Gene engineering method for raising plant useful secondary substance content

A secondary substance and useful technology, applied in the direction of plant genetic improvement, botanical equipment and methods, using micro-injection method, etc., can solve the problem of lignin content that has not been seen, and achieve the effect of increasing content and increasing yield

Inactive Publication Date: 2004-07-21
ZHEJIANG ACADEMY OF AGRICULTURE SCIENCES
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Problems solved by technology

[0004] 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. to report

Method used

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  • Gene engineering method for raising plant useful secondary substance content
  • Gene engineering method for raising plant useful secondary substance content
  • Gene engineering method for raising plant useful secondary substance content

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0042] Example 1. Cloning of lignin synthesis key enzyme gene CCR and construction of its antisense expression vector

[0043] 1) Plant material: The tested Arabidopsis thaliana L. Heynh was Columbia ecotype, donated by Mr. Xu Zhengwei, Institute of Plant Physiology and Ecology, Chinese Academy of Sciences. Genomic DNA of Arabidopsis thaliana was extracted from leaves by CTAB method.

[0044] 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.

[0045] 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...

Embodiment 2

[0065] Example 2. Transformation of Arabidopsis thaliana with antisense CCR gene

[0066] 1) Arabidopsis transformation

[0067] 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.

[0068] 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 ...

Embodiment 3

[0081] Example 3. Using antisense CCR gene to increase soybean isoflavone content

[0082] 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).

[0083] 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 4000rpm and 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...

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Abstract

A gene engineering method for increasing the content of useful secondary substance in plant, such as isoflavone, polyphenol and taxol, includes configuring chimeric gene, introducing it to plant cell to generate, the transgenic plant cell, making it grow in the condition benefiting expression, screening and discriminating the transgenic plant cell, and further derivative culture.

Description

technical field [0001] 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 [0002] 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 organ...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): A01G7/00A01H1/00A01H5/00C12N5/10C12N15/52C12N15/62C12N15/74C12N15/87C12N15/89
Inventor 陈锦清黄锐之王伏林刘智宏陈笑芸胡张华吴关庭郎春秀金卫郭晨悦
Owner ZHEJIANG ACADEMY OF AGRICULTURE SCIENCES
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