. And sequencing, analyzing and splicing to obtain the full-length sequence.
The amino acid sequence SEQ ID NO.2 expressed by the cloned gene is compared with the amino acid sequence of the reported CYP family found from NCBI, and the gene has the highest similarity with the Arabidopsis CYP94C1 gene through MEGA 7.0 construction of a phylogenetic tree, so that the gene is named FtCYP94C1 (figure 1), and the T vector plasmid containing the SEQ ID NO.1 is FtCYP94C1-T vector.
(2) FtCYP94C1 gene responds to jasmonic acid and rhizoctonia solani stress induction
To investigate the role of FtCYP94C1 in damping-off resistance, the expression of FtCYP94C1 after rhizoctonia solani infection and methyl jasmonate (MeJA) treatment for different periods of time was analyzed.
The specific method comprises the following steps:
after culturing seedlings for 21 days, 50 mu is usedM MeJA is used for treating seedlings for 1 hour, 4 hours and 12 hours, rhizoctonia solani (AG 4-HGI 3 strain is used as a strain) is used for infecting the seedlings for 6 hours, 14 hours and 22 hours, and total RNA of tartary buckwheat at different time points is extracted. And using the RNA as a templateIII 1st Strand cDNA Synthesis Kit (+gDNA wind) kit reverse transcription into cDNA was performed. The FtH gene expressed by the buckwheat component is taken as an internal reference, and the primer sequence is FtH-qF: SEQ ID NO.5; ftH3-qR: SEQ ID NO.6. Meanwhile, designing a gene specific primer qFtCYP94C1-F: SEQ ID NO.7, qFtCYP94C1-R: SEQ ID No.8, and 3 biological replicates were performed, and FtCYP94C1 expression was detected on a BAI 7500 real-time fluorescent quantitative PCR apparatus using real-time fluorescent quantitative PCR (Quantitative real-time PCR, qRT-PCR). The qRT-PCR kit was Taq Pro Universal SYBR qPCR Master Mix (Nanjinouzan Biotechnology Co., ltd.) and RQ (relative expression level) =2 in this experiment -ΔΔCT Calculating the relative expression quantity of the target gene; the expression level for 0 hour of induced expression was set to 1.
The results indicate that the expression of FtCYP94C1 was significantly up-regulated during both the rhizoctonia infection and MeJA treatment for different periods of time (fig. 2-3).
(3) FtCYP94C1 over-expression vector construction
Designing homologous recombination primers, taking FtCYP94C1-T vector as a template, taking 1302-FtCYP94C 1F/R as a primer, and amplifying the full-length sequence of FtCYP94C1 by PCR. Upstream primer 1302-ftCYP94C1-F: SEQ ID NO.9 and downstream primer 1302-FtCYP94C1-R: SEQ ID NO.10. And then, after enzyme digestion, recovery and connection transformation, the full-length sequence of FtCYP94C1 is inserted into the downstream of the CaMV35S promoter of the pCAMBIA1302 vector in the forward direction, and the overexpression vector pCAMBIA1302-FtCYP94C1 is obtained after sequencing is completed, and the vector map is shown in figure 4.
(4) Transformation of Agrobacterium
Sequencing and verification of correct pCAMBIA1302-FtCYP94C1 recombinant plasmid and pCAMBIA1302 empty vector plasmid are used for respectively transforming agrobacterium GV3101 competent cells by a heat shock method. After colony PCR identification, pCAMBIA1302-FtCYP94C1 recombinant plasmid positive bacteria and pCAMBIA1302 empty vector positive bacteria are obtained.
(5) Infection with Arabidopsis thaliana
The method comprises the steps of using pCAMBIA1302-FtCYP94C1 recombinant plasmid positive bacteria, infecting Arabidopsis thaliana with pCAMBIA1302 empty vector positive bacteria, sterilizing the infected Arabidopsis thaliana seeds with 10% sodium hypochlorite for 8 minutes, washing the seeds with sterile water for 4-5 times, then planting the treated seeds on an MS solid culture medium containing hygromycin (Hyg) resistance (Hyg concentration is 100 mg/mL), and culturing for 1-2 weeks, wherein Arabidopsis thaliana plants with true leaves can be initially identified as candidate positive plants.
qFtCYP94C1-F was used: SEQ ID NO.7, qFtCYP94C1-R: the primer of SEQ ID NO.8 and Taq Pro Universal SYBR qPCR Master Mix kit (Nanjinouzan biotechnology Co., ltd.) detect the gene expression, and the transgenic plant FtCYP94C1 gene expression level is higher than that of the wild type (FIG. 5), and the FtCYP94C1 over-expression plant is obtained.
(6) Detection of disease resistance of in vitro leaf of overexpression Arabidopsis thaliana
Sterilizing over-expression (FtASP-OE) Arabidopsis plants, non-load-transferring carrier (Ev) plants and wild type Arabidopsis seeds (Col-0), respectively planting the Arabidopsis seeds in an MS solid culture medium for 1-2 weeks, selecting plants with good growth vigor and consistency, transplanting the plants into soil, culturing for about 1-2 weeks until Arabidopsis rosette leaves grow to about 12 leaves, shearing the leaves with basically consistent sizes before bolting, placing the leaves in a culture dish with soaked filter paper, placing a rhizoctonia solani cake with the diameter of 0.5mm in the middle of the leaves, wrapping the culture dish with a preservative film, placing the culture dish in a fungus biochemical culture box with the temperature of 28 ℃ for germ infection experiment, and carrying out morbidity and disease index statistics after 2 days. The statistical method of the morbidity and the disease index is specifically as follows:
rhizoctonia solani infection arabidopsis leaf disease incidence grading standard
As can be seen from fig. 6 and 7, after the FtCYP94C1 overexpressed arabidopsis thaliana is infected by the rhizoctonia solani, the generated lesion area is far smaller than that of the control group, and the morbidity and the disease index are also lower than those of the control group, which indicates that the FtCYP94C1 overexpressed arabidopsis thaliana has stronger damping off resistance.
(7) DAB staining experiments: transgenic plants and wild type in vitro leaves were selected for rhizoctonia solani infection treatment and control was performed with blank PDB medium (beiku pacing technologies limited). After 24h incubation in a 28℃incubator, the isolated leaves were stained with DAB staining solution (Beijing Soy Bao technology Co., ltd.) for 20min, and then decolorized with chloroplast decolorization solution until the leaves were transparent. As shown in FIG. 8, after DAB staining, the leaf spots of the transgenic lines in vitro were significantly less than those of the control group, indicating that the disease resistance of FtASP-OE over-expressed plants was stronger than that of the control group.
(8) Metabolome analysis
Sterile transgenic arabidopsis seedlings grown to two weeks were subjected to extensive targeted metabolite analysis (fig. 9).
The method comprises the following steps: the lyophilized whole plant was ground and then extracted with 80% (v/v) methanol/water pre-chilled. Centrifuging at 15,000g for 20min at 4deg.C, and collecting supernatant with ExionLC TM AD System (SCIEX)6500+ Mass Spectrometry (SCIEX) analysis. The analysis conditions were as follows: chromatographic column (2.1 mm. Times.150 mm,2.5 μm); an aqueous solution (a) of 0.1% formic acid; acetonitrile solution of 0.1% formic acid (B). Gradient procedure was as follows, 2% B,2 min; 2-100% B,15.0 min; 100% B,17.0 min; 100-2% B,17.1 min; 2% B,20 minutes.
Qualitative analysis was performed using multi-reaction monitoring (multiple reaction monitoring, MRM) based on the Novogene database. The data file generated by HPLC-MS/MS was processed using SCIEX OS v1.4 to correct the peak. The area of each peak represents the relative content of the corresponding metabolite. Metabolites were further annotated with KEGG database (http:// www.genome.jp/KEGG /), HMDB database (http:// www.hmdb.ca /), and lipid profile database (https:// www.lipidmaps.org /). Differentially expressed metabolites were identified using Principal Component Analysis (PCA), partial least squares discriminant analysis (PLS-DA) and t-test. Metabolites with VIP (variable projection importance) >1, P value <0.05 and |log2fc|gtoreq 1 were considered differentially expressed metabolites.
The results show that: the significant accumulation of 12OH-JA-Ile in FtCYP94C1 overexpressing plants further demonstrates that FtCYP94C1 can cause the oxidation of JA-Ile to 12OH-JA-Ile. In addition, flavonoids such as rutin, quercetin and chalcone also accumulate in the FtCYP94C1 overexpression line, confirming that the FtCYP94C1 gene may affect the production of specific flavonoids in transgenic Arabidopsis.
The transgenic arabidopsis is obtained by adopting a transgenic engineering strategy of the Fagopyrum tataricum FtCYP94C1 gene transgenic arabidopsis, and the disease resistance of the over-expression strain is identified, so that a foundation is laid for the research on the genetic mechanism of the Fagopyrum tataricum for resisting damping-off, and the method has important significance for the genetic engineering research and innovation utilization of the Fagopyrum tataricum.
The previous description of the embodiments is provided to facilitate a person of ordinary skill in the art in order to make and use the present invention. It will be apparent to those skilled in the art that various modifications can be readily made to these embodiments and the generic principles described herein may be applied to other embodiments without the use of the inventive faculty. Therefore, the present invention is not limited to the above-described embodiments, and those skilled in the art, based on the present disclosure, should make improvements and modifications without departing from the scope of the present invention.

Claims (15)

  1. The application of the FtCYP94C1 gene in resisting rhizoctonia solani infection is characterized in that the nucleotide sequence of the FtCYP94C1 gene is shown in SEQ ID NO.1, and the amino acid sequence coded by the FtCYP94C1 gene is shown in SEQ ID NO.2.
  2. 2. The use according to claim 1, wherein the use is damping off.
  3. 3. The use according to claim 1, wherein the application object is a plant.
  4. 4. The use according to claim 3, wherein the application object is a buckwheat plant.
  5. 5. The method of claim 4, wherein the subject is Tartary buckwheat.
  6. The application of the FtCYP94C1 gene or a vector expressing the FtCYP94C1 gene or a cell expressing the FtCYP94C1 gene in constructing transgenic plants is characterized in that the nucleotide sequence of the FtCYP94C1 gene is shown in SEQ ID NO. 1.
  7. 7. The use according to claim 6, wherein said transgenic plant overexpresses the FtCYP94C1 gene.
  8. 8. The use according to claim 7, wherein the transgenic plant has damping off resistance.
  9. 9. The construction method of the transgenic plant is characterized by comprising the step of overexpressing the FtCYP94C1 gene in the plant, wherein the nucleotide sequence of the FtCYP94C1 gene is shown in SEQ ID NO. 1.
  10. 10. The method of claim 9, comprising inserting the FtCYP94C1 gene into an expression vector, and infecting a plant after transformation of the host cell to obtain a transgenic plant.
  11. 11. The construction method according to claim 10, wherein the expression vector is pCAMBIA1302 vector.
  12. 12. The method of claim 11, wherein the host cell is agrobacterium GV3101.
  13. 13. The method of construction according to claim 12, comprising the steps of:
    (1) Cloning to obtain FtCYP94C1 gene;
    (2) Operably constructing the FtCYP94C1 gene into an expression control sequence to form a plant expression vector containing the FtCYP94C1 gene;
    (3) Transforming agrobacterium tumefaciens GV3101 with the plant expression vector containing the FtCYP94C1 gene obtained in the step (2) to obtain a agrobacterium strain containing the FtCYP94C1 gene for transformation;
    (4) Genetically transforming a plant with the agrobacterium strain constructed in step (3).
  14. 14. The expression vector containing the FtCYP94C1 gene is characterized in that the nucleotide sequence of the FtCYP94C1 gene is shown in SEQ ID NO. 1.
  15. 15. A genetically engineered cell comprising the expression vector of claim 14, wherein the nucleotide sequence of the FtCYP94C1 gene is set forth in SEQ ID No. 1.
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