CN115820684A - Method for increasing caffeic acid, milk tree alkali content and/or bioluminescence intensity in plant - Google Patents

Abstract

The invention discloses a method for increasing the content of caffeic acid and milkyline and/or the intensity of bioluminescence in plants, and belongs to the field of biotechnology. The present invention analyzes the synthesis pathway of caffeic acid in plants, and enhances the synthesis pathway of endogenous caffeic acid and milkyline in plants through genetic manipulation. Specifically, the CM1 gene, PAT gene, ADT2 gene, and PAL1 gene are integrated in plants by means of genetic engineering. , C4H gene, 4CL1 gene, multi-gene co-expression can significantly improve the synthesis of caffeic acid and milky acid in plants, and coupling the caffeic acid synthesis pathway with the plant autoluminescence pathway can create plants with stronger bioluminescence brightness. The invention provides a feasible technical scheme for the breeding and production of plants with high caffeic acid and milky tree content and sustainable luminescent plants.

Description

A method for increasing caffeic acid, milkyline content and/or bioluminescent intensity in plants method

technical field

The present invention relates to the field of biotechnology, in particular to a method for increasing the content of caffeic acid, hispidin and/or bioluminescence in plants by co-expression of CM1, PAT, ADT2, PAL1, C4H and 4CL1 genes method of strength.

Background technique

Bioluminescence is a common method used in biological research to indicate gene expression and protein localization. Kotlobay et al. reported a fungal bioluminescence pathway (FBP) in 2018, which can convert caffeic acid into luciferin molecules, and luciferase Luz can directly oxidize luciferin to generate bioluminescence signals. And identified the key enzymes involved in the FBP pathway, fungal luciferase (Luz), hispidin synthase (HispS), hispidin3-hydroxylase (H3H) and caffeoylpyruvate hydrolase (CPH), CPH for the generation of autoluminescence Not required, but significantly prolongs the luminescent signal, thereby elucidating the biosynthetic cycle of fungal luciferin and the mechanism of luminescence (Kotlobay et al., 2018).

Khakhar A et al. combined the NPGA (4'-phosphopantetheinyl transferase) gene in the Aspergillus nidulans genome, the H3H (hispidin-3-hydroxylase) gene and the Hisps (Hispidin synthase) gene in the Neonothopanus nambi genome , and the fungal luciferase (Luz) gene was integrated into the Moclo plasmid, and then the plasmid was introduced into Agrobacterium tumefaciens (A. tumefaciens), and mixed with the pretreated stem explants to integrate the bioluminescence element into the plant genome. The self-luminescence mechanism of the plant is as follows: caffeic acid from the phenylalanine biosynthesis pathway in the plant is activated by Hisps protease and converted into milky acid, and milky acid is catalyzed by milky acid 3-hydroxylase (H3H) to produce 3- 3-hydroxymilanine (3-hydroxymyspidin), luciferase (Luz) will oxidize 3-hydroxymilanine to fluorescein 3-hydroxymilanine, and fluorescein 3-hydroxymyspidin releases energy into caffeoyl Visible light with a wavelength of 520nm is released during the process of pyruvic acid (caffeylpyruvic acid). Subsequently, the protease encoded by CPH (Caffey pyruvate hydrolase) converts caffeoylpyruvate into caffeic acid, thereby realizing the plant self-luminescence cycle and allowing the plant to continuously produce light (Mitiouchkina et al., 2020).

The above-mentioned system can be used to develop tools for the detection of biological macromolecules, but at present, the light-emitting system of this light-emitting system is weak in plants, which seriously affects the wide application of this light-emitting system. The study found that the most critical substances that affect the luminous intensity are caffeic acid and milkyline. Therefore, how to increase the content of caffeic acid and milkyline in plants is an important solution to improve the luminous intensity of plants.

In addition, caffeic acid widely exists in plants. As a polyphenolic substance, it has very high biological activity, can effectively remove free radicals, has good antioxidant activity, and prevents cardiovascular and cerebrovascular diseases. It can be used as a beverage additive and also Can be used as raw material medicine. Milk tree has strong anti-oxidation, anti-cancer, anti-diabetes and anti-dementia effects. Enhance the synthesis pathway of endogenous caffeic acid and milkyline in plants through genetic manipulation, increase the content of caffeic acid and milkyline in plants, and use it to extract caffeic acid and milkyline, which is important in chemical industry, medicine and food effect.

Contents of the invention

The purpose of the present invention is to provide a method that can increase the content of endogenous caffeic acid and milkyline in plants, or enhance the intensity of plant bioluminescence, and apply it to plant cultivation or enhance biological Self-illuminating plants are growing.

To achieve the above object, the present invention adopts the following technical solutions:

The invention provides the application of multi-gene co-expression in increasing the content of caffeic acid and milkythine in plants and/or increasing the intensity of plant bioluminescence, the genes comprising: CM1 gene, PAT gene, ADT2 gene, PAL1 gene and C4H gene , also includes the 4CL1 gene, the coding sequence of the 4CL1 gene is shown in SEQ ID NO.1 or has at least 70% homology with the sequence shown in SEQ ID NO.1 and the encoded protein is functionally equivalent.

The CM1 gene encodes chorismate mutase; the PAT gene encodes prephenate aminotransferase; the ADT2 gene encodes arogenate dehydratase 2; the PAL1 gene encodes benzene Alanine ammonia-lyase (phenylalanine ammonia-lyase); the C4H gene encodes cinnamate 4-hydroxylase (Cinnamate 4-hydroxylase); the 4CL1 gene encodes coenzyme A ligase (4-coumarate: coenzyme A ligase) , the amino acid sequence of the protease is shown in SEQ ID NO.2.

The integration of the CM1 gene, the PAT gene, the ADT2 gene, the PAL1 gene and the C4H gene through genetic engineering means to co-express them in plants can significantly increase the caffeic acid content in plants. On this basis, the present invention further integrates the 4CL1 gene, and the protease encoded by it participates in the synthesis pathway of caffeic acid and milky acid, which can further increase the content of caffeic acid and milky acid in plants.

Caffeic acid and milkythine are precursors in the bioluminescent metabolic pathway, and the increase in their content helps to enhance the bioluminescent intensity of self-luminous plants. Studies have shown that compared with those produced by Hisps gene, CPH gene, H3H gene, The self-luminous plants obtained by integrating the NPGA gene and the Luz gene into the plant genome can significantly increase the self-luminous intensity of the plant by further integrating the CM1, PAT, ADT2, PAL1, C4H, and 4CL1 genes after the second round of transgenics.

Further, the coding sequence of the CM1 gene is shown in SEQ ID NO.3, the coding sequence of the PAT gene is shown in SEQ ID NO.4, and the coding sequence of the ADT2 gene is shown in SEQ ID NO.5 , the coding sequence of the PAL1 gene is shown in SEQ ID NO.6, and the coding sequence of the C4H gene is shown in SEQ ID NO.7.

Further, the application includes: using biological technology means to overexpress the gene in the plant to obtain a transgenic plant that promotes the synthesis of caffeic acid and milkythine;

Alternatively, biological techniques are used to overexpress the gene in the bioluminescence recipient plant to obtain a transgenic plant with enhanced bioluminescence intensity.

Further, the plant is tobacco, rapeseed, rice, Phalaenopsis or chrysanthemum.

The present invention also provides a method for increasing the content of caffeic acid and milkythin in plants and/or increasing the intensity of plant bioluminescence, comprising the following steps:

(1) Using multi-gene assembly technology to integrate the CM1 gene, PAT gene, ADT2 gene, PAL1 gene, C4H gene and 4CL1 gene into the acceptor carrier to construct and obtain a multi-gene carrier;

(2) Using transgenic technology to introduce the target gene fragment in the multi-gene carrier into the recipient plant or bioluminescent recipient plant, and cultivate it to obtain transgenic plants that promote the synthesis of caffeic acid and milkythin or enhance the intensity of bioluminescence.

In step (1), the CM1, PAT, ADT2, PAL1, C4H and 4CL1 genes are integrated into the acceptor vector by means of genetic engineering to construct a multigene vector.

Preferably, the upstream of each target gene in the multigene vector contains a 35S promoter sequence. Specifically, the 35S promoter is a CaMV 35S promoter.

Preferably, the TransGene Stacking II system is used for multi-gene assembly. The TransGeneStacking II system is a multi-gene assembly vector system, see Chinese patent application number 2017103841977.

Preferably, pYL322d1 is used as the donor vector I, pYL322d2 is used as the donor vector II, and pYLTAC380GW is used as the acceptor vector.

Specifically, the construction method of the multigene carrier comprises the following steps:

1) Insert the CM1 gene fragment, the ADT2 gene fragment and the C4H gene fragment into the multiple cloning site of the donor vector pYL322d1 to obtain the donor vectors pYL322d1-CM1, pYL322d1-ADT2, and pYL322d1-C4H;

Insert the PAT gene fragment, the PAL1 gene fragment and the 4CL1 gene fragment into the multiple cloning site of the donor vector pYL322d2 to obtain the donor vectors pYL322d2-PAT, pYL322d2-PAL1, pYL322d2-4CL1;

2) The donor vector pYL322d1-CM1 and the acceptor vector pYLTAC380GW were mixed at a ratio of 1:1 to 2:1, co-transformed into Escherichia coli NS3529 competent cells, and coated with kanamycin and chloramphenicol (chloramphenicol) double antibody culture medium, take the positive strain to extract the plasmid;

3) Use homing enzyme I-Sce I to digest the plasmid extracted in step 2), then transform Escherichia coli strain XL10 or NEB10-β, culture, screen, and extract the plasmid to obtain a positive clone pYLTAC380GW-CM1 containing the target gene CM1 ;

4) Mix the donor vector pYL322d2-PAT and the acceptor vector pYLTAC380GW-CM1 prepared in step 3) at a ratio of 1:1 to 2:1, and transfer them into Escherichia coli NS3529 competent cells. (kanamycin) and ampicillin (ampicillin) double-antibody culture medium, take the positive strain to extract the plasmid;

5) Use the homing enzyme PI-Sce I to digest the plasmid extracted in step 4), then transform Escherichia coli strain XL10 or NEB10-β, culture, screen, and extract the plasmid to obtain a positive clone pYLTAC380GW containing the target gene CM1 and PAT -CM1-PAT;

6) Repeat steps 2)-5), the new plasmid containing the target gene obtained in the previous step is used as the acceptor vector, and the d1 and d2 donor vectors containing different genes are used for recombination until all the target genes are assembled into the acceptor vector In the last step, the BP recombination reaction was connected to remove the selection marker gene expression cassette element, and the multigene vector pYLTAC380GW-CM1-PAT-ADT2-PAL1-C4H-4CL1 was constructed.

In step (2), the multi-gene fragment constructed is introduced into the recipient plant to express it in the plant, and the expressed proteases participate in the synthesis of caffeic acid and milky acid, increasing the endogenous caffeic acid cycle of the plant and making the plant sustainably Produce caffeic acid and milky tree, which provide precursor substances for bioluminescent metabolic pathways.

The recipient plant may be, but not limited to: tobacco, rapeseed, rice, Phalaenopsis or chrysanthemum.

Preferably, Agrobacterium-mediated technology is used to introduce the multi-gene fragment into recipient plants. Specifically, the Agrobacterium uses EHA105.

The bio-self-luminescence recipient plant can be a natural self-luminescence plant, or a bio-self-luminescence transgenic plant obtained by integrating bio-self-luminescence gene elements.

Further, the bioluminescence gene elements include Hisps gene, CPH gene, H3H gene, NPGA gene and Luz gene.

Specifically, the coding sequence of the Hisps gene is shown in SEQ ID NO.8, the coding sequence of the CPH gene is shown in SEQ ID NO.9, the coding sequence of the H3H gene is shown in SEQ ID NO.10, and the coding sequence of the NPGA gene is shown in SEQ ID NO. As shown in 11, the coding sequence of Luz gene is shown in SEQ ID NO.12.

The beneficial effect that the present invention possesses:

The present invention analyzes the synthesis pathway of caffeic acid in plants, and enhances the synthesis pathway of endogenous caffeic acid and milkyline in plants through genetic manipulation. Specifically, the CM1 gene, PAT gene, ADT2 gene, and PAL1 gene are integrated in plants by means of genetic engineering. , C4H gene, 4CL1 gene, multi-gene co-expression can significantly improve the synthesis of plant caffeic acid and milky acid. And coupling the caffeic acid synthesis pathway with the plant autoluminescence pathway can create plants with stronger bioluminescence brightness. The invention provides a feasible technical scheme for the breeding and production of plants with high caffeic acid and milky tree content and sustainable luminescent plants.

Description of drawings

Figure 1 is a detection map of intermediate carrier digestion during the construction of caffeic acid and milky acid and hispidin synthesis enhanced DNA module (eCHM, enhanced caffeic acid and hispidin synthesis module) pYLTAC380MF-8G vector.

Figure 2 is the detection of target gene expression in transiently transformed tobacco leaves with the eCHM module, where EV-1, EV-2, and EV-2 represent empty vectors (empty vector) as negative controls, and eCHM-1, eCHM-2, and eCHM-3 represent Transient transformation of eCHM vector module transformants.

Figure 3 is the detection of target gene expression in leaves of tobacco transgenic positive plants mediated by Agrobacterium, in which eCAM-1, eCAM-2, eCAM-2 represent eCAM vector transgene control, eCHM-3, eCHM-17, eCHM-19 represent eCHM Vector transgenic tobacco overexpression plants, the same below; the relative expression of the target gene in the eCHM group was defined as 1 for the expression of 5 genes in the eCAM group for comparison.

Fig. 4 is the detection of caffeic acid content in leaves of tobacco transgenic positive plants mediated by Agrobacterium.

Fig. 5 is the detection of milky acid content in the leaves of tobacco transgenic positive plants mediated by Agrobacterium.

Figure 6 is a comparison of the luminous intensity of tobacco transgenic tobacco at the bolting stage.

Fig. 7 is a quantitative comparison chart of the fluorescence intensity of leaves of transgenic plants.

Detailed ways

The present invention will be further described below in conjunction with specific embodiments. The following examples are only used to illustrate the present invention, and are not intended to limit the scope of application of the present invention. Without departing from the spirit and essence of the present invention, any modifications or substitutions made to the methods, steps or conditions of the present invention belong to the scope of the present invention.

The test methods used in the following examples are conventional methods unless otherwise specified; the materials and reagents used are commercially available reagents and materials unless otherwise specified.

pYL322d1, pYL322d2, PYLMFH and pYLTAC380GW were gifts from the laboratory of Professor Liu Yaoguang of South China Agricultural University. For the construction method, please refer to the Chinese patent application number 2017103841977.

Bioluminescent transgenic tobacco (with the luminescence-related gene Hisps-CPH-H3H-NPGA-Luz integrated in the genome) seeds were constructed and cultivated by our laboratory in the early stage, and the construction method refers to Mitiouchkina et al., 2020. Use the TransGene Stacking II system for multi-gene assembly, use pYL322d1 and pYL322d2 as donor vectors, put the coding sequence as the Hisps gene shown in SEQ ID NO.8, the coding sequence as the CPH gene shown in SEQ ID NO.9, the coding sequence The H3H gene shown in SEQ ID NO.10, the NPGA gene whose coding sequence is shown in SEQ ID NO.11 and the Luz gene whose coding sequence is shown in SEQ ID NO.12 were integrated into the pYLTAC380GW plasmid, and finally passed a one-step BP recombination reaction The vector pYLTAC380GW-Hisps-CPH-H3H-NPGA-Luz was constructed by connecting and removing the selection marker gene expression cassette element, and then the plasmid was introduced into Agrobacterium, and mixed with the pretreated tobacco leaves, and the bioluminescent element integrated into the plant genome.

Example 1 Construction process of caffeic acid and milkyline synthesis enhancement (enhanced caffeic acid and hispidinsynthsis module, eCHM) module vector pYLTAC380MF-8G

The corresponding CDS of NtCM1, NtPAT, NtADT2, NtPAL1, NtC4H and Nt4CL1 genes in the tobacco (Nicotiana tabacum) genome were amplified.

In this study, the TransGene Stacking II system was used for multigene assembly. The following tobacco genes NtCM1, NtPAT, NtADT2, NtPAL1, and NtC4H driven by the 35S promoter of tobacco mosaic virus were integrated into the pYLTAC380GW plasmid, and finally the selection marker gene expression box element was removed by one-step BP recombination reaction to form an enhanced Caffeic acid synthesis pathway vector pYLTAC380GW-NtCM1-NtPAT-NtADT2-NtPAL1-NtC4H (pYLTAC380MF-7G, eCAM).

The following tobacco genes NtCM1, NtPAT, NtADT2, NtPAL1, NtC4H, and Nt4CL1 driven by the 35S promoter of tobacco mosaic virus were used to integrate into the pYLTAC380GW plasmid, and finally the one-step BP recombination reaction was used to remove the selection marker gene expression box element, thereby Constitute the enhanced caffeic acid synthesis pathway vector pYLTAC380GW-NtCM1-NtPAT-NtADT2-NtPAL1-NtC4H-Nt4CL1 (pYLTAC380MF-8G, eCHM).

For the sequence information of NtCM1, please refer to the gene accession number: XP_009768497; for the sequence information of NtPAT, please refer to the gene accession number: XP_016480150; for the sequence information of NtADT2, please refer to the gene accession number: XP_009617905; For the information, see the gene accession number: NP_001312445; for the sequence information of Nt4CL1, see the gene accession number: NM_001325738.1.

The coding sequences of the above genes were amplified by PCR to obtain the corresponding gene fragments. Specifically, the CDS sequence of the NtCM1 gene is shown in SEQ ID NO.3; the CDS sequence of the NtPAT gene is shown in SEQ ID NO.4; the CDS sequence of the NtADT2 gene is shown in SEQ ID NO.5; the CDS sequence of the NtPAL1 gene It is shown in SEQ ID NO.6; the CDS sequence of NtC4H gene is shown in SEQ ID NO.7; the CDS sequence of Nt4CL1 gene is shown in SEQ ID NO.1, and its encoded amino acid sequence is shown in SEQ ID NO.2.

The specific construction process of caffeic acid and milkythine synthesis synthesis enhancement vector pYLTAC380MF-8G is as follows:

(1) Construct donor vectors, pYL322d1-NtCM1, pYL322d2-NtPAT, pYL322d1-NtADT2, pYL322d2-NtPAL1, pYL322d1-NtC4H, pYL322d2-Nt4CL1;

(2) Mix the donor vector pYL322d1-NtCM1 and the acceptor vector pYLTAC380GW (1:1 to 2:1) in NS3529 competent medium for co-transformation, heat shock method, ice bath for 30min, heat shock for 90s, ice bath 2-3min, in LB without antibiotics, 37°C, 200rpm, revived for 2h, spread on the LA plate containing kanamycin (Km, 25mg/L) and chloramphenicol (Chl, 15mg/L), grow out after about 18h For single clones, wash all single clones into tubes with ddH ₂ O and extract the mixed plasmids.

(3) Take about 50-100ng of the mixed plasmid and digest it with 0.5uL I-Sce I (NEB) in a 10uL system for 4-5h, and transform Escherichia coli strain XL10 (Vazyme) or NEB10-β (Bomad Biotechnology Co., Ltd. ), coated on LA plates containing kanamycin (Km, 25mg/L), picked a single clone after 15 hours at 37°C, cultured in LB (containing 25mg/L Km and 0.5mM IPTG), and carried out bacterial liquid PCR identification, Use Green Taq Mix to further extract plasmids that can amplify bright bands, take 200ng of each and use 0.2uL Not l to digest and verify in a 20uL reaction system, four bands appear, and the target gene containing 0.95k bp is the desired positive Cloning pYLTAC380GW-NtCM1 (Figure 1, 380GW-1G).

(4) Mix the donor vector pYL322d2-NtPAT and the acceptor vector pYLTAC380GW-NtCM1 (1:1 to 2:1) in (3) in the NS3529 competent medium for co-transformation, transform according to the method (2), and apply on On the LA plate containing kanamycin (Km, 25mg/L) and ampicillin (Amp, 70mg/L), single clones grew after about 18 hours, and all the single clones were washed into tubes with ddH ₂ O, and the mixed plasmids were extracted.

(5) Take about 40-90ng of the mixed plasmid and digest it with 0.5uL PI-Sce I (NEB), add 0.5uL BSA in a 10uL system for 4-5h, then transform and verify according to the method in (3), and five bands appear , containing the target gene 1.4k bp NtPAT and 0.95k bp NtCM1 is the positive clone pYLTAC380GW-NtCM1-NtPAT (Figure 1, 380GW-2G).

(6) More rounds of recombination, using d1 and d2 donor vectors containing different genes to co-transform with the acceptor vector constructed in the previous round, and pYLTAC380GW-5G (Figure 1, 380GW-5G) and pYLTAC380GW were constructed -6G (Figure 1, 380GW-6G);

(7) Finally, by BP reaction, at 25°C, pYLTAC380GW-5G and pYLTAC380GW-6G (200ng) were mixed with PYLMFH-Bnmlpro (100ng) in 5μl reaction with 1μL of 5×BP enzyme mixture for 5 hours. Then 1 μL of proteinase K solution was added to terminate the reaction at 37 °C for 10 min. Transfer to NEB10-β (Bomad Biotechnology Co., Ltd.) competent state, and select single clones for identification. Use Not1 enzyme digestion test to identify the correct positive final vectors pYLTAC380MF-7G and pYLTAC380MF-8G (Figure 1, 380MF-8G), pick positive clones for full plasmid sequencing analysis, and select the correct pYLTAC380MF-7G and pYLTAC380MF-8G vectors for use in follow-up experiment.

Example 2 Analysis of Tobacco Leaf Transiently Transformed with Caffeic Acid and Milkyline Synthesis (enhanced caffeic acid and hispidinsynthsis module, eCHM)

1. Streak and activate the EHA105 bacterial solution containing the verified correct carrier plasmid pYLTAC380MF-8G on the LA+Kana+Rif plate, 28°C, 36h, pick colonies from the plate, and transfer to LB+Kana+Rif+15μM In As medium, 28°C, 200rpm culture to OD ₆₀₀ =0.8-1.0, 4000rpm, 10min to collect the cells, suspend the Agrobacterium cells with the infection solution (containing 10mM MgCl, 10mMMES, 150μM As), and let stand at room temperature for 2~ 3h.

2. For the verification of transient expression in tobacco leaves, use a 1mL needle to gently open a small opening on the surface of the tobacco leaves, then use the needle tube with the needle removed to absorb the bacterial liquid, and inject it into the leaves from the wound of the tobacco leaves. Normal cultivation was carried out indoors for 48 hours, and then some leaves were sampled for relative expression detection by qPCR to determine the overexpression of the target gene. As shown in Figure 2, the target gene was overexpressed in 3 independent transgenic families.

Example 3 Analysis of caffeic acid and milkyline content and luminescence intensity of eCHM module transgenic plants mediated by Agrobacterium

1. Streak the EHA105 bacterial solution containing the verified correct vector plasmids pYLTAC380MF-8G (eCHM) and pYLTAC380MF-7G (eCAM) on the LA+Rif+Kana plate respectively, at 28°C for 36 hours, pick a single clone into 3- In 5ml LB medium at 200rpm, 28°C, 36h, according to the ratio of 1:100-1:50, expand the culture of 50ml for 3-5h to OD=0.6, then centrifuge the bacterial liquid, and use MS0 liquid medium (MS+3%Sucrose +PH5.8, 50ml) suspended bacteria to OD=0.6 for infection;

2. Select the seeds of the successfully obtained bioluminescent transgenic tobacco FBP (there is already a luminescence-related gene Hisps-CPH-H3H-NPGA-Luz in the genome), and plant the tobacco on the sterile MS medium for 4-5 weeks until fully expanded Healthy leaves, cut into 0.5cm square size with a scalpel (cut off the leaf edge to avoid the main vein), the upper surface of the leaf is facing down in MS1 solid medium (MS+0.5mg/L IAA+2.0mg/L BA+3% sucrose+0.6-0.8% Phytagel, PH=5.8), cultured in dark at 25°C for 2-3 days;

3. Add the pre-cultured tobacco leaves to the bacterial solution, vortex to ensure that the incision of the leaf is submerged in the bacterial solution, stand still for 5-30 minutes, and use sterile filter paper to absorb the attached bacterial solution; the upper surface of the infected leaf Put it face down on MS1 solid medium at 28°C, and culture in the dark for 2 days; put the leaves with the upper surface facing up on MS1 screening medium containing Timentin and glyphosate, and culture at 25°C in light; when the leaf edge grows buds and can be separated (more than 1 cm), the buds were excised and transferred to MS2 (MS+0.5 mg/L IAA+3% sucrose+0.6-0.8% Phytagel, PH=5.8) solid medium containing antibiotics (TM+basta). Roots grow out after one week, and the lid of the seedling box is opened to train the seedlings. After one week, they are transferred to the planting soil for cultivation, and eCAM and eCHM transgene positive plants are obtained.

The leaves of three transgenic positive families of eCAM and eCHM were taken for qPCR analysis and caffeic acid content detection, and the results are shown in Figure 3. Five target genes in eCAM module and six target genes in eCHM module were overexpressed in transgenic tobacco.

The contents of caffeic acid and milkyline in the leaves of three transgenic positive families in FBP, eCAM and eCHM plants were detected by high performance liquid chromatography-mass spectrometry. The results are shown in Figure 4 and Figure 5. The caffeic acid content and the milky acid content in the leaves of the eCHM transgenic positive families were significantly higher than those in the FBP transgenic families, and the milky acid content in the leaves of the eCHM transgenic positive families was significantly higher than that in the eCAM transgenic positive families. .

4. Move the above-mentioned positive transgenic tobacco at the bolting stage to a dark room and take pictures with a Nikon D750 camera, using the lens AF-S17-35mm F2.8D ED-IF, ISO 2000, and the exposure time is 60 seconds. The results are shown in FIG. 6 , the luminescence intensity of the eCHM transgene-positive plants was higher than that of the eCAM transgene-positive plants.

Quantitative analysis of light quanta, taking photos of the above-mentioned transgenic tobacco leaves at the bolting stage fully unfolded in the in vivo imaging system NightSHADE LB985 instrument, and recording the light quanta. As shown in Fig. 7, statistical analysis found that the luminescence intensity of eCHM transgenic positive plants was higher than that of eCAM transgenic positive plants and FBP transgenic positive plants.

Claims (10)

1. Use of co-expression of multiple genes for increasing caffeic acid, milk tree alkali content and/or increasing bioluminescence intensity in a plant, said genes comprising: CM1 gene, PAT gene, ADT2 gene, PAL1 gene and C4H gene, characterized in that, also includes 4CL1 gene, the coding sequence of the 4CL1 gene is shown in SEQ ID NO.1 or has at least 70% homology with the sequence shown in SEQ ID NO.1 and the coded protein is equivalent in function.

2. The use according to claim 1, wherein the coding sequence of the CM1 gene is shown as SEQ ID No.3, the coding sequence of the PAT gene is shown as SEQ ID No.4, the coding sequence of the ADT2 gene is shown as SEQ ID No.5, the coding sequence of the PAL1 gene is shown as SEQ ID No.6, and the coding sequence of the C4H gene is shown as SEQ ID No. 7.

3. The application of claim 1, wherein the application comprises: the gene is over-expressed in a plant body by utilizing a biological technical means to obtain a transgenic plant for promoting the synthesis of caffeic acid and the milk tree alkaloid;

or the gene is over-expressed in a biological self-luminous receptor plant by utilizing a biological technical means to obtain a transgenic plant with enhanced bioluminescence intensity.

4. The use as claimed in any one of claims 1 to 3, wherein the plant is tobacco, oilseed rape, rice, moth orchid or chrysanthemum.

5. A method for increasing caffeic acid and milk tree alkali content in plants and/or increasing plant bioluminescence intensity, which comprises the following steps:

(1) Integrating the CM1 gene, PAT gene, ADT2 gene, PAL1 gene, C4H gene and 4CL1 gene described in claim 1 or 2 into a receptor vector by using a multigene assembly technology to construct a multigene vector;

(2) A transgenic technology is utilized to introduce target gene segments in a polygene vector into a receptor plant or a biological self-luminous receptor plant for cultivation, so as to obtain a transgenic plant which promotes the synthesis of caffeic acid and milk tree alkali or enhances the biological self-luminous intensity.

6. The method of claim 5, wherein in step (1), the upstream of each gene of interest in the multi-gene vector comprises a 35S promoter sequence.

7. The method of increasing caffeic acid, daidzein content in a plant and/or increasing plant bioluminescence intensity as claimed in claim 5, wherein in step (1) multigene assembly is performed using the TransGene Stacking II system.

8. The method of claim 5, wherein in step (2) the multiple gene fragments are introduced into the recipient plant using Agrobacterium mediated techniques.

9. The method according to claim 5 or 8, wherein the genome of the bioluminescent recipient plant has integrated into it a bioluminescent genetic element.

10. The method of claim 9, wherein the bioluminescent genetic element comprises: hisps gene with a coding sequence shown in SEQ ID No.8, CPH gene with a coding sequence shown in SEQ ID No.9, H3H gene with a coding sequence shown in SEQ ID No.10, NPGA gene with a coding sequence shown in SEQ ID No.11 and Luz gene with a coding sequence shown in SEQ ID No. 12.

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