CN116144701A - Application method of ginkgo bZIP transcription factor GbbZIP08 in promotion of plant flavonoid synthesis - Google Patents

Abstract

The invention discloses an application method of a gingko bZIP transcription factor GbbZIP08 in promoting plant flavonoid synthesis, belonging to the technical field of plant molecular genetic engineering, wherein the invention separates complete cDNA for encoding the GbbZIP08 transcription factor from gingko, connects the complete cDNA to a plant over-expression vector, utilizes agrobacterium to mediate and transform plants, and proves that the GbbZIP08 is positioned in plant cell nuclei through transient expression of tobacco; transgenic arabidopsis thaliana and transgenic tobacco are obtained by an inflorescence infection method and a leaf disc transformation method respectively, and the result shows that the accumulation of flavonoid substances in plants can be improved by over-expression of GbbZIP 08. The invention provides a certain research foundation and gene resources for the research of promoting the synthesis of plant flavonoids by bZIP transcription factors.

Description

Application method of ginkgo bZIP transcription factor GbbZIP08 in promotion of plant flavonoid synthesis

Technical Field

The invention belongs to the technical field of plant molecular genetic engineering, and particularly relates to an application method of a gingko bZIP transcription factor GbbZIP08 in promoting plant flavonoid synthesis.

Background

Ginkgo biloba (Ginkgo biloba L.) is the deciduous tree of Ginkgo of Ginkgoaceae, and is the oldest plant on earth, and is also a special medicinal plant resource in China. The main active ingredients in the ginkgo leaf extract comprise flavonoids, terpene lactones, long-chain phenols, proanthocyanidins, phenolic acids and other compounds. The content of flavonoid in ginkgo leaf extract EGB is regulated to 24% internationally, and the flavonoid has the functions of enhancing immunity, resisting cancer activity, resisting aging activity and the like, and has important curative effects in preventing and treating cardiovascular and cerebrovascular diseases and the like. However, the flavonoid content in most ginkgo varieties is extremely low, and this factor has become an important obstacle for intensive research and development of ginkgo leaf preparations. At present, genetic improvement is carried out on key regulatory factors of secondary metabolites by utilizing a genetic engineering technology, which has important significance for improving the content of ginkgo flavonoids, and no related report about regulation of flavonoid metabolism by using ginkgo bZIP transcription factors is currently seen.

Disclosure of Invention

The invention aims at solving the existing problems and provides an application method of a gingko bZIP transcription factor GbbZIP08 in promoting plant flavonoid synthesis.

The invention is realized by the following technical scheme:

an application method of ginkgo bZIP transcription factor GbbZIP08 in promoting plant flavonoid synthesis comprises the following steps:

step 1: the gene sequence of GbbZIP08 cloned from ginkgo by PCR amplification is shown as a sequence table SEQ ID NO.1, and the coded amino acid sequence obtained by translation is shown as a sequence table SEQ ID NO. 2;

SEQ ID NO.1：

Atggcttttggagatctcagaggagaacttaaaatgcagcccaatgatacctcaacttccgtacaacagcagtctagcggcgagaaaacgtccagttctggtgacccacttgatgagatgaacaaatatgtgaatgagagtgatgatgatatccgtagagtaccagagatggtaaaccagttaggctcttctagtaattctgttgctgttaagatggagagccaacaggctactggtacttctaatcaaaggaaaagaggcagaacacctgcagacaaagaacataaacgtctgaaaagattgctgaggaatagagtatctgctcaacaagcaagggagaggaagaaggcttacttgagtgagcttgagacaaaggccaaagagtatgagcagaagaactctgagttggaagagaaagtttctactttgcagaatgaaaatttcatgcttagacaggtaccttaa

SEQ ID NO.2：

MAFGDLRGELKMQPNDTSTSVQQQSSGEKTSSSGDPLDEMNKYVNESDDDIRRVPEMVNQLGSSSNSVAVKMESQQATGTSNQRKRGRTPADKEHKRLKRLLRNRVSAQQARERKKAYLSELETKAKEYEQKNSELEEKVSTLQNENFMLRQVP

step 2: performing first-generation sequencing on the cloned gene sequence in the step 1 to obtain a base sequence of a GbbZIP08 gene, and performing subsequent gene sequence analysis and expression vector construction primer design;

step 3: constructing GbbZIP08 obtained by cloning on a pNC-Cam1304-sub plant subcellular localization vector and a pNC-Cam2304-MCS35S plant overexpression vector by a Nimble cloning method;

step 4: transforming agrobacterium GV3101 with the pNC-Cam 1304-sub-GbbZIP 08 expression vector constructed in the step 3, using the vector for transient transformation of tobacco, observing subcellular localization of GbbZIP08 in plants, and taking empty vector as a control;

step 5: transforming the pNC-Cam2304-MCS35S-GbbZIP08 expression vector in the step 3 into agrobacterium, transforming into arabidopsis by using an arabidopsis inflorescence infection method, and transforming into tobacco by using a tobacco leaf disc transformation method;

step 6: obtaining transgenic arabidopsis homozygote by adopting resistance screening, and obtaining a transgenic tobacco regeneration system by a plant tissue culture method; positive PCR, GUS staining and qRT-PCR methods were used to verify transgenic plants;

step 7: and (3) carrying out flavonoid content detection on the transgenic plant and the wild plant obtained in the step (6), and analyzing the influence of the over-expression GbbZIP08 on flavonoid synthesis in the plant.

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

the complete cDNA for encoding the GbbZIP08 transcription factor is separated from ginkgo, is connected to a plant over-expression vector, and utilizes agrobacterium to mediate and transform plants, and proves that the GbbZIP08 is positioned on plant cell nuclei through transient expression of tobacco; transgenic arabidopsis thaliana and transgenic tobacco are obtained by an inflorescence infection method and a leaf disc transformation method respectively, and the result shows that the accumulation of flavonoid substances in plants can be improved by over-expression of GbbZIP 08. The invention provides a certain research foundation and gene resources for the research of promoting the synthesis of plant flavonoids by bZIP transcription factors.

Drawings

FIG. 1 is a thermal chart showing correlation analysis of FPKM values of bZIP genes and total flavone contents in 8 tissues of ginkgo;

FIG. 2 is a diagram of the results of phylogenetic tree analysis of the GbbZIP08 amino acid sequence;

FIG. 3 is a schematic representation of the pNC-Cam 1304-sub-pNC-GbbZIP 08 plant subcellular localization vector and Cam2304MCS-35S-GbbZIP08 overexpression vector;

FIG. 4 is a graph showing subcellular localization results of GbbZIP 08;

FIG. 5 is a diagram of a transgenic Arabidopsis homozygous screen;

FIG. 6 is a diagram obtained from a tobacco genetic transformation system;

FIG. 7 is a graph of the positive PCR identification results of transgenic plants;

FIG. 8 is a GUS staining chart of transgenic plants;

FIG. 9 is a graph of the results of quantitative analysis of transgenic plants GbbZIP 08;

FIG. 10 is a graph showing the analysis result of the total flavone content of transgenic plants;

FIG. 11 is a graph showing analysis results of kaempferol content in transgenic plants;

FIG. 12 is a graph showing the results of anthocyanin content analysis of transgenic plants.

Detailed Description

The technical solutions of the present invention will be clearly and completely described below in conjunction with the embodiments of the present invention, and it is apparent that the described embodiments are only some embodiments of the present invention, not all embodiments. All other embodiments, which can be made by one of ordinary skill in the art without undue burden on the person of ordinary skill in the art based on embodiments of the present invention, are within the scope of the present invention.

Examples

An application method of ginkgo bZIP transcription factor GbbZIP08 in promoting plant flavonoid synthesis comprises the following steps:

(1) Screening and identification of ginkgo bZIP gene families:

based on a ginkgo genome database, 40 GbbZIP genes are obtained by utilizing a local blast to screen from the database; correlation analysis of transcriptome data and flavonoid content in 8 tissues of ginkgo;

the GbbZIP08 gene and ginkgo flavonoid content were found to have a high positive correlation (figure 1);

the phylogenetic tree analysis shows that GbbZIP08 is a homologous gene of arabidopsis AtbZIP56 (fig. 2);

(2) The GbbZIP08 gene sequence in ginkgo is obtained:

grinding fresh ginkgo leaves in a mortar precooled by liquid nitrogen, extracting plant RNA according to an RNA extraction kit (TaKaRa) specification, detecting the integrity of the RNA by using gel electrophoresis after the extraction is finished, detecting the concentration of the RNA by using a Nanodrop instrument, and performing reverse transcription on an RNA sample passing detection by using an RNA reverse transcription kit (Vazyme), thereby synthesizing cDNA according to a product specification;

cloning primers were designed based on the CDs sequence of GbbZIP08 in the ginkgo genome (U: ATGGCTTTTGGAGATCTCAG; D: TTAAGGTACCTGTCTAAGCATG);

amplifying a target gene by PCR, purifying and recovering, connecting the target gene to a T carrier by using TA cloning, converting to E.coli competence, selecting a monoclonal and carrying out bacterial liquid PCR verification by using an M13 primer, storing a part of positive clone bacterial liquid by using 50% glycerol for standby, and sending a part of positive clone bacterial liquid to Shanghai biological company for sequencing to obtain a gene sequence shown as a sequence table SEQ ID NO. 1;

(3) Construction of GbbZIP08 expression vector in ginkgo:

designing a vector construction primer (U: AGTGGTCTCTGTCCAGTCCTATGGCTTTTGGAGATCTCAG; D: GGTCTCAGCAGACCACAAGTTTAAGGTACCTGTCTAAGCATG), activating a plasmid bacterial liquid, referring to a plasmid extraction kit to illustrate plasmid extraction, adopting SfiI restriction enzyme to respectively enzyme-cut a plant subcellular localization vector pNC-Cam1304-sub C and a plant overexpression vector pNC-Cam2304-MCS35S (figure 3), and purifying enzyme-cut products for subsequent vector construction;

constructing a recombinant expression vector by using a Nimble cloning kit, transforming the recombinant expression vector into competent escherichia coli, selecting a monoclonal and using a detection primer to carry out bacterial liquid PCR verification, activating positive clone bacterial liquid by liquid, extracting a recombinant plasmid, and transferring the recombinant plasmid into agrobacterium GV3101 by using an electrotransformation method;

(4) Subcellular localization of GbbZIP08 in tobacco:

activating GV3101 agrobacterium containing a recombinant vector twice, culturing until the OD600 value is 0.6-0.8, centrifuging at 5000rpm at room temperature, removing supernatant, re-suspending thalli by using MES buffer, adjusting the OD600 value to 0.6, sucking bacterial liquid by using a 1mL syringe to inject from the back of tobacco leaves, marking, culturing tobacco in the dark for 12 hours after injection is completed, transferring into an illumination incubator to culture for 3-5 days, tearing the lower epidermis of the leaves in an injection area, observing fluorescence under a laser confocal microscope, photographing, and taking the tobacco leaves with the empty vector as a contrast;

it was observed that in tobacco leaves of the empty vector, nuclei were stained blue with DAPI dye, but no green fluorescence was observed, whereas in tobacco epidermal cells transformed with the 35S: gbbZIP08-GFP fusion expression vector, green fluorescence was observed (fig. 4), and overlapped with DAPI blue fluorescence, thus concluding that GbbZIP08 was located at nuclei;

(5) GbbZIP08 transformed Arabidopsis thaliana:

the arabidopsis plants in the full bloom stage are selected for infection, water is irrigated thoroughly the day before transformation, and the existing fruit pods are trimmed; pouring GV3101 agrobacterium infection solution (OD 600 is 0.8) containing a recombinant vector into a vessel with a large opening, completely soaking an arabidopsis inflorescence in the infection solution for about 1min, sleeving a plastic bag for moisture preservation, culturing in the dark for 12h, transferring into an illumination incubator, repeatedly infecting once after one week, culturing the infected plant in the plant illumination incubator until seeds are mature, and harvesting T0 generation seeds;

after the T0 generation seeds are disinfected, uniformly spreading the seeds in an MS solid plate containing corresponding antibiotics for culturing, growing for about 10 days, and transplanting seedlings with normal cotyledons and roots into a plant illumination incubator for culturing; after the seedlings are mature, taking tender leaves to carry out crude extraction DNA, carrying out positive PCR verification by using a carrier detection primer, continuously culturing the plants which are detected to be positive until the seeds are mature, and separating the plants to collect T1 generation seeds;

further screening the T1 generation seeds after disinfecting by using a resistant MS solid culture medium, selecting strains with positive ratio of 3:1 (figure 5) for transplanting, continuing to culture until the seeds are mature, and collecting the T2 generation seeds by dividing;

continuously screening the T2 generation seeds in the same way, wherein all the surviving seedlings in the resistance plate can be considered as homozygotes, and continuously culturing until mature plants, thus obtaining T3 generation transgenic arabidopsis homozygotes;

in fig. 5: A-D represent wild-type, T0 generation, T1 generation and T2 generation transgenic Arabidopsis thaliana, respectively;

(6) GbbZIP08 transformed tobacco:

cutting the sterile tobacco leaves into squares of 1cm multiplied by 1cm in an ultra-clean bench, placing the squares in GV3101 agrobacterium infection solution (OD 600 value is 0.8) containing an over-expression vector for 10-15 min, and intermittently shaking the squares during the period to enable the heavy suspension to fully soak the leaves; after infection, sucking the surface liquid of the leaf by sterile filter paper, transferring the back of the leaf to a co-culture medium downwards, and culturing in darkness at 28 ℃ for 48 hours;

transferring the explant into a differentiation culture medium to induce callus and buds (figure 6), forming adventitious buds to be differentiated into seedling bodies, cutting the seedling bodies, transferring the seedling bodies to a rooting culture medium to induce rooting, taking out tobacco from a tissue culture bottle after plant growth is stable, cleaning the residual culture medium at the root of the tobacco, transplanting the culture medium to nutrient medium soil, and placing the culture medium in a plant illumination incubator for culturing;

(7) Transgenic plant verification:

taking 1-2 tender leaves of the transgenic plant, marking the tender leaves in a 1.5mL centrifuge tube, grinding the leaves to be uniform slurry by a plant tissue grinding machine, and extracting genomic DNA of the plant by using a plant DNA rough extraction method;

carrying out positive PCR (polymerase chain reaction) verification on a transgenic plant by using a detection primer used for constructing a vector, taking wild plant DNA as a negative control, taking escherichia coli bacterial liquid containing a recombinant expression vector as a positive control, detecting a PCR product by agarose gel electrophoresis, and finding that the length of a target strip obtained by amplifying the transgenic plant is consistent with that of bacterial liquid PCR, wherein the target strip is not amplified in the wild plant (figure 7), thus indicating that the GbbZIP08 over-expression vector is successfully transferred into arabidopsis and tobacco;

in fig. 7: p is bacterial liquid PCR positive control; c is a wild plant negative control; m is 2000bp marker;

placing wild type and transgenic plant leaves in a 1.5mL centrifuge tube, marking, adding the prepared GUS dye solution to submerge the leaves, wrapping the leaves with tinfoil paper, placing the leaves in a constant temperature incubator at 37 ℃ for incubation overnight, transferring the leaves into 75% alcohol for soaking for 16 hours for decoloring after the dyeing is finished, changing the alcohol into 95% alcohol for decoloring again, changing the alcohol once a day until the leaves become white and transparent completely, placing the decolored leaves under a split microscope for observation and recording, and finding that the transgenic plant leaves are dyed blue, and the wild type plant leaves are not dyed (figure 8), thus indicating that the GbbZIP08 expression vector is successfully expressed in the transgenic plant;

in fig. 8: a is wild type Arabidopsis thaliana; b is transgenic Arabidopsis thaliana; c is wild type tobacco; d is transgenic tobacco;

taking leaves of wild type and transgenic plants, extracting RNA of the plants by referring to a plant RNA extraction kit, reversely transcribing the RNA into cDNA, using a target gene quantitative primer (U: GTCCAGTTCTGGTGACCCAC; D: GCCTGTTGGCTCTCCATCTT), performing fluorescent quantitative PCR detection on the target gene by taking the cDNA as a template, taking the wild type plants as a control, and setting 3 biological repeats for each sample;

the Arabidopsis thaliana reference gene is AtUBQ (NM_ 178970), the tobacco reference gene is NbGADPH (Niben 101Scf05177g 02025.1), and the quantitative result is 2 ^-△△CT Performing calculation by a method;

the expression level of GbbZIP08 in transgenic plants was significantly higher than wild type (fig. 9), indicating successful overexpression of GbbZIP08 in transgenic plants;

(8) Transgenic plant flavone content analysis:

grinding wild type and transgenic plant leaves with liquid nitrogen, drying by a freeze dryer, sieving with a 40-mesh sieve, weighing 0.02g of dried sample in a centrifuge tube by a balance, adding 2mL of 75% ethanol into the centrifuge tube, oscillating at 60 ℃ for 2h, centrifuging at room temperature 10000 Xg for 10min, taking supernatant, respectively adding relevant reagents in a kit for reaction, taking reaction liquid, measuring the absorbance value at 510nm by an enzyme-labeling instrument, and calculating the content of flavonoid in the sample, wherein the result is shown in figure 10;

weighing 0.1g of freeze-dried sample, adding 2mL of 75% methanol, carrying out ultrasonic crushing for 30min, centrifuging at room temperature 10000 Xg for 10min, taking 500 mu L of supernatant, adding 500 mu L of 36% hydrochloric acid, carrying out water bath at 85 ℃ for 1h, naturally cooling to room temperature, and filtering by a 45 mu m organic filter membrane to obtain the product for High Performance Liquid Chromatography (HPLC) detection;

HPLC conditions used in this experiment: the column model was Hypersil GOLD C18 (4.6 mm. Times.250 mm,5 μm), the sample size was 20. Mu.L, mobile phase A:100% acetonitrile, mobile phase B:2% acetic acid, column temperature: 30 ℃ and the flow rate is 1.0mL/min, and the elution procedure is as follows: 85% B to 67.5% B in 0-14 min, 67.5% B to 10% B in 14-23 min, 10% B to 85% B in 23-24 min, 24-28 min,85% B;

the kaempferol content was measured at a detection wavelength of 320nm and the results are shown in FIG. 11;

in fig. 11: A-C are chromatograms of kaempferol standard samples, arabidopsis thaliana and tobacco extracts at 320nm respectively;

grinding a plant sample with liquid nitrogen, drying by a freeze dryer, sieving with a 40-mesh sieve, weighing 0.1g of freeze-dried sample powder by a balance, adding 4mL of methanol (containing 1% hydrochloric acid in volume ratio), carrying out ultrasonic treatment at room temperature for 1h, wrapping the sample with tinfoil paper to avoid light, centrifuging at 120rpm for 4h and 5000rpm for 10min, taking 500 mu L of supernatant, adding 500 mu L of distilled water, shaking and mixing uniformly, then adding 500 mu L of chloroform, shaking and mixing uniformly to remove chlorophyll, sucking an upper water phase, and measuring a light absorption value at 530nm by an enzyme-labeled instrument;

anthocyanin content was calculated as formula 10× (a 530)/g (DW) and the results are shown in fig. 12;

through the detection analysis, the over-expression of the GbbZIP08 can obviously improve the contents of total flavonoids, kaempferol and anthocyanin in transgenic arabidopsis and tobacco, and the GbbZIP08 can promote the biosynthesis of plant flavonoids.

The present invention is not limited to the above-mentioned embodiments, and any changes or substitutions that can be easily understood by those skilled in the art within the technical scope of the present invention are intended to be included in the scope of the present invention.

Claims (1)

1. An application method of a gingko bZIP transcription factor GbbZIP08 in promoting plant flavonoid synthesis is characterized by comprising the following steps:

step 1: the gene sequence of GbbZIP08 cloned from ginkgo by PCR amplification is shown as a sequence table SEQ ID NO.1, and the coded amino acid sequence obtained by translation is shown as a sequence table SEQ ID NO. 2;

step 2: performing first-generation sequencing on the cloned gene sequence in the step 1 to obtain a base sequence of a GbbZIP08 gene, and performing subsequent gene sequence analysis and expression vector construction primer design;

step 3: constructing GbbZIP08 obtained by cloning on a pNC-Cam1304-sub plant subcellular localization vector and a pNC-Cam2304-MCS35S plant overexpression vector by a Nimble cloning method;

step 4: transforming agrobacterium GV3101 with the pNC-Cam 1304-sub-GbbZIP 08 expression vector constructed in the step 3, using the vector for transient transformation of tobacco, observing subcellular localization of GbbZIP08 in plants, and taking empty vector as a control;

step 5: transforming the pNC-Cam2304-MCS35S-GbbZIP08 expression vector in the step 3 into agrobacterium, transforming into arabidopsis by using an arabidopsis inflorescence infection method, and transforming into tobacco by using a tobacco leaf disc transformation method;

step 6: obtaining transgenic arabidopsis homozygote by adopting resistance screening, and obtaining a transgenic tobacco regeneration system by a plant tissue culture method; positive PCR, GUS staining and qRT-PCR methods were used to verify transgenic plants;

step 7: and (3) carrying out flavonoid content detection on the transgenic plant and the wild plant obtained in the step (6), and analyzing the influence of the over-expression GbbZIP08 on flavonoid synthesis in the plant.

Images