CN119082129B - A coding gene of chrysanthemum transcription factor, the transcription factor and its application - Google Patents
A coding gene of chrysanthemum transcription factor, the transcription factor and its application Download PDFInfo
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- CN119082129B CN119082129B CN202411282219.5A CN202411282219A CN119082129B CN 119082129 B CN119082129 B CN 119082129B CN 202411282219 A CN202411282219 A CN 202411282219A CN 119082129 B CN119082129 B CN 119082129B
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Abstract
本发明公开了一种菊花转录因子的编码基因、该转录因子及其应用，编码基因包括如Seq_1所示的基因序列，菊花转录因子的序列如Seq_2所示；编码基因或转录因子的应用，菊花转录因子用于耐热菊花品种的培育，通过调控编码基因的表达水平，改善温度对耐热菊花品种花瓣褪色情况的影响；本发明以HSFs基因调控菊花花色表型的研究出发，研究热激转录因子CmHSFA2b对于调控菊花花色的机理，得到高温环境栽培菊花花色的培育方法。
The invention discloses a coding gene of a chrysanthemum transcription factor, the transcription factor and an application thereof. The coding gene comprises a gene sequence as shown in Seq_1, and the sequence of the chrysanthemum transcription factor is as shown in Seq_2. The coding gene or the transcription factor is applied to the cultivation of heat-resistant chrysanthemum varieties, and the influence of temperature on the fading of petals of the heat-resistant chrysanthemum varieties is improved by regulating the expression level of the coding gene. The invention starts from the research on the regulation of chrysanthemum flower color phenotype by HSFs gene, studies the mechanism of heat shock transcription factor CmHSFA2b for regulating chrysanthemum flower color, and obtains a cultivation method for chrysanthemum flower color cultivated in a high temperature environment.
Description
Technical Field
The invention relates to a coding gene of a chrysanthemum transcription factor, the transcription factor and application thereof, belonging to the technical field of molecular biology.
Background
Chrysanthemum (Chrysanthemum multiplied by morifolium Ramat.) is one of four cut flowers in the world, is also a traditional famous flower with homology of medicine and food, and is widely used in industries of ornamental flowers, tea drinks, foods, chinese medicinal materials and the like. The south China has warm climate advantage of winter and spring, and is one of the main production areas of flowers in winter and spring of China. Most of the commercial chrysanthemum varieties in the current market are still from the European-American-day region with high latitude and cool, the varieties cannot be well suitable for the high-temperature climatic environment in the regions such as south China, southwest and the like, the phenomenon of high-temperature fading exists in most varieties, and the phenomenon of petal fading exists in most red-purple-series chrysanthemum varieties in the high-temperature region due to the global greenhouse effect and the urban heat island effect, so that the market value and the ornamental value of the chrysanthemum are seriously influenced. The chrysanthemums are classified into varieties of (pink), purple (red), orange (red), yellow, white, multiple colors and the like according to colors, and petal color pigments of the varieties of red and purple series chrysanthemums mainly comprise anthocyanin (Anthocyanin, also called anthocyanin), compared with a very detailed anthocyanin synthesis pathway and regulation research thereof, the regulation research of the anthocyanin degradation pathway is still very rare at present, and the regulation mechanism of plant anthocyanin metabolism (synthesis or degradation) pathway under high-temperature stress is still unclear. The prior researches show that the high temperature stress often inhibits the accumulation of plant anthocyanin to form a fading phenotype, but the specific molecular mechanism is still unclear, so that the influence of temperature on the color of chrysanthemum cannot be improved, and the market value of flowers is limited.
Disclosure of Invention
In order to overcome the defects of the prior art, the first aim of the invention is to provide a coding gene of a chrysanthemum transcription factor.
The second object of the present invention is to provide a chrysanthemum transcription factor which has direct influence on regulating chrysanthemum flower color.
The third object of the invention is to provide an application of the coding gene and the chrysanthemum transcription factor, which is a cultivation method for cultivating chrysanthemum flower colors in a high-temperature environment.
The fourth object of the invention is to provide a cultivation reagent for heat-resistant chrysanthemum varieties.
The first object of the present invention can be achieved by adopting the following technical scheme that a coding gene (CmHSFA b) of a chrysanthemum transcription factor comprises a gene sequence shown as seq_1.
The second aim of the invention can be achieved by adopting the following technical scheme that the chrysanthemum transcription factor is a chrysanthemum transcription factor, the coding gene of the chrysanthemum transcription factor is as described above, and the sequence of the chrysanthemum transcription factor is shown as seq_2.
The third purpose of the invention can be achieved by adopting the following technical scheme that the coding gene or the chrysanthemum transcription factor is used for cultivating heat-resistant chrysanthemum varieties.
Further, the influence of temperature on the fading condition of petals of the heat-resistant chrysanthemum variety is improved by regulating and controlling the expression level of the coding gene.
Further, the temperature is at least 25 ℃ at night and at least 34 ℃ at daytime.
Further, the cultivation of the heat-resistant chrysanthemum variety comprises the step of infecting the chrysanthemum leaf disc or the head inflorescence of the bud stage with a vector with a coding gene through a mediation method.
Further, the vector is pB 2GW7 vector.
Further, the means for controlling the coding gene is an over-expression means for increasing the expression level.
Further, the means for modulating the coding gene is a gene silencing, gene editing or gene knockout means.
The fourth aim of the invention can be achieved by adopting the following technical scheme that the reagent for cultivating the heat-resistant chrysanthemum varieties regulates the expression level of the coding genes.
Further, the regulated expression level is over-expression, the breeding reagent is a proliferation culture medium, and the proliferation culture medium comprises MS, 3 mg/L6-BA, 0.5mg/L NAA and 0.2mg/L PVP.
Compared with the prior art, the invention has the beneficial effects that:
1. the invention provides a gene for coding CmHSFA b protein, an amino acid sequence of chrysanthemum heat shock transcription factor CmHSF A b protein, and application, such as a cultivation method and a reagent, a recombinant vector and a cell with a gene sequence for coding CmHSFA b protein, and a cultivation reagent;
2. According to the invention, by starting from the research of HSFs gene regulation and control of chrysanthemum flower color phenotype, the mechanism of heat shock transcription factor CmHSFA b on regulating and controlling chrysanthemum flower color is researched, and a cultivation method for cultivating chrysanthemum flower color in a high-temperature environment is obtained;
3. The research result of the invention can be used for molecular breeding of heat-resistant chrysanthemum, lays a molecular breeding theoretical foundation for breeding domestic chrysanthemum varieties with stable and non-fading color in high-temperature areas, and has important significance for promoting the high-quality development of flower industry in China.
Drawings
FIG. 1 relative expression of the CmHSFA b gene in the S1-S4 period of the head inflorescence;
FIG. 2 phenotype of high temperature fugitive 'purple red Toguy' chrysanthemum after glufosinate spraying;
FIG. 3 clones amplified bands of the high temperature non-discoloring chrysanthemum `purple windmill` CmHSFA b gene;
FIG. 4 is a map of the entry cloning vector Gateway pDONR 221;
FIG. 5 is a map of the overexpression vector pB 2GW7;
FIG. 6 DNA identification of high temperature fugitive chrysanthemum 'purple sweet osmanthus' overexpressing plants;
FIG. 7 'analysis of gene expression levels of leaves of the purple sweet osmanthus' CmHSFA b over-expressed plants;
FIG. 8 'purple sweet osmanthus' CmHSFA b over-expressed plant for flower color phenotyping;
FIG. 9 is a map of silencing expression vector pNC-TRV 2;
FIG. 10 inflorescence phenotype analysis of high temperature non-discoloring chrysanthemum `purple windmill` after CmHSFA b gene silencing;
FIG. 11 'Violet' gene silencing plant CmHSFA b gene expression levels.
Detailed Description
The invention will be further described with reference to the accompanying drawings and detailed description below:
Example 1
1. High-temperature treatment and collection of chrysanthemum experimental material
The chrysanthemum experimental material is selected from a high-temperature and fading-prone chrysanthemum variety 'purple red Togui' and a high-temperature and fading-resistant variety 'purple windmill', and the chrysanthemum experimental material is collected from a Guangzhou white cloud bamboo house and village-collecting chrysanthemum base (east longitude 113.23 degrees and North latitude 23.16 degrees). Potted chrysanthemum which contains head inflorescences in four development periods (S1 is a bud color development period, S2 is a primary flowering period, S3 is a flower containing period, S4 is a flower containing period) is selected, a normal temperature control group (day 24 ℃,10h,20000Lx; night 15 ℃,14h,0 Lx) and a high temperature experiment group (day 35 ℃,10h,20000Lx; night 26 ℃,14h,0 Lx) are set according to the day and night temperature characteristics of winter and spring in south China, and after 7 days of treatment, the head inflorescences in each development period are collected and quick-frozen by liquid nitrogen and then stored in a-80 ℃ ultralow temperature refrigerator.
2. Extraction of total RNA of chrysanthemum and expression analysis of CmHSFA b gene
The tongue petals of the head inflorescences of four developmental stages were taken and RNA extraction and purification were performed using FastPure Universal Plant Total RNA Isolation Kit (Nanjing, nanjing Biotech Co., ltd.) according to the specification. Mu.g of total RNA was taken, reverse transcribed into cDNA using HIFAIRIII 1: 1ST STRA ND CDNA SYNTHESIS SuperMix for qPCR kit (Highway, inc. of Saint Biotech Co., ltd.) and the cDNA product obtained by the reverse transcription was diluted 3-fold and used for qRT-PC R.
Downloading feverfew HSFA2b from the feverfew reference genome database (http:// mum-gardon. Kazusa. Or. Jp /) as reference sequence, primers were designed on-line via NCBI-BLAST website (http:// www.ncbi.nlm.nih.gov/to ls/primer-BLAST /):
The upstream primer qCmHSFA b-F5'-AAAGAGCACCTTCGCAGTCA-3' (seq_3);
the downstream primer qCmHSFA b-R5'-TGGGCTCGTGTGTTTTGTTG-3' (seq_4);
The internal reference gene is CmEF alpha gene (Genbank accession number: KF 305681.1):
Upstream primer qCmEF a-F: 5'-TTTTGGTATCTGGTCCTGGAG-3' (seq_5);
Downstream primer qCmEF a-R5'-CCATTCAAGCGACAGACTCA-3' (seq_6).
Primer verification, namely detecting the amplification efficiency and the correlation coefficient of the primer by using a standard curve method. The experiment used CF X Connect Real-Time fluorescent quantitative PCR System (BIO-RAD, USA) with a reaction system of 50. Mu.L containing 25. Mu. L HIEFF QPCR SYBR GREEN MASTER Mix (Highway, st. Biotech Co., ltd.), 1. Mu.L of upstream/downstream primer (10. Mu.M), 1. Mu.L of cDNA template and appropriate amount of ddH 2 O. The reaction was performed using the procedure of 95℃pre-denaturation for 5min, 95℃denaturation for 10s,60℃annealing for 20s,72℃extension for 20s,40 cycles, and finally 72℃final extension for 5min, and after amplification the reaction product was subjected to gel electrophoresis to confirm that the amplified bars were clear and single.
Expression analysis of chrysanthemum CmHSFA b gene:
The reaction procedure was divided into two steps, a first step, a 95 ℃ pre-denaturation for 5min, a second step, a 95 ℃ denaturation for 10s, a 60 ℃ annealing for 30s, which were repeated for 40 cycles, and a dissolution profile was made by the fluorescent quantitative PCR instrument default setting. The cDNA concentration was adjusted according to the number of cycles of Deltact, and the cDNA was diluted with dd H 2 O with a fold change of 2 -ΔΔct as a reference, followed by qRT-PCR to determine if the number of cycles Deltact was between 20 and 21. Carrying out real-time fluorescence quantitative detection reaction on the CmHSFA b gene primer with specificity, the internal reference CmEF alpha gene primer and the cDNA diluted 3 times (3 times of technical repetition are carried out on the same cDNA template, 3 times of biological repetition are carried out on the same treatment group, and each reaction takes the internal reference gene as a control).
The relative expression amount of the gene was calculated by the method of 2 -ΔΔCT, and the result is shown in FIG. 1. The results show that the expression level of the 'purple windmill (ZFC)' CmHSFA b gene in inflorescences at various periods after high-temperature treatment is obviously improved compared with that of normal temperature. The expression quantity of the gene 'Zihongtuogui (ZHTG)' CmHSFA b in the early flowering stage (S3) under normal temperature condition is highest, and the expression quantity of the gene CmHSFA b in the normal temperature control group plant and the high temperature treatment group plant is greatly different in the period, namely the expression level of the gene in the inflorescence in the early flowering stage after the high temperature treatment is obviously reduced.
Example 2
1. Hormone orthogonality experiment of high-temperature fading-prone chrysanthemum purple sweet osmanthus proliferation culture medium
The explant is selected from young leaves with the seedling age of about 30d and fully developed upper parts of aseptic seedlings of the 'purple red Togui' chrysanthemum, and the young leaves are cut into leaf discs with the size of 0.5cm 2 and the size of 0.5 x with flush wounds at the periphery by using a surgical knife, and are respectively inoculated into M1-M9 proliferation culture mediums, as shown in table 1:
TABLE 1 Chrysanthemum leaf disk proliferation Medium, inductivity and budding number data
10 Leaf discs were inoculated per medium, repeated 5 times, and a total of 50 leaf discs were inoculated. After the culture medium inoculated with the leaf disc is cultured for 30 days under the environment condition of 25 ℃ and 14h/10h (light/dark), the callus induction rate and the bud number of the leaf disc are counted, wherein the basic formula of the proliferation culture medium is MS+3wt% sucrose+0.7wt% agar powder, 6-benzylaminopurine (6-BA) and naphthalene acetic acid (NA A) with different proportions are added on the basis, and the calculation formula of the callus induction rate is 100% of the number of explants producing callus per the number of explants inoculated. Test results show that the optimal proliferation culture medium formula of the 'purple sweet osmanthus' She Panzu culture is MS+3 mg/L6-BA+0.5 mg/L NAA+0.2mg/L PVP+3wt% sucrose+0.7 wt% agar, and the culture medium formula is a key formula for influencing the transformation success rate of the 'purple sweet osmanthus' over-expression plant, namely indirectly influencing the flower color phenotype of the over-expression plant.
2. High-temperature easy-to-fade chrysanthemum purple red tolggui' glufosinate spraying concentration experiment
Glufosinate (also known as glufosinate) belongs to an organophosphorus herbicide and is an active ingredient of a nonselective broad-spectrum herbicide Basta, and the glufosinate strongly inhibits the activity of glutamine synthetase in plants, so that nitrogen metabolism in the plants is disturbed, ammonium is excessively accumulated, chloroplasts are disintegrated, photosynthesis is inhibited, and finally the plants die due to yellowing. The glufosinate-ammonium water agent (PPT content is 10 wt%) of the spraying experiment is purchased from the Shanghai corporation of biological engineering, and is diluted by pure water by 1000 times (PPT concentration is 100mg/L,0.01 wt%), 2000 times (PPT concentration is 50mg/L,0.005 wt%), 3000 times (PPT concentration is 33mg/L,0.0033 wt%), 4000 times (PPT concentration is 25mg/L,0.0025 wt%) and 5000 times (PPT concentration is 20mg/L,0.002 wt%) respectively for later use. Spraying glufosinate solution with different concentrations on the 'purple red Togui' chrysanthemum seedlings with similar growth vigor, treating three pots with each concentration, and spraying pure water as a control, wherein the spraying is performed once every 3d, and the total spraying is performed for 4 times. The commercial 100mL miniature plastic watering can with good atomization effect is selected, the uniform spraying of the front and back surfaces of all leaf discs of the chrysanthemum is ensured, the higher soil matrix humidity and air humidity (about 80% relative humidity) are kept during the treatment period, and the growth state of plants is recorded after 3d of the fourth treatment. Experimental results show that when the PPT concentration in the sprayed glufosinate solution is 0.01wt%, 0.005wt% and 0.0033wt%, the plants of the 'rhododendron torsemium' chrysanthemum all show different degrees of verticality wilting, wherein when the PPT concentration is 0.005wt%, the plant has the highest degree of verticality, and the result is shown in figure 2, and the PPT concentration is the key concentration for successfully screening the 'rhodotorsemium' over-expression positive plants, namely the color phenotype of the over-expression plants is indirectly influenced.
Example 3
1. Chrysanthemum CmHSFA b gene clone
The nucleotide sequence of 'purple windmill' CmHSFA b with the length in accordance with the expected length is cloned, the length of the coding sequence is 1029bp, and the result is shown in figure 3. Sequencing results show that the nucleotide sequence of the 'purple windmill' CmHSFA b gene is shown as seq_1, and the amino acid sequence of the coded protein is shown as seq_2.
ATGGATCCTTTTTACTCCGTTGTTAAAGAAGAATACCCATCTAGTGGTAGTGGTAGTGGTGATGGTAGAGGGGCAAGGCAGATGATCATGAGTGTGCAAATACCACAACCAATGGAGGGTTTACATGATGCAGGGCCACCACCATTTTTGACAAAAGTATATGATATGGTGGAAGATAAAAGTATTGATGATATTATTCGTTGGAGCAGAGGTGGTCAAAGCTTTGTTGTATTGGATCCACAGGCTTTCTCTACTAATCTCTTGCCAAGATATTTTAAGCATAATAATTTCTCTAGCTTTGTCAGGCAGCTCAATACTTATGGTTTTAGAAAGATAGATCCTGATATATGGGAGTTTGCAAATGAAACATTCGTGAGAGGCCAAAGGCATGTTTTGAAGAACATCAAGAGAAAAAGAGCACCTTCGCAGTCATTATCTCCACAAAAAACTCAGAGTCCTGGTTCCGAAACTGCAACATTAGGATCAGATGAAGTCGGCCGCCTAAAACACGAAAAAGAGGTTCTCATGATGGAATTAGTGAAACTCAGACAGAAACAACAAAACACACGAGCCCAAATTCAAGCTATGGAGGTTAGATTACAAGGAACCGAGGAAAAACAGCGAAAGATGATGAGGTTCTTGGCAAAAGCAATGCAAAATCCTGATTTTATTCGGAAGTTGGTCAAACATGGTAAAGGAAAGGAACTTCAAGAAGCTTTCATGAACCAAATTGGCGAATCAAGTGGCGGGTCAAAGCTTATTAAAGCCGAACCTGAAGACTTCACGGATGCTTCCACATTTGAAGTGTCTGAACTCGAAGCACTTGCTTTAGAAATGCAAGGGTTTGGTAGAAACCAAAGGAATCAAGAGGAAGAAAGTAACAAGCTTGAATTCAAAGGTGGCGAAAGAGAACTTGATGACGAATTCTGGGAAGAGTTATTTAGTGAACGAGTTGATGATAAATCAGGAGTTGAAGATGTGAATTTCTTGGCTGAAAAGTTGGATTTCTTGGGTTCAAGCCCAAAGTAA(Seq_1);
MDPFYSVVKEEYPSSGSGSGDGRGARQMIMSVQIPQPMEGLHDAGPPPFLTKVYDMVEDKSIDDIIRWSRGGQSFVVLDPQAFSTNLLPRYFKHNNFSSFVRQLNTYGFRKIDPDIWEFANETFVRGQRHVLKNIKRKRAPSQSLSPQKTQSPGSETATLGSDEVGRLKHEKEVLMMELVKLRQKQQNTRAQIQAMEVRLQGTEEKQRKMMRFLAKAMQNPDFIRKLVKHGKGKELQEAFMNQIGESSGGSKLIKAEPEDFTDASTFEVSELEALALEMQGFGRNQRNQEEESNKLEFKGGERELDDEFWEELFSERVDDKSGVEDVNFLAEKLDFLGSSPK(Seq_2).
2. Gene function analysis of chrysanthemum CmHSFA b over-expression
The gene over-expression experiment is carried out by using Gateway BP Cl onase II Enzyme Mix and GATEWAY LR Clonase II Enzyme Mix kits of Simer Feishier technology (China) Co., ltd., ga teway recombination can clone PCR products to a donor vector containing attP locus through BP recombination reaction to generate an entry clone, and then the entry clone is recombined to a target vector containing attR locus through LR recombination reaction to generate an expression clone. The Gateway pDONR 221 vector is purchased from Simer Feishier technology (China) limited company, the vector map is shown in figure 4, the pB 2GW7 vector is stored by a common high school tropical and subtropical flower and garden plant key laboratory of Guangdong province of Semish kai agricultural engineering college, and the vector map is shown in figure 5.
Primers were designed on-line via the NCBI-BLAST website based on the cloned 'purple windmill' CmHSFA b sequence and the HSFA2b reference sequence downloaded from the feverfew reference genome database (with attB linker sequences added at both ends of the primers according to the primer design requirements of the Gateway recombination kit, underlined below):
The upstream primer attB-CmHSFA b-F:5'-ggggacaagtttgtacaaaaaagcaggcttcATGGATCC TTTTTACTCCGTTG-3' (seq_7);
the downstream primer attB-CmHSFA b-R:5'-ggggaccactttgtacaagaaagctgggtcTTACTTTG GGCTTGAACCCAAG-3' (seq_8);
The cDNA of chrysanthemum is used as a template, a designed primer with a joint is used according to the use instruction of 2X HIEFF CANACE Plus PCR MASTER Mix (the following Santa Biotechnology Co., ltd., shanghai), an amplification system of a target gene is configured, a reaction program is set, the obtained PCR product is subjected to 2m/v% agarose gel electrophoresis detection to determine that the size of a band is correct, and then the E.Z.N.A.gel Extraction Ki t D2500 kit of Omega Bio-Tek company is used for cutting, recovering and purifying, wherein the specific steps are carried out according to the instruction.
2.1BP recombination reaction (construction of the Advance cloning vector)
The purified gel recovery product was mixed with Gateway pDONR221 vector to prepare a 10. Mu.L system, which included adding 15-150ng of the gel recovery product, 150ng Gateway pDONR221 vector, 2. Mu. LBP Clonase II Enzyme Mix, TE buffer (pH 8.0) to 10. Mu.L, and after thoroughly mixing, reacted at 25℃for 1h using ordinary PCR. The experiment was performed using DH 5. Alpha. FA ST CHEMICALLY Competent Cell F (11803 ES 80) from the company of Santa Biotechnology, inc., and by heat shock, the specific steps were performed according to the instructions. After transformation of the reaction products into competent cells, positive transformants were screened using LB medium containing kanamycin, and bacterial liquid PCR was performed using the universal primers M13F and M13R, and sequencing was performed on bacterial liquids whose agarose gel electrophoresis band results were consistent with the expected sizes (all sequencing works in this experiment were performed by the company Shanghai Biotechnology Co., ltd.), and bacterial liquids which showed successful transfer of the target genes were extracted using the E.Z.N.A.plasmid Mini KitI (D6943) kit from Omega Bio-Tek company, and the specific procedures were performed according to the specification.
2.2LR recombination reactions (construction of expression cloning vector)
The plasmid containing the entry clone was mixed with pB 2GW7 vector to prepare a 10. Mu.L system, which included adding 50-150ng of the plasmid containing the entry clone, 150ng of pB 2GW7 vector, 2. Mu. L LR Clonase II Enzyme Mix, TE buffer (pH 8.0) to 10. Mu.L, and after thoroughly mixing, the reaction was performed at 25℃for 1 hour using ordinary PCR, and the reaction product was transformed into DH 5. Alpha. FAST CHEMICALLY Competent Cell F by a heat shock method. After transformation of the reaction products into competent cells, positive transformants were screened using LB medium containing spectinomycin and verified by bacterial liquid PCR with attB1 and attB2 primers, the sequences of which were as follows:
attB1:5’-GGGGACAAGTTTGTACAAAAAAGCAGGCT-3’(Seq_9);
attB2:5’-GGGGACCACTTTGTACAAGAAAGCTGGGT-3’(Seq_10);
Sequencing and verifying the bacterial liquid with the same expected size as the agarose gel electrophoresis band result, wherein the sequencing result shows that the bacterial liquid successfully transferred into the target gene is extracted by using an E.Z.N.A.plasmid Mini KitI kit to obtain the recombinant plasmid containing the expression clone.
2.3 Transformation of recombinant plasmids into Agrobacterium tumefaciens
The experiment was performed by selecting GV3101 CHEMICALLY COMPETENT CELL (CAT#: AC 1001) from Shanghai Biotechnology Co., ltd and transforming by freeze thawing, and the specific steps were performed according to the specification. After transformation of recombinant plasmids containing expression clones into competent cells, positive transformants were screened using LB medium containing rifampicin and spectinomycin and bacterial liquid PCR verification was performed with attB1 and attB2 primers, verifying that the correct bacterial liquid was mixed with 50% v/v glycerol 1:1 for seed preservation and stored in a-80℃ultra-low temperature refrigerator.
2.4 Acquisition of acceptor materials
Selecting young stem segments of healthy 'purple sweet osmanthus' single plants, removing terminal buds and leaves, washing for 30min in running water, soaking chrysanthemum stem segments in a super clean bench with 75% v/v alcohol (50 mu L Tween-20) for 30s, washing with sterile water for 5 times, then soaking with 2wt% NaClO solution for 6min (50 mu L Tween-20), and washing with sterile water for 5 times. Placing chrysanthemum stem on sterile filter paper, sucking surface liquid, cutting into stem with bud point of about 1cm by using sterile surgical knife, inoculating the lower end of morphology into differentiation culture medium, cutting when the bud of stem in differentiation culture medium is 1cm long, inoculating into culture medium without antibiotic root to make rooting induction, and obtaining the aseptic seedling of purple sweet osmanthus with good rooting of plant. The formula of the differentiation medium is MS+3 mg/L6-BA+0.5 mg/L NAA+3wt% sucrose+0.7wt% agar+200 mg/L carbenicillin, and the formula of the root-free medium is 1/2MS+0.1mg/L NAA+3wt% sucrose+0.7wt% agar.
2.5 Agrobacterium-mediated genetic transformation of the 'Violet Tonna' leaf discs
2.5.1 Pre-incubation of acceptor materials
Taking the aseptic seedling leaf of 'Violet Tonggui' with seedling age of about 30d, cutting into leaf discs with the size of 0.5cm 2 and the periphery with flush wound, horizontally placing leaf backs on a preculture medium, and culturing in dark for 48h. According to example 2, the formula of the pre (co) culture medium, namely the proliferation culture medium, is MS+3mg/L6-BA+0.5 mg/L NAA+0.2mg/L PVP+3wt% sucrose+0.7wt% agar, and PVP is added into the culture medium in the step and the subsequent steps, so that the phenomenon of browning of chrysanthemum leaf discs after infection by agrobacterium can be effectively prevented.
2.5.2 Preparation of the dye liquor
Taking out the stored agrobacterium liquid from a-80 ℃ ultralow temperature refrigerator, streaking the agrobacterium liquid into a YEB culture medium containing rifampicin and spectinomycin, after culturing for 48 hours in a 28 ℃ incubator, picking single colonies, performing initial shaking in 4mL of the YEB liquid culture medium containing the corresponding antibiotics, after culturing for 24 hours in a constant temperature shaker at 28 ℃ and 240rpm, taking 1mL of the single colonies, adding 100mL of the YEB liquid culture medium containing the corresponding antibiotics, culturing to OD 600nm of about 0.6 in the constant temperature shaker under the same condition, transferring the bacterial liquid into a sterile 50mL centrifuge tube, collecting the bacterial liquid in a refrigerated centrifuge at 4 ℃ and 6000rpm for 5min, removing supernatant in an ultra-clean workbench after centrifugation, and adding MS heavy suspension to adjust the OD 600nm to 0.6. MS heavy suspension formula is MS+3wt% sucrose+100 mu M acetosyringone.
2.5.3 Infestation
Pouring the invasion solution into a sterile container, immersing the pre-cultured leaf disc in the invasion solution for 8min, continuously shaking to enable the bacterial solution to be in full contact with the leaf disc, taking the leaf disc out of the invasion solution after infection, and sucking the surface liquid by using sterile filter paper.
2.5.4 Co-cultivation
The infected leaf discs were inoculated leaf-back down onto pre (co) medium and dark cultured at 25 ℃ for 48h.
2.5.5 Delay culture
The co-cultured explant is soaked in sterile water containing 250mg/L of timentin for 10min, the explant is continuously shaken to enable the liquid to be fully contacted with the leaf disc, the surface liquid is sucked by sterile filter paper for 2 times, the leaf disc is inoculated on a delay culture medium in a mode of downward leaf back, and the culture is carried out in darkness for 48h under the condition of 25 ℃. The formula of the delay medium comprises MS+3 mg/L6-BA+0.5 mg/L NAA+0.2mg/L PVP+3wt% sucrose+0.7 w/v% agar+250 mg/L timentin.
2.5.6 Decarboxylation culture
The leaves after the delay culture were inoculated back to back on the decarboxylation medium, and subjected to decarboxylation culture for 7d. The decarboxylation culture medium comprises MS+3 mg/L6-BA+0.5 mg/L NAA+0.2mg/L PVP+3wt% sucrose+0.7 w/v% agar+500 mg/L carbenicillin.
2.5.7 Induced culture
The explant after decarboxylation culture is soaked in sterile water containing 500mg/L of carbenicillin for 10min, the liquid is continuously shaken to be fully contacted with the leaf disc, the leaf disc is washed by sterile water for 2 times, surface liquid is sucked by sterile filter paper, the leaf disc is inoculated on an induction culture medium in a mode that the leaf back of the leaf disc faces downwards, and the culture is carried out under the environment condition of 25 ℃ and 14h/10h (light/dark), and the new induction culture medium is replaced every 10 d. The formula of the induction culture medium comprises MS+3 mg/L6-BA+0.5 mg/L NAA+0.2mg/L PVP+3wt% sucrose+0.7 w/v% agar+250 mg/L carbenicillin.
2.5.8 Rooting induction
Cutting when the buds differentiated from the leaf discs in the induction culture medium grow to 1-2cm, and inoculating the buds into a rooting culture medium for rooting induction. The rooting culture medium comprises 1/2MS+0.1mg/L NAA+3wt% sucrose+0.7w/v% agar+250 mg/L carbenicillin.
2.5.9 Hardening off and transplanting
After rooting induction for 15d, the well-grown plants of the root system can be opened to smelt seedlings for 7d, and then the plants are removed from the culture flask, excess agar in the root system is washed away, transplanted into a clean universal gardening matrix, and normal flowering culture in the field is maintained.
2.6 Screening and identification of transgenic Chrysanthemum
The pB2GW7 vector contains a glufosinate-ammonium resistance Bar gene which codes phosphinothricin acetyl transferase PAT, PAT can acetylate free amino of PPT, so that the activity of glutamine synthetase is not inhibited, and plants containing the gene have resistance to glufosinate herbicide. The 'purple red Toguy' plants obtained by screening and transformation were sprayed with a glufosinate aqueous solution (PPT concentration 0.005 wt%) diluted 2000-fold with pure water, and the specific spraying process is described in example 2. And (3) taking a third leaf of the plant which grows normally after the treatment, quickly freezing the third leaf in liquid nitrogen, extracting DNA from the collected sample by using FastPure PLANT DNA IS olation Mini Kit kit of Nanjinouzan biotechnology Co., ltd, carrying out PCR identification by using attB1 and attB2 primers, and obtaining the plant corresponding to the DNA of the target strip by agarose gel electrophoresis, wherein the plant is the transgenic positive plant. Three transgenic positive plants were obtained in total in this experiment, and the results are shown in FIG. 6.
2.7 Expression analysis of transgenic Chrysanthemum
Total RNA extraction and purification are carried out on chrysanthemum transgenic plant leaves, and cDNA is synthesized by reverse transcription. The cDNA product obtained by reverse transcription was diluted 3 times and used for qRT-PCR, cmHSFA2b gene primer was qCmHSFA b-F/qCmHSFA b-R, internal reference gene primer qCmEF1 a-F/qCmEF 1 a-R, and the reaction procedure was as described in example 1.
The calculation of the relative expression amount of the gene was performed by the method of 2 -ΔΔCT, and the result is shown in FIG. 7. The results showed that the expression level of CmHSFA b in the over-expressed plants (pB 2GW7 -HSFA2 b) was increased compared to the wild type plants (WT) and the empty control plants (pB 2GW7), wherein the expression level of CmHSFA b in the over-expressed plant 1 was significantly increased. This shows that the 35S promoter in pB 2GW7 vector can raise the transcription level of CmHSFA b well, so as to raise the expression of the gene in purple sweet osmanthus plant.
2.8 Phenotypic identification of transgenic Chrysanthemum
Phenotypic observation of the chrysanthemum overexpressing plants and the empty control plants at 10h/14h (light/dark) short sunlight environment condition until flowering shows that the colors of the tongue petals of the head inflorescences of the 'purple red Tonggui' plants overexpressing CmHSFA b are obviously deepened, and the result is shown in figure 8.
Example 4
1. Virus-induced Gene silencing (VIGS) verification of Chrysanthemum CmHSFA b
Gene silencing experiments were performed using Nimble Cloning kit from Hainan-Biotechnology, inc., nimble Cloning (NC cloning) enabled cloning of PCR products into circular expression vectors of NC system via Nimble Mix. The pNC-TRV2 vector was given away by the doctor of the language (Yan et al 2020), and the vector map is shown in fig. 9.
Primers were designed on-line via the NCBI-BLAST website based on the cloned 'purple windmill' CmHSFA b sequence and the HSFA2b reference sequence downloaded from the feverfew reference genome database (20 bp universal adaptor sequences were added at both ends of the primers according to the primer design requirements of the Nimble Clonin g kit, the following are underlined):
The upstream primer NC-CmHSFA2b-F:5'-agtggtctctgtccagtcctTACTCCGTTGTTAAAG AAGAATACCAATC-3' (seq_11);
The downstream primer NC-CmHSFA2 b-R5'-ggtctcagcagaccacaagtATAAGTATTGAGCTG CCTGACAAAG-3' (seq_12).
The designed adaptor-carrying primer is used for preparing an amplification system of a target gene according to the use instruction of 2X HIEFF CANACE Plus PCR MASTER Mix and setting a reaction program, the obtained PCR product is purified by using an E.Z.N.A.cycle-Pure Kit (D6492) Kit of Omega Bio-Tek company, and the specific steps are carried out according to the instruction since target fragments are smaller than 200bp and 6 times of CP buffers are added.
1.1TRV2-HSFA2b recombinant vector construction
The purified PCR product was mixed with NC system expression vector TRV2 to prepare a 10. Mu.L system, which included adding 10-80ng of the PCR product, 20-120ng of NC system expression vector, 5. Mu L Nimble Mi x, ddH 2 0 to 10. Mu.L, after thoroughly mixing, the reaction was carried out at 50℃for 45min using ordinary PCR, and the reaction product was converted to DH 5. Alpha. FAST CHEMICALLY Competent Cell F by a heat shock method. After transformation of the reaction products into competent cells, positive transformants were selected using LB medium containing kanamycin and verified by bacterial liquid PCR with PE GFP-C5 and NosR primers, the sequences of which were as follows:
PEGFP-C5:5’-catggtcctgctggagttcgtg-3’(Seq_13);
NosR:5’-accggcaacaggattcaatc-3’(Seq_14)。
Sequencing and verifying the bacterial liquid with the same expected size as the agarose gel electrophoresis band result, wherein the sequencing result shows that the bacterial liquid successfully transferred into the target gene is extracted by using an E.Z.N.A.plasmid Mini KitI kit to obtain the plasmid containing the recombinant vector.
1.2 Transformation of recombinant plasmids into Agrobacterium tumefaciens
The plasmid containing the recombinant vector is transformed into GV3101 CHEMICALLY COMPETENT CE LL by a freeze thawing method, positive transformants are screened by using LB culture medium containing rifampicin and kanamycin, bacterial liquid PCR verification is carried out by using PEGFP-C5 and NosR primers, and correct TRV2-HSFA2b bacterial liquid is verified to be mixed with 50% v/v glycerol 1:1 for seed preservation and stored in an ultralow temperature refrigerator at-80 ℃.
1.3 Agrobacterium-mediated Chrysanthemum plant infestation
The experiment refers to the method of Wang Yu and the like (2018), adopts an improved vacuum infection method to infect the plants of high-temperature non-fading chrysanthemum 'purple windmill', and carries out three groups of experiments, namely a pure water control group (CK), an empty load control group (TRV 2) and a VIGS experiment group (TRV 2-HSFA2 b).
1.3.1 Preparation of the dye liquor
Taking out the stored TRV2, TRV2-HSFA2b and TRV1 (GenBank: AF406990, namely pTRV1 vector without exogenous gene transfer) from a-80 ℃ ultralow temperature refrigerator, respectively streaking the TRV2, TRV2-HSFA2b and TRV1 (GenBank: AF406990, namely pTRV1 vector without exogenous gene transfer) in a YEB culture medium containing rifampicin and kanamycin, culturing for 48 hours in a 28 ℃ incubator, picking up single colony, initially shaking in 10mL of the YEB liquid culture medium containing corresponding antibiotics, culturing for more than 24 hours in a constant temperature shaking table at 28 ℃ and 240rpm until the bacterial liquid is saturated, taking 5mL of the solution, adding 500mL of the YEB liquid culture medium containing the corresponding antibiotics, culturing to OD 600nm of about 0.8 in the constant temperature shaking table under the same condition, respectively transferring the bacterial liquid to 50mL of aseptic for multiple times, collecting bacterial bodies in a freezing centrifuge at 4 ℃ and 6000rpm and 5min, removing the supernatant in an ultra-clean workbench, adding MMA solution, fully mixing until OD 600nm is 1.0, and the method can be used for infection. MMA solution was formulated with 10mM MgCl 2 +10mM MES (pH 5.6) +100. Mu.M acetosyringone. After the three bacterial solutions are resuspended by MMA solution to meet the requirement, the TR V1 bacterial solution is uniformly mixed with the TRV2 bacterial solution and the TRV2-HSFA2b bacterial solution according to equal volumes, and the prepared dyeing solution is required to be used after being kept stand for 4 hours at normal temperature and away from light.
1.3.2 Infestation
The plant material is a plant in which flower buds are differentiated from chrysanthemum 'purple windmill', the plant material is subjected to water-break treatment 2d in advance, and is subjected to dark treatment for 4h before infection, and after marking, a bag is used for covering the plant flowerpot to prevent flower mud from pouring out. After the infection liquid is kept stand at normal temperature and away from light, 30 mu L of Silwet L-77 surfactant is added and fully mixed. Inverting the plant material, immersing the flower buds of the plant in the dyeing liquid for 1min (pure water control group is immersed in ultrapure water), then placing the plant material into a vacuum pump for vacuumizing to 0.7atm, carrying out negative pressure treatment for 10min, slowly exhausting air, taking out the plant, and repeating the operations of bacterial liquid immersion and negative pressure treatment on the plant. And (5) fully watering after plant material infection, culturing for 24 hours in a dark place, and recovering normal field flowering culture after dark place is finished.
Phenotypic identification and expression analysis of 1.4CmHSFA2b gene silencing plants
After 21d infestation, plant material was placed in an intelligent light incubator for 5d (day 35 ℃,10h,20000lx; night 26 ℃,14h,0 lx) with approximately 70% humidity, and watered once every 2d to keep the soil moist. After the treatment is completed, the plant material is photographed and recorded, and the head inflorescences at the early stage of flower filling are taken and quick frozen by liquid nitrogen and then stored in an ultralow temperature refrigerator at-80 ℃. Total RNA extraction and reverse transcription treatment are carried out on the tongue petals of the chrysanthemum samples to obtain cDNA, cmEF alpha is taken as an internal reference gene, the expression level of CmHSFA b in three groups of experimental plants is verified through qRT-PCR experiments, the dissolution curve and the standard curve of the experiment are analyzed through relative quantitative software, and the relative expression level is compared and analyzed through a2 -ΔΔCt method.
The results show that the rapid fading of the tongue-shaped petals of the head inflorescence of the 'purple windmill' appears after the flower buds of the chrysanthemum 'purple windmill' are silenced CmHSFA b and subjected to 5d high temperature (day 35 ℃,10h,20000Lx; night 26 ℃,14h,0 Lx), and the results are clearly compared with the pure water control group and the no-load control group, and as a result, as shown in figure 10, the expression analysis of qRT-PCR experiments is carried out on the tongue-shaped petals of the flower-bearing initial stage of the three treatment groups respectively, the expression quantity of CmHSFA b genes after the silencing treatment is obviously reduced compared with the pure water control group and the no-load control group, and the results are shown in figure 11.
The invention discovers for the first time that the expression of the gene CmHSFA b for encoding the heat shock transcription factor in the chrysanthemum can stabilize the color of the chrysanthemum reed petals at high temperature without fading. When the gene is expressed excessively, the lingual side flowers of the chrysanthemum varieties which are easy to fade can not fade or fade obviously after high-temperature treatment, and when the expression of the gene is restrained, the lingual side flowers of the chrysanthemum varieties which are easy to fade at high temperature fade from purple to pink in a high-temperature environment, which shows that the transcription factor plays an important role in stabilizing the chrysanthemum colors and not fading at high temperature, lays a theoretical foundation for high Wen Juhua color regulation research and molecular improvement breeding, and has important application value.
Various other corresponding changes and modifications will occur to those skilled in the art from the foregoing description and the accompanying drawings, and all such changes and modifications are intended to be included within the scope of the present invention as defined in the appended claims.
Claims (6)
1. The coding gene of the chrysanthemum transcription factor is characterized in that the coding gene is a gene sequence shown as SEQ ID NO. 1.
2. A chrysanthemum transcription factor is characterized in that the coding gene of the chrysanthemum transcription factor is as set forth in claim 1, and the sequence of the chrysanthemum transcription factor is shown as SEQ ID NO. 2.
3. The application of the coding gene of the chrysanthemum transcription factor is characterized in that the expression level of the coding gene in claim 1 is improved through an over-expression tool, and the influence of temperature on the fading condition of petals of a heat-resistant chrysanthemum variety is improved.
4. The application of the chrysanthemum transcription factor is characterized in that the expression level of the coding gene of the chrysanthemum transcription factor in claim 2 is improved through an over-expression tool, and the influence of temperature on the fading condition of petals of a heat-resistant chrysanthemum variety is improved.
5. The use according to claim 3 or 4, wherein the temperature is at night temperature not less than 25 ℃ and at daytime temperature not less than 34 ℃.
6. The use according to claim 3 or 4, wherein the cultivation of the heat-resistant chrysanthemum cultivar comprises the step of infecting the chrysanthemum leaf disc or the flower bud stage of the head inflorescence with the vector carrying the coding gene by a mediation method.
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