CN116790626A - Application of MhMYB1 gene in improving total flavone content of Mirabilis himalaica - Google Patents
Application of MhMYB1 gene in improving total flavone content of Mirabilis himalaica Download PDFInfo
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Abstract
The application discloses application of MhMYB1 genes in improving total flavonoid content of Mirabilis himalaica, wherein the nucleotide sequence of the MhMYB1 genes is shown as SEQ ID NO.1, the coded amino acid sequence is shown as SEQ ID NO.2, and overexpression of the MhMYB1 genes can improve the flavonoid content in Mirabilis himalaica hairy roots, so that discovery of the MhMYB1 genes of Mirabilis himalaica provides related gene resources for molecular breeding for improving the flavonoid content of medicinal plants, and can be applied to mass production of flavonoid compounds in Mirabilis himalaica, thereby having great application value.
Description
Technical Field
The application relates to the technical field of genetic engineering, in particular to application of an MhMYB1 gene in improving total flavone content of Mirabilis himalaica.
Background
Mirabilis himalaica is a superior product of five Tibetan medicines, is widely used as a tonic medicine in Tibetan medicine prescription preparations such as twenty-five-flavor catechu pills, three-flavor Mirabilis himalaica soup and the like, has continuously increased market demands, and becomes one of the most promising characteristic varieties in Tibetan medicines. Because the living environment of the Mirabilis himalaica distribution is very narrow, and the roots are used as medicines, the extinct influence can be caused to the Mirabilis himalaica endangered by picking and digging in the wild. However, compared with wild resources, the artificially cultivated Mirabilis himalaica has lower flavonoid component content. The flavonoid compound is used as a main active ingredient in Mirabilis himalaica, and the content of the flavonoid compound directly determines the quality of Mirabilis himalaica. Therefore, cultivating Mirabilis jalapa with high yielding flavonoid components has been a long-sought goal in the industry.
The existing research shows that MYB transcription factor is the main effective regulator for biosynthesis of flavonoid compounds, can regulate the expression of a plurality of enzyme genes in the synthetic route of the flavonoid compounds, and has very important research value in the cultivation of novel medicinal plant varieties of high-yield flavonoid compounds. Through deep analysis of genome and transcriptome information of Mirabilis himalaica, MYB transcription factors for regulating and controlling accumulation of flavonoid compounds of Mirabilis himalaica are excavated, and a great amount of flavonoid compounds can be synthesized in Mirabilis himalaica by utilizing a genetic engineering means. Therefore, the identification of the transcription factor capable of regulating the biosynthesis pathway of the flavonoid compound has important significance for cultivating the Himalayan mirabilis jalapa with high flavonoid yield.
Disclosure of Invention
Therefore, one of the purposes of the application is to provide an application of over-expressing MhMYB1 gene in improving total flavone content of Mirabilis himalaica; the second object of the application is to provide a method for increasing the total flavone content in Mirabilis himalaica.
In order to achieve the above purpose, the present application provides the following technical solutions:
1. application of over-expressed MhMYB1 gene in improving total flavone content of Mirabilis himalaica
The MhMYB1 gene is from Mirabilis himalaica, the nucleotide sequence of the MhMYB1 gene is shown as SEQ ID NO.1, and the coded amino acid sequence of the MhMYB1 gene is shown as SEQ ID NO. 2.
2. A method for improving the content of total flavonoids in Mirabilis himalaica comprises the following steps: through over-expression of MhMYB1 gene in Mirabilis himalaica, the nucleotide sequence of the MhMYB1 gene is shown as SEQ ID NO.1, and the coded amino acid sequence of the MhMYB1 gene is shown as SEQ ID NO. 2.
Preferably, the method for over-expressing the MhMYB1 gene comprises the following steps: cloning MhMYB1 genes, constructing a plant expression vector to obtain a recombinant plant expression vector containing the MhMYB1 genes, transforming agrobacterium rhizogenes with the obtained recombinant plant expression vector to obtain engineering bacteria, transforming Mirabilis jalapa explants with the engineering bacteria, screening Mirabilis jalapa hairy roots, and identifying positive hairy roots by PCR to obtain Mirabilis jalapa hairy roots with improved total flavone content.
Preferably, the method for cloning the MhMYB1 gene comprises the following steps: PCR amplification is carried out by taking Mirabilis jalapa cDNA as a template and sequences shown as SEQ ID NO.3 and SEQ ID NO.4 as primers, electrophoresis detection is carried out, and the MhMYB1 gene with homology arms is obtained after recovery.
Preferably, the recombinant plant expression vector is prepared by taking a pBI121 vector as a framework and replacing a GUS gene on the pBI121 vector with an MhMYB1 gene with a homology arm in a homologous recombination mode.
Preferably, the agrobacterium rhizogenes is C58C1.
The application has the beneficial effects that:
the application discloses a Mirabilis himalaica MhMYB1 gene, the nucleotide sequence of which is shown as SEQ ID NO.1, and the coded amino acid sequence is shown as SEQ ID NO. 2. The overexpression of the Mirabilis himalaica MhMYB1 gene can improve the content of flavonoid compounds in the Mirabilis himalaica hairy roots. Therefore, the discovery of the Mirabilis himalaica MhMYB1 gene provides related gene resources for molecular breeding for improving the content of flavonoid compounds in medicinal plants, can be applied to mass production of flavonoid compounds in Mirabilis himalaica, and has great application value.
Drawings
In order to make the objects, technical solutions and advantageous effects of the present application more clear, the present application provides the following drawings for description:
FIG. 1 is a schematic diagram of the structure of a plant expression vector pBI121-MhMYB 1;
FIG. 2 is a result of quantitative PCR detection of the relative expression level of MhMYB1 in Mirabilis himalaica hairy roots in a preferred embodiment of the present application (WT: mirabilis himalaica hairy roots; mhMYB1-1, mhMYB1-4, mhMYB1-6, mhMMYB 1-7, mhMMYB 1-9: representing different transgenic MhMMMMMYB 1 gene Mirabilis himalaica hairy root lines);
FIG. 3 shows the results of spectrophotometric detection of total flavonoids in Mirabilis himalaica hairy roots in a preferred embodiment of the present application (WT: mirabilis himalaica hairy roots; mhMYB1-1, mhMYB1-4, mhMYB1-6, mhMYB1-7, mhMMYB 1-9: representing different transgenic MhMMMMMYB 1 genes Mirabilis himalaica hairy root lines).
Detailed Description
The present application will be further described with reference to the accompanying drawings and specific examples, which are not intended to limit the application, so that those skilled in the art may better understand the application and practice it.
The experimental procedure, which does not specify specific conditions in the examples below, is generally followed by routine conditions, such as molecular cloning by Sambrook et al: the laboratory manual is described in 1989 edition of New York: cold Spring Harbor Laboratory Press or according to the manufacturer's recommendations. The various chemicals commonly used in the examples are commercially available.
EXAMPLE 1 obtaining and identification of Mirabilis jalapa hairy root over-expressed by MhMYB1 Gene
The inventor of the present application isolated and cloned a MYB type transcription factor by deeply analyzing genome and transcriptome information of Mirabilis himalaica, and named MhMYB1. The coding sequence (CDS) of the MhMYB1 gene is shown as SEQ ID No.1, the length is 1122bp, 373 amino acids are coded, and the coded amino acid sequence is shown as SEQ ID No. 2.
1. Construction of pBI121-MhMYB1 recombinant vector
Taking a proper amount of rhizome and leaf tissues of Mirabilis himalaica, grinding the rhizome and leaf tissues in liquid nitrogen, extracting RNA by a Trizol method, and performing reverse transcription into cDNA by using a StarScript II reverse transcription kit A234; the gene specific primer is designed according to the sequence of the MhMYB1 gene, and the specific primer is as follows:
MhMYB1-F:5’-acgggggactctagaggatccatgggccgagcaccgtgt-3’(SEQ ID No.3)；
MhMYB1-R:5’-cgatcggggaaattcgagctcctaggatagaagccaagctaccatag-3’(SEQ ID No.4)。
then, the MhMYB1 gene is amplified from the total cDNA by PCR with high-fidelity DNA polymerase, and the amplified fragment is detected by 1% agarose gel electrophoresis and recovered and purified for standby.
The pBI121 vector was digested with BamH I and Sac I, subjected to 1% agarose gel electrophoresis, and the linearized vector backbone was recovered using a DNA purification recovery kit.
The recovered MhMYB1 amplified fragment and the linearized pBI121 vector are subjected to splice cloning by using a BM seamless cloning kit according to a molar ratio of 3:1, and the expression vector pBI121-MhMYB1 (figure 1) is obtained after sequencing.
In the embodiment, the Mirabilis himalaica MhMYB1 gene is operably connected with the plant expression vector pBI121, so that the expression vector pBI121-MhMYB1 can be used for regulating and controlling the content of flavonoid compounds in the Mirabilis himalaica by a transcription regulation strategy.
2. Transformation of Agrobacterium rhizogenes
The successfully connected vector plasmid pBI121-MhMYB1 is transformed into agrobacterium rhizogenes C58C1 strain to obtain recombinant agrobacterium tumefaciens C58C1/pBI121-MhMYB1, the expected band (the band with the size of 1122 bp) exists through PCR verification, and then the bacterial liquid is mixed with 50% glycerol in equal proportion and is put in a refrigerator at the temperature of minus 80 ℃ for standby.
3. Agrobacterium rhizogenes mediated MhMYB1 over-expression vector genetic transformation of Mirabilis himalaica to obtain transgenic Mirabilis himalaica hairy root
(1) Preparation of Mirabilis jalapa explant
Selecting mature and full Mirabilis jalapa seeds, washing overnight with running water, soaking in 75% ethanol for 1min, soaking in 2% NaClO for 10min, washing with sterilized water for 3 times, absorbing water, inoculating onto MS solid culture medium, and culturing in a 20 deg.C illumination incubator to obtain Mirabilis jalapa aseptic seedlings. After 5-6 true leaves grow out of the seedling, the aseptic seedling leaf explant is cut for transformation.
(2) Co-culture of Agrobacterium and explants
Adding the obtained explant into the activated heavy suspension (1/2MS+AS100 mu mol/L) of agrobacterium rhizogenes engineering bacteria containing pBI121-MhMYB1 expression vector, co-culturing the explant and bacterial liquid for 15 minutes, transferring to a co-culture medium, and culturing in dark at 28 ℃ for 2 days.
(3) Screening and detection of resistant hairy roots
The Mirabilis himalaica explants co-cultured for 2 days were transferred to kanamycin screening medium supplemented with 200mg/L cephalosporin and 100mg/L, and dark culture was continued until resistant hairy roots appeared. After the well-grown hairy roots were cultured to be completely sterile, the hairy root genomic DNA was extracted using the CTAB method. PCR identification is carried out by using the extracted genome DNA as a template and using a primer pair (35S promoter-F and pBI121-MhMYB 1-R) to obtain the transgenic hairy root of the over-expressed MhMYB1 gene.
35S promoter-F：5’-gacgcacaatcccactatcc-3’(SEQ ID No.5)；
pBI121-MhMYB1-R：5’-ctaggatagaagccaagctaccatag-3’(SEQ ID No.6)。
(4) Detection of MhMYB1 Gene expression in transgenic hairy root Using qRT-PCR
Taking a proper amount of transgenic hairy roots with consistent growth vigor, extracting RNA, performing reverse transcription to form cDNA, and using MhMYB12-eq-F and MhMYB12-eq-R primers to perform qRT-PCR to detect the expression quantity of the MhMYB1 gene in the sampled hairy roots. The reference gene is 18S rRNA, and the primers are 18SQ-F and 18SQ-R.
MhMYB12-eq-F：5’-gaatggcaatccaagcgga-3’(SEQ ID No.7)；
MhMYB12-eq-R：5’-tgcattgcagctcggctag-3’(SEQ ID No.8)。
18SQ-F：5’-atgataactcgacggatcgc-3’(SEQ ID No.9)；
18SQ-R：5’-cttggatgtggtagccgttt-3’(SEQ ID No.10)。
As shown in the figure 2, the expression level of the transgenic Mirabilis jalapa hairy root MhMYB1 is obviously higher than that of the common hairy root, which indicates that the MhMYB1 gene is successfully transferred into Mirabilis jalapa and the Mirabilis jalapa hairy root with the over-expressed MhMYB1 gene is obtained.
(5) Determination of total flavone content in hairy roots of transgenic Himalayan Mirabilis jalapa
a. Pretreatment of plant materials: the plant material is sufficiently ground after being dried at 60 ℃, and is sieved by a No.4 sieve, and 0.1g of the plant material is accurately weighed.
b. Extraction of flavonoids: adding 2mL of 60% ethanol as an extracting agent into each sample, performing ultrasonic extraction for 30min, centrifuging 12000g for 15min, taking the supernatant into a centrifuge tube, and using 60% ethanol to fix the volume to 2mL; in the color development process, 0.4mL of 10% sodium nitrite is added into the extracting solution, the extracting solution is uniformly shaken and then is kept stand for 6min, 0.4mL of 10% aluminum nitrate is added, the extracting solution is uniformly shaken and then is kept stand for 6min again, 4mL of 1mmol/L sodium hydroxide is added, 30% ethanol is used for fixing the volume to 10mL, and the extracting solution is kept stand for 15min after thorough uniform mixing.
c. Detection of flavonoids: OD values were determined for all samples at 510nm using uv spectrophotometry, and each experiment was repeated 3 times. And calculating the total content of the flavonoid compounds according to a standard solution regression curve.
As shown in FIG. 3, in the application, the MhMYB1 gene is overexpressed in Mirabilis himalaica, and the content of total flavonoids is remarkably improved. In the Mirabilis himalaica hairy root with MhMYB1 over-expressed, the relative content of total flavonoids is 1.41-2.11 times of that in the common hairy root at the same time.
The above-described embodiments are merely preferred embodiments for fully explaining the present application, and the scope of the present application is not limited thereto. Equivalent substitutions and modifications will occur to those skilled in the art based on the present application, and are intended to be within the scope of the present application. The protection scope of the application is subject to the claims.
Claims (6)
1. The application of the over-expressed MhMYB1 gene in improving the total flavone content of Mirabilis himalaica is characterized in that the MhMYB1 gene is from Mirabilis himalaica, the nucleotide sequence of the MhMYB1 gene is shown as SEQ ID NO.1, and the coded amino acid sequence of the MhMYB1 gene is shown as SEQ ID NO. 2.
2. A method for increasing the total flavone content in mirabilis jalapa, comprising the following steps: through over-expression of MhMYB1 gene in Mirabilis himalaica, the nucleotide sequence of the MhMYB1 gene is shown as SEQ ID NO.1, and the coded amino acid sequence of the MhMYB1 gene is shown as SEQ ID NO. 2.
3. The method of claim 2, wherein the method of overexpressing the MhMYB1 gene is: cloning MhMYB1 genes, constructing a plant expression vector to obtain a recombinant plant expression vector containing the MhMYB1 genes, transforming agrobacterium rhizogenes with the obtained recombinant plant expression vector to obtain engineering bacteria, transforming Mirabilis jalapa explants with the engineering bacteria, screening Mirabilis jalapa hairy roots, and identifying positive hairy roots by PCR to obtain Mirabilis jalapa hairy roots with improved total flavone content.
4. The method according to claim 3, wherein the method for cloning the MhMYB1 gene is: PCR amplification is carried out by taking Mirabilis jalapa cDNA as a template and sequences shown as SEQ ID NO.3 and SEQ ID NO.4 as primers, electrophoresis detection is carried out, and the MhMYB1 gene with homology arms is obtained after recovery.
5. The method according to claim 4, wherein the recombinant plant expression vector is prepared by replacing the GUS gene on the pBI121 vector with the MhMYB1 gene with a homology arm by homologous recombination.
6. A method according to claim 3, wherein the agrobacterium rhizogenes is C58C1.
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