CN116375829B - Application of Osmanthus fragrans OfWRKY36 gene in enhancement of synthesis of plant dihydro-beta-ionone - Google Patents

Abstract

The invention discloses an application of an osmanthus fragrans OfWRKY36 gene in enhancing synthesis of plant dihydro-beta-ionone, and belongs to the field of plant molecular biology. The invention discloses an application of an Osmanthus fragrans OfWRKY36 gene in enhancing synthesis of plant dihydro-beta-ionone, wherein the nucleotide sequence of the OfWRKY36 gene is shown as SEQ ID NO. 1. According to the invention, flowers in the flowering period of dixiang Gui Cheng are taken as materials, the full-length gene of the osmanthus fragrans OfWRKY36 is obtained through cloning, an overexpression vector pBI121-OfWRKY36 is constructed on the basis, the transgenic plants are obtained after the expression vector pBI121-OfWRKY36 is transferred into osmanthus fragrans petals, and a gene function identification result shows that the OfWRKY36 gene is an important transcription factor in the process of enhancing the synthesis of dihydro-beta-ionone in plants, and can be used for improving ornamental characters of the floral fragrance in osmanthus fragrans genetic engineering and breeding works such as genetic quality.

Description

Application of Osmanthus fragrans OfWRKY36 gene in enhancement of synthesis of plant dihydro-beta-ionone

Technical Field

The invention belongs to the field of plant molecular biology, and particularly relates to an application of an osmanthus fragrans OfWRKY36 gene in enhancing dihydro-beta-ionone synthesis in plants.

Background

Osmanthus fragrans (Osmanthus fragrans) belongs to the genus Oleaceae (Oleaceae), is one of ten traditional flowers in China, and is one of the most important garden plants at present. The osmanthus fragrans essence is favored by people with pleasant fragrance and graceful tree shape. The osmanthus fragrans is one of a few flowers which are obtained by China and are internationally registered with cultivars, and the important position of the osmanthus fragrans in the flowers in China is highlighted.

WRKY transcription factors are one of the ten large families of transcription factors of plants, and are widely involved in plant responses to biotic, abiotic and hormonal stresses, as well as in plant growth and development and physiological processes. At present, the research on regulating and controlling the synthesis of plant secondary metabolites by WRKY transcription factors is mainly focused on terpenes, phenols and alkaloids. Terpenes are hydrocarbons and oxygen-containing derivatives thereof which exist in nature and have a molecular formula which is a multiple of isoprene units, and common terpenes can be divided into monoterpenes, sesquiterpenes, diterpenes, sesterterpenes, tetraterpenes and polyterpenes, such as linalool, myrcene, trans-beta-ocimene, farnesene, nerol and the like, according to the amount of isoprene units, and are all important components for forming floral fragrance.

The WRKY transcription factor can play a role in regulating plant secondary metabolism independently or through interaction with other transcription factors. Different WRKY transcription factors can be combined with different genes in the secondary metabolic pathway, and can also have different regulation modes for the same secondary metabolic product. Therefore, it is highly desirable to discover important related genes in the genome of osmanthus fragrans, which regulate the synthesis of floral substances.

Disclosure of Invention

Aiming at the problems existing in the prior art, the invention aims to provide the application of the Osmanthus fragrans OfWRKY36 gene in enhancing the synthesis of dihydro-beta-ionone in plants.

In order to solve the technical problems, the technical scheme adopted by the invention is as follows:

the application of the osmanthus fragrans OfWRKY36 gene in enhancing the synthesis of dihydro-beta-ionone in plants is provided, and the nucleotide sequence of the osmanthus fragrans OfWRKY36 gene is shown as SEQ ID NO. 1.

Further, the application comprises the following steps:

(1) Constructing a vector of an OfWRKY36 gene;

(2) Transforming the constructed vector of the OfWRKY36 gene into a plant or plant tissue;

(3) And culturing and screening to obtain transgenic plants or plant tissues with enhanced aroma substance synthesis.

Further, the plant is osmanthus fragrans.

Further, the plant tissue is osmanthus flower petals.

Further, the transformation is transient transformation by agrobacterium.

Further, the vector is a plant expression vector.

Further, the plant expression vector is pBI121-OfWRKY36.

Compared with the prior art, the invention has the beneficial effects that:

the invention takes flowers in the flowering period of day-lily Gui Cheng as materials, and obtains the full-length gene of the osmanthus fragrans OfWRKY36 through cloning. Meanwhile, an overexpression vector pBI121-OfWRKY36 is constructed on the basis, and is transferred into osmanthus petals, and the OfWRKY36 is efficiently expressed in osmanthus. After the transgenic osmanthus flower petals and the non-transgenic osmanthus flower petals are cultivated for 48 hours in a dark place, the content of terpene aroma substances in the non-transgenic osmanthus flower petals is not obviously improved. The alpha-ocimene, (S) -linalool oxide, dihydro-beta-ionone and 2,6, 10-trimethyltridecane in the transgenic osmanthus flower petals are obviously higher than the control, wherein the content of the dihydro-beta-ionone is obviously improved. The OfWRKY36 gene is an important transcription factor in the process of enhancing the synthesis of terpene aroma substances of osmanthus fragrans, and can be used for improving ornamental properties of the osmanthus fragrans in osmanthus fragrans genetic engineering, and breeding works such as genetic quality.

Drawings

FIG. 1 is an agarose electrophoresis chart of a target gene amplification product;

FIG. 2 is a diagram of agarose electrophoresis of a vector after double digestion of the recombinant OfWRKY36 vector (lanes 1-2-3-4 are vector strips after double digestion; lane 5 is an empty vector control diagram);

FIG. 3 is a graph of positive single colony detection after ligation transformation;

FIG. 4 is a agarose electrophoresis chart of a bacterial sample after transformation of Agrobacterium GV 3101;

fig. 5 is a positive detection chart of the transiently transformed osmanthus petals, and the following is noted: a is 35S, namely a GUS staining condition chart (CK: wild type, NC: empty) of the OfWRKY36 fusion protein, B is an expression condition chart of the OfWRKY36 after the OfWRKY36 instantaneously infects the petals of the osmanthus fragrans, and C is a growth condition chart of the OfWRKY36 on a culture medium after the OfWRKY36 instantaneously infects the petals of the osmanthus fragrans;

fig. 6 is a substance metabolism diagram of the gene OfWRKY36 after transiently infecting petals of osmanthus fragrans, and is annotated: a is PCA-X scatter plot, B is OPLS-DA scatter plot, C is load analysis scatter plot of OfWRKY36 transgenic plants and wild-type flower volatile matter metabolites: red and yellow arrows represent the substance components of VIP >1, D is a relative content analysis chart of the substances marked by the arrows in fig. C;

FIG. 7 is a graph (A) of GO entries in OfWRKY 36-transformed flower petals and control annotation DEGs and a differential gene KEGG pathway annotation (B).

Detailed Description

The invention is further described below in connection with specific embodiments. In the following examples, the procedures not described in detail are all routine biological experimental procedures, and can be performed with reference to molecular biology laboratory manuals, journal literature published in the prior art, and the like, or according to the kit and product instructions. Materials, reagents and the like used in the examples described below are commercially available unless otherwise specified.

Example 1: cloning of Pimenta dioica OfWRKY36 gene, construction of super-expression vector and transformation

1. Total RNA extraction and cDNA Synthesis

The material used in the application is flowers in the flowering period of dixiang Gui Cheng, and the osmanthus flower petals used for instant conversion are the golden osmanthus variety 'Bo leaf golden osmanthus' growing in the university campus of Nanjing forestry.

Total plant RNA was extracted using TIANGEN plant RNA extraction kit (DP 432).

Using TaKaRaPrimeScript ^TM RTMasterMix (PerfectRealTime) reverse transcription kit for extracting RNAReverse transcription to cDNA, diluting the final cDNA with water 10 times, and storing at-20deg.C.

2. Obtaining the target gene fragment

According to the full genome database of osmanthus published by the earlier task group (Xiulian Yang, yuanzheng Yue, et al, the chromoname-level quality genome provides insights into the evolution of the biosynthesis genes for aroma compounds ofOsmanthus clusters), 1 gene sequence is obtained by screening, and compared with the sequence of model plant Arabidopsis thaliana, the gene is judged to belong to the WRKY gene family, and the gene family member is named as OfWRKY36 according to the position of the gene family member on a chromosome.

2. Obtaining the target gene fragment

The full-length nucleotide sequence of the gene was subjected to restriction enzyme site analysis using the BioXM software, and XbaI and BamHI were selected as restriction enzyme sites. Primers were designed using CE design software. Filling relevant information according to the requirement, specifically comprising sequences near the enzyme cutting sites on the carrier, the whole length of the target gene, and filling the enzyme cutting sites according to the sequence of the 5 'end and the 3' end, thus obtaining the amplification primer. The designed sequences were synthesized by the JieRui biosystems. The primer sequences were as follows:

pBI121-OfWRKY36-F：

5′-GAGAACACGGGGGACTCTAGAATGGAAATTAATGAAGCAGGGAA-3′，

pBI121-OfWRKY36-R：

5′-GGACTGACCACCCGGGGATCCAGTTGCTGATTTATTCTTCATACTTGTCT-3'。

PCR amplification was performed using 10-fold dilution of the Pimenta racemosa OfWRKY36cDNA as a template, and the reaction system (20. Mu.L) was: the forward primer pBI121-OfWRKY 36-F1. Mu.L; reverse primer pBI121-OfWRKY 36-R1. Mu.L; 1 μl of cDNA; prime STAR 10. Mu.L (high-fidelity PCR enzyme purchased from Bao Ri doctor Material technology (Beijing) Co., ltd., high-speed high-fidelity PCR enzyme R045A); ddH ₂ O7. Mu.L. The PCR reaction procedure was: denaturation at 98℃for 10s; annealing at 58 ℃ for 15s; extending at 72 ℃ for 2min for 35 cycles; extending at 72deg.C for 10min; the reaction was terminated at 16 ℃.

And (3) carrying out agarose electrophoresis detection on the amplified product (shown in figure 1) to obtain a target gene fragment, and then carrying out gel cutting recovery on the target gene fragment by using a kit.

3. Ligation of the Gene fragment of interest to the vector

1) The pBI121 vector was taken out of the-80℃ultra-low temperature refrigerator in advance for activation and shaking, and the plasmid of the pBI121 vector was extracted according to a plasmid extraction kit (Beijing Tiangen Biochemical technology Co., ltd.), followed by a double enzyme digestion experiment, and the reaction system (20. Mu.L) was: 1 μl of restriction enzyme BamHI; restriction enzyme XbaI 1. Mu.L; buffer 2. Mu.L (Buffer is attached to Takara restriction endonuclease); vector plasmid X. Mu.L; ddH ₂ O was made up to 20. Mu.L.

Where X (μl) =1000 ng/vector plasmid concentration (ng/μl). The centrifuge tube is slightly vibrated, evenly mixed, instantaneously centrifuged for 6s and placed in a water bath kettle at 37 ℃ for culturing for 1h. The double digested vector was subjected to agarose electrophoresis (FIG. 2), and then subjected to gel-cutting recovery using a kit (Hunan Ai Kerui Biotechnology Co., ltd.), as follows.

2) The ligation system (20. Mu.L) was as follows: x mu L of target gene recovery product; the pBI121 vector is subjected to double digestion to recover a product Y mu L; 2. Mu.L of ligase; buffer 1. Mu.L; ddH ₂ O was made up to 20. Mu.L (ligase and Buffer from homologous recombination reagent c112, nanjinouzan Biotechnology Co., ltd.).

Wherein X (μl) =200 ng/target gene recovery product concentration (ng/μl), Y (μl) =200 ng/plasmid double cleavage recovery product concentration (ng/μl). And (3) slightly vibrating the centrifuge tube, uniformly mixing, instantaneously centrifuging for 6s, and placing the centrifuge tube in a water bath at 37 ℃ for culturing for 30min and ice for 2min.

3) Conversion: in a super clean bench, 5. Mu.L of ligation product was removed with a pipette onto 50. Mu.L of Trelief ^TM 5. Alpha. Competent cells were gently flicked, mixed well, ice-bathed for 5min, water-bath at 42℃for 60s, ice-bathed for 2min, and incubated in a shaker at 37℃and 200rppm for 30min with 250. Mu.L of liquid LB (without Kana).

4) Coating: 200 mu L of the incubated bacterial liquid is taken, uniformly coated on LB solid medium (containing 50mg/L Kana) by using a sterilized glass rod, and then dried, and after sealing, the bacterial liquid is inversely cultured in a constant temperature incubator at 37 ℃ for 12-14h.

4. Recombinant plasmid selection

After bacteria grow out on the culture medium, single colony detection is carried out in an ultra-clean workbench. 8 full single colonies are picked for each gene, backup is carried out on an LB solid medium containing Kana resistance in sequence, and corresponding single colonies are dipped into the following 20 mu L system by using a sterile toothpick for bacterial detection: pBI121-OfWRKY 36-F1. Mu.L, pBI121-OfWRKY 36-R1. Mu.L, green Mix 10. Mu.L (Greenmix available from Nanjinopran Biotech Co., ltd.) and ddH ₂ O8μL。

The PCR reaction conditions were: pre-denaturation at 94℃for 3min; denaturation at 94℃for 30s; annealing at 58 ℃ for 30s; extending at 72 ℃ for 2min for 35 cycles; extending at 72deg.C for 10min; the reaction was terminated at 16 ℃. And (3) carrying out agarose electrophoresis on the obtained amplified product, picking 3 positive single colonies with correct length, and carrying out measurement, wherein the nucleotide sequence of the measured OfWRKY36 gene is shown as SEQ ID NO.1 in a sequence table, the fragment length is 1374bp, protein with 457 amino acids is encoded, and the amino acid sequence of the protein is shown as SEQ ID NO. 2. The positive single colony extracted plasmid with the lowest base mismatch rate and repeated sequencing was selected for subsequent experiments (fig. 3).

Example 2: transformation and functional identification of OfWRKY36 gene

1. Recombinant plasmid transformed agrobacterium

(1) GV3101 in-80 deg.c refrigerator is taken out and thawed on ice. Adding 1 μl plasmid into each 33 μl of the mixture, sucking, beating, mixing, sequentially ice-bathing for 20min, quick-freezing with liquid nitrogen for 5min, water-bathing at 37deg.C for 5min, and ice-bathing for 5min; adding 500 mu L of non-resistant LB liquid medium, and culturing for 1h on a 200rppm shaking table at 28 ℃;

(2) After the culture is completed, the bacterial liquid is centrifuged for 6000r for 1min, partial supernatant is removed, 100 mu L of the supernatant is reserved and evenly coated on LB solid culture medium (50 mg/LKana is contained), sealing film is used for sealing, and the culture medium is placed in a 28 ℃ incubator for culture for 40-48h;

(3) Bacterial inspection and backup: as shown in FIG. 4, the target strips in the bacterial inspection are correct and consistent in brightness, the corresponding colonies in the backup plates are picked into LB liquid culture medium (50 mg/LKana is contained) for shaking, bacterial liquid and 50% glycerol are preserved according to the volume ratio of 3:7, and the bacterial liquid and the glycerol are quickly frozen in liquid nitrogen and then are preserved in an ultralow temperature refrigerator at-80 ℃.

2. Agrobacterium mediated transformation of 'Boleaf golden osmanthus' osmanthus flower petals

(1) Shaking: pBI121 empty load, P19 auxiliary expression vector and GUS transferred into agrobacterium are prepared through melting the bacterial liquid of pBI121-OfWRKY36 target gene fusion expression vector at-80 deg.c to ice water mixed state, adding into 30mLLB liquid culture medium (containing Kana 10. Mu.g.mL) according to 300. Mu.L bacterial liquid ^-1 ) Shake culturing at 28deg.C and 200rpm in dark place until the bacterial liquid OD ₆₀₀ Between=0.6-0.8;

(2) Preparing a mixed bacterial liquid: 0.0196g of Acetosyringone (AS) powder is weighed, dimethyl sulfoxide is used for assisting dissolution (operation is carried out in a fume hood), a proper amount of sterile water is added to fix the volume to 100mL, mother liquor is obtained, 15mL of mother liquor is taken, and 85mL of sterile water is added, thus 150 mu mol.L is obtained ^-1 Is of (2); 0.2035g of MgCl was weighed out ₂ And 0.2135g of 2- (N-morpholino) ethanesulfonic acid Monohydrate (MES) were added to the 150. Mu. Mol.L formulation ^-1 Is contained in AS solution; centrifuging (4 ℃ at 5000rpm for 10 min), discarding the supernatant, collecting thalli, re-suspending by using a buffer solution prepared on the same day, mixing (P19 auxiliary vector: target gene (V: V) =5:7) according to the optimal proportion, fully vibrating and uniformly mixing, and activating for 3h;

(3) Infection: selecting osmanthus flower petals with good growth state, and injecting mixed bacterial liquid into the osmanthus flower petals by using a vacuum pump;

(4) Culturing: and (3) culturing the transient transformed plant injected with the target gene mixed bacterial liquid and the transient transformed plant injected with the empty vector bacterial liquid for 48 hours in a dark place, wherein the growth condition of the transient transformed plant on the culture medium after the transient infection of the OfWRKY36 on the flower petals of the osmanthus fragrans is shown in figure 5C.

3. GUS dyeing for instant conversion of osmanthus flower petals

Soaking the wild osmanthus flower petals (CK), the no-load osmanthus flower petals (NC) and GUS in a fresh GUS dye solution of pBI121-OfWRKY36, shading from light, dyeing for 1-2 days, decolorizing with 75% alcohol for 3-5 times, decolorizing with 95% absolute alcohol until the plants are completely decolorized, and photographing.

As shown in FIG. 5A, the GUS staining condition shows that after the fusion expression vector of pBI121 and 35S: ofWRKY36 infects the petals of osmanthus fragrans, the petals are all obviously blue after GUS staining, and the petals are not blue after wild type staining. On the basis, it was confirmed that the foreign gene was introduced into the petals of osmanthus fragrans.

4. Fluorescent quantitative analysis: total plant RNA was extracted using TIANGEN plant RNA extraction kit (DP 432). Using TaKaRa PrimeScript ^TM Reverse transcription of the extracted RNA into cDNA using RT Master Mix (Perfect Real Time) kit, diluting the resulting cDNA 10-fold with water, taking 1. Mu.L as template, adding F0.4. Mu.L, R0.4. Mu.L, TBGreen Mix 5. Mu.L, rox dye solution 0.2. Mu.L, ddH ₂ O3. Mu.L was formulated into a 10. Mu.L system for fluorescent quantitation assay TaKaRa TB Green Premix Ex Taq (RR 420A) using the following thermal cycling conditions: for 30s at 95℃and then for 15s at 95℃after 40 cycles of 5s at 95℃and 30s at 60℃and 15s at 95℃and 1min at 60 ℃.2 used for calculation of relative expression level ^-△△CT A method of manufacturing the same.

As a result, FIG. 5B shows that the expression of the target gene after transient infection of the control, ofWRKY36 is about 6 times that of the control.

5. Determination of aroma substances of osmanthus flower petals converted instantaneously

Clamping 0.4g of osmanthus flower petals by forceps at room temperature (25+/-2 ℃), recording the weight of the leaves, placing the leaves in a sealed 1mL extraction bottle, adding an internal standard solution (1/10000 ethyl decanoate: 1 micro ethyl decanoate standard sample is added into 10mL of methanol), shaking and uniformly mixing, and balancing for 15min; in a water bath kettle with the temperature of 45 ℃, an extraction head of 65 mu mDB/5MS is inserted into the middle part of an extraction bottle after sealing, the extraction head is inserted into a chromatographic column TRACE TR-5MS C30m multiplied by 0.25mm multiplied by 0.25gym after extracting for 30min, carrier gas is high-purity helium (He), the flow rate of the helium is 1mL/min, sample introduction is not split, the temperature of a sample inlet is 250 ℃, and the desorption time is 3min; the temperature program is as follows: firstly, keeping the temperature at 60 ℃ for 2min, wherein the first temperature-raising program is that the temperature is raised to 150 ℃ at a speed of 5 ℃/min, and the second temperature-raising program is that the temperature is raised to 250 ℃ at a speed of 10 ℃/min, and then keeping the temperature for 1min; the mass spectrum condition is that the temperature of an ion source is 250 ℃, the ionization mode EI and the electron energy are 70eV;

as shown in fig. 6, after removing the silicon oxide impurities in the data, the relative content of each substance is calculated, the relative content of each substance is introduced into SIMCA5.0, VIP >1 substances are selected for load analysis according to the OPLS-DA cluster and VIP value size, VIP >1 substances are selected for specific content analysis, VIP >1 substances comprise alpha-ocimene, (S) -linalool oxide, dihydro-beta-ionone and 2,6, 10-trimethyltridecane, and the result shows that the content of dihydro-beta-ionone in the petals of the osmanthus fragrans transformed with OfWRKY36 is significantly higher than that of the control, and the OfWRKY36 has an important regulation effect on the aroma substances with the difference p < 0.05.

Example 3: analysis of transcript levels of the full genome of the OfWRKY36 transiently overexpressed petal RNA-seq

The sequenced samples were pBI121 empty and its corresponding pBI 121:OfWRKY 36, repeated 6 times per gene, construction of RNA library and RNA-seq sequencing were performed in three replicates consistent with the result of metabolite clustering. RNA library construction and sequencing was done by Shanghai European company using Illumina HiSeq X Ten platform. Gene expression level calculations were performed using the FPKM method. GO and KEGG enrichment analysis was performed using GOseqRplatage and KOBAS software, respectively. Differential transcripts with fold difference greater than 2 were selected and subjected to GO and KEGG enrichment analysis of differential transcripts, screening for differentially expressed genes to determine the biological function or pathway that was predominantly affected by differential transcripts. Meanwhile, carrying out unsupervised hierarchical clustering on the differential transcripts, and displaying the expression modes of the differential transcripts among different samples in a heat map mode.

As shown in FIG. 7, the OfWRKY36 transgenic osmanthus flower petals T-OfWRKY36 and the RNA-seq data of the empty transgenic osmanthus flower petals NC were compared (T-OfWRKY 36 vsNC), and a total of 322 DEGs were identified. The GO enrichment analysis is carried out on the differential expression genes, and the DEGs are mainly concentrated under the category of biological processes, and the enriched GO items are as follows: metabolic processes, cellular processes, stress responses, and the like. KEGG differential gene analysis indicates that DEGs are primarily involved in metabolic processes such as terpenoid metabolism, carbohydrate metabolism, and other secondary metabolite biosynthesis. The detailed analysis that follows is largely focused on the identified expression differential genes involved in secondary metabolite synthesis and transport.

Claims (5)

1. The application of the osmanthus fragrans OfWRKY36 gene in enhancing the synthesis of dihydro-beta-ionone in osmanthus fragrans bodies is provided, and the nucleotide sequence of the osmanthus fragrans OfWRKY36 gene is shown as SEQ ID NO. 1.

2. The use according to claim 1, characterized by the steps of:

(1) Constructing a plant expression vector of an OfWRKY36 gene;

(2) Transforming the plant expression vector of the constructed OfWRKY36 gene into a plant or plant tissue;

(3) And culturing and screening to obtain transgenic plants or plant tissues with enhanced aroma substance synthesis.

3. The use according to claim 2, wherein the plant tissue is osmanthus flower petals.

4. The use according to claim 2, wherein said transformation is transient transformation by agrobacterium.

5. The use according to claim 2, wherein the plant expression vector is pBI121.