CN116083451A - Method for synthesizing betanin from carrots - Google Patents

Abstract

The invention discloses a method capable of synthesizing betaine in carrot fleshy roots through genetic engineering technology. The present invention optimizes the structure of the cDOPA5GT gene derived from Mirabilis mirabilis and the DODA1 and CYP76AD1 genes of red beet, the nucleotide sequences of which are shown in SEQ ID No 1, SEQ ID No 2 and SEQ ID No 3 respectively. Each gene is connected with an independent 35S promoter and NOS terminator, which is used to construct a plant multi-gene expression vector. Through genetic transformation mediated by Agrobacterium, betaine can be synthesized in the fleshy roots of carrot-positive plants, and the content reaches 63.4±9μg/g fresh weight.

Description

A method for synthesizing betaine in carrots

technical field

The invention belongs to the field of genetic engineering, and specifically relates to a gene cluster sequence comprising three genes for biosynthesizing betaine after structure optimization.

Background technique

Betalains are a class of nitrogen-containing, water-soluble plant pigments named after their first discovery in beetroot. According to its structure and spectroscopic features, betalain can be divided into red-purple betalain and yellow-brown betaaxanthin. Common betalains mainly include: betaine, probetaine, cockscomb anthocyanin, sipilin, amaranthin, malonyl betalain, mesembrolin and leaf flower purple glycoside, etc. Betaine with the simplest structure is betalain, which is produced by replacing the C5 position of betaglycone with a glucosyl group. It is the main component of betalain in red beet, accounting for about 75% to 95%.

Betaine has outstanding antioxidant capacity and health care functions, such as antibacterial, anticancer, antilipidemia, neuroprotection, liver protection and other functions. At present, the source of betalain is limited to Caryophyllales, mainly red beet. Red beets can be used both as an edible vegetable and to extract betalains. Other edible sources of betalain include amaranth, dragon fruit, etc. However, due to differences in eating habits around the world, there are not many opportunities to ingest betalain in daily life. Other components in the beetin plant will also have an impact on the application of beetin. For example, although a pokeweed can also be used as a source of beetin, it cannot be used as a food additive because it contains toxic saponins and lectins.

In the biosynthetic pathway of betalain, tyrosine first generates dopa (L-DOPA) under the action of tyrosinase or cytochrome P450 enzyme. Under the action of L-DOPA-4,5-dioxygenase, L-DOPA generates the core structure of betaine - betaine aldoline. Betaine aldehydes are naturally condensed with different amino acids or amines to produce different types of betaflavins. L-DOPA can also generate cyclodopa ( cyclo -DOPA) under the action of cytochrome P450 enzymes, and cyclo -DOPA spontaneously condenses with betaine aldehyde to generate betaine aglycone, which is the basis of all betalains structure, adding various groups at different positions to generate a variety of betalains. Betaine can generate betalain under the action of 5- O -glucosyltransferase, which is the most common and simple structure of betalain. Cyclo -DOPA can also first generate cyclo -DOPA-5- O -glycoside under the catalysis of cyclodopasyltransferase, and then react with betaine aldehyde to directly generate betaine. Betaine can be further modified by glycosylation or acetylation. Therefore, the enzymatic reactions involved in the biosynthesis of betaine include: ① hydroxylation of tyrosine to generate L-DOPA; ② conversion of L-DOPA to cyclo -DOPA; ③ conversion of L-DOPA to betaine aldoline; Glycosylation modification of cyclo -DOPA or betaglycone. The genes involved in each step of the enzymatic reaction have also been cloned one after another, which makes it possible for us to synthesize betaine in non-betalain plants by means of genetic engineering or metabolic engineering technology.

Carrot is a vegetable crop with fleshy roots as edible parts, and it is one of the top ten vegetables in the world. Carrots have strong environmental adaptability and can be planted in Northeast, North, Central, Southwest and Northwest China. China is the main planting country of carrots. The cultivated area reached 476,000 hm ² in 2012, accounting for about 40% of the world's total planted area, with an annual output of 16.406 million tons. With the advancement of cultivation technology, the annual supply of carrots has basically been realized at present. However, due to differences in varieties, natural environments, and field management, the yield per unit area of carrots varies greatly. The average yield per unit area of carrots in China is about 36.5 t/hm ² , which is significantly higher than that of major food crops such as rice, corn, and wheat. Therefore, carrots are an ideal target for genetic engineering. The synthesis of betaine in carrots can help to improve the nutritional and economic value of carrots.

Contents of the invention

The technical problem to be solved by the present invention is to provide three genes related to betaine biosynthesis that can be expressed in carrot fleshy roots: CYP76AD1S , DODA1S and cDOPA5GTS .

On the basis of our previous experiments, we finally screened three genes for the construction of the betalain synthesis pathway: Mirabilis cyclodopa 5- O -glucosyltransferase cDOPA5GT gene (GenBank No.: AB182643.1 ), red beet dopa dioxygenase DODA1 gene (GenBank No.: HQ656021.1) and cytochrome P450 enzyme CYP76AD1 gene (GenBank No.: HQ656023.1). According to the original sequence of each gene, their structure is optimized and transformed, and only the coding region is retained. The nucleotide sequences of the optimized three genes are shown as SEQ ID No 1, SEQ ID No 2 and SEQ ID No 3. And connect them with independent 35S promoter and NOS terminator respectively. The full-length sequence was synthesized by Sangon Bioengineering (Shanghai) Co., Ltd., with specific restriction endonuclease sites at both ends of the gene:

CYP76AD1S : BamH I and Kpn I.

DODA1S : Kpn I and Xba I.

cDOPA5GTS : Xba I and Sac I.

The three optimized genes were digested and connected, and inserted into the vector pCamBIA-1301 one by one (the multiple cloning sites were modified). Finally, a vector with three genes was obtained, which was named pYB- CDD .

The vector pYB- CDD was used to transform Agrobacterium LBA4404, and the genetic transformation of carrot was carried out according to the method reported in the literature (Xu ZS, Feng K, Xiong AS. CRISPR/Cas9-Mediated Multiply Targeted Mutagenesis in Orange and Purple Carrot Plants. Mol Biotechnol. 2019 Mar;61 (3): 191-199. doi: 10.1007/s12033-018-00150-6.). It was found that betalain could be synthesized in the fleshy roots of the positive plants, and it was mainly betaine identified by mass spectrometry.

Beneficial effect:

The present invention optimizes and synthesizes a gene cluster containing three betaine biosynthesis-related genes, and can synthesize betaine in the fleshy roots of carrot-positive plants through genetic transformation mediated by Agrobacterium, with a content of 63.4±9 μg/g fresh Heavy. It shows that the gene cluster constructed in the present invention can be used to transform carrots, which is expected to increase the nutritional and economic value of carrots.

Description of drawings:

Fig. 1 Schematic diagram of plant expression vector pYB- CDD .

Figure 2 Comparison of positive plants for betaine synthesis and wild-type plants.

Figure 3 Mass spectrometric identification of betaine in the fleshy roots of positive plants.

Detailed ways:

The present invention will be further described below in combination with specific embodiments. The embodiment is only used to illustrate the technical solution of the present invention rather than limit it. Although the present invention has been described in detail with reference to the preferred implementation, those skilled in the art should understand that the technical solution of the invention can be modified or equivalently replaced without Any deviation from the spirit and scope of the technical solutions of the present invention shall be covered by the scope of the claims of the present invention.

The experimental methods not specified in the implementation of the present invention, such as connection, transformation, preparation of relevant medium, etc., were carried out with reference to the third edition of the Molecular Cloning Experiment Guide (translated by Huang Peitang et al., China, Science Press, 2002). The strains and carrot seeds used were preserved by the Plant Genetic Engineering Laboratory of the Institute of Biotechnology, Shanghai Academy of Agricultural Sciences, and various restriction enzymes and ligases were purchased from Shanghai Haojia Company. Unspecified chemicals were of analytical grade and purchased from Sangon Bioengineering (Shanghai) Co., Ltd. or Shanghai Sinopharm Group Co., Ltd.

Example 1

Structural Optimization of Three Genes for Betanin Biosynthesis and Construction of Plant Expression Vectors

Based on the original sequence of mirabilis cDOPA5GT gene, red beet DODA1 gene and CYP76AD1 gene, the structure was optimized, introns were removed, and the recognition sites of common restriction endonucleases inside the genes were eliminated. The identities of optimized CYP76AD1S , DODA1S and cDOPA5GTS genes to the original sequences were 79.38%, 82.13% and 72.40%, respectively.

The synthesized CYP76AD1S gene fragment was recovered by gel, digested with BamH I and Kpn I, and then ligated with the same digested pCamBIA-1301 (its multiple cloning site was modified) with T ₄ DNA ligase to obtain One gene vector pYB- CYP76AD1S . Similarly, the resulting DODA1S gene fragment was digested with Kpn I and Xba I, and then ligated with pYB- CYP76AD1S that had undergone the same digestion to obtain a vector pYB- CYP76AD1S-DODA1S with two genes. Subsequently, the cDOPA5GTS fragment was connected to the vector pYB- CYP76AD1S-DODA1S by the same method, and finally the vector pYB- CDD with three genes was obtained (Figure 1). Sequencing identified the completeness and accuracy of each gene sequence.

Example 2

Mass Spectrometric Identification and Quantitative Analysis of Betaine in Various Parts of Positive Plants

The genetic transformation of carrots is carried out with reference to the method in the above literature. Carrot-positive seedlings are grown in a culture room at a temperature of 23°C, a light/dark ratio of 16 hours/8 hours, a relative humidity of 60%, and a growth period of 2-3 months. .

Detection of betaine in plant tissues by liquid chromatography-mass spectrometry (LC-MS) (Figure 2):

1) Take 0.5 g sample of fresh plant tissue (leaf, petiole, fleshy root) and put it into a mortar, grind it into a fine powder with liquid nitrogen, transfer it to a glass bottle, add 3 mL of 0.1% formic acid-water solution, and place it in an ultrasonic instrument ( Shanghai Bannuo Biotechnology Co., Ltd.) for ultrasonic extraction for 1 hour.

2) Centrifuge at 10,000 rpm at 4°C for 20 minutes, discard the sediment and retain the supernatant.

3) The supernatant was vacuum-dried and concentrated in a freeze dryer (Marin Christ, Germany), and reconstituted with 200 μL of 0.1% formic acid-water solution.

4) The sample is detected by a Thermo TSQ Quantum liquid chromatography-triple quadrupole mass spectrometer (Thermo Fisher, USA, equipped with an electrospray ionization source):

Chromatographic column: C18 chromatographic column (ACE UltraCore 2.5 Super C18, 150 × 2.1 mm, British ACE Company).

Mobile phase conditions: eluent A (1% acetonitrile, 0.1% formic acid) and B (100% acetonitrile, 0.1% formic acid) in the following conditions: 95% eluent A/5% eluent B in 0-3 minutes Isocratic elution, 3-18 minutes gradient to 100% B, and hold for 5 minutes, the column temperature and flow rate were set at 35°C and 0.3 mL/min, respectively.

Mass spectrometry conditions: SRM mode is adopted, the precursor ion is 551 in positive ion mode, and the product ion is 389 under the condition of collision energy of 10 eV.

Claims (2)

1. A betanin biosynthetic gene cluster useful for constructing a plant expression vector, wherein said artificial gene cluster comprises three related genes:CYP76AD1S、DODA1SandcDOPA5GTSthe saidCYP76AD1S、DODA1SAndcDOPA5GTSthe nucleotide sequences of the genes are respectively shown as SEQ ID No.1, SEQ ID No.2 and SEQ ID No. 3.

2. Using the method of claim 1CYP76AD1S、DODA1SAndcDOPA5GTSplant expression vector pYB for gene constructionCDDCharacterized in that each gene sequence is connected with a 35S promoter and a NOS terminator to form an expression unit, and the three expression units are combined according to the following stepsCYP76AD1S、DODA1SAndcDOPA5GTSis inserted into expression vector in serial order, and can synthesize betaine in fleshy root of positive plant after carrot is transformed.

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