A long-tube Lycoris radiata LlMYB4 gene and its encoded protein and application

Abstract

The invention discloses a long-tube Lycoris radiata LlMYB4 gene and its encoded protein and application, and belongs to the technical field of plant molecular biology. The long-tube Lycoris radiata LlMYB4 gene of the invention has a nucleotide sequence as shown in SEQ ID NO.1 and an amino acid sequence as shown in SEQ ID NO.2. The invention uses the perianth pieces of the long-tube Lycoris radiata in full bloom as materials, obtains the long-tube Lycoris radiata LIMYB4 gene by cloning, constructs its overexpression vector Super1300‑LlMYB4 on this basis, transfers it into Nicotiana benthamiana leaves, obtains transgenic plants, and the results of gene function identification show that the long-tube Lycoris radiata LlMYB4 gene is an important transcription factor that inhibits anthocyanin synthesis or promotes anthocyanin degradation, and has good application prospects in plant growth and development and breeding.

Description

Technical Field

[0001] The invention belongs to the technical field of microorganisms, and more specifically relates to a long-tube Lycoris radiata LlMYB4 gene and its encoding protein and application. Background Art

[0002] Lycoris longituba belongs to the genus Lycoris of the family Amaryllidaceae. It is mainly distributed in Jiangsu and Anhui and is endemic to my country. Its flower color variation is extremely rich, including white, pink, yellow, orange, purple, red and blue, and some of them can also emit a faint fragrance, so it is of great ornamental value and has broad prospects for garden application. The color of the pink group in Lycoris longituba gradually becomes lighter during the three stages of tepal development (early bud stage, middle bud stage, and full flowering stage). Research on this phenomenon is conducive to improving the mechanism of the formation of rich flower colors of Lycoris longituba.

[0003] Anthocyanin is one of the important pigments that determines the formation of plant flower color. Its synthesis and accumulation in plants are not only affected by some environmental factors, but also regulated by some key enzyme genes and transcription factors in the metabolic pathway. MYB transcription factors are one of the most influential families in plant life activities. Its regulation of anthocyanin synthesis pathways has been systematically verified in many plants. Not only have MYB transcription factors that can positively regulate anthocyanin synthesis and accumulation been found, but also MYB transcription factors that can negatively regulate anthocyanin synthesis and accumulation to lighten flower color have been found. At present, the research on anthocyanin synthesis and accumulation in Lycoris radiata is focused on structural genes, and there are relatively few studies on transcriptional regulation. Summary of the invention

[0004] In view of the above problems existing in the prior art, the technical problem to be solved by the present invention is to provide a long-tube Lycoris radiata LlMYB4 gene to meet the use requirements. Another technical problem to be solved by the present invention is to provide a protein encoded by the long-tube Lycoris radiata LlMYB4 gene. Another technical problem to be solved by the present invention is to provide an application of the long-tube Lycoris radiata LlMYB4 gene.

[0005] In order to solve the above technical problems, the technical solution adopted by the present invention is as follows:

[0006] A Lycoris radiata LlMYB4 gene, the nucleotide sequence of which is shown in SEQ ID NO.1.

[0007] The amino acid sequence of the expressed protein of the Lycoris radiata LlMYB4 gene is shown in SEQ ID NO.2.

[0008] A vector containing the LlMYB4 gene of Lycoris radiata.

[0009] Furthermore, the vector is a plant expression vector.

[0010] Furthermore, the plant expression vector is Super1300-LlMYB4.

[0011] Application of Lycoris radiata LlMYB4 gene in inhibiting anthocyanin synthesis or promoting anthocyanin degradation.

[0012] Furthermore, the use of the Lycoris radiata LlMYB4 gene in inhibiting anthocyanin synthesis or promoting anthocyanin degradation comprises the following steps:

[0013] (1) constructing a vector of the Lycoris radiata LlMYB4 gene;

[0014] (2) transforming the constructed vector of Lycoris radiata LlMYB4 gene into plants or plant tissues;

[0015] (3) Cultivate and screen transgenic plants with lighter flower colors.

[0016] Furthermore, the plant is Nicotiana benthamiana.

[0017] Furthermore, the plant tissue is Nicotiana benthamiana leaves.

[0018] Compared with the prior art, the present invention has the following beneficial effects:

[0019] 1) The present invention uses the perianth pieces of the long-tube Lycoris radiata in full flowering period as materials, obtains the long-tube Lycoris radiata LlMYB4 gene by cloning, constructs its overexpression vector Super1300-LlMYB4 on this basis, and transfers it into Nicotiana benthamiana leaves, so that the long-tube Lycoris radiata LlMYB4 gene is efficiently expressed in the plant.

[0020] 2) The expression of the wild-type and transgenic Lycoris radiata LlMYB4 gene positive strains was analyzed respectively, and finally three T0 generation positive plants with phenotypes were obtained. Representative strains 4-15 were selected for comparison with the wild type. It was found that the overall color of the transgenic tobacco corolla was lighter, but there was no obvious difference between the plant as a whole, filaments, anthers and other parts and the wild type. BRIEF DESCRIPTION OF THE DRAWINGS

[0021] Figure 1 This is the result of LlMYB4 expression analysis in different developmental stages of pink long-tube Lycoris (S1 early bud stage, S2 middle bud stage, S3 full flowering stage);

[0022] Figure 2The subcellular localization map of LlMYB4 in Nicotiana benthamiana leaves (35s::GFP is the expression of lower epidermal cells of leaves after injection of Agrobacterium GV3101 carrying the empty vector Super1300 into Nicotiana benthamiana, 35s::GFP-LlMYB4 is the expression of lower epidermal cells of leaves after injection of Agrobacterium GV3101 carrying the recombinant plasmid Super1300-LlMYB4 into Nicotiana benthamiana);

[0023] Figure 3 Comparison diagram of the phenotypes of wild-type tobacco and transgenic strains (a: strain 4-15, b: strain 4-3, c: strain 4-10, d: top view and side view of wild-type, e: top view and side view of positive strain 4-15);

[0024] Figure 4 A comparison of anthocyanin content in wild-type tobacco and transgenic line 4-15 (A is the wild-type, B is the transgenic plant);

[0025] Figure 5 The results of fluorescence quantitative analysis of LlMYB4 expression in wild-type tobacco and transgenic line 4-15 (A is wild-type and B is transgenic plants). DETAILED DESCRIPTION

[0026] In order to make the purpose, technical scheme and advantages of the present invention clearer, the present invention is further described below in conjunction with specific embodiments. In the following embodiments, unless otherwise specified, the technical means used are conventional means well known to those skilled in the art.

[0027] The materials used in this experiment were collected from the Lycoris Germplasm Resource Bank of the Garden Plant Discipline of Nanjing Forestry University. In August 2020, the perianth segments of pink long-tube Lycoris flowers at three different developmental stages (S1 early bud stage, S2 middle bud stage, S3 flowering stage) were selected, placed in sterilized centrifuge tubes, and immediately quick-frozen in liquid nitrogen, and then stored in a -80°C refrigerator.

[0028] Example 1

[0029] 1. Total RNA extraction from plant tissues and cDNA synthesis

[0030] In this experiment, Aidlab's EASYspinPlus Plant RNA Rapid Extraction Kit was used to extract total RNA. The One-Step RT-PCR SuperMix reverse transcription kit reverse transcribes the extracted RNA into cDNA, and then the obtained cDNA is diluted 10 times with water and stored in a -20°C refrigerator.

[0031] 2. Target gene amplification

[0032] Based on the existing transcriptome database information of Lycoris longituba petals (Yue YZ, Liu JW, Shi TT, et al. Integrating Transcriptomic and GC-MS Metabolomic Analysis to Characterize Color and Aroma Formation during Tepal Development in Lycoris longituba. Plants, 2019, 8(3): 53.), we screened out a MYB gene family member that was differentially expressed in three different developmental stages of the pink Lycoris longituba tepals and named it LlMYB4.

[0033] Primer Premier 5.0 software was used to design the full-length amplification primers of LlMYB4. The primer sequences are as follows:

[0034] F: 5′-GGGCATTTTGTGTAATGGGG-3′,

[0035] R: 5′-GCACAATCACCAAAAGGTAAAAC-3′.

[0036] The cDNA of the S3 tepals was diluted 10-fold and used as a template using Takara's MaxDNAPolymerase high-fidelity enzyme was used for PCR amplification. The reaction system was as follows: PrimeSTARMaxPremix (2×) 10 μL, ForwardPrimer 1 μL, ReversePrimer 1 μL, 1stStrandcDNA 1 μL, ddH 2 O 7 μL, Total Volume 20 μL.

[0037] Reaction conditions: 94°C, 4 min; (98°C, 10 s; 59°C, 30 s; 72°C, 35 s, 35 cycles); 72°C, 10 min; 16°C to terminate the reaction. The obtained product was detected by 1.2% agarose gel electrophoresis and recovered using the EasyPure Quick Gel Extraction Kit.

[0038] 3. Connect the cloning vector

[0039] Use Quanshijin -Blunt Cloning Vector cloning vector was connected to the target fragment, and the vector and target fragment were mixed in a molar ratio of 1:7, connected at 37°C for 15 minutes, and then placed on ice.

[0040] 4. Transformation of E. coli

[0041] Add the ligation product to the Trans1-T1 competent cells and place on ice for 20 minutes; incubate at 42°C for 30 seconds, remove and place on ice for 2 minutes; add 250 μL of LB liquid culture medium (no resistance), incubate at 200 rpm and 37°C for 1 hour; then centrifuge the cultured bacterial solution and discard 200 μL of the supernatant, pipette and mix the remaining bacterial solution, then smear it on an LB solid plate (kana resistance), and incubate at 37°C overnight.

[0042] 5. Bacterial testing and sequencing

[0043] White monoclonal colonies were selected from the plates cultured overnight for 12 h for bacterial detection. The reaction system was as follows: Green Taq Mix 10 μL, M13 Forward Primer 1 μL, M13 Reverse Primer 1 μL, ddH 2 O 8μL, Total Volume 20μL.

[0044] Reaction conditions: 94°C, 5 min; (94°C, 30 s; 59°C, 30 s; 72°C, 2 min, 35 cycles); 72°C, 10 min; 16°C to terminate the reaction.

[0045] The PCR product was detected by 1.2% agarose gel, and three positive clones with the correct length were selected for sequencing. The nucleotide sequence of the LlMYB4 gene was shown in the sequence table SEQ ID NO.1, and the amino acid sequence was shown in the sequence table SEQ ID NO.2. The positive single colony with the lowest base mismatch rate and repeated sequencing was selected to extract the plasmid for subsequent experiments.

[0046] Example 2

[0047] 1. Design fluorescent quantitative primers in the non-conserved region based on the full-length cDNA sequence of LlMYB4. The primer sequences are as follows:

[0048] F: 5′-GGGAGAGGAGGCTACAAACTGAT-3′,

[0049] R: 5′-CGCTCTCAGAAATCTCGGACA-3′.

[0050] The eIF gene of Lycoris radiata was used as the internal reference gene, and the primer sequences were as follows:

[0051] F: 5′-CGAAAGAACGGGTACTTAGTCA-3′,

[0052] R: 5′-ATGCGGAACATCACAGTTATG-3′,

[0053] The cDNA of the pink long-tube Lycoris radiata tepals at three developmental stages was diluted 10 times and used as a template for real-time fluorescence quantification. The reaction system of 10 μL was as follows: TB Green Premix ExTaq 5 μL, Forward Primer 0.4 μL, Reverse Primer 0.4 μL, ROX Reference Dye 0.2 μL, cDNA 1 μL, ddH 2 O 3μL, Total Volume 10μL.

[0054] The reaction program was 95°C, 30 s; (95°C, 5 s; 60°C, 30 s, 40 cycles); 95°C, 15 s; 60°C, 1 min.

[0055] Each sample was repeated three times biologically and three times technically to ensure good repeatability of the experimental data. The reliable data were used for 2 -ΔΔCT The expression difference of target genes was calculated by the SPSS 20.0 software.

[0056] 2. The results are as follows Figure 1 As shown in the figure, the expression pattern of LlMYB4 gene in the three different developmental stages of the pink long-tube Lycoris radiata showed a trend of first decreasing and then increasing, and the expression level in the full flowering stage was significantly higher than that in the first two stages. This trend is opposite to the rule that the color of the pink long-tube Lycoris radiata is darker in the early bud stage and gradually fades in the middle and late stages. It can be seen that the function of LlMYB4 gene is mainly to inhibit anthocyanin synthesis or promote anthocyanin degradation in the full flowering stage.

[0057] Example 3

[0058] 1. Construction of GFP::Super1300-LlMYB4 fusion vector

[0059] BioXM software was used to analyze the restriction sites of the complete ORF sequence of the LlMYB4 gene, and the available restriction endonucleases Sam1 and Kpn1 were selected to design primers containing restriction sites. The primer sequences are shown below:

[0060] F: 5′-aagcttctgcaggggcccgggATGGGGAGGCCACCTTGCTG-3′,

[0061] R: 5′-gcccttgctcaccatggtaccTAACGAAAGAGACTCATTATCCATTGA-3′,

[0062] The pEASY-Blunt-LlMYB4 plasmid with correct sequencing was used as a template for amplification and the PCR product was recovered. The LlMYB4 target fragment was inserted into the Super1300 vector containing the reporter gene GFP using the double enzyme digestion method and then transformed into Escherichia coli Trans1-T1. The transformed Escherichia coli was spread on LB solid medium (containing 50 mg / L Kana) and cultured overnight at 37°C, and positive single colonies were picked for bacterial detection and sequencing.

[0063] 2. Freeze-thaw method to transform Agrobacterium tumefaciens GV3101

[0064] After double enzyme digestion identification of the correctly sequenced recombinant plasmid, 1 μL of the recombinant plasmid was added to 33 μL of Agrobacterium GV3101 competent cells, pipetted and mixed, and then ice-bathed for 20 minutes, liquid nitrogen quick-frozen for 5 minutes, 28°C water bath for 5 minutes, and ice-bathed for 5 minutes; then 250 μL of LB liquid culture medium (without resistance) was added, and cultured at 200 rpm and 28°C for 1 hour; after the culture was completed, the bacterial solution was centrifuged at 6000r for 1 minute, part of the supernatant was discarded, and 100 μL was evenly spread on LB solid culture medium (containing 50 mg / L Kana), inverted in a 28°C incubator for 40-48 hours, and positive single colonies were picked for bacterial inspection, and the single colonies with correct bacterial inspection results were kept for later use.

[0065] 3. Transient expression injection of Nicotiana benthamiana

[0066] Agrobacterium containing no Super1300, Agrobacterium containing GFP::Super1300-LlMYB4 fusion vector, and Agrobacterium containing auxiliary expression vector P19 were cultured in LB liquid medium (containing 50 mg / L Kana) in the dark at 28°C, 200 r·min -1 ) to the bacterial solution OD 600 Reach 0.6-0.8. The bacterial solution was heated at 4℃ and 5000r·min -1 After centrifugation for 10 min, the cells were collected and washed with a buffer solution (containing 10 mmol / L -1 MgCl 2 , 10mmoL·L -1 MES, 150 μmol·L -1 The resuspended bacterial solution was mixed according to the proportion (V P19辅助载体 :V 空载体 =5∶7, V P19辅助载体 :V 融合载体 =5:7), after sufficient shaking and mixing, activate at 28°C in the dark for 3 h.

[0067] Use a 1mL medical syringe to inject the mixed bacterial solution into the back of the leaves of Nicotiana benthamiana that had been watered 3 days in advance, water thoroughly after injection, and place in an incubator for 2 days. Then take the tobacco leaves injected with bacterial solution, add DYPI dye solution and prepare slides, and observe the fluorescence display position of the lower epidermal cells of tobacco injected with Agrobacterium carrying GFP::Super1300-LlMYB4 fusion vector under LSM710 laser confocal microscope, and take the lower epidermal cells of tobacco injected with Agrobacterium carrying Super1300 empty vector as control.

[0068] The results are as follows Figure 2 As shown, GFP signal can only be detected in the nuclei of the lower epidermal cells of Nicotiana benthamiana, which preliminarily proves that LlMYB4 protein is mainly localized in the nucleus and can play a role in transcriptional regulation in the nucleus.

[0069] Example 4

[0070] 1. Construction of LIMYB4 overexpression vector and transformation of Agrobacterium

[0071] KpnI and XbaI were selected as two restriction sites, and the target gene fragment on the cloning vector and the overexpression vector pCAMIA2300 were connected by double restriction digestion, and then transformed into Escherichia coli Trans1-T1 according to the method of Example 1. After the results of bacterial inspection, sequencing and double restriction digestion were correct, the overexpression vector containing the target gene was transferred into Agrobacterium EHA105 by freeze-thaw method, and single colonies were selected for detection, and the single colonies with correct detection results were propagated and preserved for future use.

[0072] 2. Stable transformation of tobacco by leaf disc method

[0073] Agrobacterium containing pCAMIA2300-L1MYBs recombinant plasmid was inoculated into 50 mL of LB containing 50 mg / mL Kan, and the culture was shaken at 28°C and 200 rpm until OD = 0.4 for infection. The explants were young tobacco leaves before flowering, and the collected tobacco leaves were placed under running water for 20 minutes. The subsequent steps were completed in the clean bench, first soaked in 75% alcohol for 30 seconds, and then sterilized with ddH 2 O for 3-5 times, then disinfect with 0.1% disinfectant for 8 min, and finally with sterilized ddHO 2O Wash 3-5 times. After disinfection, remove the edges and main veins of the leaves, cut into 0.5cm×0.5cm leaves and place them in the bacterial solution for infection for 10 minutes. Shake them continuously during the period to facilitate better infection. After 10 minutes, take out the leaves from the infection solution, place them on sterile filter paper to absorb the excess bacterial solution, dry the surface bacterial solution and transfer them to the symbiotic medium for dark culture for 3 days. After the dark culture is completed, transfer them to the screening medium and culture them under 25℃ light conditions. After the callus tissue around the leaf disc differentiates into resistant buds, transfer them to the strong bud medium for continued culture, and change the medium every 15 days. When the resistant buds grow to about 2cm, cut them off and transfer them to the rooting medium. After the roots grow to a certain extent, harden the seedlings and transplant them to the greenhouse for growth.

[0074] 3. Positive detection of genetically modified tobacco

[0075] The leaves of transgenic plants were collected, and the leaf DNA was extracted using the Tiangen Plant Genomic DNA Extraction Kit. The transformed plants were identified by PCR using 35S-F and the reverse primer of the target gene.

[0076] 4. Extraction and preservation of anthocyanins

[0077] The transgenic tobacco corolla material stored at -80°C was fully ground in liquid nitrogen and 100 mg of dry powder was weighed. 1.5 ml of 1% hydrochloric acid methanol extract was added and fully shaken for 1 min. Extraction was carried out at 4°C in the dark and shaken once every 8 h. After 24 h of extraction, centrifugation was performed at 4°C, 10000 rpm, and the supernatant was filtered with a 0.22 μm organic phase filter membrane and stored in a 1.5 ml brown chromatographic bottle and stored at -20°C.

[0078] 5. Determination of anthocyanin content

[0079] The content of anthocyanin in transgenic tobacco was determined by high performance liquid chromatography (HPLC). The chromatographic column used for liquid chromatography was C18, the diode array detector was, the mobile phase A was acetonitrile, the mobile phase B was 0.4% phosphoric acid solution by volume, the elution was isocratic, the volume ratio of A to B was 20:80, the flow rate was 1.0 ml / min, the column temperature was 30°C, the injection volume was 10 μL, and the detection wavelength was 526 nm.

[0080] Using cyanidin-3-O-glucoside as a standard, it was dissolved in 1% hydrochloric acid methanol solution to prepare standard solutions of 0.01, 0.025, 0.05, 0.075, and 0.1 mg / ml, respectively. The peak area was measured by HPLC, and a standard curve was plotted with the mass concentration of the standard (X) as the abscissa and the peak area (Y) as the ordinate. The linear regression equation and correlation coefficient were obtained for quantitative analysis of the total anthocyanin content in the transgenic tobacco corolla.

[0081] 6. Fluorescence quantitative analysis

[0082] The wild type and the positive strain with phenotype were respectively extracted from the corolla RNA, and then reverse transcribed to obtain cDNA. The expression of LlMYB4 gene in the wild type and the positive strain was analyzed with tobacco Actin as the internal reference, and the specific method was the same as in Example 2.

[0083] 7. The results are as follows Figure 3 As shown, three lines of T0 generation positive plants with phenotypes were finally obtained. Representative lines 4-15 were selected for comparison with the wild type. It was found that the overall flower color of the transgenic tobacco corolla was lighter, but there were no obvious differences between the plant as a whole, filaments, anthers and other parts and the wild type.

[0084] The results are as follows Figure 4 As shown in the figure, the corollas of the transgenic strains 4-15 with significant phenotypes and the wild-type control were collected and preserved. After anthocyanin extraction and determination, it was found that a certain amount of anthocyanin could be detected in the corolla of the wild-type tobacco. However, due to the light color of the transgenic plants, the HPLC spectrum measured was at the baseline level as a whole, and almost no anthocyanin accumulation was detected. The results are shown in the figure. Figure 5 As shown, the fluorescence quantification results showed that the expression level of LlMYB4 was higher in the transgenic strain 4-15, while there was no LlMYB4 expression in the wild type. The difference in expression level further verified that LlMYB4 was the key to the difference in flower color phenotype between the transgenic strain and the wild type, confirming that LlMYB4 has the function of inhibiting anthocyanin synthesis or promoting the degradation of anthocyanins.

Claims

1. A long-tube Lycoris LlMYB4 The gene has a nucleotide sequence as shown in SEQ ID NO.

1.

2. The long-tube Lycoris radiata according to claim 1 LlMYB4 The amino acid sequence of the expressed protein of the gene is shown in SEQ ID NO.

2.

3. A plant containing the long-tube Lycoris radiata according to claim 1 LlMYB4 Gene vector.

4. The long-tube Lycoris radiata according to claim 1 LlMYB4 The application of genes in inhibiting the synthesis of anthocyanins in tobacco or promoting the degradation of anthocyanins in tobacco.

5. The use according to claim 4, characterized in that: The following steps are involved: (1) Construction of Lycoris radiata LlMYB4 Gene carrier; (2) The constructed long-tube Lycoris radiata LlMYB4 The gene vector is transformed into tobacco; (3) Cultivate and screen transgenic tobacco plants with lighter flower color.

Images