CN112322641A - Xanthophyll synthesis related gene and application thereof - Google Patents

Abstract

The invention discloses a lutein synthesis related gene and application thereof. Wherein the nucleotide sequence of the gene TeCHX is shown as SEQ ID NO.1, and the amino acid sequence thereof is shown as SEQ ID NO. 2. The TeCHX gene is cloned from marigold. The real-time quantitative analysis result shows that the expression level of TeCHX in mature flowers of marigold with significantly increased lutein content is obviously increased compared with that in immature flowers. When TeCHX is expressed in tobacco, the lutein content of the tobacco can be increased. The gene SEQ ID NO.1 plays a role in lutein biosynthesis, and the carotene hydroxylase gene TeCHX cloned from the pigment marigold can be used for improving the lutein content of plants.

Description

Xanthophyll synthesis related gene and application thereof

Technical Field

The invention relates to the technical field of plant genetic engineering, in particular to a gene obtained by cloning marigold and application of the gene in plant lutein synthesis.

Background

Carotenoids are an important pigment component in plants, not only play a role in photosynthesis, but also are used by plants for stress protection, attraction of pollinating animals, and synthesis and metabolism to form other regulating substances. Tagetes erecta L (Tagetes erecta L) which is an annual herb of Compositae and is native to Mexico and America is an ornamental plant, because the Tagetes erecta L is bright in color, has various colors from yellow to orange red, has a long flowering phase and is easy to cultivate. The main pigment component in marigold is carotenoid and has high content, so that the ornamental plant also becomes a main plant resource for extracting the carotenoid, especially lutein.

Most xanthophylls are present in the plant in the form of esters. Lutein cannot be synthesized in the human body, but can be converted in vivo into free lutein by using lutein esters. The lutein can prevent cardiovascular sclerosis, coronary heart disease, tumor and the like caused by aging of human body, and has unique effect on preventing vision loss and blindness caused by pathological changes of senile eyeball macular areas. Since lutein cannot be synthesized in human body and is difficult to be synthesized by chemical method, it needs to be extracted from natural plant. Although lutein is widely found in fruits and vegetables, it is generally present in minor amounts, resulting in inadequate daily dietary intake. Therefore, the natural pigment component is extracted from the plant resource rich in the lutein and then is widely applied to food, medicine and livestock breeding as a supplement additive.

Xanthophylls are oxygenated polyterpenes in carotenoids. In plant body, synthetic precursor isopentenyl pyrophosphate is condensed for many times to generate the first carotenoid-phytoene, and then is converted into other carotenoid components through dehydrogenation, cyclization, hydroxylation, epoxidation and the like, and the whole process is completed through multiple enzymes. Through researches on model plants such as arabidopsis thaliana and tomatoes, enzymes catalyzing reactions in all steps of carotenoid biosynthesis are well known, and genes of partial key enzymes are obtained through cloning. However, some important genes of the synthetic pathway of marigold derived from important plant sources of lutein have not been isolated and reported. Such as a carotene hydroxylase gene that catalyzes the final synthesis of lutein.

Disclosure of Invention

The invention aims to solve the technical problem of providing a lutein synthesis related gene.

Another technical problem to be solved by the invention is to provide the application of the gene in the synthesis of plant lutein.

For the genes related to lutein synthesis, the technical scheme provided by the invention is that the nucleotide sequence is shown as SEQ ID NO.1, and the amino acid sequence is shown as SEQ ID NO. 2.

The invention also comprises the application of the gene in the synthesis of the plant lutein.

The invention clones related genes for synthesizing lutein from marigold, and the genes are named as Tagetes erecta carotene hydroxyylase (TeCHX for short).

The invention clones TeCHX gene from marigold: performing data assembly on the TeCHX gene according to the high-throughput transcriptome sequencing of marigold; then, Primer design software Primer Premier is used for designing a nested PCR specific Primer, total RNA is extracted from the mature marigold flowers, and a TeCHX gene is obtained by reverse transcription-PCR (reverse transcription-PCR, RT-PCR) cloning.

The real-time quantitative analysis result of the TeCHX gene shows that the expression level of TeCHX in mature flowers with significantly increased lutein content of marigold is obviously increased compared with that in immature flowers. When TeCHX is expressed in tobacco, the lutein content of the tobacco can be increased. The gene SEQ ID NO.1 plays a role in lutein biosynthesis, and the carotene hydroxylase gene TeCHX cloned from the pigment marigold can be used for improving the lutein content of plants.

The invention has the beneficial effects that:

the marigold gene TeCHX provides a new gene resource for a synthesis way of lutein, and can be used for improving the lutein content in plants.

Detailed Description

In the following examples, the specific experimental conditions, where not indicated, are according to conventional conditions well known to those skilled in the art, such as those described in the molecular cloning Laboratory Manual of Sambrook J. and Russell, D.W. (New York: Cold Spring Harbor Laboratory Press,2001), or according to the manufacturer's recommendations.

Example 1: high throughput transcriptome sequencing of marigold

As the genome of marigold is not determined, in order to obtain a functional gene transcript sequence, a tissue sample of a marigold cultivated variety is utilized, and high-throughput transcriptome sequence determination and assembly annotation are carried out on 9 samples in total by respectively extracting leaf, immature flower and mature flower RNA of three individual plants.

1. Reagent

The plant RNA extraction reagent TRIzol is purchased from Invitrogen company, DNase I (dnase I) is purchased from TaKaRa company, the RNA Library preparation Kit (RNA Library Prep Kit) is from Beijing Baimaike biotechnology limited, and other reagents are imported subpackaged or domestic analysis pure products.

2. Plant material

Marigold (Tagetes erectaL.) cultivar Juwang is provided by Chifengxin flower horticulture.

3. Method of producing a composite material

3.1RNA extraction

1) Crushing 100mg of plant tissues by using a liquid nitrogen grinding method, transferring the plant tissues into a 1.5mL centrifuge tube, adding 1mL of TRIzol, violently shaking, and standing at room temperature for 5 min;

2) adding 200 μ L chloroform into the centrifuge tube, shaking for 30s, mixing, and standing at room temperature for 5 min;

3) centrifuging at 12000rpm for 15min at 4 deg.C;

4) transferring 700 mu L of the supernatant into a 1.5mL centrifuge tube, wherein the lower organic phase and the middle layer have protein and other impurities to avoid touching and absorbing;

5) adding equal volume of isopropanol into the supernatant, mixing, and standing at room temperature for 10 min;

6) centrifuging at 4 deg.C and 12000rpm for 15min, and removing supernatant;

7) adding 1mL of 70% ethanol, gently oscillating the centrifugal tube, and suspending and precipitating;

8) centrifuging at 4 deg.C and 12000rpm for 5min, and removing supernatant;

9) drying at room temperature for 5-10 min;

10) adding 50. mu.L RNase-free H water₂O), dissolving RNA;

11) mu.g of RNA was taken according to the concentration of the RNA solution, and 5. mu.L of 10 Xbuffer (400mM Tris-HCl, pH 7.5, 80mM MgCl)₂50mM DTT), 5. mu.L of Dnase I and 2. mu.L of RNase inhibitor, and reacting at 37 ℃ for 30 min;

12) adding 2.5 μ L0.5M EDTA, inactivating Dnase I at 80 deg.C for 2 min;

13) adding 10 μ L of 3M sodium acetate and 250 μ L of precooled ethanol, standing at-80 deg.C for 20 min;

14) centrifuging at 4 deg.C and 12000rpm for 10min, and removing supernatant;

15) adding 1mL of 70% ethanol to clean RNA;

16) centrifuging at 4 deg.C and 12000rpm for 5min, and removing supernatant;

17) drying at room temperature for 5-10 min;

18) adding 50 mu L of RNase-free water to dissolve RNA;

19) and detecting the purity and concentration of the RNA sample.

3.2 transcriptome sequencing Assembly and Annotation

Transcriptome sequencing was performed using the RNA Library Prep Kit via Illumina HiSeq high throughput sequencing platform, according to the following steps:

1) enriching eukaryotic RNA by magnetic beads with oligo (dt), and randomly breaking mRNA;

2) taking mRNA as a template, synthesizing a first cDNA chain by using a hexabasic random primer, then adding dNTPs, RNase H and DNA polymerase I to synthesize a second cDNA chain, and purifying the cDNA by using beads (beads);

3) carrying out end repair on the purified double-stranded cDNA, connecting a sequencing joint, then carrying out fragment size selection by using microbeads, and obtaining a cDNA library through PCR enrichment;

4) detecting the concentration of the library and the size of the insert;

5) sequencing the cDNA library by using an Illumina HiSeq high-throughput sequencing platform, wherein the sequencing read length is PE 125;

6) cutting sequencing joints and primer sequences of sequencing fragments (reads), and filtering low-quality value data to obtain high-quality sequencing data;

7) extending the high-quality sequencing read into longer fragments (contigs) by using Trinity assembly software, obtaining fragment sets (components) by using the overlapping of the fragments, and finally obtaining a transcript sequence (unigene) by using a method of a De Bruijn graph;

8) aligning transcript sequences to the NR (NCBI non-redundant database), Swiss-Prot (database maintained by the European bioinformatics institute), GO (Gene ontology), COG (Clusters of organizations), KOG (eKaryotic organizations), KEGG (Kyoto Encyclopedia of Genes and genomes) databases using BLAST software;

9) the coding region sequence of unigene and the corresponding amino acid sequence are predicted by using TransDecoder software, and the annotation information of unigene is obtained by comparing HMMER software with Pfam (protein family) database.

4. Results

RNA extraction, library construction and high-throughput transcriptome sequencing are carried out on marigold tissue materials, and then assembly and gene function prediction are carried out on sequencing sequences. Through quantitative and differential expression analysis of the expression level of the assembled genes, the differentially expressed genes in mature flowers are enriched, and the expression level of the Tagetes erecta carotenes hydroxyylase (TeCHX) gene in the mature flowers of marigold is obviously improved by about 3 times compared with that in the immature flowers.

Example 2: cloning of Tagetes erecta TeCHX gene

According to the data assembly of the TeCHX gene of marigold in example 1, PCR specific primers are designed by using Primer Premier as Primer design software, total RNA is extracted from mature flowers of marigold, and the TeCHX gene is obtained by reverse transcription-PCR (reverse transcription-PCR, RT-PCR) cloning.

1. Reagent

The plant RNA extraction reagent TRIzol is purchased from Invitrogen company, DNase I (dnase I) is purchased from TaKaRa company, reverse transcriptase, dNTP and high-fidelity DNA polymerase are purchased from Beijing Quanjin biotechnology limited company, the primers are synthesized by the company Limited in the biological engineering (Shanghai), and other reagents are imported subpackaged or domestic analysis pure products.

2. Vectors and strains

Cloning Vector pEASY-Blunt Simple Cloning Vector is available from Beijing Quanji Biotechnology Ltd, and Escherichia coli (Escherichia coli) strain DH 5. alpha. is available from Beijing Quanji Biotechnology Ltd.

3. Culture medium and reagent

LB culture medium, tryptone 10g/L, yeast powder 5g/L, NaCl 10 g/L. Adjusting pH to 7.0 with NaOH, and autoclaving.

100×Mg²⁺Solution 20.33g MgCl₂.6H₂O、24.65g MgSO₄.7H₂O constant volume in 100mL H₂And O, autoclaving.

SOC culture medium including tryptone 20g/L, yeast powder 5g/L, NaCl 0.58g/L, KCl 0.19g/L, 100 XMg²⁺10 mL. Adjusting pH to 7.0 with NaOH, and autoclaving. Then 2mL of filter sterilized 1mol/L glucose was added.

1000 Xampicillin: 100mg/mL, dissolved in sterile deionized water and stored at-20 ℃.

4. Method of producing a composite material

4.1 extraction of RNA from mature flower tissue of marigold

The procedure was as described in 3.1 of example 1.

4.2 RT-PCR

4.2.1 RT

1) Taking 1. mu.g of total RNA and 1. mu.L of polyT₁₈(10. mu.M) primer mix with RNase-free ddH₂O to complement to 12.75μ L, mixing gently;

2) preserving heat at 65 ℃ for 5min, immediately transferring to an ice bath, and standing for 2 min;

3) adding 5 × reaction buffer 4 μ L, 10mM dNTP 2 μ L, RNA inhibitor 0.25 μ L (40U/μ L), reverse transcriptase 1 μ L (100U/μ L), 42 deg.C for 1h, and synthesizing first chain cDNA;

4) heating at 95 deg.C for 5min, inactivating reverse transcriptase, and terminating reaction.

4.2.2 PCR

According to the sequence presumed by the TeCHX gene obtained in example 1, primers are designed by using Primer Premier software, the sequences are respectively shown as SEQ ID NO.3, SEQ ID NO.4, SEQ ID NO.5 and SEQ ID NO.6, and the specific sequences are as follows:

TeCHXF1:5’taacacctacaaaccaccaacca 3’

TeCHXR1:5’ctcgttttttaaaagattttcatac 3’

TeCHXF2:5’atttcatctttcatcatcctcac 3’

TeCHXR2:5’cgttagatttatggcctaaactgt 3’

the cDNA of the mature flower tissue of marigold obtained in step 4.2.1 of this example was subjected to cloning of the TeCHX gene.

Place 200 μ L EP tube on ice, add reagents:

amplification was performed according to the following procedure: 2min at 98 ℃ (pre-denaturation); 10s at 98 ℃ (denaturation), 20s at 55 ℃ (renaturation), 90s at 72 ℃ (extension), and the denaturation-renaturation-extension is performed for 30 cycles; 5min at 72 ℃ (total extension).

Using the PCR product as a template, a second round of PCR was performed using primers TeCHXF2 and TeCHXR2, at a renaturation temperature of 56 ℃ under the same conditions as above.

Through the above operation, a TeCHX gene PCR amplification product is obtained.

4.3 ligation of PCR amplification products to pEASY-Blunt vector

The PCR amplification product of the TeCHX gene obtained in step 4.2 of this example was ligated to the Cloning Vector pEASY-Blunt Simple Cloning Vector in a molar ratio of 1:4 (25 ℃, 15min) in the following manner:

pEASY-Blunt Simple Cloning Vector(50μg/μL) 4μL

PCR product (. about.150. mu.g/. mu.L) 1. mu.L

4.4 transformation of E.coli

1) Taking out the frozen Escherichia coli (Escherichia coli) strain DH5 alpha competent cells for ice bath thawing;

2) gently mixing the ligation product of 4.3 with escherichia coli competent cells uniformly, and carrying out ice bath for 30 min;

3) thermally shocking for 90s at 42 ℃, and immediately carrying out ice bath for 1-2 min;

4) adding 0.8mL of SOC, mixing uniformly, and carrying out mild shaking culture at 37 ℃ for 1 h;

5) after centrifugation at 13000rpm for 1min at room temperature, a part of the supernatant was discarded to leave about 200. mu.L of the supernatant, which was then mixed with the cells by a pipette tip, spread on LB plates containing ampicillin (100. mu.g/mL), and cultured at 37 ℃ for 12 hours.

4.5 colony PCR identification

The Escherichia coli described in step 4.4 of this example was further subjected to colony PCR assay to confirm the insertion of the target fragment, and the reaction system was as follows:

reaction conditions are as follows: 3min at 94 ℃ (pre-denaturation); 30s at 94 ℃ (denaturation), 20s at 56 ℃ (renaturation), 90s at 72 ℃ (extension), and 26 cycles of denaturation-renaturation-extension; 5min at 72 ℃ (total extension).

The recombinant vector identified by colony PCR, named pEASY-TeCHX, was sequenced. Sequencing results show that the full-length sequence of the TeCHX gene connected with the pEASY-Blunt cloning vector is obtained. Wherein, the nucleotide sequence of the TeCHX gene is shown as SEQ ID NO.1, and the specific nucleotide sequence is as follows:

the amino acid sequence of the TeCHX gene is shown as SEQ ID NO.2, and the specific amino acid sequence is as follows:

example 3: HPLC method for measuring lutein content in marigold flowers

1. Reagent

The lutein standard is purchased from Shanghai Allan Biotechnology corporation, 2, 6-di-tert-butyl-4-methylphenol is purchased from chemical reagents of national medicine group, and other reagents are imported subpackaged or domestic analytical pure products.

2. Method of producing a composite material

Taking immature flowers and mature flower products of marigold, drying at 45 ℃, grinding the dried samples by liquid nitrogen, weighing 0.15g, adding 1mL of ethanol (containing 0.1% of 2, 6-di-tert-butyl-4-methylphenol), swirling for 15s, adding 400mL of 50% KOH aqueous solution, swirling for 15s, and carrying out water bath at 50 ℃ for 60min, and swirling once every 15 min. Extracting with 3.3mL n-hexane for 3 times, mixing extractive solutions, and sampling 50 μ L filtrate. Chromatographic conditions are as follows: high performance liquid chromatography (WATERS corporation, usa); column SymmetryC18(250mm × 4.6mm, 5 μm); mobile phase: acetonitrile: dichloromethane: the methanol content is 70: 20: 10 (v: v: v); the flow rate is 1 mL/min; detecting the wavelength of 475 nm; the column temperature was 30 ℃.

3. Results

According to the standard curve of the lutein standard and the HPLC measurement result, the lutein content in the immature flower of marigold is 3.6mg/g dry weight, and the lutein content in the mature flower is 16.3mg/g dry weight.

Example 4: expression analysis of TeCHX gene in flower tissues of marigold at different periods

Quantitative PCR primers were designed using Primer Premier, a Primer design software, according to the full-length sequence of the TeCHX gene cloned in example 2. Respectively extracting total RNA from immature flowers and mature flowers of marigold, carrying out reverse transcription to obtain cDNA, and carrying out quantitative analysis on the expression level of the TeCHX gene.

1. Reagent

RNA extraction, reverse transcription reagents as described in example 2; a real-time quantitative PCR reagent TransStart Green qPCR SuperMix is purchased from Beijing all-purpose gold biotechnology, Inc.; the primers are synthesized by the company of Biotechnology engineering (Shanghai) and other reagents are imported split charging or domestic analytical pure products.

2. Method of producing a composite material

Respectively taking tissue samples of immature flowers and mature flowers of marigold, grinding by liquid nitrogen, extracting RNA, and carrying out reverse transcription, wherein the operation steps are as described in 3.1 in example 1 and 4.2 in example 2.

According to the high-throughput sequencing result, quantitative PCR analysis of the expression level of TeCHX was performed using the transformation Initiation Factor 6(TIF6) stably expressed in different tissues of marigold as an internal control gene. TeCHX primers were TeCHXRTF and TeCHXRTR, and TIF6 primers were TIF6F and TIF 6R. The primer sequences are respectively shown as SEQ ID NO.7, SEQ ID NO.8, SEQ ID NO.9 and SEQ ID NO.10, and the specific sequences are as follows:

TeCHXRTF:5’gctacgtgatgaccttttgtcaat 3’

TeCHXRTR:5’cggtcaacttcctcttgtgcct 3’

TIF6F:5’taagacctggtggtggaaataga 3’

TIF6R:5’cagcaccatgaggacgaaga 3’

the quantitative PCR reaction system is as follows:

reaction conditions are as follows: 30s at 95 ℃; 5s at 95 ℃, 15s at 60 ℃, 10s at 72 ℃ and 40 cycles. The cDNA was diluted 30-fold with the cDNA template obtained as described in example 2, 4.2.1, and used for quantitative PCR. After amplification, melting curve analysis was performed at 65 ℃ for 5s, with 0.5 ℃ increase per cycle, and 60 cycles. Each sample was replicated three times. The PCR reaction was run on Bio-Rad CFX 96.

4. Results

The real-time quantitative PCR analysis result shows that the expression level of the TeCHX gene in mature flower tissue of the pigment marigold is about 5.5 times of that of the immature flower and is obviously higher than that in the immature flower. The TeCHX gene which can code carotene hydroxylase can play a role in the synthesis of marigold lutein, and the corresponding transcription expression is increased along with the developmental maturity of marigold flowers and the increase of the synthesis amount of the lutein in the flowers.

Example 5: construction of tobacco expression vector of TeCHX gene

PCR was performed using the pEASY-TeCHX plasmid obtained in example 2 as a template, and Kpn I and Stu I cleavage sites were introduced. The PCR product and the tobacco expression vector pBTEX-HA are respectively cut by Kpn I and Stu I, the cut product glue is recovered and then connected, and the cut product glue is transformed into escherichia coli and agrobacterium.

1. Reagent

The plasmid extraction kit and the gel recovery kit are purchased from Omega company; pfu high fidelity DNA polymerase, T4 DNA ligase, available from Beijing Quanjin Biotechnology, Inc.; restriction enzymes Kpn I and Stu I were purchased from Fermentas, Inc. The other reagents are imported split charging or domestic analytical pure products.

2. Escherichia coli strains and agrobacterium strains

Escherichia coli (Escherichia coli) strain DH5 alpha, Agrobacterium tumefaciens (Agrobacterium tumefaciens) strain GV2260 was purchased from Beijing Quanji Biotech Ltd.

3. Culture medium and antibiotics

The LB liquid medium, LB solid medium, SOC medium were prepared as described in example 2.

1000 kanamycin (Kan): 100mg/mL, dissolving in sterilized deionized water, performing sterile suction filtration, and packaging at-20 deg.C.

500 × rifampicin (Rif): 50mg/mL, dissolved in sterile deionized water and stored at-20 ℃.

4. Method of producing a composite material

4.1 plasmid extraction

Plasmid extraction was performed on the pEASY-TeCHX vector obtained in example 2 and the plant expression vector pBTEX-HA, and the experimental procedures were performed as described by the kit manufacturer.

1) Column balancing: adding 500 μ L of balance liquid BL into adsorption column, centrifuging at 12000rpm for 1min, and discarding waste liquid;

2)12000rpm for 1min, centrifugally collecting bacterial precipitates, and abandoning the supernatant as much as possible; adding 250 mu L P1 (RNase A is added), blowing, sucking and mixing evenly until the bacterial sediment is suspended completely;

3) adding 250 mu L P2, and gently turning the centrifugal tube up and down for 8 times to fully crack the thalli;

4) adding 350 mu L P3, immediately and gently turning the centrifuge tube up and down for 8 times at 12000rpm, and centrifuging for 10 min;

5) sucking out the supernatant to a new centrifuge tube, centrifuging at 12000rpm for 5 min;

6) carefully transferring the supernatant to an adsorption column, centrifuging at 12000rpm for 1min, and discarding the waste liquid;

7) adding 500 μ L PD at 12000rpm, centrifuging for 1min, and discarding the waste liquid;

8) adding 600 μ L PW (added with anhydrous ethanol), 12000rpm, centrifuging for 1min, discarding waste liquid, and repeating the operation once;

9) centrifuging at 12000rpm for 2min to remove residual PW;

10) the adsorption column was transferred to a fresh centrifuge tube and 50. mu.L of sterile ddH was added to the center of the column₂O, standing at room temperature for 2min, then centrifuging at 12000rpm for 2min, and eluting DNA.

4.2 PCR

PCR amplification is carried out by taking the obtained pEASY-TeCHX plasmid as a template, and Kpn I and Stu I enzyme cutting sites are introduced. The obtained PCR amplification product of the expected size is purified.

4.3 enzyme digestion

The pBTEX-HA plasmids and TeCHX gene amplification products obtained in the above-mentioned 4.1 and 4.2 were digested with restriction enzymes Kpn I and Stu I. The reaction system is as follows, 37 ℃, 1 h:

4.4 glue recovery

And (3) carrying out gel recovery on the plasmid subjected to enzyme digestion and the TeCHX gene amplification product, and carrying out the experimental steps according to the specifications of a kit manufacturer.

1) Adding 500 μ L of balance liquid BL into the gel recovery adsorption column, centrifuging at 12000rpm for 1min, and pouring the waste liquid at the bottom of the column.

2) The electrophoretically separated fragments were recovered on a UV gel cutting instrument with plastic gloves, and the cut fragments were placed into a pre-prepared EP tube.

3) The volume of the sol solution PN is determined according to the mass of the gel, and the sol solution PN is added according to the volume of 1: 1. Putting the EP tube into a heater, and dissolving at 50 ℃ for 10-15min until the glue is completely dissolved.

4) After the sol is completely dissolved, cooling to room temperature, transferring the dissolved liquid into a gel recovery adsorption column, and standing for 3 min.

5) Centrifuging at 12000rpm for 1min, and pouring to collect waste liquid at the bottom of the column. 600. mu.L of washing solution PW was added to the adsorption tube.

6) Centrifuging at 12000rpm for 1min, and pouring to remove waste liquid at the bottom of the collecting column. The washing was repeated once.

7) Placing the adsorption column in a centrifuge at 12000rpm for 3min, and standing for 15 min.

8) Adding 30 μ L eluant EB onto the adsorption film in the center of the adsorption column, standing for 3min, and centrifuging at 12000rpm for 3min to obtain recovered gel product.

4.5 ligation of the enzyme fragments

The TeCHX gene fragment and the pBTEX-HA plasmid fragment which are recovered by the 4.4 enzyme digestion are connected by T4 ligase, and the connection reaction system is as follows, at 25 ℃ and for 3 h:

4.6 transformation of E.coli

The experimental procedure was as described in 4.4 of example 2.

4.7 colony PCR identification of recombinant plasmids

The experimental procedure was as described in 4.5 of example 2.

The colony PCR identified the recombinant vector (named pBTEX-TeCHX) and sequenced. Sequencing results showed that the TeCHX coding sequence was ligated into the pBTEX vector. The recombinant E.coli with correct sequencing was stored at-80 ℃.

4.8 Agrobacterium transformation

1) The pBTEX-TeCHX plasmid was extracted as described in 4.1 of this example.

2) The pBTEX-TeCHX plasmid was added to 50. mu.L of competent cells of Agrobacterium strain GV2260, mixed well with gentle stirring, and allowed to ice-cool for 30 min.

3) Placing in liquid nitrogen for cold shock for 1 min.

4) The EP tube was transferred to a constant temperature heater at 37 ℃ and heated for 5 min.

5) Adding 800 μ L SOC culture solution, and culturing in shaker at 28 deg.C and 200rpm/min for 4-5 h.

6) The bacterial liquid was centrifuged at 4000rpm/min for 5 min.

7) The supernatant was aspirated in a clean bench, and about 100. mu.L of the supernatant was left, and the cells were gently pipetted, suspended, and mixed.

8) The bacterial liquid is evenly coated on LB + Rif + Kana solid culture medium by using a sterilized glass ball, and cultured for 48h in a constant temperature incubator at 28 ℃.

9) Colony PCR was identified by the same method as 4.5 in example 2, and the positive Agrobacterium transformed with the identified recombinant plasmid was stored at-80 ℃.

Example 6: expression of TeCHX in tobacco

Expressing the TeCHX gene in tobacco leaves by an agrobacterium-mediated method, detecting the expression of TeCHX encoding protein, and determining the content of lutein in a tobacco sample expressing the TeCHX.

1. Reagent

Acetosyringone and HA-tagged mouse monoclonal antibody were purchased from Sigma, USA; PageRuler Plus Prestated Protein Ladder, Western Blot ECL Substrate, available from Thermo Scientific Inc., USA. The other reagents are imported split charging or domestic analytical pure products.

2. Plant material

Tobacco (Nicotiana benthamiana) was planted in a phytotron.

3. Culture media and solutions

IM solution:

475mL of IM solution: 4.88g of 2-morpholine ethanesulfonic acid (MES); 2.5g of glucose; NaH₂PO₄0.126 g. Add MES to ddH first₂In O, adjusting the pH value to 5.6, and then adding glucose and NaH₂PO₄Stirring, and sterilizing at high temperature.

1L 20 × AB salt solution: NH (NH)₄Cl 20g；MgSO₄，6g；KCl，3g；FeSO₄ 0.05g，CaCl₂And 2 g. Sequentially adding the components, completely and uniformly dissolving, and sterilizing at high temperature.

200mM acetosyringone (1000 ×): 39mg of the powdered acetosyringone was dissolved in 1mL of DMSO and stored at-20 ℃ in the dark.

20mL of induction medium: IM solution 19mL, 20 × AB salt solution 1mL, 200mM acetosyringone 20 μ L, 25mg/mL kanamycin 20 μ L.

1L 5 × protein electrophoresis buffer: 15.1g of Tris (hydroxymethyl) aminomethane (Tris), 72.0g of glycine and 5.0g of SDS.

1L of 10 × western blot membrane transfer buffer: 144g of glycine and 30.2g of Tris. Dissolving Tris, glycine in 0.9L ddH₂Adding ddH into O, stirring and mixing uniformly₂And O is metered to 1L. When 1L of 1 Xtransmembrane buffer was prepared, 100mL of 10 Xtransmembrane buffer, 100mL of methanol and ddH were added₂And O is metered to 1L.

1L of 10 × TBS (Trish-buffered saline) buffer: 80g of NaCl, 2g of KCl and 30g of Tris. The components were dissolved in 0.8L ddH₂In O, adjust pH to 7.4, add ddH₂And O is metered to 1L. When 1L of 1 XTSST (Trish-buffered saline with Tween) buffer was prepared, 100mL of 10 XTSST, 202.5 mL of 20% Tween, and ddH were added₂And O is metered to 1L.

Protein extraction buffer: 50mM Tris-HCl (pH 7.5), 150mM NaCl, 5mM ethylenediaminetetraacetic acid, 10% glycerol, 1% polyvinylpyrrolidone, 20. mu.M dithiothreitol, 1mM phenylmethylsulfonyl fluoride, plant protease inhibitors (plant protease inhibitors, 100. mu.L/10 mL extraction buffer).

4. Method of producing a composite material

4.1 tobacco transient expression

1) Agrobacterium transformed with pBTEX-TeCHX recombinant plasmid and pBTEX-HA empty vector was streaked on LB plates with Rif and Kana, respectively, and cultured in a 28 ℃ incubator for 48 hours.

2) The monoclonal was picked and cultured at 28 ℃ for 12h, and 300. mu.L of the strain was transferred to 2.7mL of LB + Rif + Kana medium and cultured at 28 ℃ for 6-8 h.

3) Centrifuging at 3000rpm for 6min at room temperature, removing the supernatant, and adding 3mL of IM solution to resuspend the thallus; repeatedly centrifuging once, resuspending the thallus with 3mL induction culture medium solution, and culturing at 28 ℃ and 250rpm for 5-14 h;

4) centrifuge at 3000rpm for 6min, discard the supernatant, add 10mM MES 2mL (pH 5.7, 200. mu.L with 200mM acetosyringone), resuspend the cells, vortex and shake. Repeating the steps once;

5) measurement of bacterial liquid concentration (OD)₆₀₀) Preparing a staining solution;

6) injecting the staining solution into the tobacco leaf from the lower epidermis of the leaf by using a disposable injector, and marking the injection range;

7) placing the injected plant in shade for 0.5h, growing in the light for 36-48h, collecting the sample, rapidly cooling in liquid nitrogen, and storing at-80 deg.C.

4.2Western hybridization

1) The sample was taken out of the freezer at-80 ℃, ground in a mortar with liquid nitrogen, ground to a powder and transferred to a pre-cooled 1.5mL EP tube.

2) Adding 300 μ L protein extract, shaking on vortex instrument to mix the extract and sample, and standing for 10 min.

3) Centrifuge at 12000rpm for 10min in a 4 ℃ centrifuge.

4) Pipette 200. mu.L of supernatant into a new EP tube, add 100. mu.L of 2 XProte loading buffer, and heat at 95 ℃ for 5 min.

5) The sample was spotted on a polypropylene gel and electrophoresed for 2 h.

6) Sealing with 5% skimmed milk for 1 hr after membrane conversion. HA-tagged immune mouse antibodies were added and incubated for 1h at room temperature.

7) Washing the membrane for 3 times, adding horseradish peroxidase-conjugated secondary antibody, and incubating for 1h at room temperature.

8) Washing the membrane for 3 times, adding a reaction substrate (western blotting ECL substrate), and detecting the protein expression condition in a chemiluminescence apparatus.

4.3 assay of lutein

The determination of the lutein content of TeCHX expressing tobacco leaves was performed as described in example 3. Tissue samples injected with pBETX-HA empty vector served as controls.

5. Results

Western blot Western blot experiment results show that the constructed marigold TeCHX gene plant expression vector is mediated by agrobacterium, so that TeCHX encoding protein is expressed in tobacco, and a protein band with the expected size of about 60KD is obtained. The TeCHX-expressing tobacco leaf tissue was elevated in lutein from 0.19mg/g dry weight to 0.37mg/g dry weight compared to the control sample transferred with the empty vector. The gene SEQ ID NO.1 plays a role in the synthesis of plant lutein, and the cloning of the carotene hydroxylase gene TeCHX from the pigment marigold can be used for improving the content of the plant lutein.

The above-described embodiments of the present invention do not limit the scope of the present invention. Any modification, equivalent replacement, and improvement made within the spirit and principle of the present invention should be included in the scope of the claims of the present invention.

Sequence listing

<110> university of fertilizer industry

<120> xanthophyll synthesis related gene and application thereof

<160> 10

<170> SIPOSequenceListing 1.0

<210> 1

<211> 1990

<212> DNA

<213> marigold (Tagetes erecta)

<400> 1

taacacctac aaaccaccaa ccatttcatc tttcatcatc ctcacaacat gctttcacta 60

ctacactcac aaccaccact tataaccacc actcaccacc gcatcttcgc cgtaacacct 120

acaaaccacc gcttcatcac aatcaaatcc tcaattgaaa acaattcaaa accaaaacct 180

aaaagttcat catcaaactc aaaccctaga aacacgtcat ggatcagtcc tgattggctc 240

acaggcttct tccgctcaat ttccttcaca caaaccgacg attcgaacat accgattgca 300

aatgcacagc tcgatgacgt gtcggatttg ttaggtggag ctttatttct tccgttgttc 360

aaatggatga atgattatgg tccgatttat agattagcag ctggaccgag gaactttgtg 420

attgttagtg atccggttat tgctaaacat gtgttgagaa attatggaag catttatgct 480

aaaggacttg ttgctgaagt ttctgagttt ttgtttggtt ctggttttgc aatcgctgaa 540

ggttcacttt ggacggcaag gcgcagggct gttgttccat cacttcatag gaagtactta 600

tcagtgatag ttgatcgcgt gttttgcaaa tgctctgaga gatttgtgga aaagcttaaa 660

ttgtatgcgc gcaatggcac ggctgtcaac atggagcaac aattttctca gttaactctt 720

gatgttattg gtctagcagt atttaactac aattttgatt cacttactgc tgatagccct 780

gtaattgaat ctgtttatac tgcattgaaa gaagctgaag cccgttcaac tgatctttta 840

ccatattgga agataagtgc attatgtaag attattccaa gacagataaa agcagaacaa 900

gcagttactg taattagaga aaccgttgaa gaacttatta tgaaatgcaa ggaaatcgtt 960

gaaaaagaag gtgaaaaaat tgatgatgaa gattatgtaa atgatgcaga cccaagtatc 1020

cttcgattcc tgcttgctag cagggaagag gtttcaagtc aacagctacg tgatgacctt 1080

ttgtcaatgt tggttgctgg acatgaaacc actggttcag tgttgacttg gactacgtat 1140

cttctaagta aggacccatc ttctttagtg aaggcacaag aggaagttga ccgagtttta 1200

caaggccgtc aaccaactta tgaagacata aaaaacctca aatttataac acgatgcata 1260

aacgagtcta tgcgtcttta cccacatccc ccagtcttga taagaagagc tatagtcgca 1320

gacgagcttc ccggaaatta caaggtcaac cctggtcaag atataatgat atccgtttat 1380

aacattcatc attcatccaa ggtttgggat agagcagaag aattcatacc tgaaaggttt 1440

ggattagacg ggcctgtacc aaatgaaacc aacaccgatt acaagtatat cccgttcagt 1500

ggtggcccgc ggaaatgtgt aggtgatcag tttgctatgc tggaagcaat tgttgcacta 1560

gcaatctttt tgcagaacat tgaattcgag ttggttccaa atcaaaagat taacatgact 1620

actggtgcca ccattcacac aacaagtggt ttgtacatga aagtcaaaga acgtaaagtc 1680

aaatctatgg cggtgtcgtc aacgtaggcc gcttacttct acatacaagc attcatggta 1740

aatttaaatt aatgtctata atttttcccc gtagtttaag ttgtagagca tctatgtaac 1800

atactacggt gttgtgtacc taaacatgct ttttgttctt gatgtgagaa caaaaacagt 1860

ttaggccata aatctaacgt gtatatcaaa tgcaatagta cacttgagat atatcttatc 1920

ttgaaatgta gtgacgtaaa tcatttataa tcatattgtg atggcgtatg aaaatctttt 1980

aaaaaacgag 1990

<210> 2

<211> 552

<212> PRT

<213> marigold (Tagetes erecta)

<400> 2

Met Leu Ser Leu Leu His Ser Gln Pro Pro Leu Ile Thr Thr Thr His

1 5 10 15

His Arg Ile Phe Ala Val Thr Pro Thr Asn His Arg Phe Ile Thr Ile

20 25 30

Lys Ser Ser Ile Glu Asn Asn Ser Lys Pro Lys Pro Lys Ser Ser Ser

35 40 45

Ser Asn Ser Asn Pro Arg Asn Thr Ser Trp Ile Ser Pro Asp Trp Leu

50 55 60

Thr Gly Phe Phe Arg Ser Ile Ser Phe Thr Gln Thr Asp Asp Ser Asn

65 70 75 80

Ile Pro Ile Ala Asn Ala Gln Leu Asp Asp Val Ser Asp Leu Leu Gly

85 90 95

Gly Ala Leu Phe Leu Pro Leu Phe Lys Trp Met Asn Asp Tyr Gly Pro

100 105 110

Ile Tyr Arg Leu Ala Ala Gly Pro Arg Asn Phe Val Ile Val Ser Asp

115 120 125

Pro Val Ile Ala Lys His Val Leu Arg Asn Tyr Gly Ser Ile Tyr Ala

130 135 140

Lys Gly Leu Val Ala Glu Val Ser Glu Phe Leu Phe Gly Ser Gly Phe

145 150 155 160

Ala Ile Ala Glu Gly Ser Leu Trp Thr Ala Arg Arg Arg Ala Val Val

165 170 175

Pro Ser Leu His Arg Lys Tyr Leu Ser Val Ile Val Asp Arg Val Phe

180 185 190

Cys Lys Cys Ser Glu Arg Phe Val Glu Lys Leu Lys Leu Tyr Ala Arg

195 200 205

Asn Gly Thr Ala Val Asn Met Glu Gln Gln Phe Ser Gln Leu Thr Leu

210 215 220

Asp Val Ile Gly Leu Ala Val Phe Asn Tyr Asn Phe Asp Ser Leu Thr

225 230 235 240

Ala Asp Ser Pro Val Ile Glu Ser Val Tyr Thr Ala Leu Lys Glu Ala

245 250 255

Glu Ala Arg Ser Thr Asp Leu Leu Pro Tyr Trp Lys Ile Ser Ala Leu

260 265 270

Cys Lys Ile Ile Pro Arg Gln Ile Lys Ala Glu Gln Ala Val Thr Val

275 280 285

Ile Arg Glu Thr Val Glu Glu Leu Ile Met Lys Cys Lys Glu Ile Val

290 295 300

Glu Lys Glu Gly Glu Lys Ile Asp Asp Glu Asp Tyr Val Asn Asp Ala

305 310 315 320

Asp Pro Ser Ile Leu Arg Phe Leu Leu Ala Ser Arg Glu Glu Val Ser

325 330 335

Ser Gln Gln Leu Arg Asp Asp Leu Leu Ser Met Leu Val Ala Gly His

340 345 350

Glu Thr Thr Gly Ser Val Leu Thr Trp Thr Thr Tyr Leu Leu Ser Lys

355 360 365

Asp Pro Ser Ser Leu Val Lys Ala Gln Glu Glu Val Asp Arg Val Leu

370 375 380

Gln Gly Arg Gln Pro Thr Tyr Glu Asp Ile Lys Asn Leu Lys Phe Ile

385 390 395 400

Thr Arg Cys Ile Asn Glu Ser Met Arg Leu Tyr Pro His Pro Pro Val

405 410 415

Leu Ile Arg Arg Ala Ile Val Ala Asp Glu Leu Pro Gly Asn Tyr Lys

420 425 430

Val Asn Pro Gly Gln Asp Ile Met Ile Ser Val Tyr Asn Ile His His

435 440 445

Ser Ser Lys Val Trp Asp Arg Ala Glu Glu Phe Ile Pro Glu Arg Phe

450 455 460

Gly Leu Asp Gly Pro Val Pro Asn Glu Thr Asn Thr Asp Tyr Lys Tyr

465 470 475 480

Ile Pro Phe Ser Gly Gly Pro Arg Lys Cys Val Gly Asp Gln Phe Ala

485 490 495

Met Leu Glu Ala Ile Val Ala Leu Ala Ile Phe Leu Gln Asn Ile Glu

500 505 510

Phe Glu Leu Val Pro Asn Gln Lys Ile Asn Met Thr Thr Gly Ala Thr

515 520 525

Ile His Thr Thr Ser Gly Leu Tyr Met Lys Val Lys Glu Arg Lys Val

530 535 540

Lys Ser Met Ala Val Ser Ser Thr

545 550

<210> 3

<211> 23

<212> DNA

<213> Artificial sequence (Artificial sequence)

<400> 3

taacacctac aaaccaccaa cca 23

<210> 4

<211> 25

<212> DNA

<213> Artificial sequence (Artificial sequence)

<400> 4

ctcgtttttt aaaagatttt catac 25

<210> 5

<211> 23

<212> DNA

<213> Artificial sequence (Artificial sequence)

<400> 5

atttcatctt tcatcatcct cac 23

<210> 6

<211> 24

<212> DNA

<213> Artificial sequence (Artificial sequence)

<400> 6

cgttagattt atggcctaaa ctgt 24

<210> 7

<211> 24

<212> DNA

<213> Artificial sequence (Artificial sequence)

<400> 7

gctacgtgat gaccttttgt caat 24

<210> 8

<211> 22

<212> DNA

<213> Artificial sequence (Artificial sequence)

<400> 8

cggtcaactt cctcttgtgc ct 22

<210> 9

<211> 23

<212> DNA

<213> Artificial sequence (Artificial sequence)

<400> 9

taagacctgg tggtggaaat aga 23

<210> 10

<211> 20

<212> DNA

<213> Artificial sequence (Artificial sequence)

<400> 10

cagcaccatg aggacgaaga 20

Claims (3)

1. A xanthophyll synthesis related gene has a nucleotide sequence shown in SEQ ID NO. 1.

2. The gene of claim 1, wherein: the coded amino acid sequence is shown in SEQ ID NO. 2.

3. Use of the gene of claim 1 in the synthesis of lutein in plants.