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Method for improving caffeic acid content and/or biological self-luminous intensity of plant

A technology of self-luminescence and caffeic acid, which is applied in the biological field, can solve the problems of low caffeic acid content and weak luminescence of bio-self-luminous tobacco, and achieve the effect of improving the intensity of self-luminescence

Active Publication Date: 2022-06-07
ZHEJIANG UNIV HANGZHOU GLOBAL SCI & TECH INNOVATION CENT
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

[0005] However, the luminescence of bioluminescent tobacco in published articles is weak, and an important limiting factor is the low content of endogenous caffeic acid in plants. Therefore, it is necessary to enhance the endogenous caffeic acid synthesis pathway of plants through genetic manipulation and make the caffeic acid synthesis The pathway is coupled with the plant self-luminescence pathway to create plants with stronger bioluminescent brightness, which plays an important role in the application of self-luminous plants

Method used

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  • Method for improving caffeic acid content and/or biological self-luminous intensity of plant
  • Method for improving caffeic acid content and/or biological self-luminous intensity of plant
  • Method for improving caffeic acid content and/or biological self-luminous intensity of plant

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0049] Example 1 Construction process of enhanced caffeic acid synthesis (eCAS) module vector pYLTAC380GW-7G

[0050] This study used the TransGene Stacking II system for multigene assembly. The NtCM1, NtPAT, NtADT2, NtPAL1, NtC4H genes in the tobacco (Nicotianatabacum) genome corresponding to CDS were amplified. 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 connected to remove the selection marker gene expression cassette element through a one-step BP recombination reaction, thereby constituting an enhanced Caffeic acid synthesis pathway vector pYLTAC380GW-NtCM1-NtPAT-NtADT2-NtPAL1-NtC4H (pYLTAC380GW-7G).

[0051] For the sequence information of NtCM1, please refer to the gene accession number: XP_009768497; the sequence information of NtPAT, please refer to the gene accession number: XP_016480150; the sequence information of NtADT2, please refer...

Embodiment 2

[0061] Example 2 Analysis of transient transformation of tobacco leaves with enhanced caffeic acid synthesis (eCAS) module vector

[0062] 1. The EHA105 bacterial solution containing the verified correct vector plasmid pYLTAC380GW-7G was streaked and activated on the LA+Kana+Rif plate, 28°C, 36h, and the colonies were picked from the plate and transferred to LB+Kana+Rif+15μM As medium, 28°C, 200rpm to OD 600 = 0.8-1.0, 4000 rpm, 10 min to collect the cells, suspend the Agrobacterium cells with an infection solution (containing 10 mM MgCl, 10 mM ES, 150 μMAs), and let stand at room temperature for 2-3 h.

[0063] 2. To verify the transient expression in tobacco leaves, use a 1mL needle to gently make a small opening on the surface of the tobacco leaves, and 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. Indoor normal culture for 48h, and then sample some leaves for relative ex...

Embodiment 3

[0064] Example 3 Analysis of caffeic acid content and luminous intensity in eCAS module transgenic plants mediated by Agrobacterium

[0065] 1. Streak the EHA105 bacterial solution containing the verified correct vector plasmid pYLTAC380GW-7G on the LA+Rif+Kana plate, 28°C, 36h, pick and clone into 3-5ml LB medium at 200rpm, 28°C, 36h , according to the ratio of 1:100-1:50 to expand and cultivate 50ml for 3-5h to OD=0.6, then centrifuge the bacterial liquid, use MS0 liquid medium (MS+3%Sucrose+PH5.8, 50ml) to suspend the bacterial cells to OD=0.6 for infection;

[0066] 2. Select the successfully obtained bioluminescent transgenic tobacco (there is a luminescence-related gene Hisps-CPH-H3H-NPGA-Luz in the genome) seeds, and plant them on sterile MS medium for 4-5 weeks to fully expanded tobacco health Leaves, cut into 0.5cm square size with a scalpel (cut the leaf margins to avoid the main veins), with the upper surface of the leaves facing down in MS1 ​​solid medium (MS+0.5m...

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Abstract

The invention discloses a method for improving caffeic acid content and / or biological self-luminous intensity of plants, and belongs to the technical field of biology. The method comprises the following steps: (1) sequentially integrating an NtCM1 gene, an NtPAT gene, an NtADT2 gene, an NtPAL1 gene and an NtC4H gene into a receptor vector by utilizing a multi-gene assembly technology, and constructing to obtain a multi-gene vector; and (2) introducing the target gene segment into a receptor plant by using a transgenic technology to obtain a transgenic plant with enhanced caffeic acid content and / or biological self-luminous intensity. The gene participates in synthesis of caffeic acid in the plant for the first time, and co-expression of the gene and a self-luminous Hisps gene, a CPH gene, an H3H gene, an NPGA gene and a Luz gene is carried out, so that the biological self-luminous intensity of the plant can be remarkably improved. The invention provides a feasible technical scheme for breeding and production of high caffeic acid plants and sustainable luminous plants.

Description

technical field [0001] The invention relates to the field of biotechnology, in particular to a method for improving plant caffeic acid content and bioluminescence intensity by utilizing the co-expression of tobacco NtCM1, NtPAT, NtADT2, NtPAL1 and NtC4H genes. Background technique [0002] Caffeic acid, also known as "3,4-dihydroxycinnamic acid", molecular formula C 9 H 8 O 4 , molecular weight 180.15. Caffeic acid is generally found in plants. As a polyphenolic substance, caffeic acid 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 or as an API (Ekeuku et al. al., 2021), the latest research found that caffeic acid is also the most important precursor in the bioluminescence metabolic pathway (Reuter et al., 2020), so the synthesis and application of caffeic acid has broad application prospects. [0003] It has long been ...

Claims

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Application Information

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IPC IPC(8): C12N15/53C12N15/54C12N15/60C12N15/61C12N15/82A01H5/00A01H6/82
CPCC12N9/90C12N9/1096C12N9/88C12N9/0073C12N15/8243C12N15/8261C12Y504/99005C12Y206/01C12Y402/01091C12Y403/01024C12Y114/13011Y02A50/30
Inventor 都浩郑鹏葛洁瑜
Owner ZHEJIANG UNIV HANGZHOU GLOBAL SCI & TECH INNOVATION CENT
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