CN1318579C - Gene engineering method for raising plant useful secondary substance content - Google Patents

Abstract

The invention discloses a genetic engineering method for increasing the content of useful secondary matter in plants, which is realized through the following technical solutions: 1. Construct a chimeric gene, including a nucleic acid fragment of a promoter and a nucleic acid encoding a plant lignin synthesis key enzyme CCR 2. Introduce the chimeric gene into plant cells to produce transgenic plant cells; 3. Grow the transgenic plant cells under conditions suitable for expression; 4. Screen and identify transgenic plant cells; 5. Transgenic plant cells Further derived and cultured to become transgenic plants or transgenic callus or transgenic cell lines. The method can effectively inhibit the synthesis of plant lignin, change the flow direction of its substrate, and achieve the purpose of increasing the content of secondary substances such as isoflavones or paclitaxel.

Description

A Genetic Engineering Method for Improving the Content of Useful Secondary Matter in Plants

technical field

The invention relates to the technical field of plant breeding, in particular to a method for improving useful secondary metabolites of plants by using genetic engineering technology.

Background technique

Plant secondary metabolism produces many natural products with practical value, which are the raw materials of many medicines, cosmetics, spices, pigments, etc. Most of the active ingredients contained in traditional medicinal materials are secondary metabolites, but in natural plants, the content of these useful secondary substances is usually very low, and even lower in cultivated and tissue cultured plants. Therefore, increasing the content of active ingredients in medicinal plants is an urgent problem to be solved by effectively utilizing plant resources, ensuring the quality of traditional Chinese medicines and the sustainable utilization of traditional Chinese medicine resources. Lignin is the second largest organic substance in plants after cellulose, accounting for more than 95% of the total plant secondary matter (Anterola A, Lewis N. Trends in Lignin modification: a comprehensive analysis of the effects of genetic manipulations /mutations on lignification and vascular integrity. Phytochemistry. 2002, 61(3): 221-294). Lignin and many other important secondary substances such as flavonoids, lignans, phenols, alkaloids, and anthocyanins, which are important components of herbal medicines, are synthesized by the Phenylpropanoid Pathway ( http://www.genome. ad.jp/kegg/pathway/map/map00940.html ), they have a competitive utilization relationship for common precursors (substrates). Although lignin has certain physiological functions in plants, its excessive synthesis not only consumes a large amount of carbon frame structure and energy, but also pollutes the paper industry and increases its production energy consumption; excessive lignin content in forage grass High will reduce its digestion and absorption rate. Therefore, in recent years, people have paid more attention to the regulation of lignin content, which has become an important direction of plant genetic engineering research.

Hydroxycinnamoyl CoA reductase (cinnamoyl CoA reductase, CCR) can reduce three kinds of COA esters of hydroxycinnamic acid to generate corresponding cinnamaldehyde (Pichon M, courbou I, Beckert M et al. Cloning and characterization of two maize cDNAsencoding cinnamoyl -CoA reductase (CCR) and differential expression of the corresponding genes. Plant Mol. Bio. 1998, 38(4): 671-676). The enzyme catalyzes the first specific step in the biosynthetic pathway of lignin monomers and thus has a regulatory effect on the flow of carbon into the lignin synthesis pathway. Abbott et al. (Abbott JC, Barakate A, PinconG et al.Simultaneous suppression of multipe genes by singletransgenes.Down-regulation of three unrelated lignin biosynthetic genes in tobacco.Plant Physiol.2002,128:844～853) research on tobacco proved that Inhibition of CCR gene expression can significantly reduce plant lignin content. These research results show the possibility of genetic engineering technology to regulate the expression of CCR gene to change the lignin content of plants. In addition, transgenic plants with altered lignin content or composition have been obtained from Arabidopsis, tobacco, poplar and other plants. The key enzyme genes that have been proven to control the lignin content include: PAL (L-phenylalanine ammonia-lyase, phenylalanine ammonia-lyase) (Bate N, Orr J, Ni W. et a;. Quantitytive relationship between phenylalanine ammonia -lyase levels and phenylpropanoid accumulation in transgenic tobacco identifies a rate-determining step in natural products synthesis.1994, PNAS, 91:7608-7612), C4H (cinnamate 4-hydroxylase, cinnamate-4-hydroxylase) (Blee K, Choi JW, O'Connell AP et al. Antisense and sense expression of cDNA coding for CYP73A15, a class II cinnamate4-hydroxylase, leads to a delayed and reduced production of lignin intobacco. Phytochemistry 2001, 57(7): 1149CL-66), (4-coumarate: coenzyme A ligase, hydroxycinnamic acid: coenzyme A ligase) (Kajia S, KatayamaY, Omori S. Alteration in the biosynthesis of ligninin transgenic plants with chimeric genes for 4-coumarate: coenzyme Aligase. Plant Cell Physiol. 1996, 37:957-965), COMT (caffeic acidO-methyltransferase, caffeic acid methyltransferase) (Atanassova R, Favet N, Martz F, Chabbert b, Tollier M-T, Monties B, Fritig B, Legrand M(1995) Altered lignin composition in transgenic tobacco expressing O-methyltransferase sequences in sense and antisense orientation. Plant J8: 465-477), CCoAOMT (caffeoyl-CoA 3-O-methyltransferase, 5-adenosyl-methionine: caffeoyl-CoA/ 5-Hydroxyferuloylcoenzyme A-O-methyltransferase) (ZhongR, Morrison WH, Himmelsbach DS et al. Essential role of caffeoylcoenzyme A O-methyltransferase in lignin biosynthesis in woody poplarplants. Plant Physiol. 2000, 124: 563-577 ), CCR (cinnamony-CoAreductase, hydroxycinnamoyl coenzyme reductase). By inhibiting the expression of these enzyme genes, the lignin content of plants can be reduced.

However, the research on lignin genetic engineering is currently mainly focused on the reduction of plant lignin content and the change of its components, and there is no systematic research report on the impact of lignin content regulation on the synthesis of other secondary metabolic pathway products; in addition, in In cell and tissue culture, the use of inhibitors to reduce the metabolic flux of competing pathways to increase the content of target products has been successful, but the study of providing more substrates for the synthesis pathways of desired secondary products by inhibiting the expression of key enzyme genes of competing pathways has not been seen. reports. The invention utilizes the "substrate competition" mechanism, adopts genetic engineering technology to regulate the expression of the CCR gene of the specific enzyme system related to lignin synthesis, reduces the amount of lignin biosynthesis, and provides more substrates for the synthesis of useful secondary substances such as medicinal plant active ingredients , thereby increasing the content of useful secondary matter in plants.

Contents of the invention

The purpose of the present invention is to provide a genetic engineering method for increasing the content of useful secondary substances in plants, that is, to apply genetic engineering technology to regulate the gene expression of key enzymes in CCR lignin synthesis, reduce the biological content of lignin, and facilitate the synthesis of other useful secondary substances. Provide more substrates, thereby increasing the content of useful secondary substances in plants.

The present invention improves the genetic engineering method of plant useful secondary matter content, comprises the following steps:

1. Construct a chimeric gene, including a nucleic acid fragment encoding a promoter that functions in plant cells, and a nucleic acid fragment encoding a plant lignin synthesis key enzyme CCR that is reversely connected to the promoter, and a transcription termination region;

2. When the chimeric gene is introduced into a non-transformed plant, it can produce and accumulate useful secondary substances in the plant cells selected from soybean or yew in its organs or tissues to produce transgenic plant cells;

3. Growing the transgenic plant cells under conditions suitable for chimeric gene expression;

4. Screening and identification of transgenic plant cells. Compared with similar non-transgenic plant cells, the lignin synthesis key enzyme CCR and lignin content are reduced, while the content of at least one useful secondary substance is increased;

5. Further culturing the transgenic plant cells to become transgenic plants, or transgenic callus, or transgenic cell lines.

The chimeric genes constructed in the present invention are antisense gene structures (antisense constructs).

The chimeric gene is constructed in the present invention, wherein the promoter is a constitutive expression promoter or an organ-specific expression promoter, wherein the constitutive expression promoter is CaMV 35S, and the organ-specific expression promoter is a xylem-specific expression promoter.

The key enzyme of plant lignin synthesis in the present invention is cinnamoyl CoA reductase (cinnamoyl CoA reductase, CCR).

When the plant of the present invention is an untransformed plant, it can accumulate useful secondary substances in its organs or tissues, and this type of plant can be selected from soybean or yew.

The chimeric gene of the present invention is introduced into the plant cell through the method mediated by Agrobacterium, electric shock method, microprojectile bombardment method, microinjection method or liposome technology.

The useful secondary substances in the present invention are products that provide precursors (substrates) through the phenylpropanoid pathway (Phenylpropanoid pathway), including active ingredients of medicinal plants, which may specifically be isoflavones or paclitaxel.

The transgenic plant cell of the present invention contains the constructed chimeric gene, and the cell can be further cultivated to become a transgenic plant, a transgenic callus or a transgenic cell line.

A transgenic plant cell with increased useful secondary substance content obtained by the method of the present invention contains the constructed chimeric gene.

A transgenic plant cell with increased useful secondary substance content obtained by the method of the present invention is further cultured to form a transgenic cell line or a transgenic callus or a transgenic plant.

The beneficial effect of the present invention is that the constructed chimeric gene is introduced into plant cells by genetic engineering methods, and further cultivated to form transgenic plants, callus or cell lines, and their offspring can stably inhibit lignin synthesis-specific enzyme CCR The expression of lignin reduces the amount of biosynthesis, so that the substrate of lignin biosynthesis——aromatic amino acid, and the products of the phenylpropane pathway flow into the synthesis pathway of secondary substances such as isoflavones or paclitaxel to increase its production. This provides a new technical approach for increasing the content of useful secondary metabolites such as active ingredients of medicinal plants.

Description of drawings

Figure 1: Construction of CCR antisense plant expression vector;

Figure 2: PCR identification of the recombinant plasmid pUCm-T-CCR

P: PCR molecular weight standard; 1: amplification product of plasmid pUCm-T-CCR

Figure 3: Recombinant plasmid: pUCm-T-CCR PstI enzyme digestion identification

M: λDNA/Hind III;

E: Plasmid: pUCm-T-CCR digested with PstI

Figure 4: PCR Identification of Plasmid pACCR

4-Amplified product of plasmid pACCR using CCPF+CCRR as primers

P: PCR molecular weight marker;

1: Amplified product of plasmid pUCm-T-CCR;

2: Amplified product of plasmid pIG121;

3: Plasmid pACCR amplification product;

4-b The amplified product of plasmid PACCR using P _35S + CCRF as primers.

P: PCR molecular weight marker;

1: amplified product of plasmid pIG121; 2---3: amplified product of plasmid pACCR.

Figure 5: Plasmid pACCR enzyme digestion identification

M: λDNA/Hind III; L: DNA Ladder

5a: SacI digestion result of pACCR plasmid.

1: Plasmid pACCR;

2: pACCR plasmid SacI digestion;

5b: BamHI digestion result of pACCR plasmid.

M: λDNA/HindIII

1: BamHI digestion of pIG121 plasmid;

2: BamHI digestion of pACCR plasmid.

Detailed ways

The following examples illustrate the implementation of the present invention in detail, but the present invention is not limited to the application provided below, and the following description does not constitute any sense of limitation to the protection scope of the present invention.

Example 1. Cloning of lignin synthesis key enzyme gene CCR and construction of its antisense expression vector

1) Plant material: The tested Arabidopsis thaliana L. Heynh was a Columbia ecotype, donated by Mr. Xu Zhenghuang, Institute of Plant Physiology and Ecology, Chinese Academy of Sciences. Genomic DNA of Arabidopsis thaliana was extracted from leaves by CTAB method.

2) Strains and plasmids: The cloning vector pUCm-T vector is a product of Bocai Biotechnology Co., Ltd. Plant expression binary vector pIG121, helper plasmid pRK2013, Escherichia coli DH5α, HB101, Agrobacterium tumefacious EHA105 were preserved by the Crop Quality Improvement Genetic Engineering Laboratory of Zhejiang Academy of Agricultural Sciences.

3) Enzymes and chemical reagents: restriction endonuclease, T4 DNA ligase, calf intestinal alkaline phosphatase (CIAP) and other tool enzymes, λDNA/HindIII were purchased from MBI Company, DNA molecular weight marker PCR Marker was purchased from Huamei Biotechnology Company, DNA Ladder was purchased from Dingguo Biotechnology Development Center. Taq DNA polymerase, agarose and other biochemical reagents were produced by Shanghai Sangon Bioengineering Company; PCR primers were synthesized by Shanghai Sangon Bioengineering Company.

4) Amplification and cloning of CCR gene: According to the CCR gene sequence in Genbank, a pair of specific primers were designed and synthesized, and PCR amplification was performed using Arabidopsis genomic DNA as a template.

5′ end primer:

Primer CCRF: 5′-GCTGGTGGATACATCGCTTCT-3′

3′ end primer:

Primer CCRR: 5′-AGGAGAAGCCGTGTGAAAGAC-3′

PCR amplification program: the amplification conditions are 94°C pre-denaturation for 5 minutes; 94°C, 30s, 55°C, 1min, 72°C, 1.5min 30 cycles. The last cycle was followed by an extension at 72°C for 10 min.

The PCR amplification product was subjected to 2% agarose gel electrophoresis, and an amplified band of about 343bp was obtained ( FIG. 2 ), which was consistent with the expected size.

After the PCR product with a single A was ligated with the pUCm-T vector by T4 DNA ligase, it was transformed into Escherichia coli DH5α competent bacteria. Screen with X-Gal/IPTG/LB plates containing 100 μg/mL ampicillin. A small amount of plasmid DNA from positive clones was extracted, and two DNA electrophoresis bands were obtained after digestion with PstI, one was similar in size to the vector pUCm-T (about 2.8 kb), and the other was similar in size to the PCR amplified fragment (about 343 bp) ( Fig. 3) proves that the PCR product has been cloned into the pUCm-T vector, and this clone is named pUCm-T-CCR.

5) Sequence determination and analysis: The positive recombinant plasmids were automatically sequenced using the T7 promoter sequence located upstream of the multiple cloning site of the cloning vector pUCm-T Vector. The sequencing work was completed by Boya Biotechnology Company. After sequence determination, the cloned fragment consists of 343 nucleotides, and its sequence is as follows:

GCTGGTGGAT ACATCGCTTC TTGGATTGTT AAGATACTTC

TCGAGAGAGG TTACACAGTC AAAGGAACCG TACGGAATCC

AGGTACTTGT CCATTTTTAT ATATACTTTT TTGGTCACTT

GTGATAATTA TCTGAAGTTG TTGAGGTTAT TTTTATTGTT

TTGATTGACA ATTTGGATTT CTTTATAATT ATTTTTCTTG

CAGATGATCC GAAGAACACA CATTTGAGAG AACTAGAAGG

AGGAAAGGAG AGACTGATTC TGTGCAAAGC AGATCTTCAG

GACTACGAGG CTCTTAAGGC GGCGATTGAT GGTTGCGACG

GCGTCTTTCA CACGGCTTCT CCT

Nucleotide sequence homology comparison was carried out in GenBank+EMBL+DDBJ+PDB with BLAST program, and the result showed that the homology between this fragment and the partial sequence of CCR gene on chromosome 1 of Arabidopsis was 100%.

6) Construction and identification of the antisense CCR gene plant expression binary vector: the plant expression structure of the antisense CCR gene is shown in FIG. 1 . To facilitate gene cloning, Sac I sites were introduced at both ends of the target fragment by PCR. The pUCm-T-CCR plasmid DNA was used as a template for PCR amplification, the amplified product was digested with Sac I, and a fragment of about 343 bp was recovered; the expression vector pIG121 was digested with Sac I, and CIAP was dephosphorylated. Vector: target fragments are mixed at a ratio of 1:3 and ligated with T4 DNA ligase. Transform Escherichia coli HB101 competent cells, spread on LB plates containing 100 μg/mL kanamycin to select recombinants. Clones were screened by PCR with CCRF and CCRR primers, and the amplified fragments of positive clones were in the same size as the fragments amplified from pUCm-T-CCR (Fig. 4a). To determine the reverse connection between the target fragment and the CaMV 35S promoter, the 5' primer CCRF of the target gene and a nucleotide sequence corresponding to the CaMV 35S promoter are used as primers (P _35S : 5'-CGTAAGGGATGATGACGCACAAT-3'), After PCR screening, some clones had amplified fragments, which were slightly longer than those amplified with P _35S and GUS gene 3' primers ( _PGUS : 5'-CGCAAGACCGGCAACAGG-3') (Fig. 4b). The plasmid DNA of the positive clone was identified by enzyme digestion, and a fragment of about 349 bp was obtained by digestion with Sac I, which was consistent with the length of the insert after adding the Sac I site to the CCR gene (Figure 5a); a fragment was obtained by digestion with Bam HI A fragment of about 4.1 kb (Fig. 5b), that is, GUS gene fragment + CCR gene fragment + NOS terminator fragment + 35S promoter fragment + HPT II gene fragment. The results of PCR and enzyme digestion showed that the CCR gene fragment had been inserted behind the GUS gene in the expression vector, and the direction of connection with 35S was antisense. The antisense CCR plant expression vector plasmid was named pACCR.

7) Transformation of the antisense CCR plant expression binary vector into Agrobacterium: the antisense CCR plant expression vector pACCR was introduced into Agrobacterium tumefaciens EHA105 by triparental mating method and pRK2013 as the helper plasmid. Transformants were screened on YEB medium containing Rif (25 μg/mL) and Km (100 μg/mL), PCR amplification was performed on positive clones of triparental hybridization using CCRF, CCRR primers and P _35S , CCRF primers, and alkaline method Plasmids were extracted for enzyme digestion identification. The enzyme digestion results combined with the PCR identification results indicated that the antisense CCR plant expression vector had successfully transformed Agrobacterium tumefaciens.

Example 2. Transformation of Arabidopsis thaliana with antisense CCR gene

1) Arabidopsis transformation

Use the inflorescence spray method. Select plump Arabidopsis thaliana (Columbia ecotype) seeds, sow them after vernalization at 4°C for 2-3 days, cultivate them at 22-24°C with a photoperiod of 16h light/8h dark, and cut off the main stems above the rosette leaves after bolting. rachis to promote bolting of secondary rachis. When the secondary flower axis is 2-10 cm long, it is used for transformation.

Cultivation of Agrobacterium: Pick a single colony of Agrobacterium EHA105 (containing plasmid pACCR) and inoculate it into 5 mL of YEB culture solution containing 25 mg/mL Rif+100 mg/mL Km, culture at 200 rpm at 25-28°C overnight for preactivation. Inoculate 50 μL of the bacterial solution into 50 mL of the same culture solution, and cultivate until the logarithmic growth phase. The bacterial solution was centrifuged at 5500 g for 20 min at room temperature. The cells were diluted with transformation solution (1/2MS, pH5.7 containing: 5% sucrose, 0.02-0.05% Silwet L-77, 44mM benzylamino purine) to _OD600-0.80 . Spray the bacterial solution evenly on the Arabidopsis plants until the inflorescences are completely wet. The seeds of the transformed plants were collected and screened on a 1/2 MS plate containing 50 mg/mL Km, and the resistant seedlings were transplanted into the soil for molecular detection. Validated transgenic plants were analyzed for lignin and secondary product content.

2) Identification of transgenic plants

PCR detection of transgenic plants: The PCR method can quickly detect the integration of foreign genes in transformed plants or callus. Since the plant itself has an endogenous CCR gene, the sequence of the CCR gene itself cannot be used to design primers or probes when detecting transgenic plants by PCR or Southern method. In this study, P _35S and CCRF primers were used for PCR amplification.

Extract the total DNA of transgenic plants or callus, and use it as a template for PCR. The transgenic plants amplified a band with the same molecular weight as the amplified fragment of the recombinant plasmid pACCR containing the Anti-CCR gene, while the control plants did not amplify the corresponding band.

Southern hybridization analysis of transgenic plants or callus: The total DNA of the regenerated plants was completely digested with restriction endonucleases and then subjected to Southern hybridization. The DNA probe is a fragment with hygromycin gene in the super binary vector pIG121. Labeling was carried out according to the instructions of the random primer labeling kit from TaKaRa Company. Southern hybridization was carried out according to the protocol described in "Molecular Cloning" (Sambrook T, TanaKa K, Monma T. Molecular Cloning: A Laboratory Manual. Cold Spring Harbor Laboratory après, New York, 1989).

3) Determination of lignin content: refer to Pochinnock method (Pochinnock XH. Plant biochemical analysis method. 1983, Beijing: Science Press. 165-166, 178-180.) Weigh plant material (fresh material ) 0.2-0.5 g, treated with 1% acetic acid to separate sugar, organic acid and other soluble compounds. Then treat it with acetone to separate chlorophyll, fat and other fat-soluble compounds, and then use 72% (specific gravity 1.63) sulfuric acid to separate fibers and hemifibres. Centrifuge (2500rpm), discard the supernatant. After the precipitate was washed with distilled water, the lignin in the hydrolyzate was oxidized with potassium dichromate in the presence of sulfuric acid, and the excess potassium dichromate was determined by iodometric method. Add 1mL of starch indicator before the end point, and then titrate to green. Calculation formula of lignin content:

$\mathrm{<span\; class="notranslate">\; x</span>}<span\; class="notranslate">\; =</span>\frac{\mathrm{<span\; class="notranslate">\; 0.433</span>}<span\; class="notranslate">\; \&times;</span>\mathrm{<span\; class="notranslate">\; K</span>}<span\; class="notranslate">\; (</span>\mathrm{<span\; class="notranslate">\; a</span>}<span\; class="notranslate">\; -</span>\mathrm{<span\; class="notranslate">\; b</span>}<span\; class="notranslate">\; )</span>}{\mathrm{<span\; class="notranslate">\; DW</span>}}$

x: lignin content ₍ %); K: concentration of _NaS2SO3 ; DW: sample weight (g);

a: the volume of 0.5mol/L NaS ₂ SO ₃ used in the control solution (mL);

b: The volume (mL) of 0.5 mol/L NaS ₂ SO ₃ used to titrate the sample.

4) Determination of plant phenolic content: Refer to Zhu Guanglian (Zhu Guanglian. Plant Physiological Experiments. Peking University Press, 1990: 229-231) Folin-phenol reagent color development method, and the content is calculated in (μg/g fresh weight.

Table 1. Effect of antisense CCR gene on lignin and phenolic content of Arabidopsis plants

sample Lignin content (% dry weight) Phenolic content (μg/g, FW) Increased value of phenolic content (%) Control (untransformed plants) 9.2 118.20 transgenic plant 1 8.3 142.36 20.4 transgenic plant 2 6.9 254.78 115.5 transgenic plant 3 7.5 201.78 70.7

5) Obtaining homozygous antisense CCR transgenic Arabidopsis plants: the above-mentioned antisense CCR transgenic plants can be bred to obtain homozygous antisense CCR gene transgenic plants after selfing and molecular tracking detection of offspring such as Southern and PCR.

Example 3. Using antisense CCR gene to increase soybean isoflavone content

1) Plant transformation: Agrobacterium-mediated method was used to carry out genetic transformation of soybean with soybean cotyledon nodes as explants. The transformation process referred to the method of Zhou Sijun et al. (Zhou Sijun, Li Xichen, Liu Zhaojun, etc. Bt( cryIA) gene into soybean (Soybean Science, 2001, 20(1): 157-163).

Bacterial solution preparation: Pick a single colony from a fresh plate, inoculate it into YEP medium containing corresponding antibiotics, and cultivate it with shaking at 27°C until the logarithmic growth phase (OD ₆₀₀ ≈0.5). The bacterial solution was centrifuged at 4000 rpm at 4°C for 10 minutes, and then the bacteria were resuspended in 1/10 B5 medium (Gamborg et al., 1968) (OD ₆₀₀ ≈0.5). The resuspension medium was supplemented with 1.7 mg/LBA, 0.25 mg/LGA3, 100 μM AS (acetosyringone) and 3% sucrose, and AS was added after autoclaving. The pH of the medium was adjusted to 5.4 before autoclaving, and 20 mM M ES was added as a pH buffer.

Explant preparation: select disease-free and plump soybean seeds for disinfection; 70% alcohol for 1 minute → 0.1-0.2% HgCl for 15 minutes → rinse 4-5 times with sterile water, at least 5 minutes each time → soak in sterile water for 2 hours or more. The sterilized seeds were inoculated on B5 medium (containing 2 mg/L BA) to germinate. Cut off the cotyledon node explants from sterile seedlings that have germinated for 5-7 days: cut off the hypocotyl at about 3 mm from the ion leaf node, then cut off 1/3 of the cotyledon, and then cut the hypocotyl longitudinally between the two cotyledons Open, remove terminal bud, draw 5-7 knives in the scope of the about 3mm diameter of cotyledon and hypocotyl junction with scalpel. After the above operations, each sterile seedling can produce 2 cotyledonous node explants for transformation.

Transformation and screening: soak the prepared cotyledon node explants in the resuspended bacterial solution for 5-30 minutes. Pour off the bacterial liquid, put the explants in a large Petri dish covered with sterile filter paper above and below, and absorb the excess bacterial liquid. Then inoculate the explants adaxially on a co-cultivation medium covered with a layer of sterile filter paper, the co-cultivation medium is the same as the bacterial suspension medium, and 0.5% agar is added for solidification. After the culture was co-cultivated at 24-25°C for 3 days in the dark or under low light, the explants were transferred to a sterile Erlenmeyer flask, and liquid B5 medium (containing 1.7mg/L BA, 0.25mg/LGA3, 3% sucrose , 500mg/L carbenicillin, 20-30mg/L hygromycin, pH5.6) Shake the Erlenmeyer flask to wash twice, or shake and culture on a shaker at 150rpm for 2-3 days, and replace fresh medium every day. Then transfer to solid culture, the composition of the medium is the same, the adaxial side of the cotyledon is facing upward, and the hypocotyl is inserted into the medium. Transplant once every two weeks, and cut off the aging tissue at the base of the hypocotyl. When buds are visible (about 4 weeks or so), the explants are transferred to shoot elongation medium. The composition of the medium was roughly the same as that of the adventitious bud induction medium: the concentration of BA was reduced to 1.0mg/L or 1.0mg/L zeatin was used instead, the concentration of GA3 was increased to 0.5mg/L, 0.2mg/LIBA was added, and Gln and Asn were added respectively. 50mg/L. After 4-6 weeks, the regenerated seedlings can grow to a height of 2.5-3cm. Cut off the regenerated shoots and transfer to 1/2 B5 medium (additional 0.5-2.0 mg/L IBA or NAA, 2% sucrose, 0.8 agar) to induce rooting. The culture conditions are all 22-26°C, relative humidity 70-75%, photoperiod 16/8 hours. It takes about 2 weeks to take root.

Regenerated plant transplanting: After the regenerated plant takes root and grows more than two compound leaves, open the lid of the triangular flask, and exercise for 5-7 days in a climate chamber (relative humidity 85%, light intensity 3000Lux, temperature 22-26°C). Then take out the small plants, wash the medium of the roots, plant them in small pots filled with sterilized sand and soil (1:1) mixture, and continue to cultivate them in the climate box for 1-2 weeks. Gradually reduce the relative humidity to 75% in the previous week, so that the small plants are gradually acclimated. Then planted in large pots to grow to maturity.

2) Molecular identification of transgenic plants: see Example 2.

3) Determination of isoflavone content in soybean: using reversed-phase high-performance liquid phase method (Sun Meijun, Luo Lian, Shi Changying, etc., Determination and Analysis of Flavonoid Content in Chinese Soybean Products, Food and Fermentation Industry, 2000, 26(3) 14- 19) Determination.

Materials: Standard samples of genistin and daidzein are Sigma products. HPLC uses methanol and acetic acid as chromatographic grade, water as high-purity water, and other reagents as analytical grade.

Main instruments: high performance liquid chromatography (Shimadzu LC-9A, Japan).

Chromatographic column: Shim-pack clc-ods C-18 0.15m×6.0mmФ; UV detector: SPD-6AV; mobile phase: methanol: 5% acetic acid = 30:70; flow rate: 1.0mL/mim (before 8.5min) , 1.5mL/min (after 8.5min); wavelength: 224nm; detection sensitivity: 0.08AUFS; column temperature: 50°C.

Grind the soybean seeds and pass through a 40-mesh sieve, then degrease with n-hexane, and extract with 80% methanol under reflux. , qualitative according to the retention time of the standard, and quantitative according to the peak area of the standard.

Table 2. Effect of antisense CCR gene on isoflavone content of soybean seeds

sample Isoflavone content in seeds (μg/g) Isoflavone content increase value (%) Control (untransformed soybean seeds) 1890.80 Transgenic plant 1 soybean seed 1955.02 3.4 Transgenic Plant 2 Soybean Seeds 2022.30 7.0

Transgenic Plant 3 Soybean Seeds 2414.63 27.7

4) Obtaining homozygous antisense CCR transgenic soybean plants: the above-mentioned antisense CCR transgenic plants can be bred to obtain transgenic soybean lines homozygous for the antisense CCR gene after selfing and progeny undergoing molecular tracking detection such as Southern and PCR.

Example 4. Utilizing antisense CCR gene to increase paclitaxel content in Taxus chinensis callus

1) Obtaining of antisense CCR transgenic Taxus chinensis calli: by gene gun transformation method.

Disinfection of explants: select 1-2 year old twigs of Taxus chinensis with green skin and in a dark place as the test material, wash the branches and cut them into small sections, and sterilize them with 70% ethanol and 0.1% HgCl ₂ for 2 minutes and 20 minutes respectively . After rinsing with sterile water for 6 times, the stems were cut into small pieces about 1.5-2.0 cm long and obliquely inserted on the induction medium. The callus induction medium is the inorganic salt of _B5 medium, supplemented with 100.0mg/l inositol, 1.25mg/L nicotinic acid, 1.0mg/L vitamin _B1 , 0.5mg/L vitamin _B6 , 20.0g/L Sucrose, 1.0g/L hydrolyzed milk protein, 0.1mg/L BAP, 2.0mg/L 2,4-D, 8.0g/L agar, and the pH value of the medium is 5.8. Grow in a dark culture room. The culture temperature is 22±2°C. Healing occurs in about 20 days. At the end of the induction, the larger piece of callus (more than 0.5 cm in diameter) formed on the explant was peeled off, and inoculated on the proliferation medium for subculture. The callus proliferation medium is the same as the induction medium, but without hydrolyzed milk protein, and the concentration of 2,4-D is 1.0mg/L. Cultivate in a light culture room, the culture temperature is 22±2°C, the light intensity is about 1500 lx, and the photoperiod is 14h.

The callus after 1-2 subcultures was used for gene gun transformation. 24 hours before the bombardment, the callus was transferred to a high-sugar medium (containing 5% sucrose, and the remaining components were the same as the proliferation medium). Microprojectile DNA preparation: Plasmid pCCRA was extracted by alkaline method, the concentration of plasmid DNA was estimated according to its OD ₂₆₀ value, and the concentration was adjusted to 1 μg/μL with TE. The particle diameter of the gold powder used is 1.6 μm. Prepare the ethanol solution of gold powder under sterile conditions, the concentration is 60 μg/μL. 50 μL of the suspension was divided into Eppendorf tubes, and 50 μg of plasmid, 50 μL of 2.5 μg CaCl ₂ and 20 μL of 1.0 M spermidine solution were added to each tube. Fully shake for 3 minutes, centrifuge at 10,000 rpm for 10 minutes, discard the supernatant, and resuspend the pellet in 50 μL of absolute ethanol. Use 10 μL per bombardment.

Gene gun bombardment: use Bio-Rad's PDS-1000/He pneumatic gene gun for transformation, and operate according to the product instructions. The vacuum degree is 26-30 inches of mercury, the bombardment pressure is 1100psi, and each dish is bombarded with one shot.

After bombardment, the callus material was cultured overnight on a high-sugar medium, and then transferred to a solid proliferation medium without antibiotics. After 4-6 days of culture, it was transferred to a resistant proliferation medium containing hygromycin (20mg/L) , transferred once every 4 weeks, and selected resistant tissues with normal growth.

2) Molecular identification of transgenic callus: see Example 2.

3) Determination of paclitaxel content in yew callus: using high performance liquid chromatography (Sun Jun, Hu Zhenghai. Culture of yew callus and production of paclitaxel. Journal of Northwest University. 2000, 30(1) 55~59 ) to detect the paclitaxel content of the yew culture.

Instruments and reagents: Shimadzu Corporation LC-9A high performance liquid chromatograph; Shim Pack CLC-ODS chromatographic column; CR-9A machine for recording and calculation. Methanol and chloroform are analytically pure. HPLC uses methanol, acetonitrile as the liquid chromatography eluent; Water is high-purity water; Paclitaxel and harringtonine standard products are all products of Sigma; Mobile phase is methanol-acetonitrile-water (25:24:41); flow rate 1mL /min; detection wavelength is 227nm.

Preparation of standard curve: Accurately weigh paclitaxel standard substance and prepare 1mg/mL methanol solution. Then accurately draw 2.00, 4.00, 6.00, 8.00, 10.00μL of the standard solution into the liquid chromatograph, record the chromatogram, measure the peak area A, repeat 3 times, and take the average value. Take the peak area A as the abscissa and the injection volume m as the ordinate to draw a straight line, which is the standard curve. The regression equation can be obtained from the curve: m=bA+c, where b and c are constants, and the linear correlation coefficient of the equation should be r=0.999~1.000.

Sample processing and determination of paclitaxel content: the callus after 30 days of culture was dried at 60°C to constant weight, and ground to 0.25mm. Reflux extraction with chloroform:methanol=6:4 for 3 times, concentration, deesterification with petroleum ether, and vacuum drying under reduced pressure at low temperature to constant weight.

Accurately weigh an appropriate amount of the sample to be tested, add mobile phase to dissolve it, and make a solution to be tested with a concentration of 0.3 mg/mL, and inject it precisely for analysis. Repeat 3 times, measure the peak area A _i , take the average value, and substitute it into the regression equation to obtain m _i . The percentage content P _i % is calculated according to the following formula:

Pi-=m _i /m _w × 100%,

where _mw is the sample mass.

Table 3. Effect of antisense CCR gene on paclitaxel content in Taxus chinensis callus

sample Paclitaxel content in callus (% dry weight) Paclitaxel content increase value (%) Control (untransformed yew callus) 0.019 Transgenic yew callus 1 0.042 121.7 Transgenic yew callus 0.036 89.5

Weave 2 Transgenic yew callus 3 0.063 231.6

Example 5. Using the antisense CCR gene to increase the paclitaxel content of the Taxus chinensis cell line

1) Induction of Taxus chinensis callus: see Example 4.

2) Obtaining the suspension cell line of Taxus chinensis: compare and select the induced Taxus chinensis callus culture, and initially detect the paclitaxel content in the culture. Clonal tissues with faster growth and higher paclitaxel content were obtained for follow-up research.

Inoculate the fine clone callus material into _B5 (containing 2,4-D1.0mg/L, IAA1.0mg/L, KT 0.1mg/L) medium and carry out suspension culture, and every 250mL Erlenmeyer flask is divided into 60mL About 150 mg (dry weight) of the inoculum of each Erlenmeyer flask. The shaker speed is 120r/min, and other culture conditions are the same as solid culture. Two weeks later, under aseptic conditions, first use a sieve to filter out larger particles, and then filter out fine cell clusters. Select 3-5 small cell clusters of cell aggregation, spread out and inoculate on the agar medium of the petri dish for culture. After about 20 days, after the small cell clusters grow up, select the cell clusters with different color, shape and growth state, cut them separately and inoculate them into 50ml small Erlenmeyer flasks for culture. Choose well-growing cell clusters for further observation and selection. The cell clusters selected for further observation and comparison were equally divided into two: one part continued to be cultured and used as selection material; the other part was expanded to be cultured and tested for content. After observation and selection, the cell clusters with slow growth and low paclitaxel content were removed from the selected culture materials; the remaining cell clusters were divided into small pieces and inoculated on new medium for a new round of observation, Compare and test. After multiple rounds of selection, the cell line material with high paclitaxel content and good growth was finally selected, and it was expanded and propagated and subcultured, and further impurities were removed to ensure its purity. After the properties of the culture material were stable, it was used as an excellent cell line. For cultivation experiments and expanded cultivation.

3) Transformation of Taxus chinensis suspension cells with antisense CCR gene: Transformation by gene gun, microprojectile DNA preparation and gene gun bombardment conditions refer to Example 4. After transformation, the yew suspension cells were transferred to a liquid medium containing hygromycin (20mg/L) for screening, and the untransformed yew suspension cells were cultured in the same way as a control. The surviving cells after screening were expanded and cultured, and PCR detection was performed.

4) Determination of paclitaxel content in Taxus chinensis suspension cells: Harvest the culture to be tested, dry it at 60° C. to constant weight, grind it finely, and pass it through a 40-mesh sieve. The paclitaxel content was determined by high-pressure liquid chromatography, and the method is referred to in Example 4.

Table 4. Effect of antisense CCR gene on paclitaxel content in Taxus chinensis suspension cell line

sample Paclitaxel content in yew suspension cell line (% dry weight) Paclitaxel content increase value (%) Control (untransformed taxus suspension cell line) 0.096 Transgenic Taxus Suspension Cell Line 1 0.135 40.6 Transgenic Taxus Suspension Cell Line 2 0.126 31.3 Transgenic Taxus Suspension Cell Line 3 0.168 75.0

The implementation process of the present invention has been described in detail above by means of examples, but it is obvious to those skilled in the art that many equivalent modifications and substitutions can be made during the implementation process. Accordingly, the scope of the present invention is limited only by the appended claims.

Claims (7)

1. A genetic engineering method for improving the content of useful secondary substances in plants, characterized in that it comprises the steps of: a) constructing a chimeric gene, including a nucleic acid fragment encoding a promoter functioning in plant cells, and a nucleic acid fragment encoding a plant lignin synthesis key enzyme hydroxycinnamoyl-CoA reductase CCR that is reversely connected to the promoter, and transcription termination region; b) introducing the chimeric gene into a plant cell selected from soybean or yew that can produce and accumulate useful secondary substances in its organs or tissues when it is a non-transformed plant, to produce a transgenic plant cell; c) growing the transgenic plant cells under conditions suitable for expression of the chimeric gene; d) Screening and identifying transgenic plant cells, compared with similar non-transgenic plant cells, the lignin synthesis key enzyme CCR and lignin content are reduced, while the content of at least one useful secondary substance is increased; e) further culturing the transgenic plant cells into transgenic plants, or transgenic callus, or transgenic cell lines. 2. The method according to claim 1, characterized in that said useful secondary substances are precursor-provided products by the phenylpropanoid pathway. 3. The method according to claim 1, characterized in that said useful secondary substances are active ingredients of medicinal plants. 4. The method according to claim 1, characterized in that the useful secondary substance is isoflavone or paclitaxel. 5. A transgenic plant cell with increased useful secondary substance content obtained by the method of claim 1, characterized in that it contains the constructed chimeric gene. 6. A transgenic plant cell line with increased useful secondary substance content obtained by the method of claim 1, characterized in that it is formed by further culturing the transgenic plant cells. 7. A transgenic plant callus with increased useful secondary substance content obtained by the method of claim 1, characterized in that it is formed by further culturing the transgenic plant cells.

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