CN103820475B - The protein of Fructus Lycii yak base geranylpyrophosphate synthase gene and coding thereof and application - Google Patents

Abstract

The present invention relates to a wolfberry geranyl geranyl pyrophosphate synthase gene and its encoded protein and application thereof, specifically geranyl in wolfberry (<i>Lycium</i><i>chinense Miller</i>) Cloning of yak pyrophosphate synthase gene <i>LmGGPS</i><i>2</i>. The geranylgeranyl geranyl pyrophosphate synthase gene <i>LmGGPS2</i> in Lycium barbarum was cloned by extracting total RNA from fresh Lycium barbarum leaves, and the complete sequence of the gene was 1246bp; the Escherichia coli expression vector pET28a- LmGGPS2, through the heterologous expression system of E. coli, the LmGGPS2 expression protein was obtained; and the binary plant expression vector pCAMBIA2300-LmGGPS2 was constructed, and the vector was transferred into Agrobacterium C58 cells by electric shock method, and the cells were used to transform tobacco to obtain transgenic tobacco. Through testing, it was found that the genetically modified tobacco greatly increased the content of plant lycopene, and the content of carotenoids also increased.

Description

Lycium barbarum geranyl geranyl pyrophosphate synthase gene and its encoded protein and application

technical field

The invention relates to a wolfberry geranyl geranyl pyrophosphate synthase gene and its coded protein and application thereof, in particular to the cloning of the geranyl geranyl pyrophosphate synthase gene LmGGPS2 in Lycium chinense Miller.

Background technique

Geranylgeranyl pyrophosphate (GGPP) is an important intermediate metabolite ubiquitous in the biological world. In plants, GGPP participates in chlorophyll, carotenoids, gibberellins, plastoquinones, vitamin E, monoterpenes and benzoquinones, etc. The synthesis of products has an important impact on plant photosynthesis, growth and development, and product quality. The product was directly catalyzed by GGPS. GGPP is not only the most direct precursor of carotenoid biosynthesis, but also the precursor of gibberellin, chlorophyll and plastoquinone in plants. The synthesis of GGPP is the rate-limiting process in carotenoid synthesis. Through a series of enzymatic reactions, the lycopene produced not only gives tomatoes a bright color, but also has important nutritional and health value. Medical research shows that high levels of lycopene in serum can significantly reduce the incidence of various cancers. The incidence of cancer is negatively correlated. Studies have shown that high lycopene content in the human body can significantly reduce the incidence of prostate cancer. The role of lycopene in protecting cardiovascular and cerebrovascular is mainly through its antioxidant effect, reducing the peroxidation of serum lipids and low-density lipoproteins, thereby reducing the incidence of arteriosclerosis and coronary heart disease. High intake of lycopene can Significantly reduce the incidence of cardiovascular and cerebrovascular diseases. Lycopene also has the functions of improving immunity and delaying aging.

Lycopene can quench singlet oxygen, scavenge free radicals, and prevent protein and DNA from being damaged by oxygen. The ability of lycopene to scavenge singlet oxygen is 10 times that of commonly used antioxidant vitamin E and 2 times that of β-carotene. Lycopene molecule contains the largest number of conjugated double bonds among all carotenoids, and its singlet oxygen quenching rate constant is 100 times that of vitamin E, making it the carotenoid with the strongest antioxidant capacity. The chemical structure of lycopene determines that it has a strong ability to remove active oxygen and free radicals, and is an effective antioxidant. Moreover, carotenoids, especially β-carotene, can inhibit and scavenge free radicals in the body, which can delay aging and prevent diseases such as tumors, thrombosis, and atherosclerosis. 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.

In plants, the GGPS gene works with other enzymes to produce diterpenes, tetraterpenes and polyterpenes. GGPS is a key enzyme in the synthesis of diterpenes, tetraterpenes and polyterpenes. Tobacco terpenoids are closely related to the aroma of tobacco leaves. They not only exist in tobacco leaves as important tobacco aroma precursors, but also found most of the terpenoids in tobacco leaves in smoke. For example, diterpene is the main component of the glial secretions of foliage glands, and its main component is cembratriene diol, and its degradation product solanone and its derivatives are important aroma substances; tetraterpene compound carotenoids are Important aroma precursors in tobacco leaves, and their degradation products such as damascenone, ionone, and macrostigmatrienone are important aroma components in tobacco leaves. In the production of tobacco leaves in my country, increasing the content of terpenoids in tobacco leaves, especially the accumulation of aroma precursors such as diterpenes and carotenoids, which are closely related to aroma quality, can increase the aroma of tobacco leaves and improve the aroma quality of tobacco leaves. Therefore, it is of great significance to study transgenic GGPS tobacco.

The steps of plant carotenoid synthesis are as follows: 3-methyl-3,5-dihydroxyvaleric acid (MVA) synthesizes isopentenyl pyrophosphate (IPP) under the catalysis of enzymes, and isopentenyl pyrophosphate (IPP) first Under the action of isopentenyl pyrophosphate isomerase (IPI), the isomer dimethylpropylene pyrophosphate (DMAPP) with propenyl structure is generated, which is an essential precursor substance in the synthesis pathway of isopentene , DMAPP and IPP undergo continuous condensation to generate geranyl pyrophosphate (GPS), farnesyl pyrophosphate (FPP), and geranyl geranyl pyrophosphate (GGPP). Two molecules of GGPP are catalyzed by phytoene synthase (PSY) to form phytolycopene. Octahydrolycopene undergoes continuous dehydrogenation reactions to generate cis-structure prolycopene, and prolycopene is isomerized into trans-structure lycopene under the action of carotene isomerase (CRTISO). Lycopene is then catalyzed by the corresponding cyclase to generate α, β-carotene, and further generate other types of pigments (Tanaka et al., 2008).

With the continuous discovery of the medicinal value and health care function of lycopene and carotenoids, the demand for the types and production of lycopene and carotenoids will also increase. However, lycopene carotenoids It is difficult to synthesize chemically. With the development of modern molecular biology research methods, a series of key enzyme genes in the biosynthetic pathway of lycopene and carotenoids have been isolated and identified one after another, providing a basis for the regulation and production of lycopene and carotenoids through DNA recombination technology and genetic engineering. The road has been opened up, especially the "golden rice" and "golden rapeseed" obtained through carotenoid genetic engineering, which greatly enhanced people's confidence in carrying out plant carotenoid genetic engineering.

Terpenoids are the most numerous class of compounds in plant metabolites, which are composed of isoprene as a structural unit. It plays an important role in plant respiration, photosynthesis, growth, development, reproduction, signal transduction and defense. Some terpenoids have important economic value, and some can be used as natural fragrances and aroma compounds or anti-tumor chemotherapy drugs. In plants, the biosynthesis of terpenoids occurs in the cytoplasm and plastids, and its precursor is isopentenyl pyrophosphate (IPP) with C5 structure and its isomer - dimethylene Dimethylallylpyrophosphate (DMAPP). IPP synthesized via the mevalonic acid (MVA) pathway is the precursor for the synthesis of farnesyl pyrophosphate (FPP, C15), and FPP is finally synthesized into sesquiterpenes, triterpenes and sterols in the cytoplasm. IPP and DMAPP synthesized by the methyl-erythritol-phosphate (MEP) pathway in the plastid are precursors for the formation of geranylpyrophosphate (GPP, C10). GPP generates monoterpene under the action of monoterpene synthase; under the action of geranylgeranylpyrophosphate synthase (GGPPS), it generates geranylgeranylpyrophosphate (GGPP, C20), Then, under the action of enzymes, diterpenes, tetraterpenes and polyterpenes are generated.

Contents of the invention

The object of the present invention is to provide a wolfberry geranyl geranyl pyrophosphate synthase 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 geranyl geranyl pyrophosphate synthase gene LmGGPS2, which is composed of the nucleotide sequence shown in SEQ ID NO.1 in the sequence listing.

The present invention provides a protein encoded by the wolfberry geranyl geranyl pyrophosphate synthase gene LmGGPS2, 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 pMD18-T-LmGGPS2 of the wolfberry geranyl geranyl pyrophosphate synthase gene LmGGPS2.

The recombinant vectors containing the above-mentioned wolfberry geranyl geranyl pyrophosphate synthase gene LmGGPS2 include plasmids.

The plasmid expression vector is the Escherichia coli expression vector pET28a-LmGGPS2.

The recombinant vectors containing the above-mentioned wolfberry geranyl geranyl pyrophosphate synthase gene LmGGPS2 include plasmids.

The plasmid expression vector is the Escherichia coli expression vector pET28a-LmGGPS2.

The plasmid expression vector is a binary plant expression vector pCAMBIA2300-LmGGPS2.

A host cell containing the complete coding reading frame sequence of the Lycium barbarum geranyl geranyl pyrophosphate synthase gene LmGGPS2, such as a host cell containing the above recombinant vector, also belongs to the scope of protection of the present invention.

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

The invention provides an engineering bacterium containing the LmGGPS2 gene.

The application of the wolfberry geranyl geranyl pyrophosphate synthase gene LmGGPS2 includes the application of the protein encoded by the gene in plants; using the recombinant vector, such as plant expression vector, to transform corn cells; or using the gene containing the gene Agrobacterium is co-cultured with cells such as corn, soybean, sunflower, potato, cotton, millet, barley, flowers and vegetables to obtain transgenic regenerated plants; or use the LmGGPS2 genetic transformation to obtain transgenic plants of the above species.

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

Cloning method of the present invention comprises the following steps:

Total RNA was extracted from fresh leaves of Lycium barbarum, and the upstream primers LmGGPS2-F1:5'-GTCATGTCTATGCTAATAGGTGT-3', LmGGPS-R1 were designed according to the nucleotide sequence of Lycium barbarum geranyl geranyl pyrophosphate synthase gene LmGGPS2 in the transcriptome Unigene sequence :5'-GGTCA

GTTTCTGATGGAGAAATT-3', the full-length sequence of the gene was 1246bp obtained by PCR amplification.

The present invention constructs the Escherichia coli expression vector pET28a-LmGGPS2 containing geranyl geranyl pyrophosphate synthase gene LmGGPS2 and the plant expression vector pCAMBIA2300-LmGGPS2, consisting of the following steps:

1) Construct the intermediate vector pMD18-T-LmGGPS2 containing the geranyl geranyl pyrophosphate synthase gene.

Using LmGGPS-F1/LmGGPS-R1 as primers and Lycium barbarum cDNA as a template, perform PCR amplification, connect the PCR amplification product to the pMD18-T vector, and obtain an intermediate vector containing the LmGGPS2 gene shown in SEQ ID NO.1 in the sequence table pMD18-T-LmGGPS2.

2) Construction of Escherichia coli expression vector pET28a-LmGGPS2

Using LmGGPS-F1/LmGGPS-R1 as primers, using Lycium barbarum cDNA as template, PCR amplification was carried out, the PCR amplification product was connected to pMD18-T vector, and transformed into competent Escherichia coli TOP10, after colony PCR verification, extraction For the plasmid, the plasmid was double-digested with BamHI and SalI, and the pET28a empty vector was double-digested with BamHI and SalI, purified and then ligated to obtain the Escherichia coli expression vector pET28a-LmGGPS2.

3) Construction of plant expression vector pCAMBIA2300-LmGGPS2

Using LmGGPS-F1/LmGGPS-R1 as primers, using Lycium barbarum cDNA as template, PCR amplification was carried out, the PCR amplification product was connected to pMD18-T vector, and transformed into competent Escherichia coli TOP10, after colony PCR verification, extraction For the plasmid, the plasmid was double-digested with BamHI and SalI, and the pCAMBIA2300-35S-OCS empty vector was double-digested with BamHI and SalI, respectively purified and ligated to obtain the plant expression vector pCAMBIA2300-LmGGPS2.

The invention provides a wolfberry geranyl geranyl pyrophosphate synthase gene and its encoded protein and application. For the first time, the complete cDNA encoding geranyl geranyl pyrophosphate synthase gene was isolated from Lycium barbarum, connected to the expression vector of Escherichia coli, and the expression protein of Lycium barbarum LmGGPS2 gene was verified by using the exogenous expression system; then connected to the plant expression vector, Tobacco is transformed by using the Agrobacterium infection method to obtain transgenic plants, and the research shows that the carotenoid content of the transgenic plants is increased, that is, the invention has wide application in enhancing plant carotenoid content and the like.

The wolfberry geranyl geranyl pyrophosphate synthase gene LmGGPS2 of the present invention is expected to be used to prepare transgenic corn, soybean, rice, peanut, sunflower, potato, cotton, millet, barley, flower and vegetable plants.

Description of drawings

Figure 1 is the electrophoresis diagram of the full-length PCR amplification of LmGGPS2.

Figure 2 is a schematic diagram of the pMD18-T-LmGGPS2 vector.

Figure 3. Schematic diagram of pET-28a-LmGGPS2 vector.

Figure 4. Schematic diagram of pCAMBIA2300-LmGGPS2 vector.

Figure 5. Genome PCR for transgenic tobacco.

Figure 6. Genome PCR for transgenic corn, soybean, rice, peanut, sunflower, millet, wheat, and cotton.

Figure 7 shows the genome PCR of transgenic rose, eustoma, anthurium, and Phalaenopsis.

Figure 8. Genome PCR for transgenic poplar.

detailed description

Example 1

Cloning of the geranylgeranyl pyrophosphate synthase gene LmGGPS2 from Lycium barbarum

Total RNA was extracted from fresh leaves of Lycium barbarum, and the RNA was reverse-transcribed into cDNA using the 3'FULLRACECoreSetVer.2.0 (TaKaRa, Japan) kit. The specific steps refer to the instructions. The reaction system is as follows:

RNA 2μL. OligdT 1μL 5×M-MLV Buffer 2μL dNTP Mixture 1μL RNase Inhibitor 0.25 μL Reverse Transcriptase M- MLV 0.25 μL RNase Free ddH ₂ O 3.5μL

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

Design upstream primers LmGGPS-F1:5'-GTCATGTCTATGCTAATAGGTGT-3', LmGGPS-R1:5'-GGTCAGTTTCTGATGGAGAAATT-3', Using the synthesized cDNA as a template, the PCR reaction system is as follows:

1st Strand cDNA 1μL LmGGPS2-F1 0.5μL LmGGPS2-R1 0.5μL4 --> 2.5mM dNTP Mixture 2μL 10×LA Taq PCR buffer 2.5μL TaKaRa LA Taq DNA Polymerase 0.25 μL ddH ₂ O 18.25μL Total volume 25 μL

A total of 4 tubes of reaction solution were prepared, and the reaction conditions were as follows: 94°C, 4min; 94°C, 30Sec; 56°C, 30Sec; 72°C, 1min20Sec; 72°C, 10min; 30 cycles. After the reaction, the PCR reaction product was purified using the common DNA product purification kit of Tiangen Company to obtain the purified LmGGPS PCR fragment, as shown in Figure 1, and the operation of the kit instruction manual.

Example 2

Construction process of cloning vector pMD18-T-LmGGPS2

The LmGGPS2 gene shown in the sequence listing is connected to the pMD19-T carrier, and the reaction system is as follows:

LmGGPS2 full-length recovery product 4μL pMD18-T vector 1μL SolutionI 5μL

The LmGGPS2PCR fragment is the recovered GGPS full-length product in Example 1.

Reaction conditions: 16°C, 30min. The ligation product was transformed into competent E-Coli.TOP10, spread on LB agar plate medium containing 40ml25mg/ml X-Gal, 16ml50mg/ml IPTG, 100mg/LAmp and cultured to form a single colony. Select white colonies, colony PCR method to confirm the length of the insert in the T vector, as shown in Figure 2, as expected, the vector was sent to Huada Gene Company for sequencing, and we obtained the 1074bp base sequence of the gene, which was carried out at NCBI blast, has high homology with Solanaceae, indicating that the gene was successfully cloned.

Example 3

Construction process of Escherichia coli expression vector pET28a-LmGGPS2

The Escherichia coli containing the pMD19-T-LmGGPS2 plasmid obtained in the expanded culture experiment example 2 was extracted, and the plasmid was double-digested with BamHI and SalI, and the pET28a empty vector was double-digested with BamHI and SalI, purified and ligated , to obtain the Escherichia coli expression vector pET28a-LmGGPS2, and connect the digested products of the two.

The connection system is as follows:

pET28a empty vector fragment 2μL pMD19-T-LmGGPS2 restriction fragment 5μL 5×T4 DNA ligase buffer 2μL T4 DNA ligase 1μL

The ligation product was transformed into competent Escherichia coli BL21. Spread on LB plates containing 100 mg/L kana resistance, and culture at 37°C. After 12 hours, a single colony was picked for colony PCR verification, as shown in Figure 3. The bacteria that were positive for colony PCR verification were shaken to extract plasmids, digested and identified to obtain the target band, and finally sent to Huada Gene Sequencing Company for sequencing. The results showed that the vector pET28a- LmGGPS2 builds correctly.

Example 4

Construction of Binary Plant Expression Vector pCAMBIA2300-LmGGPS2

The Escherichia coli containing the pMD19-T-LmGGPS2 plasmid obtained in the expanded culture experiment example 2 was extracted, and the plasmid was double-digested with BamHI and SalI, and the pCAMBIA2300 empty vector plasmid was also double-digested with BamHI and SalI. The enzyme digestion products were connected, and the connection system was as follows:

pCAMBIA2300 empty vector fragment 2μL pMD19-T-LmGGPS2 restriction fragment 5μL 5×T4 DNA ligase buffer 2μL T4 DNA ligase 1μL

The ligation product was transformed into E-Coli.DH5α, and spread on LB plates containing 100 mg/L kana resistance. Cultivate at 37°C, pick a single colony after 12 hours for colony PCR verification, shake the bacteria that are positive in the colony PCR verification, extract the plasmid, and identify the target band by enzyme digestion, as shown in Figure 4.

Example 5

Construction of Agrobacterium engineering strain C58:pCAMBIA2300-LmGGPS2 for plant transgenesis.

Agrobacterium Competent Cell Preparation

1. Inoculate a single colony of Agrobacterium C58 in 5mL YEP liquid medium, culture at 28°C with shaking at 180r/min.

2. Transfer the above bacterial liquid into 100mLYEP liquid medium, culture at 28°C, 180r/min, with shaking until (OD ₆₀₀ value is about 0.5).

3. After 30 minutes in ice bath, centrifuge at 4000 r/min for 10 minutes at 4°C, collect the bacteria, and resuspend in 20 mL of pre-cooled H ₂ O.

4. Centrifuge at 4000r/min for 10min at 4°C, collect the bacteria, resuspend in pre-cooled 10% glycerol, quickly freeze in 200μL per tube, and store at -80°C for later use.

Electroporation Transformation of Plant Expression Vectors

1. Put the C58 competent cells taken out at -80°C on ice and let it melt slowly;

2. Add 4 μL of plasmid, mix well, and ice bath for 5 minutes;

3. Transfer to the electric shock cup;

4. Set the parameters of the electric shock conversion instrument: 1500V, 0.2s, electric shock conversion;

5. After standing at room temperature for 2 minutes, add 500 μL YEB liquid medium, 28 ° C, 180 r/min shaking culture for 3 hours;

6. Centrifuge at 4000r/min at room temperature for 10min, suck out 400μL of the supernatant, mix the remaining bacterial solution, spread it on a YEB plate containing 100mg/L kanamycin and 100mg/L rifampicin resistance, and invert plate, cultured at 28°C for 48 hours, until a clear single colony was seen.

7. Pick a single colony, colony PCR verification.

Example 6

Agrobacterium-mediated genetic transformation of tobacco

The aseptic culture of tobacco seedlings: choose plump, healthy tobacco seeds, soak 1min with 75% ethanol, 25% antiformin (available chlorine 2.5%) aqueous solution sterilization 8min, rinse three times with sterile water, put the seeds in In MS medium, light culture at 25°C, 16h/8h photoperiod.

The media used in this experiment are shown in the table below

Agrobacterium transformation of tobacco

Preparation of the infection solution

1. Pick a single positive Agrobacterium colony, inoculate it into 5ml of YEP liquid medium containing 100mg/L kanamycin, and culture it overnight on a shaker at 28°C and 200r/min.

2. The next day, take 3ml of the bacterial solution and inoculate it into 50ml of YEP liquid medium containing 100mg/L kanamycin. When the bacterial solution is in the vigorous growth phase (OD ₆₀₀ =0.6-0.9), pour the bacterial solution into 50ml The centrifuge tube was centrifuged at 3500r/min, 4°C for 10min, the supernatant was discarded, and the bacteria were collected. Resuspend with an equal amount of MS liquid medium to make OD ₆₀₀ =0.9-1.

explant infection

1. Remove the main veins and leaf edges from the tobacco leaves, then cut the leaves into 0.5cm×0.5cm size, immerse in the prepared Agrobacterium bacteria solution, soak for 15-20min, shake 2-3 times during the period, so that the leaves are fully exposed to the bacteria Remove the leaves, absorb the excess bacterial solution with sterile filter paper, inoculate in the co-cultivation medium with the leaf face down and the leaf back up, and culture in dark at about 25°C for 2 days.

2. Transfer the leaves to the screening medium, replace the medium every 20 days or so, and induce the production of resistant buds. When the resistant buds grow to about 1 cm from the callus, cut the resistant buds from the callus and inoculate them on Resistant seedling rooting medium.

Transgenic seedlings transplanted

Take out the tobacco tissue culture seedlings with good root growth and vigorous vitality from the tissue culture bottle, rinse the medium with tap water (to minimize root damage), plant it in the culture soil of frog stone and humus, cover it with a film to keep it warm for 15 days, Then uncover the film, water and fertilize regularly to make it grow normally in the greenhouse.

Example 7

Molecular Detection of Transgenic Tobacco and Determination of Carotene Content

PCR detection of genomic DNA in transgenic tobacco: 1. CTAB method to extract total tobacco DNA; 2. PCR detection using genomic DNA as a template, primers GGPS-F3 and GGPS-R-FULL, reaction conditions: 94 ° C, 4 min; 94 ℃, 30Sec; 54℃, 30Sec; 72℃, 1min10Sec; 72℃, 10min; 30 cycles. 5 μl of the above PCR product was taken for electrophoresis detection, as shown in Figure 5, indicating that the LmGGPS2 gene has been successfully transferred into tobacco.

The carotenoids of the transgenic tobacco were extracted, and the content of carotenoids in the transgenic tobacco was significantly higher than that of the wild-type tobacco as determined by HPLC, indicating that the gene plays an important role in increasing the content of carotenoids.

Example 8

Our laboratory transgenes (LmGGPS2) mature embryos of corn, soybean, rice, peanut, sunflower, millet, barley, wheat, cotton and other crops by growing point infection method, and successfully obtained transgenic corn, soybean, rice, peanut, sunflower, millet , barley, wheat, cotton plants, genome PCR electrophoresis, as shown in Figure 6, B: negative control, 1-8 are respectively the genome PCR of corn, soybean, rice, peanut, sunflower, millet, barley, wheat, cotton.

Lycopene and carotenoids were extracted from the transgenic seedlings and wild-type control seedlings grown normally in the greenhouse, and the contents of lycopene and carotenoids in the transgenic seedlings were significantly higher than those in the wild-type group as determined by HPLC. Not only that, but the transgenic group seedlings grew vigorously, while the wild type grew relatively slowly and had relatively less biomass.

Example 9

In our laboratory, we use the Agrobacterium infection method to transgene (LmGGPS1) rose, Eustoma, Anthurium, Phalaenopsis and other flowers, and obtain transgenic rose, Eustoma, Anthurium, Phalaenopsis plants, genome PCR electrophoresis, as shown in Fig. 7, B: Negative control, 1-4 are respectively the genome PCR of Chinese rose, eustoma, anthurium, Phalaenopsis. The transgenic flower seedlings normally grown in the greenhouse grew stronger than the wild type, the flower color was bright, and the content of carotenoids in the petals increased.

Example 10

In our laboratory, we use the Agrobacterium infection method to transgene (LmGGPS2) poplar and other arbor trees to obtain transgenic poplar plants. The genome PCR electrophoresis map is the poplar genome PCR, as shown in Figure 8.

The transgenic saplings and wild-type control seedlings normally grown in the greenhouse, the transgenic plants grew better than the wild-type plants, and the contents of lycopene and carotenoid in the transgenic plants were significantly higher than those of the wild-type plants.

sequence listing

<110> Tianjin University

<120> Lycium barbarum geranyl geranyl pyrophosphate synthase gene and its encoded protein and application

<130>20131025

<160>2

<170>PatentInversion3.3

<210>1

<211>1246

<212>DNA

<213> Artificial series

<220>

<221> gene

<222>(1)..(1246)

<400>1

gtcatgtctatgctaataggtgtaatccacaatcttgcaagaggtactataaataggtcc60

agatctgcaggaacaaagttgcttctttcttcagaggaaacaccagaagctctattgttc120

ctcccaaaagcaagaaccttttgtaatagcaaagatgtttccaagaatgaggctgaagtt180

atcaaacatgaaaacacattcagacaagctggtgtatttgatttcaaaagttacatgctt240

caaaagatcaagtctgtcaatacagccttagatgctgctgtcccaatcagagaaccaatc300

aagttccatgaagcaatgagatattcgctcctttctgaaggtaagcgggtttgtcctgta360

ctctgcatagccacctgtgagcttgttggtggccaagaatcaacagcaatgcctgctgct420

tgcggaatggagatgatacatgctatgtgtatgatgcacgacgaccttccgtgcatggac480

aatgatgatctccgtcgaggaaagttgtcacatcacaaggtttatggcgaaaatgtcact540

gttctagctggttatcccttgttgccttagcatttgagcatatcgcaatagccactaaa600

ggggtccacccgaaaacaatggttcgtgctgttggagaactagcaagattgataggacca660

gagggggcgacagctggccaggtggttgacttgctgtgtgggaggtaaatcgggtaccaga720

ttagaagagctcgagtatattcatcgtcacaagacagcggactttgcagaggcagcgtcc780

attgtaggagcaattcttggtggtgcttccgaggatgagattaatagacttaggaaattc840

tcccagtgtattgggctgctgtttcaggttgtggatgacattcttgatgtgactaaatcc900

tctgagcaattaggaaagacagcagggaaggatttgttggctaacaagttgacgtacccg960

aagatgattggcattgacaagtctaaaaaatatgctcaaaaacttagcaaggaggctaag1020

gagcagcttgtcggttttgatccagaaaaggcagctccactactttctatggcggatttc1080

gttcttcatcggcaaaaatgatctccttaggatgataatacctgtactatttaacattag1140

ttgtgcgcatttatgtaaggacatagggggggagtgaagctattccagttccttgagatat1200

atcaggaaatatggttgctgcttaatttctccatcagaaactgacc1246

<210>2

<211>365

<212>PRT

<213> MUTAGEN

<220>

<221> MUTAGEN

<222>(1)..(365)

<400>2

MetSerMetLeuIleGlyValIleHisAsnLeuAlaArgGlyThrIle

151015

AsnArgSerArgSerAlaGlyThrLysLeuLeuLeuSerSerGluGlu

202530

ThrProGluAlaLeuLeuPheLeuProLysAlaArgThrPheCysAsn

354045

SerLysAspValSerLysAsnGluAlaGluValIleLysHisGluAsn

505560

ThrPheArgGlnAlaGlyValPheAspPheLysSerTyrMetLeuGln

65707580

LysIleLysSerValAsnThrAlaLeuAspAlaAlaValProIleArg

859095

GluProIleLysPheHisGluAlaMetArgTyrSerLeuLeuSerGlu

100105110

GlyLysArgValCysProValLeuCysIleAlaThrCysGluLeuVal

115120125

GlyGlyGlnGluSerThrAlaMetProAlaAlaCysGlyMetGluMet

130135140

IleHisAlaMetCysMetMetHisAspAspLeuProCysMetAspAsn

145150155160

AspAspLeuArgArgGlyLysLeuSerHisHisLysValTyrGlyGlu

165170175

AsnValThrValLeuAlaGlyTyrSerLeuValAlaLeuAlaPheGlu

180185190

HisIleAlaIleAlaThrLysGlyValHisProLysThrMetValArg

195200205

AlaValGlyGluLeuAlaArgLeuIleGlyProGluGlyAlaThrAla

21021520

GlyGlnValValAspLeuLeuCysGlyGlyLysSerGlyThrArgLeu

225230235240

GluGluLeuGluTyrIleHisArgHisLysThrAlaAspPheAlaGlu

245250255

AlaAlaSerIleValGlyAlaIleLeuGlyGlyAlaSerGluAspGlu

260265270

IleAsnArgLeuArgLysPheSerGlnCysIleGlyLeuLeuPheGln

275280285

ValValAspAspIleLeuAspValThrLysSerSerGluGlnLeuGly

290295300

LysThrAlaGlyLysAspLeuLeuAlaAsnLysLeuThrTyrProLys

305310315320

MetIleGlyIleAspLysSerLysLysTyrAlaGlnLysLeuSerLys

325330335

GluAlaLysGluGlnLeuValGlyPheAspProGluLysAlaAlaPro

340345350

LeuLeuSerMetAlaAspPheValLeuHisArgGlnLys

355360365

Claims (4)

1. A wolfberry geranylgeranylpyrophosphate synthase gene, characterized in that the gene is the nucleotide sequence shown in SEQ ID NO.1. 2. The protein encoded by Lycium barbarum geranylgeranylpyrophosphate synthase gene according to claim 1, characterized in that said protein is the amino acid sequence shown in SEQ ID NO.2. 3. A recombinant vector, characterized in that it contains the complete sequence of the Lycium barbarum geranylgeranyl pyrophosphate synthase gene according to claim 1. 4. The wolfberry geranylgeranylpyrophosphate synthase gene according to claim 1 is applied to the preparation of transgenic corn, soybean, rice, peanut, sunflower, cotton, millet, barley, flowers and vegetable plants.