阅读说明：本技术 一种诱导中国水仙产生花青素的方法及其应用 (Method for inducing Chinese narcissus to generate anthocyanin and application thereof ) 是由 彭嘉宇 曾黎辉 于 2019-12-13 设计创作，主要内容包括：本发明公开了一种诱导中国水仙产生花青素的方法及其应用,属于植物转基因技术领域。该方法包括以下步骤：(1)百合ANS基因编码区全长序列的克隆；(2)构建ANS基因pC2300-35S::ANS植物表达载体；(3)基因枪法将pC2300-35S::ANS植物表达载体导入中国水仙受体材料中；(4)受体材料中ANS基因表达结果鉴定。本发明方法使原本不含有花青素的中国水仙产生了花青素,表现出红色现象。(The invention discloses a method for inducing Chinese narcissus to generate anthocyanin and application thereof, belonging to the technical field of plant transgenosis. The method comprises the following steps: (1) cloning the full-length sequence of the coding region of the lily ANS gene; (2) constructing an ANS gene pC2300-35S, namely an ANS plant expression vector; (3) introducing an ANS plant expression vector into a Chinese narcissus receptor material by a gene gun method, wherein the expression vector is pC 2300-35S; (4) and identifying ANS gene expression results in the receptor material. The method of the invention leads the Chinese narcissus which originally does not contain anthocyanin to generate anthocyanin and show red phenomenon.)

1. A method for inducing Chinese narcissus to generate red anthocyanin is characterized by comprising the following steps:

(1) cloning the full-length sequence of the coding region of the lily ANS gene;

(2) constructing an ANS gene pC2300-35S, namely an ANS plant expression vector;

(3) introducing an ANS plant expression vector into a Chinese narcissus receptor material by a gene gun method, wherein the expression vector is pC 2300-35S;

(4) identification of ANS gene expression induced anthocyanin production in receptor material.

2. The method for inducing the Chinese narcissus to produce red anthocyanin according to claim 1, wherein the method comprises the following steps: the cloning of the full-length sequence of the coding region of the lily ANS gene in the step (1) specifically comprises the following steps: designing a specific primer containing a restriction enzyme site according to a coding region sequence of the ANS gene of the lily in NCBI, and cloning the full length of the ANS gene by taking lily cDNA as a template.

3. The method for inducing the Chinese narcissus to produce red anthocyanin according to claim 2, wherein the method comprises the following steps: the sequence of the coding region of the lily ANS gene is shown as SEQ ID NO. 1; the specific primers are as follows: SEQ ID NO: 2: ANSF: 5'-GGATCCATGCCGACCGAGATCATGCCGTT-3', respectively; SEQ ID NO: 3 ANSR: 5'-CTGCAGCCTCACTTGGGAGAAGTGAAGTCCTCC-3' are provided.

4. The method for inducing the Chinese narcissus to produce red anthocyanin according to claim 1, wherein the method comprises the following steps: the ANS gene pC2300-35S is constructed in the step (2), wherein an ANS plant expression vector specifically comprises: ANS full-length gene and pC2300-35S plasmid, using BamHIAnd PstIDouble enzyme digestion, then connection, obtaining the recombinant plasmid pC2300-35S, ANS plant expression vector.

5. The method for inducing the Chinese narcissus to produce red anthocyanin according to claim 1, wherein the method comprises the following steps: the gene gun method in the step (3) introduces the pC2300-35s into an ANS plant expression vector into a Chinese narcissus receptor material, and comprises the following specific steps:

1) pretreating the receptor material;

2) preparing gene gun bullets;

3) bombarding acceptor material by a gene gun;

4) and (5) performing bombardment post-treatment.

6. The method for inducing the Chinese narcissus to produce red anthocyanin according to claim 5, wherein the method comprises the following steps: the pretreatment of the receptor material in the step 1) comprises the following steps: the receptor material is inoculated in an osmotic culture medium for pretreatment 4-8h before bombardment; the permeation culture medium is as follows: MS +2mg/L2,4-D +0.2mol/L sorbitol +0.2mol/L mannitol +30g/L sucrose +6g/L agar, Ph5.8; the receptor material is: one of Chinese narcissus petal, auxiliary crown and bulb dish.

7. The method for inducing the Chinese narcissus to produce red anthocyanin according to claim 5, wherein the method comprises the following steps: the gene gun bullet preparation in the step 2) comprises the following steps: 60mg/ml gold powder or tungsten powder suspension 50ul, 1ug/ul plant expression vector DNA5ul, 2.5M CaCl₂50ul, 0.1M spermidine 20 ul.

8. The method for inducing the Chinese narcissus to produce red anthocyanin according to claim 5, wherein the method comprises the following steps: and 3) bombarding the receptor material by the gene gun in the step 3), wherein the bombardment parameters of the bulb plate are bombardment distance of 6cm and air pressure of 1350psi, and the bombardment parameters of the petals and the auxiliary crowns are bombardment distance of 9cm and 1100 psi.

9. The method for inducing the Chinese narcissus to produce red anthocyanin according to claim 5, wherein the method comprises the following steps: the bombardment post-treatment in the step 4) comprises the following steps: after bombardment the receptor material is placed on an infiltration medium for infiltration treatment.

10. The method for inducing the Narcissus tazetta to produce the red anthocyanin, according to claim 1, is applied to the cultivation of new Narcissus tazetta species, wherein the anthocyanin produced by the Narcissus tazetta which cannot produce the anthocyanin appears red.

Technical Field

The invention belongs to the technical field of plant transgenosis, and particularly relates to a method for inducing Chinese narcissus to generate anthocyanin and application thereof.

Background

The Chinese narcissus is a traditional famous flower in China, and is often used as a flower for the annual night in winter, so that the Chinese narcissus has great ornamental value and economic value. However, the color of the Chinese narcissus is single, and only yellow and white colors exist. One of the main reasons for the singleness of the flower color of Chinese narcissus is the lack of anthocyanins in the flower. Our studies found that the main reason why Narcissus tazetta fails to synthesize anthocyanin is the lack of expression of the gene for ANS (anthocyanin synthase), a key enzyme in the pathway for anthocyanin synthesis. Therefore, the introduction and effective expression of a proper exogenous ANS gene into the Chinese narcissus is an effective way for inducing the Chinese narcissus to generate red anthocyanin.

Chinese narcissus is a triploid plant, is highly sterile, can only be subjected to vegetative propagation by bulblet generally, and is difficult to breed a new variety by using the traditional crossbreeding mode. Therefore, the method is an effective way for the color innovation of the Chinese narcissus.

The present invention utilizes gene gun technology to transfer constructed 35S, wherein ANS carrier is transferred into Chinese narcissus to induce the Chinese narcissus to produce anthocyanin and change the color of the Chinese narcissus to make the Chinese narcissus produce red character.

Disclosure of Invention

The invention provides a method for inducing Chinese narcissus to generate red anthocyanin and application thereof, aiming at overcoming the defects that the Chinese narcissus only has yellow and white flowers and has single variety color. The lily ANS gene is cloned, 35S is constructed, wherein ANS chimeric gene is used for establishing gene gun transformation parameters, and the gene gun is introduced into a receptor material of the Chinese narcissus to express ANS, so that the Chinese narcissus generates anthocyanin and shows red character.

In order to achieve the purpose, the invention adopts the following technical scheme:

a method for inducing Chinese narcissus to produce red anthocyanin comprises the following steps:

(1) cloning the full-length sequence of the coding region of the lily ANS gene;

(2) constructing an ANS gene pC2300-35S, namely an ANS plant expression vector;

(3) introducing an ANS plant expression vector into a Chinese narcissus receptor material by a gene gun method, wherein the expression vector is pC 2300-35S;

(4) identification of ANS gene expression induced anthocyanin production in receptor material.

The cloning of the full-length sequence of the coding region of the lily ANS gene in the step (1) specifically comprises the following steps: designing a specific primer containing a restriction enzyme site according to a coding region sequence of the ANS gene of the lily in NCBI, and cloning the full length of the ANS gene by taking lily cDNA as a template.

Furthermore, the sequence of the coding region of the lily ANS gene is shown as SEQ ID NO. 1; the specific primers are as follows: SEQ ID NO: 2: ANSF: 5' -GGATCCATGCCGACCGAGATCATGCCGTT-3’；SEQ ID NO：3 ANSR：5’-CTG CAGCCTCACTTGGGAGAAGTGAAGTCCTCC-3’。

The ANS gene pC2300-35S is constructed in the step (2), wherein an ANS plant expression vector specifically comprises: ANS full-length gene and pC2300-35S plasmid, first using BamHIAnd PstIDouble enzyme digestion, then connection, obtaining the recombinant plasmid pC2300-35S, ANS plant expression vector.

The acceptor material in the step (3) is: chinese narcissus petals, assistant crowns and bulbar plates.

The gene gun method in the step (3) introduces the pC2300-35S into an ANS plant expression vector into a Chinese narcissus receptor material, and comprises the following specific steps:

1) pretreating the receptor material;

2) preparing gene gun bullets;

3) bombarding acceptor material by a gene gun;

4) and (5) performing bombardment post-treatment.

Further, the pretreatment of the receptor material in the step 1) is as follows: the receptor material is inoculated in an osmotic culture medium for pretreatment 4-8h before bombardment; the permeation culture medium is as follows: MS +2mg/L2,4-D +0.2mol/L sorbitol +0.2mol/L mannitol +30g/L sucrose +6g/L agar, Ph5.8.

Further, the gene gun bullet in the step 2) is prepared by: 60mg/ml gold powder or tungsten powder suspension 50ul, 1ug/ul plant expression vector DNA5ul, 2.5M CaCl₂50ul, 0.1M spermidine 20 ul.

Further, the gene gun in the step 3) bombards the receptor material, wherein the bombardment parameters of the bulb dish are bombardment distance of 6cm and air pressure of 1350psi, and the bombardment parameters of the petals and the secondary crowns are bombardment distance of 9cm and 1100 psi.

Further, the bombardment post-treatment in the step 4) comprises: after bombardment the receptor material is placed on an infiltration medium for infiltration treatment.

The method for inducing the Chinese narcissus to generate the red anthocyanin is applied to the generation of the anthocyanin by the Chinese narcissus which cannot generate the anthocyanin.

The invention has the advantages that:

the invention provides a method for inducing Chinese narcissus to generate red anthocyanin, which can be applied to the cultivation of red Chinese narcissus varieties (including red balls and red flowers) and overcomes the problems that the Chinese narcissus has single flower color and is difficult to innovate the flower color by a conventional breeding means.

Drawings

FIG. 1 is an electrophoresis diagram of the PCR clone of ANS gene. M is DL2000DNAmarker, 1,2 is PCR product.

FIG. 2 is a pMD19-T-ANS dicrotic map. M is DL15000DNAmarker, 1,2 is pMD19-T-ANS gene double enzyme digestion.

FIG. 3 shows pC2300-35S: ANS double enzyme cleavage map. M is DL15000DNAmarker, 1,2 is pC2300-35S, ANS gene double enzyme digestion.

Figure 4 is a graph of the effect of bombardment of the narcissus bulb-disk by different parameters. A: 6cm, 1100 psi; b: 9cm, 1100 psi; c: 9cm, 1350 psi; d: 6cm, 1350 psi.

FIG. 5 is a diagram showing the effect of bombardment of paraspinal canopy with different parameters. A: 6cm, 1100 psi; b: 9cm, 1100 psi; c: 9cm, 1350 psi; d: 6cm, 1350 psi.

Figure 6 is a graph of the effect of different parameters on the bombardment of narcissus petals. A: 6cm, 1100 psi; b: 9cm, 1100 psi; c: 9cm, 1350 psi; d: 6cm, 1350 psi.

FIG. 7 shows pC2300-35S showing the effect of ANS plasmid after being injected into petals by a gene gun, wherein the bombardment parameters are 9cm and 1100 psi.

FIG. 8 shows pC2300-35S showing the effect of ANS plasmid after gene gun injection into accessory crown, the bombardment parameter is 9cm, 1100 psi.

FIG. 9 shows pC2300-35S showing the effect of ANS plasmid gene gun driven into the bulb dish, with bombardment parameters of 6cm and 1350 psi.

FIG. 10 shows the effect of the blank vector pC2300-35S (control) after the petal injection.

FIG. 11 is a graph showing the effect of the empty vector pC2300-35S (control) after the injection into the accessory crown.

FIG. 12 is a graph showing the effect of the empty vector pC2300-35S (control) after being inserted into the bulb dish.

Fig. 13 is a Total ion flow diagram (TIC, i.e., a spectrum obtained by continuously plotting the sum of intensities of all ions in a mass spectrum at each time point) of a quality control QC sample of a narcissus mixture sample after introduction of an ANS gene.

FIG. 14 is a MRM metabolite detection map of Narcissus tazetta samples after introduction of ANS gene. A: a target detection object cyanidin peak pattern; b: ion flow spectrum of multi-substance extraction.

FIG. 15 is a graph of the results of the quantitative analysis integral correction of cyanidin in a sample.

Detailed Description

The following examples further illustrate the present invention but are not to be construed as limiting the invention. Modifications or substitutions to methods, procedures, or conditions of the invention may be made without departing from the spirit and scope of the invention.