CN102321649A - Lycium chinense miller lycopene beta-cyclase gene, recombinant vector containing gene, host cell and application - Google Patents

Abstract

A wolfberry lycopene β-cyclase gene, a recombinant vector including the gene, a host cell and application thereof. The lycopene β-cyclase gene LmLycB was cloned by 3'RACE technology by extracting the total RNA of fresh Lycium barbarum leaves, and the complete gene sequence was 1506bp. The Escherichia coli expression vector pMON-LmLycB was constructed, and the activity of the enzyme encoded by the cloned LmLycB gene was identified by using the Escherichia coli heterologous expression system. LmLycB can add two β-ionone rings to the end of lycopene to generate β -carotene. And the genetic transformation system of immature embryos of maize inbred lines was optimized, and the plant expression vector pCAMBIA2300-LmLycB-Bar containing the target gene was transformed into maize callus, and finally transgenic positive plants were obtained. The total carotenoids in transgenic maize leaves were detected by HPLC. The content is 165.70 μg/g FW, which is 12.76% higher than that of wild-type maize, and the content of β-carotene is 80.17 μg/g FW, which is 54.83% higher than that of wild-type maize.

Description

Lycium barbarum lycopene β-cyclase gene, recombinant vector including the gene, host cell and application

technical field

The invention relates to a wolfberry (Lycium chinense Miller) lycopene β-cyclase gene, a recombinant vector comprising the gene, a host cell and application thereof.

Background technique

In the biosynthetic pathway of plant carotenoids, the cyclization reaction of lycopene is the most important branch point. Symmetrical cyclization forms two β-ionone rings at both ends of linear lycopene, and then produces β-carotenoids white. Lycopene β-cyclase (LycB) is an important rate-limiting enzyme in the carotenoid biosynthesis pathway, which catalyzes the conversion of lycopene to β-carotene [Bartley GE, Scolnik PA, Plant carotenoids: Pigments for photoprotection, visual attraction, and human health, Plant Cell, 1995, 7: 1027-1038]. So far, LycB genes have been isolated from plants such as soybean, tomato, pepper, Arabidopsis, corn, daffodil, and wild tobacco.

Carotenoids are C40 hydrocarbons, fat-soluble pigments widely present in plants, some photosynthetic bacteria and algae, mainly including carotene and their oxidized derivatives, xanthophylls. Carotenoids play a vital role in plant photosynthesis. They are indispensable structural components of photosynthetic antennae and photoreaction center complexes, and can also protect chlorophyll from photooxidative damage caused by strong light. Carotenoids are the precursors of vitamin A, which have the functions of enhancing immunity, anti-oxidation, promoting communication between cells, preventing, delaying and treating cancer. Aluru et al. connected the bacterial crtB (pds) and crtI genes together, specifically expressed in the endosperm of maize, the carotenoid content in the endosperm increased by 34 times, and accumulated a large amount of vitamin A precursor: β-carotene [Aluru M, Xu Y, GuoR, et al, Generation of transgenic maize with enhanced provitamin A content, J Exp Bot, 2008, 59(13): 3551-3562].

Once highly active and damaging free radicals are formed in biological systems, they will strongly damage the genetic material and cell membranes of cells, resulting in the decline of cell functions, aging of the body and the occurrence of diseases. Carotenoids, especially β-carotene, can inhibit and scavenge free radicals in the body, delay aging and prevent tumors, thrombosis, atherosclerosis and other diseases. Carotenoids can increase the activity of B cells in the immune system, eliminate foreign invading pathogens, increase the activity of lymphatic helper T cells, assist B cells to produce antibodies, and improve the activity of other immune components; they can also increase natural killer cells The number of infected cells or cancer cells in the body to eliminate. According to reports, in addition to β-carotene, lycopene, etc. also have the function of increasing immunity.

With the continuous discovery of the medicinal value and health care function of carotenoids, the demand for the types and yields of carotenoids will also increase. However, carotenoids are difficult to synthesize by chemical methods. The development of modern molecular biology research methods has enabled a series of key enzyme genes in the carotenoid biosynthesis pathway to be isolated and identified one after another, opening up a way for the production of carotenoids to be regulated by DNA recombination technology and genetic engineering, especially through carotenoid biosynthesis. The "Golden Rice" and "Golden Rapeseed" obtained by carotene genetic engineering have greatly enhanced people's confidence in carrying out plant carotenoid genetic engineering.

Contents of the invention

The object of the present invention is to provide a wolfberry lycopene β-cyclase gene.

The second object of the present invention is to provide the protein encoded by the gene.

The object of the present invention is also to provide a recombinant vector and host cell containing the gene.

Another object of the present invention is to provide the application of the gene.

The invention provides a wolfberry lycopene β-cyclase gene LmLycB, which is composed of the nucleotide sequence shown in SEQ ID NO.1 in the sequence table.

The present invention provides a protein encoded by the above Lycopene β-cyclase gene LmLycB, such as the protein with the amino acid sequence shown in SEQ ID NO.2 in the sequence listing.

The invention provides a recombination cloning vector pBS-T-LmLycB of the wolfberry lycopene β-cyclase gene LmLycB.

The recombinant vector containing the above-mentioned wolfberry lycopene β-cyclase gene LmLycB, these recombinant vectors include plasmids and plant expression vectors.

The plasmid expression vector is the Escherichia coli expression vector pMON-LmLycB.

The recombinant plant expression vector pCAMBIA2300-LmLycB-Bar.

A host cell containing the complete coding reading frame sequence of the wolfberry lycopene β-cyclase gene LmLycB, such as a host cell containing the above-mentioned recombinant vector, also belongs to the protection scope of the present invention.

The host cell is selected from Escherichia coli cells, Agrobacterium cells or corn cells.

The invention provides two genetic engineering bacteria containing LmLycB.

The application of the above-mentioned wolfberry lycopene β-cyclase gene LmLycB includes the application of the protein encoded by the LmLycB gene in Escherichia coli and plants; use the recombinant vectors, such as plant expression vectors, to transform corn cells; or use the described recombinant vectors, such as plant expression vectors, to transform corn cells; The Agrobacterium of this gene is co-cultured with cells such as corn, soybean, rice, peanut, sunflower, potato, cotton, millet, barley, flowers and vegetables to obtain transgenic regenerated plants; or use the wolfberry lycopene β-ring Transgenic plants of the above species were obtained by genetic transformation of the enzyme gene LmLyc.

Technical scheme of the present invention is specifically summarized as follows:

A wolfberry lycopene β-cyclase gene LmLycB, such as the nucleotide sequence shown in SEQ ID NO.1 in the sequence listing, also includes the addition, substitution, and insertion of the nucleotide sequence shown in SEQ ID No.1 Or more than 70% homologous sequence or its allele and its derived nucleotide sequence missing one or more nucleotides.

An Escherichia coli DH5α containing the recombinant vector pMON-LmLycB of the lycopene β-cyclase gene LmLycB.

Agrobacterium C58 containing recombinant vector pCAMBIA2300-LmLycB-Bar of lycopene β-cyclase gene LmLycB.

A protein encoded by the lycopene β-cyclase gene LmLycB, the amino acid sequence shown in SEQ ID NO.2 in the sequence listing.

The expression application of a protein encoded by lycopene β-cyclase gene LmLycB in Escherichia coli.

Application of a protein encoded by lycopene β-cyclase gene LmLycB in maize.

The wolfberry lycopene β-cyclase gene is also applied to the preparation of transgenic plants such as soybeans, rice, peanuts, sunflowers, potatoes, cotton, millet, barley, flowers and vegetables.

Cloning method of the present invention is made up of following steps:

Total RNA was extracted from Lycium barbarum leaves, and a degenerate primer P1 was designed according to the amino acid sequence of lycopene β-cyclase in other plants in GenBank, which is characterized by the sequence shown in SEQ ID NO.3 in the sequence table. Then the complete gene sequence of 1506bp was amplified by 3'RACE method.

The present invention constructs the Escherichia coli expression vector pMON-LmLycB containing the lycopene β-cyclase gene LmLycB and the plant expression vector pCAMBIA2300-LmLycB-Bar, consisting of the following steps:

1) Construction of the intermediate vector pBS-T-LmLycB containing the lycopene β-cyclase gene LmLycB:

Design the upstream primer P1 shown by SEQ ID NO.3, and the downstream primer P2 shown by SEQ ID NO.4, take the cDNA of wolfberry lycopene β-cyclase gene as template, carry out PCR amplification, will The PCR amplification product was connected to the pBS-T vector to obtain the intermediate vector pBS-T-LmLycB containing the LmLycB gene shown in SEQ ID NO.1 in the sequence listing.

2) Construct the Escherichia coli expression vector pMON-LmLycB:

Design the upstream primer P3 shown by SEQ ID NO.5, and the downstream primer P4 shown by SEQ ID NO.6, use the plasmid pBS-T-LmLycB as a template, carry out PCR amplification, and pass the PCR amplification product through BamHI After digestion with SalI, the Escherichia coli expression vector pMON-LmLycB was digested with BamHI and SalI, and the two were ligated to obtain the Escherichia coli expression vector pMON-LmLycB.

3) Construct the plant expression vector pCAMBIA2300-LmLycB-Bar:

Design the upstream primer P5 shown by SEQ ID NO.7, and the downstream primer P6 shown by SEQ ID NO.8, use the plasmid pBS-T-LmLycB as a template to perform PCR amplification, and pass the PCR amplification product through BamHI After digestion with SalI, the plant expression vector pCAMBIA2300-35S-OCS was digested with BamHI and SalI, and the two were ligated to obtain the plant expression vector pCAMBIA2300-LmLycB.

Then, using the XhoI restriction site, the NPTII gene on the pCAMBIA2300-LmLycB vector was removed, and the glufosinate-resistant Bar gene was inserted from the 5' to 3' direction to obtain the pCAMBIA2300-LmLycB-Bar plant expression vector.

The invention provides a wolfberry lycopene β-cyclase gene, a recombinant vector including the gene, a host cell and its application. For the first time, a complete cDNA encoding lycopene β-cyclase is isolated from wolfberry and connected to On the Escherichia coli expression vector, the exogenous expression system was used to verify that the product expressed at the protein level of Lycium barbarum LmLycB gene had enzyme activity. Then connect it to the plant expression vector, use the Agrobacterium infection method to transform maize, and the obtained transgenic plants are detected by HPLC. The results show that the total carotenoid content of transgenic maize leaves is 165.70 μg/g FW, which is 12.76% higher than that of wild type maize , in which the content of β-carotene was 80.17μg/g FW, which was 54.83% higher than that of wild-type maize. See Table 2.

The invention isolates the gene LmLycB encoding lycopene β-cyclase from wolfberry, and the overexpression of the gene can lead to the increase of β-carotene biosynthesis in transgenic corn. Change the metabolic pathway through genetic engineering breeding methods to increase the production of β-carotene to better and more accurately achieve the purpose of crop nutrition breeding, or use microorganisms as bioreactors to biosynthesize related β-carotene to meet people's nutritional needs.

Description of drawings

Fig. 1 Schematic diagram of pMON-LmLycB vector.

Figure 2 pMON-LmLycB PCR, enzyme digestion verification results.

Fig. 3 Schematic diagram of pCAMBIA2300-LmLycB-Bar vector.

Figure 4 pCAMBIA2300-LmLycB-Bar PCR, enzyme digestion verification results.

Fig. 5 Expression of LmLycB in Escherichia coli.

Fig. 6 HPLC detection results of Escherichia coli expressing LmLycB.

Fig. 7 PCR verification electrophoresis results of transgenic maize genome.

Fig. 8 RT-PCR verification electrophoresis results of transgenic maize.

Fig. 9 HPLC detection results of carotenoids in leaves of wild-type maize and transgenic maize.

Detailed ways

Example 1

For the experimental methods that do not specify specific conditions in the examples, usually follow the conventional conditions and the conditions described in the manual, or follow the conditions suggested by the manufacturer.

Cloning of lycopene β-cyclase gene LmLycB from Lycium barbarum:

With RNeasy Plant Mini Kit (QIAGEN, German) kit, Total RNA was extracted from 100 mg of fresh Lycium barbarum leaves. Degenerate primer P1 was designed for the amino acid sequence, as shown in SEQ ID NO.3 in the sequence listing. The complete gene sequence was amplified using the 3′-FULLRACE Core Set Ver.2.0 (TaKaRa, Japan) kit. Specific steps: ① Using Total RNA as a template, use 3′RACE Adapter primers for reverse transcription reaction to synthesize 1st Strand cDNA. The reaction system is as follows:

RNA: 2μl

3′RACEAdaptor: 1μl

5×M-MLV Buffer: 2μl

dNTP Mixture: 1 μl

RNase Inhibitor: 0.25μl

Reverse Transcriptase M-MLV: 0.25μl

RNase Free _dHO : 3.5 μl

Reaction conditions: 42°C, 60min; 70°C, 15min.

②Using the upstream primer P1 shown in SEQ ID NO.3 and the downstream primer P2 shown in SEQ ID NO.4, use 1st StrandcDNA as a template to perform a PCR reaction. The reaction system is as follows:

1st PCR product: 2μl

dNTP Mixture: 8μl

P1: 2μl

P2: 2 μl

10×LA PCR BufferII: 4μl

MgCl ₂ : 3 μl

TaKaRa LATaq: 0.25μl

_dH2O : 28.75 μl

Reaction conditions: 94°C, 3min; 94°C, 30sec; 55°C, 30sec; 72°C, 2min30sec; 72°C, 10min, 30 cycles.

Example 2

Construction process of intermediate vector pBS-T-LmLycB

The LmLycB gene shown in SEQ ID NO.1 is connected to the pBS-T vector, and the reaction system is as follows:

Target PCR fragment: 3 μl

pBS-T vector: 1μ1

2×T4DNA Rapid Ligation Buffer: 5μl

T4DNA Ligase: 1μl

Reaction conditions: 23°C, 10 min. The ligation product was transformed into E-Coli.TOP10 and spread on LB plates containing ampicillin. PCR was carried out with the target gene as a primer (reaction conditions: 94°C, 3min; 94°C, 30sec; 54°C, 30sec; 72°C, 2min30sec; 72°C, 10min, 30 cycles.) to obtain a 1800bp product, and the extracted plasmids were subjected to BamHI Double enzyme digestion with PstI: 37°C, 16hrs, digestion (15μl plasmid DNA, 3μl R buffer, 0.5μl BamHI exonuclease, 0.5μl PstI exonuclease, 13μl ddH2O) to obtain the target band, and finally sent to Huada Gene Sequencing Company Sequencing results showed that the vector was constructed correctly.

Example 3

Construction process of Escherichia coli expression vector pMON-LmLycB

First, use pBS-T-LmLycB as a template, and P3 and P4 as upstream and downstream primers to amplify the LmLycB fragment respectively. The reaction conditions are 94°C, 3min; 94°C, 30sec; 55°C, 30sec; 72°C, 2min30sec; 72°C , 10min, 30 cycles. A BamHI restriction site (GGATCC) and a base A were introduced into P3, and a SalI restriction site (GTCGAC) was introduced into P4. Then, the PCR product and the pMON38201 plasmid were digested with BamHI and SalI respectively, and the digested products were ligated: 16°C, 16hrs, ligation (2μl 10×T4buffer, 0.5μl T4DNA ligase, 5μl carrier DNA, 7.5μl exogenous DNA, 5 μl ddH ₂ O). The ligation product was transformed into Ecoli.DH5α, and spread on the LB plate containing Amp, as shown in Figure 1, which is a schematic diagram of the constructed pMON-LmLycB vector. The target gene was used as a primer to perform PCR to obtain a 1500bp product, and the target band was identified by enzyme digestion, and finally sent to Huada Gene Sequencing Company for sequencing. The results showed that the vector pMON-LmLycB was constructed correctly, as shown in Figure 2, which is the verification result of pMON-LmLycBPCR and enzyme digestion .

Transform the constructed plant expression vector pMON-LmLycB into Escherichia coli DH5α:

Example 4

Construction process of plant expression vector pCAMBIA2300-LmLycB-Bar

First, use pBS-T-LmLycB as a template, and P5 and P6 as upstream and downstream primers to amplify the LmLycB fragment respectively. The reaction conditions are 94°C, 3min; 94°C, 30sec; 55°C, 30sec; 72°C, 2min30sec; 72°C , 10min, 30 cycles. A BamHI restriction site (GGATCC) was introduced into P5, and a SalI restriction site (GTCGAC) was introduced into P6. Then, the PCR product and the pCAMBIA2300-35S-OCS plasmid were digested with BamHI and SalI respectively, and the digested products were ligated: 16°C, 16hrs, ligation (2μl 10×T4buffer, 0.5μl T4 DNA ligase, 5μl carrier DNA, 7.5 μl exogenous DNA, 5 μl ddH ₂ O). The ligation product was transformed into E-Coli.TOP10 and spread on LB plates containing kanamycin. The target gene was used as a primer to perform PCR to obtain a 1500bp product, and the target band was obtained by enzyme digestion and identification. Finally, it was sent to Huada Gene Sequencing Company for sequencing. The results showed that the vector pCAMBIA2300-LmLycB was constructed correctly, as shown in Figure 3. Cut verification results.

Using the XhoI (CTCGAG) restriction site, the NPTII gene on the pCAMBIA2300-LmLycB vector was removed, and the glufosinate-resistant Bar gene was inserted from the 5' to 3' direction to obtain the pCAMBIA2300-LmLycB-Bar plant expression vector, as shown in the figure 4.

The constructed plant expression vector pCAMBIA2300-LmLycB-Bar was transformed into Agrobacterium C58. The specific steps were as follows: ① put the C58 competent cells taken out at -80℃ on ice and let it melt slowly; ② add 2 μL of plasmid and mix well; ③Transfer to the electric shock cup, and the above operations are all carried out on ice; ④Set the parameters of the electric shock transformation instrument: 25μF, 400olm, 1500V, 5ms, electric shock transformation; Shake culture at 28°C for 4 hours; ⑥ Spread 50 μL of the bacterial solution on a YEB plate containing 100 mg/L Ka resistance, invert the plate, and incubate at 28°C for 48 hours until a clear single colony is seen.

Example 5

Functional verification of LmLycB gene in Escherichia coli

Co-transform the plasmid pACCRT-EBI _Eu (chloramphenicol resistance) and the expression vector pMON-LmLycB (ampicillin resistance) in Escherichia coli, the specific steps are: ① Add two kinds of 1 μL of each plasmid DNA, mix well, and place on ice for 30 minutes; ② Heat shock at 42°C for 90 seconds, then place on ice for 2-3 minutes; ③ Add 800 μL, 37°C preheated LB liquid medium (without antibiotics), 37°C, 150r Cultivate with shaking/min for 45 minutes; ④ Pipette 50 μL of the above culture solution onto LB solid medium containing corresponding antibiotics, and smear on the plate; ⑤ Invert the plate, and incubate at 37°C for 12 hrs. The enzyme encoded by Lycium barbarum LmLycB can convert red lycopene into yellow β-carotene, and the color of E. coli colonies changes from red to yellow, as shown in Figure 5. The composition of LB liquid medium is: 10g/L peptone, 10g/L NaCl, 5g/L yeast extract.

Escherichia coli carotenoid extraction: ①Centrifuge to collect the bacteria, the weight of the bacteria should not exceed 0.8g; ②Add 20ml methanol to the bacteria and resuspend; ③Add 2ml, 60% KOH, and resuspend; ④Incubate at 60°C for 20min, Then cool the sample to room temperature; ⑤ transfer the sample and 20ml of petroleum ether (bp.40-60°C) containing 10% ether to a separatory funnel, mix thoroughly, and extract; ⑥If the sample is separated from ether/petroleum ether Not good, you can add an appropriate amount of saturated NaCl solution and a small amount of ethanol; ⑦ Collect the upper organic phase; ⑧ Blow nitrogen to dry the sample.

HPLC detection of carotenoids in Escherichia coli: Dissolve the sample in 40 μL acetone, use nucleosil 100-3c250×4.6mm (MN, Germany) chromatographic column, Lambo (imported SSI) quaternary gradient pump. According to Sander (Lane C. Sander, Katherine Epler Sharpless, et al, Development of Engineered Stationary Phases for the Seperation of Carotenoid Isomers, Anal. Chem, 1994, 66: 1667-1674) etc. (1994) method for high performance liquid chromatography analysis. The mobile phase is acetonitrile:methanol:isopropanol=85:10:5, and the flow rate is 1.0mL/min. At the same time, a Thermo diode array (diode-array detector, DAD) detector is used to scan the carotenoid spectrum at full wavelength, such as Figure 6 shows the results of HPLC detection of Escherichia coli expressing LmLycB.

Example 6

Agrobacterium-mediated genetic transformation of maize

1. Obtaining type II corn callus: The young corn embryos used come from the corn inbred line 7922 in the experimental field of our laboratory. After 10-15 days of pollination, the young ears are taken, the bract leaves are peeled off, and the corn is used first in the ultra-clean bench. Disinfect the surface with 75% ethanol, then soak it in 0.1% mercuric chloride for 15 minutes, and wash it with sterile water three times. Pick the complete immature embryos with a size of about 1-2mm in the middle of the ear, and inoculate them on four callus induction media with the scutellum facing up (see Table 1 for the composition of the media), culture them in the dark, and induce them in 3-4 weeks. Type II callus. The induced well-growing type II callus was divided into 2-3mm tissue pieces, cultured in dark on the subculture medium (the medium composition is shown in Table 1), and subcultured once in about 2-3 weeks, and the type II callus was carried out. Expansion of injured tissue.

2. Cultivation of Agrobacterium: Agrobacterium C58 containing the plant expression vector pCAMBIA2300-LmLycB-Bar was plated to isolate a single colony; a single colony was picked and placed in 5mL YEB liquid medium containing 100mg/L Ka at 28°C. Cultivate at 180r/min for 12hrs; inoculate the above-mentioned bacterial solution into fresh 30mL YEB liquid medium containing 100mg/L Ka at a ratio of 1:100, at 28°C, 180r/min, shake culture until the OD600 value is about 0.6-0.8 ;Use a 50mL centrifuge tube to collect the bacterial solution, centrifuge at 4000r/min for 10min; resuspend and wash the bacterial cells twice with the infection solution, add acetosyringone to a final concentration of 100μmol/L, and infect for later use.

3. Genetic transformation of corn: Cut the Type II callus of the well-growing corn inbred line 7922 into small pieces of 3-5mm, place it in a petri dish, add an appropriate amount of resuspension (N6 in large quantities+B5 Trace amount + iron salt + N6 organic + 1.5mg/L2, 4-D + 0.7g/L L-Pro + 36g/L glucose + 68.4g/L sucrose). The bacterial solution prepared in step 2 was poured into a petri dish, and the bacterial solution was poured out after the infection, and the callus was placed on filter paper, and the excess bacterial solution was blotted.

The infected immature embryo callus was inoculated on the co-cultivation medium (see Table 1 for the composition of the medium) with the shield side up, and cultured in the dark at 28° C. for 3-4 days. The immature embryo callus after co-cultivation was inoculated on (the medium composition is shown in Table 1), and cultured in the dark at 28° C. for 7 days. At the same time of degerming, the exogenous vector just transformed into the plant can be fully expressed in the plant cell.

After delayed cultivation, transfer to the selection medium containing PPT 5mg/L (medium composition is shown in Table 1), 25 ℃, dark culture for 2 weeks, then subculture to the selection medium that has improved the selection pressure, 2 Zhou Jidai once. After 6 weeks, new callus tissue was produced, and most of the immature embryos browned and died at the same time. These new calluses can be regarded as transformants and continue to be subcultured.

The embryogenic callus was picked and inoculated on the regeneration medium I (the medium composition is shown in Table 1), and cultured in the dark at 25°C for 2-3 weeks. During this process, most of the somatic embryos swelled and turned white. Some have formed coleoptiles, and the part of these callus long roots is cut off, and then it is inoculated on the regeneration medium II (substrate composition is shown in Table 1), 25 ℃ of light culture (light intensity is 5000Lux, light The period is 16h/8h). Within a week, the somatic embryos can grow leaves, and they can be transplanted in about 10 days.

When the corn seedlings grow to a size of 5 cm, the lid of the culture bottle is opened, and the seedlings are hardened in the cultivation room for 3-5 days. Then carefully take the seedlings out of the bottle with tweezers, rinse off the agar with clean water, and avoid damaging the roots and leaves during the process. Move the plants into small flower pots, fill them with culture soil, water them thoroughly, and move them into the greenhouse.

Table 1 Plant medium composition

^a medium pH value is 5.8; ^b medium pH value is 5.2; CH: casein; MES: 2-(N-morpholine) ethanesulfonic acid

Example 7

Molecular detection and HPLC detection of transgenic maize

PCR detection of genomic DNA of transgenic corn: ①CTAB method to extract total corn DNA; ②Take genomic DNA as a template for PCR detection, primers P5 and P6, reaction conditions: 94°C, 3min; 94°C, 30sec; 55°C, 30sec; 72°C, 2min30sec; 72°C, 10min, 30 cycles, the electrophoresis results are shown in Figure 7.

RT-PCR detection of transgenic maize: ① extract the total RNA of maize leaves; ② use the total RNA of maize leaves as a template, use the Reverse Transcription System (Promega) kit for reverse transcription, and synthesize the first strand of cDNA. Reaction conditions: 42°C, 50min; 95°C, 5min, 4°C, 5min; ③Take 1μL of the reaction solution as a template for PCR reaction with primers P7 and P8. Reaction conditions: 94°C, 3min; 94°C, 30sec; 55°C, 30sec; 72°C , 45s; 72°C, 10min, 30 cycles, the electrophoresis results are shown in Figure 8.

Extraction of carotenoids from corn leaves: ①Take about 50mg of fresh corn leaves (the second leaf from the top), put them in a mortar, grind them into powder with liquid nitrogen, and transfer them to a 50ml centrifuge tube; ②Add 20ml of methanol to In a centrifuge tube, mix well; ③ add 2ml, 60% KOH, mix well; ④ incubate at 60°C for 20min, then cool the sample to room temperature; Transfer to a separatory funnel, mix well, and extract; ⑥If the layering effect of the sample and ether/petroleum ether is not good, you can add an appropriate amount of saturated NaCl solution and a small amount of ethanol; ⑦Collect the upper organic phase; ⑧Blow nitrogen to dry the sample .

Determination of total carotenoid content in transgenic corn: dissolve the extracted carotenoids in 10 mL of acetone, and measure the absorbance at 450 nm with a UV spectrophotometer, as shown in Figure 9, which is the HPLC detection result of carotenoids in wild-type corn and transgenic corn leaves . And calculate the total carotenoid content of corn leaves by the following formula:

X(μg/g)＝(A×1×V×l0 ⁶ )/(ε×100×W)

In the formula: X is the carotenoid content (μg), A is the absorbance OD value of the sample measured at 450nm, V is the volume of the extract (10mL), ε is the average absorption coefficient of carotenoid molecules 2100, and W is the corn leaf The weight (g).

HPLC detection of transgenic corn leaves: at a wavelength of 450nm, the standard curve method was used to determine the content of β-carotene in the sample, and the formula was as follows:

X(μg/g FW)＝(C×V)/(A×W)

In the formula: X is the content of β-carotene, C is the content of β-carotene obtained from the standard curve (μg); V is the total volume of the extract (40 μL); A is the volume used in the determination (20 μL); W is the sample weight (g). The results are shown in Table 2.

Table 2

Claims (8)

1. matrimony vine lycopene beta cyclase gene is characterized in that being selected from one of following nucleotide sequence:

1) has the nucleotide sequence shown in the SEQ ID No.1;

2) nucleotide sequence shown in the SEQ ID No.1 add, replace, insert or delete 70% homologous sequence or its allelotrope and the deutero-nucleotide sequence thereof of one or more Nucleotide.

2. the protein of the described matrimony vine lycopene beta cyclase of claim 1 genes encoding is characterized in that described protein has the aminoacid sequence shown in the SEQ ID No.2.

3. a recombinant vectors is characterized in that containing the described matrimony vine lycopene beta cyclase of claim 1 gene complete sequence or part fragment.

4. the described recombinant vectors of claim 3 is characterized in that it is plasmid expression vector coli expression carrier pMON-LmLycB or recombinant plant expression vector pCAMBIA2300-LmLycB-Bar.

5. the described recombinant vectors of claim 3 is characterized in that it is the bacillus coli DH 5 alpha of recombinant vectors.

6. a host cell is characterized in that containing the described matrimony vine lycopene beta cyclase of claim 1 gene complete sequence or part fragment.

7. the described host cell of claim 6 is characterized in that it is Bacillus coli cells, agrobatcerium cell or maize cell.

8. the described matrimony vine lycopene beta cyclase of claim 1 gene is applied to prepare plant such as transgenic corns, soybean, paddy rice, peanut, Sunflower Receptacle, yam, cotton, millet, barley and flowers and vegetables.

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