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 improving caffeic acid and milk tree alkali content and/or bioluminescence intensity in plants, belonging to the technical field of biology. The synthesis path of caffeic acid in the plant is analyzed, the synthesis path of the caffeic acid and the milk tree alkaloid endogenous to the plant is enhanced through genetic manipulation, specifically, a CM1 gene, a PAT gene, an ADT2 gene, a PAL1 gene, a C4H gene and a 4CL1 gene are integrated in the plant body through a genetic engineering means, the synthesis of the caffeic acid and the milk tree alkaloid in the plant is obviously promoted through multi-gene co-expression, and the synthesis path of the caffeic acid and the self-luminous path of the plant are coupled, so that the plant with stronger biological self-luminous brightness can be created. The invention provides a feasible technical scheme for breeding and producing plants with high caffeic acid and milk tree alkali contents and sustainable luminous plants.

Description

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

Technical Field

The invention relates to the technical field of biology, in particular to a method for improving the content of plant caffeic acid (caffeic acid), milk tree alkaloid (hispidin) and/or biological spontaneous light intensity by co-expression of CM1, PAT, ADT2, PAL1, C4H and 4CL1 genes.

Background

Bioluminescence (Bioluminescence) is a common method used in biological research to indicate gene expression and protein localization. Kotlobay et al 2018 reported a fungal bioluminescence metabolic pathway (FBP) which can convert caffeic acid into luciferin molecules, luciferase Luz can directly oxidize luciferin to generate a bioluminescent signal, and identified key enzymes involved in the FBP pathway, fungal luciferase (Luz), hispidin synthase (HispS), hispidin 3-hydroxylase (H3H) and caffeoylacetone acid hydrolase (CPH), which is not necessary for the generation of spontaneous luminescence but significantly prolongs the luminescent signal, thereby elucidating the biosynthetic cycle of fungal luciferin and the luminescent mechanism (Kotlobay et al, 2018).

Khakhar A et al integrated NPGA (4' -phosphopanthenyl transferase) gene in the genome of Aspergillus nidulans (Aspergillus nidulans), H3H (hispin-3-hydroxylase) gene and Hisps (hispin synthase) gene in the genome of Neotophus (Neonophenus nambi), and fungal luciferase (Luz) gene into Moclo plasmid, introduced the plasmid into Agrobacterium tumefaciens (A. Tumefaciens), and cultured in mixture with pretreated stem explants, and integrated the bioluminescent element into the plant genome. The self-luminous mechanism of the plant is as follows: caffeic acid from a phenylalanine biosynthesis pathway in a plant body is activated by Hisps protease and then converted into milk tree alkali, the milk tree alkali is catalyzed by milk tree alkali 3-hydroxylase (H3H) to generate 3-hydroxycarnitine (3-hydroxyhispidin), luciferase (Luz) oxidizes the 3-hydroxycarnitine into luciferin 3-hydroxymilk tree alkali, and the luciferin 3-hydroxymilk tree alkali releases visible light with the wavelength of 520nm in the process of releasing energy to become caffeoylpyruvic acid. Subsequently, the protease encoded by CPH (Caffey pyruvate) converts caffeoyl pyruvate into caffeic acid, thereby achieving a self-luminous cycle of the plant, allowing the plant to continuously produce light (mitouchkina et al, 2020).

The system can be used for developing a tool for detecting biological macromolecules, but the current light-emitting system emits light weakly in plants, so that the wide application of the light-emitting system is seriously influenced. The research finds that the most key substances influencing the luminous intensity are caffeic acid and daidzein, so how to increase the content of the caffeic acid and the daidzein in plants is an important solution for increasing the luminous intensity of the plants.

In addition, caffeic acid is widely existed in plants, has very high biological activity as a polyphenol substance, can effectively remove free radicals, has good antioxidant activity, and can prevent cardiovascular and cerebrovascular diseases, and can be used as beverage additive or raw material medicine. The obtained milk tree alkali has strong effects in resisting oxidation, cancer, diabetes and dementia. The synthesis way of the caffeic acid and the milk tree alkaloid endogenous to the plants is enhanced through genetic manipulation, the contents of the caffeic acid and the milk tree alkaloid in the plants are improved, and the caffeic acid and the milk tree alkaloid are used for extracting the caffeic acid and the milk tree alkaloid and have important functions in chemical industry, medicines and foods.

Disclosure of Invention

The invention aims to provide a method capable of increasing the content of caffeic acid and the content of the dairy creamer or enhancing the biological self-luminous intensity of plants, which is applied to plant cultivation for increasing the content of the caffeic acid and the dairy creamer or plant cultivation for enhancing the biological self-luminous intensity.

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

the invention provides application of polygene co-expression in improving the content of caffeic acid and milk tree alkali in plants and/or improving the bioluminescence intensity of plants, wherein the gene comprises the following components: CM1 gene, PAT gene, ADT2 gene, PAL1 gene and C4H gene, also include 4CL1 gene, the coding sequence of the 4CL1 gene is shown as SEQ ID NO.1 or has at least 70% homology with the sequence shown as SEQ ID NO.1 and the coded protein is equivalent in function.

The CM1 gene encodes chorismate mutase (chorismate mutase); the PAT gene encodes prephenate transaminase (prephenate aminotransferase); the ADT2 gene encodes an aronate dehydratase (Arogenate dehydratase 2); the PAL1 gene encodes phenylalanine ammonia lyase (phenylalanine ammonia-lyase); the C4H gene encodes Cinnamate 4-hydroxylase (Cinnamate 4-hydroxylase); the 4CL1 gene codes coenzyme A ligase (4-coumarate: coenzyme A ligase), and the amino acid sequence of the protease is shown as SEQ ID NO. 2.

The CM1 gene, PAT gene, ADT2 gene, PAL1 gene and C4H gene are integrated by gene engineering means to make them co-express in plant body, and can obviously raise caffeic acid content in plant. The invention further integrates the 4CL1 gene on the basis, and the coded protease participates in the synthesis route of caffeic acid and milk tree alkali, so that the content of caffeic acid and milk tree alkali in the plant can be further improved.

The research shows that compared with self-luminous plants obtained by integrating Hisps genes, CPH genes, H3H genes, NPGA genes and Luz genes into plant genomes, the self-luminous plant intensity can be obviously improved by further integrating CM1, PAT, ADT2, PAL1, C4H and 4CL1 genes through a second round of genes.

Furthermore, 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.

Further, the application includes: the gene is over-expressed in the 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.

Further, the plant is tobacco, rape, rice, phalaenopsis or chrysanthemum.

The invention also provides a method for improving the content of caffeic acid and the content of the daidzein in the plant and/or improving the bioluminescence intensity of the plant, which comprises the following steps:

(1) Integrating the CM1 gene, the PAT gene, the ADT2 gene, the PAL1 gene, the C4H gene and the 4CL1 gene into a receptor vector by utilizing a polygene assembly technology to construct a polygene 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.

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

Preferably, the multi-gene vector contains a 35S promoter sequence upstream of each gene of interest. Specifically, the 35S promoter is a CaMV 35S promoter.

Preferably, multigene assembly is performed using the TransGene Stacking II system. The TransGene Stacking II system is a multigene assembly vector system, which is referred to in Chinese patent application No. 2017103841977.

Preferably, pYL d1 is used as donor vector I, pYL d2 is used as donor vector II, and pYLTAC380GW is used as acceptor vector.

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

1) Respectively inserting the CM1 gene fragment, the ADT2 gene fragment and the C4H gene fragment into the multiple cloning sites of a donor vector pYL d1 to obtain donor vectors pYL d1-CM1, pYL d1-ADT2, pYL d1-C4H;

inserting the PAT gene fragment, the PAL1 gene fragment and the 4CL1 gene fragment into the multiple cloning site of a donor vector pYL d2 to obtain donor vectors pYL d 322d2-PAT, pYL322d2-PAL1 and pYL d2-4CL1;

2) Mixing a donor vector pYL d 322d1-CM1 and a receptor vector pYLTAC380GW according to 1:1-2:1, co-transferring into Escherichia coli NS3529 competent cells, coating the competent cells in a double-antibody culture medium containing kanamycin (kanamycin) and chloramphenicol (chloramphenicol), culturing, and extracting plasmids from positive strains;

3) Carrying out enzyme digestion on the plasmid extracted in the step 2) by using a homing enzyme I-Sce I, then transforming an escherichia coli strain XL10 or NEB 10-beta, culturing, screening and extracting the plasmid to obtain a positive clone pYLTAC380GW-CM1 containing a target gene CM1;

4) Mixing a donor vector pYL d 322-PAT and a receptor vector pYLTAC380GW-CM1 prepared in step 3) according to 1:1-2:1, co-transferring into an Escherichia coli NS3529 competent cell, coating the competent cell in a double-antibody culture medium containing kanamycin (kanamycin) and ampicillin (ampicilin) for culture, and extracting plasmids from positive strains;

5) Carrying out enzyme digestion on the plasmid extracted in the step 4) by using a homing enzyme PI-Sce I, then transforming an escherichia coli strain XL10 or NEB 10-beta, culturing, screening and extracting the plasmid to obtain a positive clone pYLTAC380GW-CM1-PAT containing target genes CM1 and PAT;

6) Repeating the steps 2) -5), taking the new plasmid containing the target gene obtained in the previous step as an acceptor vector, alternately using d1 and d2 donor vectors containing different genes for recombination until all the target genes are assembled on the acceptor vector, and finally, continuously adding a selection marker gene expression cassette element in BP recombination reaction to construct a multi-gene vector pYLTAC380GW-CM1-PAT-ADT2-PAL1-C4H-4CL1.

In the step (2), the constructed polygene segments are introduced into a receptor plant and are expressed in the plant body, and each expressed protease participates in the synthesis of caffeic acid and the milk tree alkaloid, so that the circulation of the plant endogenous caffeic acid is increased, the plant continuously generates the caffeic acid and the milk tree alkaloid, and precursor substances are provided for a biological self-luminous metabolic pathway.

The recipient plant may be, but is not limited to: tobacco, rape, rice, moth orchid or chrysanthemum.

Preferably, the multiple gene fragments are introduced into the recipient plant using Agrobacterium-mediated techniques. Specifically, the agrobacterium is EHA105.

The biological self-luminous recipient plant can be a natural self-luminous plant and can also be a biological self-luminous transgenic plant obtained by integrating biological self-luminous gene elements.

Further, the biological self-luminous gene element comprises a Hisps gene, a CPH gene, an H3H gene, an NPGA gene and a Luz gene.

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

The invention has the following beneficial effects:

the synthesis approach of caffeic acid in plants is analyzed, the synthesis approach of the caffeic acid and the milk tree alkaloid in the plants is enhanced through genetic manipulation, and particularly, a CM1 gene, a PAT gene, an ADT2 gene, a PAL1 gene, a C4H gene and a 4CL1 gene are integrated in the plants through a genetic engineering means, and the synthesis of the caffeic acid and the milk tree alkaloid in the plants is obviously improved through the coexpression of multiple genes. And the synthesis path of the caffeic acid is coupled with the self-luminous path of the plants, so that the plants with stronger biological self-luminous brightness can be created. The invention provides a feasible technical scheme for breeding and producing plants with high caffeic acid and milk tree alkali contents and sustainable luminous plants.

Drawings

FIG. 1 is a diagram showing the detection of the enzyme digestion of an intermediate vector in the process of constructing a pYLTAC380MF-8G vector by using a caffeic acid and milk tree alkaloid synthesis enhanced DNA module (eCHM).

FIG. 2 is a diagram showing the detection of target gene expression levels of tobacco leaves transiently transformed by the eCMM module, in which EV-1, EV-2 and EV-2 represent empty vectors (empty vectors) as negative controls, and eCMM-1, eCMM-2 and eCMM-3 represent transformants of the eCMM module transiently transformed.

FIG. 3 is the detection of the target gene expression level in the leaves of Agrobacterium-mediated tobacco transgenic positive plants, wherein eCAM-1, eCAM-2 and eCAM-2 represent eCAM vector transgenic control, eCAM-3, eCAM-17 and eCAM-19 represent eCAM vector transgenic tobacco over-expression plants, the same applies below; the relative expression of the target genes in the eCAM group is compared by defining the expression of 5 genes in the eCAM group as 1.

FIG. 4 shows the detection of caffeic acid content in leaves of Agrobacterium-mediated tobacco transgenic positive plants.

FIG. 5 is the determination of the content of daidzein in leaves of Agrobacterium-mediated tobacco transgenic positive plants.

FIG. 6 is a comparison of the luminescence intensity of tobacco transgenic tobacco at bolting stage.

FIG. 7 is a graph showing the quantitative comparison of fluorescence intensity of leaves of transgenic plants.

Detailed Description

The present invention is further illustrated by the following specific examples. The following examples are merely illustrative of the present invention and are not intended to limit the scope of the invention. It is intended that all modifications or alterations to the methods, procedures or conditions of the present invention be made without departing from the spirit or essential characteristics thereof.

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

pYL322d1, pYL d2, PYLMFH and pYLTAC380GW are gifts for the laboratory of the university of south China Liu Yaoguang professor, and the construction method is disclosed in Chinese patent with the application number of 2017103841977.

Biological self-luminous transgenic tobacco (Hisps-CPH-H3H-NPGA-Luz genes integrated in genomes) seeds are constructed and cultured in the laboratory at the early stage, and the construction method is disclosed in Mitiouchkina et al, 2020. The multiple gene assembly is carried out by using a TransGene Stacking II system, and by using pYL d1 and pYL d2 as donor vectors, hisps genes with the coding sequences shown as SEQ ID NO.8, CPH genes with the coding sequences shown as SEQ ID NO.9, H3H genes with the coding sequences shown as SEQ ID NO.10, NPGA genes with the coding sequences shown as SEQ ID NO.11 and Luz genes with the coding sequences shown as SEQ ID NO.12 are integrated into pYLTAT 380GW plasmids, and finally screening marker gene expression cassette elements are removed through one-step BP recombination reaction, so that the vector pYLTAT 380GW-Hisps-CPH-H3H-NPGA-Luz is formed, and the plasmids are introduced into agrobacterium and are mixed with pretreated tobacco leaves for culture, so that biological self-luminous elements are integrated into plant genomes.

Example 1 caffeic acid and hispidin synthesis enhancing (eCPM) Module vector pYLTAC380MF-8G construction Process

The genes NtCM1, ntPAT, ntADT2, ntPAL1, ntC H, nt4CL1 in the tobacco (Nicotiana tabacum) genome are amplified corresponding to CDS.

This study used the TransGene Stacking II system for multigene assembly. The following genes NtCM1, ntPAT, ntADT2, ntPAL1, ntC H derived from the 35S promoter of tobacco mosaic virus were integrated into the pYLTAC380GW plasmid, and finally the selection marker gene expression cassette element was removed by one-step BP recombination to construct the caffeic acid synthesis pathway-enhancing vector pYLTAC380GW-NtCM1-NtPAT-NtADT2-NtPAL1-NtC H (pYLTAC 380MF-7G, eCAM).

The following genes NtCM1, ntPAT, ntADT2, ntPAL1, ntC H, nt4CL1 driven by 35S promoter derived from tobacco mosaic virus are integrated into pYLTATC 380GW plasmid, and finally a screening marker gene expression cassette element is removed by one-step BP recombination reaction, so as to form a caffeic acid synthesis pathway enhancing vector pYLTATC 380GW-NtCM1-NtPAT-NtADT2-NtPAL1-NtC H-Nt4CL1 (pYLTATC 380 MF-G, eCMM).

Wherein the sequence information of the NtCM1 is shown in a gene registration number: XP _009768497; sequence information for ntpos is found in gene accession numbers: XP _016480150; sequence information for NtADT2 is found in gene accession number: XP _009617905; sequence information of NtPAL1 is found in gene accession numbers: XP _009629066.1; sequence information for NtC H is given in gene accession number: NP _001312445; sequence information of Nt4CL1 is shown in gene accession number: NM _001325738.1.

And carrying out PCR amplification on the coding sequence of the gene to obtain a corresponding gene fragment. Specifically, the CDS sequence of the NtCM1 gene is shown as SEQ ID NO. 3; the CDS sequence of the NtPAT gene is shown as SEQ ID NO. 4; the CDS sequence of the NtADT2 gene is shown as SEQ ID NO. 5; the CDS sequence of the NtPAL1 gene is shown as SEQ ID NO. 6; the CDS sequence of NtC H gene is shown in SEQ ID NO. 7; the CDS sequence of the Nt4CL1 gene is shown in SEQ ID NO.1, and the coded amino acid sequence is shown in SEQ ID NO. 2.

The specific construction process of the synthesis enhanced vector pYLTAC380MF-8G by caffeic acid and daidzein is as follows:

(1) Constructing donor vectors, pYL d1-NtCM1, pYL d2-NtPAT, pYL322d1-NtADT2, pYL d2-NtPAL1, pYL d1-NtC H, pYL d2-Nt4CL1;

(2) Donor vector pYL d1-NtCM1 and acceptor vector pYLTAC380GW (1:1 to 2:1) were mixed in NS3529 competed for co-transformation using heat shock method, ice bath 30min, heat shock 90s, ice bath 2-3min, reactivated in LB without antibiotic at 37 ℃,200rpm,2h, spread on LA plates containing kanamycin (Km, 25 mg/L) and chloremphenicol (Chl, 15 mg/L), and after about 18h a single clone was grown up using ddH ₂ O all the monoclonals are washed into tubes and the mixed plasmids are extracted.

(3) Taking about 50-100ng mixed plasmid, using 0.5uL I-Sce I (NEB) to carry out enzyme digestion for 4-5h in a10 uL system, transforming an escherichia coli strain XL10 (Vazyme) or NEB 10-beta (Bomeide biotechnology limited), coating the mixed plasmid on a LA plate containing kanamycin (Km, 25 mg/L), picking single clone after 15h at 37 ℃, culturing in LB (containing 25mg/L Km and 0.5mM IPTG), carrying out bacteria liquid PCR identification, using Green Taq Mix, further extracting the plasmid capable of amplifying a bright band, taking 200ng each, using 0.2uL Not L to carry out enzyme digestion verification in a 20uL reaction system, generating four bands, and using 0.95 kbp containing a target gene, namely, the required positive clone pYLTAC 380-NtCM 1 (figure 1, 380-1G).

(4) The donor vector pYL d 2-NtPTA and the recipient vector pYLTAC380GW-NtCM1 of (3) (1:1 to 2:1) were mixed in NS3529 competence for co-transformation, transformed according to method (2), spread on LA plates containing kanamycin (Km, 25 mg/L) and ampicilin (Amp, 70 mg/L), and after about 18h a single clone was grown, using ddH ₂ O all the monoclonals are washed into tubes and the mixed plasmids are extracted.

(5) About 40-90ng of mixed plasmid is taken, 0.5uL PI-Sce I (NEB) is used, 0.5uL BSA is added to carry out enzyme digestion for 4-5h in a10 uL system, then the five bands are generated by transformation and verification according to the method in (3), and the positive clone pYLTAC380 GW-NtCM1-NtPTA (figure 1, 380 GW-2G) is obtained by using 1.4k bp NtPTA and 0.95k bp NtCM1 containing the target genes.

(6) Performing more rounds of recombination, and performing cotransformation by alternately using d1 and d2 donor vectors containing different genes and the receptor vector constructed in the previous round to construct pYLTATC 380GW-5G (figure 1, 380 GW-5G) and pYLTATC 380GW-6G (figure 1, 380 GW-6G);

(7) Finally, pYLTAC380GW-5G and pYLTAC380GW-6G (200 ng) were mixed with PYLMFH-Bnmlpro (100 ng), respectively, by BP reaction for 5 hours at 25 ℃ in a 5. Mu.l reaction with 1. Mu.L of 5 XBP enzyme mixture. Then, 1. Mu.L of protease K solution was added to terminate the reaction at 37 ℃ for 10 minutes. Transferring to NEB 10-beta (Bomaide Biotechnology limited) competence, and selecting single clone for identification. Detecting with Not1 enzyme digestion, identifying correct positive final vectors pYLTAC380MF-7G and pYLTAC380MF-8G (figure 1, 380 MF-8G), selecting positive clones for whole plasmid sequencing analysis, and selecting correct pYLTAC380MF-7G and pYLTAC380MF-8G vectors for subsequent experiments.

Example 2 caffeic acid and hispidin synthesis enhanced (eCPM) transient transformation tobacco lamina analysis

1. The EHA105 bacterial liquid containing the vector plasmid pYLTAC380MF-8G which is verified to be correct is streaked and activated on LA + Kana + Rif plates at 28 ℃ for 36 hours, colonies are picked from the plates, transferred into LB + Kana + Rif +15 mu M As culture medium and cultured at 28 ℃ and 200rpm until OD is reached ₆₀₀ The cells were collected at 4000rpm for 10min and then suspended in an infection solution (containing 10mM MgCl., 10mM MES, 150. Mu.M As) and allowed to stand at room temperature for 2 to 3 hours.

2. And (3) performing transient expression verification in the tobacco leaves, opening a small opening on the surface of the tobacco leaves by using a 1mL needle head, sucking bacterial liquid by using a needle tube without the needle head, and injecting the bacterial liquid into the leaves from the wound of the tobacco leaves. Indoor normal culture is carried out for 48h, then relative expression detection is carried out on partial leaves of a sample by qPCR, the overexpression of the target gene is determined, and the result is shown in figure 2, and the target genes in3 independent transgenic families are all overexpressed.

Example 3 Agrobacterium-mediated analysis of caffeic acid and milk tree alkaloid content and luminescence intensity in eCPM transgenic plants

1. EHA105 bacterial fluid containing vector plasmids pYLTATC 380MF-8G (eCMM) and pYLTATC 380MF-7G (eCAM) which are verified to be correct is streaked on LA + Rif + Kana plates respectively at 28 ℃ for 36h, and the single clones are picked into 3-5ml LB culture medium at 200rpm,28 ℃ for 36h, and 50ml is enlarged and cultured for 3-5h to OD =0.6 according to the proportion of 1;

2. selecting a transgenic tobacco FBP (genome of which has a luminescence related gene Hisps-CPH-H3H-NPGA-Luz) seed which is obtained successfully and is self-luminous, planting healthy tobacco leaves for 4-5 weeks to be completely unfolded on a sterile MS culture medium, cutting the leaves into 0.5cm square (cutting off leaf edges to avoid main veins) by using a surgical knife, placing the upper surface of each leaf downwards on an MS1 solid culture medium (MS +0.5mg/L IAA +2.0mg/L BA +3 + sucrosose +0.6-0.8 Phytagel, PH = 5.8), and carrying out dark culture at 25 ℃ for 2-3 days;

3. adding the pre-cultured tobacco leaves into the bacterial liquid, performing vortex oscillation to ensure that the cut of the leaves is immersed by the bacterial liquid, standing for 5-30min, and absorbing the attached bacterial liquid by using sterile filter paper; placing the infected leaves with the upper surface facing downwards on an MS1 solid culture medium at 28 ℃ for dark culture for 2d; putting the upper surface of the leaf upwards on an MS1 screening culture medium containing Timentin and glyphosate, and performing light culture at 25 ℃; when the leaf margins are germinated and can be separated (more than 1 cm), the shoots are cut and transferred to MS2 (MS +0.5mg/L IAA +3% sucrose +0.6-0.8% Phytagel, PH = 5.8) solid culture medium containing antibiotic (TM + basta), roots are grown after two weeks, the cover of the seedling raising box is opened, seedlings are trained for one week, and then the seedlings are transferred to planting soil for culture, so that eCAM and eCAHM transgenic positive plants are obtained.

3 transgenic positive family leaves of eCAPM and eCAPM are taken for qPCR analysis and caffeic acid content detection, and the results are shown in FIG. 3. 5 target genes in the eCAM module of the transgenic tobacco and 6 target genes in the eCAM module are over-expressed.

The caffeic acid and the daidzein content in3 transgenic positive family leaves in FBP plants, eCAM plants and eCAM plants are detected by a high performance liquid chromatography-mass spectrometry instrument. As a result, as shown in fig. 4 and 5, the caffeic acid content and the milk alkaloid content in the leaves of the eCHM transgenic positive family are significantly increased compared with those of the FBP transgenic family, and the milk alkaloid content in the leaves of the eCHM transgenic positive family is significantly increased compared with that of the eCAM transgenic positive family.

4. And (3) moving the positive transgenic bolting stage tobacco to a darkroom, taking a picture by using a Nikon D750 camera, and using a lens AF-S17-35mm F2.8D-IF, ISO 2000 and exposure time of 60 seconds. The results are shown in fig. 6, and it can be seen that the emission intensity of the eCAM transgenic positive plants is higher than that of the eCAM transgenic positive plants.

And (3) carrying out light quantum quantitative analysis, taking the completely unfolded new leaves of the tobacco in the transgenic bolting stage, taking a picture in an in vivo imaging system NightSHADE LB985 instrument, and recording light quanta. As shown in fig. 7, statistical analysis found that the light emission intensity of the eCHM transgenic positive plants was higher than that of the eCAM transgenic positive plants and the 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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