CN114525284B - Red skin longan anthocyanin biosynthesis regulatory gene DlMYB1-HP and application thereof - Google Patents
Red skin longan anthocyanin biosynthesis regulatory gene DlMYB1-HP and application thereof Download PDFInfo
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Abstract
The invention relates to the technical field of biological gene engineering, in particular to a red skin longan anthocyanin biosynthesis regulatory gene DlMYB1-HP and application thereof. The nucleotide sequence of the red skin longan anthocyanin biosynthesis regulatory gene DlMYB1-HP is shown as SEQ ID No. 1; the amino acid sequence of the red skin longan anthocyanin biosynthesis regulating gene DlMYB1-HP is shown as SEQ ID No. 2. The invention also discloses application of the red skin longan anthocyanin biosynthesis regulating gene DlMYB1-HP, which is used for regulating anthocyanin biosynthesis; the gene has a very strong anthocyanin synthesis promoting function, so that the gene DlMYB1-HP is used for regulating and promoting anthocyanin synthesis, and has good prospects and commercial value.
Description
Technical Field
The invention relates to the technical field of biological gene engineering, in particular to a red skin longan anthocyanin biosynthesis regulatory gene DlMYB1-HP and application thereof.
Background
Anthocyanin is a water-soluble flavonoid compound widely existing in plants, is synthesized in cytoplasm and accumulated in vacuoles, so that the flower color, fruits and part of leaves of the plants are different in color, has strong antioxidant capacity and certain anti-inflammatory, antibacterial and antiviral activities, and plays an important role in physiological activities of the plants. In recent decades, with the intensive research of anthocyanin, the principles of physiological activity, functions and action mechanisms of anthocyanin are gradually becoming hot points of research of a plurality of scholars, and meanwhile, anthocyanin has been widely paid attention to and accepted by people due to its edible and medicinal values, including the effects of oxidation resistance, anti-inflammation, bacteriostasis, anti-aging, anti-cancer and the like and the protective effects of anthocyanin on liver, cardiovascular and cerebrovascular diseases and vision.
MYB transcription factors are widely found in eukaryotes, a large number of MYB transcription factor genes are found in plant genomes, for example 199 MYB transcription factor members are found in arabidopsis thaliana, 191 MYB genes are identified from poplar, 200 MYB transcription factors are found in cotton, and corresponding R2R3-MYB transcription factors are also found in other plants. The common characteristics are that a specific conservation DNA binding structural domain at the N end is widely involved in physiological and biochemical processes of plants, including differentiation of plant epidermal cells, development of stomata, biosynthesis of flavonoids, abiotic stress, pathogen resistance and the like. The first MYB transcription factor Colorless (C1) in plants was isolated from maize, and post-studies found that C1 was primarily involved in regulating maize anthocyanin biosynthesis, followed by successive isolation of AtPAP1 (AtMYB 75) and AtPAP2 (AtMYB 90) involved in anthocyanin biosynthesis in the model plant Arabidopsis.
The invention discovers a DlMYB-HP gene from red skin longan, and the DlMYB-HP gene expressed independently has very strong anthocyanin biosynthesis regulation function in various plants.
Disclosure of Invention
The invention aims to provide a red skin longan anthocyanin biosynthesis regulating gene DlMYB1-HP and application thereof, so that the red skin longan anthocyanin biosynthesis regulating gene DlMYB1-HP can be applied to regulating anthocyanin biosynthesis.
In order to achieve the above purpose, the present invention adopts the following technical scheme:
the application of the red skin longan anthocyanin biosynthesis regulating gene DlMYB1-HP is provided, and the synthetic gene DlMYB1-HP is used for regulating anthocyanin biosynthesis.
The nucleotide sequence of the red skin longan anthocyanin biosynthesis regulatory gene DlMYB1-HP is shown as SEQ ID No. 1.
The amino acid sequence of the red skin longan anthocyanin biosynthesis regulating gene DlMYB1-HP is shown as SEQ ID No. 2.
The invention has at least the following beneficial effects:
the gene DlMYB1-HP for regulating anthocyanin biosynthesis is discovered from red skin longan, is expressed in each tissue of red skin longan, can accumulate a large amount of anthocyanin in leaves which do not contain anthocyanin originally through constructing an over-expression vector for transient expression in tobacco or stable transgenosis, and has a very strong anthocyanin synthesis promoting function, so that the gene DlMYB1-HP has good play prospect and commercial value in regulating and promoting anthocyanin synthesis.
Drawings
In order to more clearly illustrate the technical solutions of the embodiments of the present invention, the drawings required for the description of the embodiments will be briefly described below, and it is obvious that the drawings in the following description are some embodiments of the present invention, and other drawings may be obtained according to these drawings without inventive effort for a person skilled in the art.
FIG. 1 is an amino acid sequence alignment diagram of red skin longan DlMYB1-HP and Dan longan DlMYB 1-XS;
FIG. 2 is a schematic diagram of target bands amplified from the DlMYB1-HP gene and the DlMYB1-XS gene;
FIG. 3 is a schematic diagram of phenotype and anthocyanin content of transient expression of DlMYB1-XS and DlMYB1-HP in tobacco leaves, A is phenotype of transient expression of Dan longan DlMYB1-XS and DlMYB1-HP genes in tobacco leaves, and B is anthocyanin content.
FIG. 4 is a schematic representation of DlMYB1-XS and DlMYB1-HP longan tobacco phenotypes, A being control and transgenic tobacco leaf phenotypes, B being anthocyanin content.
Detailed Description
The present invention will be described in further detail with reference to the drawings and examples, in order to make the objects, technical solutions and advantages of the present invention more apparent. It should be understood that the specific embodiments described herein are for purposes of illustration only and are not intended to limit the scope of the invention.
The invention relates to a red skin longan anthocyanin biosynthesis regulatory gene DlMYB1-HP and application thereof.
1. Materials and methods
1.1. Material
The red skin longan and Dan longan have different tissues including pericarp, pulp, leaf, seed and stem.
RNA extraction and reverse transcription
Total RNA of different tissues of longan is extracted by using a polysaccharide polyphenol plant total RNA extraction kit (product number: DP 441) of Tiangen biochemical technology (Beijing) limited company, and then cDNA is synthesized by reverse transcription according to a HiScript III 1st Strand cDNA Synthesis Kit (+gDNA wind) kit (product number: R312-01) of Nanjinouzan company.
1.3. Gene overexpression vector construction and sequencing analysis
Specific primers CTAGTGGATCCAAAGATGTCGCATTTACTTGGTGTAAG and ACTCTAGAAGTACTCCTACTTTGCATTGTCTTCTTCTGAAC are designed, and the gene sequence of the red skin longan DlMYB1-HP is amplified by taking the red skin longan peel cDNA as a template; specific primers CTAGTGGATCCAAAGATGTTGGATTTACTTGATGCAAG and ACTCTAGAAGTACTCCTAAATTAGATTATTGTCTTCTTCTG are designed, and Dan longan DlMYB1-SX gene sequences are amplified by taking the cDNA of the young leaves of the shi-shi longan as a template.
After the PCR amplification is completed, the amplified target DNA is recovered by agarose gel electrophoresis detection, then the amplified target DNA is recovered by a gel DNA recovery kit (product number: 2001050) of the Simmen company, dlMYB1-HP and DlMYB1-HP-SX are assembled on pSAK277 vectors which are digested by EcoRI and Xho I by a seamless cloning method, the pSAK277-DlMYB1-HP and pSAK277-DlMYB1-SX super-expression vectors are named respectively, and then the products assembled by Gibsion are transformed into escherichia coli, plasmids are extracted by a rapid plasmid DNA minikit (product number: 1005250) of the Simmen company after bacterial liquid PCR detection, and are sent to the Hua macrogene company for sequencing. Nucleotide and amino acid sequence differences between the two genes were aligned using cluster software after sequencing results were returned.
Referring to FIG. 1, the sequence alignment shows that the amino acid sequences of DlMYB1-HP and DlMYB1-SX are different, and the amino acid sequences of the key anthocyanin regulatory gene LcMYB1 of the litchi of the same family are also different.
2. Transient expression and transgenic experiments in tobacco
2.1. The obtained pSAK277-DlMYB1-HP and pSAK277-DlMYB1-XS super-expression vectors are transformed into agrobacterium GV3101 by electrotransformation, and the agrobacterium GV3101 is detected correctly by PCR with specific primers AGAAGACGTTCCAACCACGTCT and TCATAGGCGTCTCGCATATCTC. Agrobacterium containing pSAK277-DlMYB1-HP and pSAK277-DlMYB1-XS overexpression vectors were selected. Agrobacterium of pSAK277-DlMYB1-HP and pSAK277-DlMYB1-XS super expression vector was inoculated in liquid LB medium containing kananamycin (100 mg/L), cultured at 28℃at 300rpm to OD 600 About 2, and centrifuging the cultured bacterial liquid at 4000rpm for 5min, and collecting bacterial cells with MAA (10 mM MES (2- [ N-morpholino)]ethanesulfonic acid)pH 5.6，10mM MgCl 2 100. Mu.M Acetostingone) suspension cell OD 600 To about 1.0; agrobacterium containing pSAK277-DlMYB1-HP and pSAK277-DlMYB1-XS expression vectors were injected into tobacco backside separately using a syringe without needle. And 5-6 d after injection, observing and photographing, and collecting a sample to analyze anthocyanin content.
Referring specifically to FIG. 3, wherein A in FIG. 3 is the phenotype of tobacco leaves transiently expressing Dan longan DlMYB1-XS and DlMYB1-HP genes, and B is anthocyanin content.
2.2. Stable genetic transformation of tobacco
Preparing agrobacterium: and taking YEP liquid culture medium on an ultra-clean workbench, adding corresponding antibiotics, and then sub-packaging into 50mL centrifuge tubes with about 5mL each tube. 100 mu L of correctly detected agrobacterium is taken in the culture medium and placed on a shaking table at 28 ℃ for overnight culture. Taking out the bacterial liquid, and centrifuging the bacterial liquid at 6000rpm for 5min. The supernatant was decanted and the cells resuspended in approximately 50mL of MSB broth for use.
(1) The healthy mature medium-sized leaves are taken and placed in tap water.
(2) 10min was sterilized with 0.5% sodium hypochlorite.
(3) Washing with sterilized water for 3 times.
(4) Pouring a small amount of sterilized water (thin and one-layer) into a culture dish, cutting the leaf into about 0.5cm pieces in sterilized water 2 About 90 explants are required for the size of the explant (removing the edge portion of the leaf and the main vein).
(5) The cut explants were dip-stained in the resuspended bacterial solution for about 10min.
(6) Spreading a piece of sterilized filter paper on the prepared co-culture medium, and spreading the impregnated explant on the co-culture medium with the front surface facing upwards.
(7) Marking and recording, placing the culture medium with the experiment in a light-proof paper box at 28 ℃ for dark culture for 2d, taking out, and making the explant edge micro-roll visible and slightly whitened.
(8) After 2d co-cultivation, the explants were transferred to selection medium for selection cultivation (note also that the leaf front was facing upwards).
(9) 2 weeks later, the leaves are transferred to a screening medium for culture, and then every 2 weeks for subculture.
If the incision is found to be upturned in the culture process, the blade is required to be replaced, and the incision is contacted with the surface of the culture medium as much as possible. So as to fully absorb the nutrients in the culture medium. And periodically observing during the period, and timely replacing after pollution.
After about four weeks, resistant shoots are induced on the selection medium, and when the resistant shoots grow to 1-2cm (which may be slightly larger), they are excised and transferred to rooting medium to induce root formation. When the resistant buds root and the height of the seedlings is more than 2/3 of the height of the bottle, taking out the seedlings, cleaning the culture medium at the roots, putting the culture medium into clear water, and hardening the seedlings on a windowsill. So that the water is in contact with the natural environment, is suitable for illumination, temperature and humidity in natural environment. After hardening seedlings for about 2-3 days, transplanting the seedlings into basin soil of a greenhouse, and watering thoroughly.
The culture at each stage is as follows:
(1) MSB liquid medium: 4.4g/L MS+30g/L sucrose+2.0 mg/L6-BA+0.1 mg/L NAA+100. Mu.M As pH 5.7. And (5) sterilizing a plurality of bottles for later use.
(2) Co-culture medium: 4.4g/L MS+30g/L sucrose+8 g/L agar+2.0 mg/L6-BA+0.1 mg/L NAA+100. Mu.M As pH5.7, sterilized and packaged for use.
(3) First screening medium: 4.4g/L MS+30g/L sucrose+8 g/L agar+2.0 mg/L6-BA+0.1 mg/L NAA+100mg/L Km+400mg/L Cef pH5.7, sterilized and sub-packaged for use.
(4) Rooting medium: 4.4g/L MS+30g/L sucrose+8 g/L agar+100 mg/L Km+400mg/L Cef pH 5.7.
The anthocyanin content of the portion of the transient transformed DlMYB1-HP gene in tobacco leaves was 0.016mg/g, while the anthocyanin content of the portion of the transient transformed DlMYB1-XS gene was: 0.001mg/g.
The DNA of the resistant candidate plants was extracted with Hangzhou New Endoconcha plant DNA kit (3201050), and the target genes were amplified using specific primers ATGCCGGAAAAGCTGTAGATTG and CATGCGATCATAGGCGTCTCGCAT to determine whether the target genes had been integrated into tobacco. As can be seen from FIG. 2, we have chosen three tobacco DNAs transformed with DlMYB1-HP gene and DlMYB1-SX gene, each of which is capable of amplifying the target band size, whereas wild-type W38 and H 2 The O control was not amplified, indicating successful transfer of both the DlMYB1-HP gene and the DlMYB1-SX gene into W38 tobacco.
The leaves of the plant stably transforming the DlMYB1-HP gene accumulate a large amount of anthocyanin which is 0.0262mg/g red, while the leaves of the stably transforming the DlMYB1-SX gene accumulate a small amount of anthocyanin which is only 0.0042mg/g, and the anthocyanin content of the leaves of the plant stably transforming the DlMYB1-HP gene is 62 times that of the leaves of the plant stably transforming the DlMYB1-SX gene.
The results are shown in FIG. 4. (anthocyanin content determination method comprises measuring anthocyanin content by pH differential method, referring to Wrolitad et al (1982), specifically comprises collecting 0.1g of radix Raphani tissue in 3mL of leaching solution (methanol; water: concentrated hydrochloric acid 85:12:3), leaching thoroughly at room temperature in the dark (about 5-6 hours), preparing Buffer 1 and Buffer 2,Buffer 1:0.2mol/L KCl-0.2mol/L HCl (25:67), and measuring pH=1 Buffer 2:1mol/L NaAc-0.4mol/L HCl (100:150), and pH=5. Collecting 0.5mL of leaching solution in test tubes, adding 2mL of Buffer 1 and Buffer 2, respectively, and measuring their absorbance at 530nm with ultraviolet spectrophotometer.
The result is calculated according to an empirical formula, which is as follows: anthocyanin content (mg/g) =Δod 530 X 5 x 3 x 445.2/29600 x 0.1, wherein 5 is the dilution factor, 3 is the extract volume, 445.2 is the relative molecular mass of the cyanidin-3-glucoside; 29600 is the molar ratio absorption coefficient (mol) -1 .mL -1 ) 0.1 represents the sample weight. )
Thus, it can be seen from the above that:
the gene DlMYB1-HP for regulating anthocyanin biosynthesis is discovered from red skin longan, is expressed in each tissue of red skin longan, can accumulate a large amount of anthocyanin in leaves without anthocyanin by constructing an over-expression vector to be expressed in tobacco in a transient mode or to be stably transgenic, shows that the gene has a strong anthocyanin synthesis promoting function, and provides theoretical and technical basis for utilizing the gene to carry out molecular genetic concepts.
The foregoing has shown and described the basic principles, principal features and advantages of the invention. It will be understood by those skilled in the art that the present invention is not limited to the embodiments described above, and that the above embodiments and descriptions are merely illustrative of the principles of the present invention, and various changes and modifications may be made therein without departing from the spirit and scope of the invention, which is defined by the appended claims. The scope of the invention is defined by the appended claims and equivalents thereof.
Sequence listing
<110> Yangtze Master school
<120> red skin longan anthocyanin biosynthesis regulatory gene DlMYB1-HP and application thereof
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catccaaatc tttgcacggc gtcatcatca acttcactgg atacaccaga taataatgaa 600
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agaacaatgt gtgcgggtag cgatggtttt gaagtgaacc agaactgctt ggacaaacat 780
catgacttgg attttgatgc gaccctttgg aatcttctga gttcagaaga agacaatgca 840
aagtag 846
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Claims (2)
1. The application of the red-skin longan anthocyanin biosynthesis regulating gene DlMYB1-HP is characterized in that the red-skin longan anthocyanin biosynthesis regulating gene DlMYB1-HP is used for regulating anthocyanin biosynthesis;
the nucleotide sequence of the red skin longan anthocyanin biosynthesis regulatory gene DlMYB1-HP is shown as SEQ ID No. 1.
2. The use according to claim 1, wherein the amino acid sequence of the red skin longan anthocyanin biosynthesis regulating gene DlMYB1-HP is shown in SEQ ID No. 2.
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| CN202210073997.8A CN114525284B (en) | 2022-01-21 | 2022-01-21 | Red skin longan anthocyanin biosynthesis regulatory gene DlMYB1-HP and application thereof |
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