Method for improving vitamin C content of tomato quality component by multi-gene polymerization

A technology of gene aggregation and vitamins, applied in biochemical equipment and methods, genetic engineering, plant genetic improvement, etc., can solve problems such as inability to cultivate high-quality tomatoes and resistance breeding, susceptible to external environmental influences, complex synthesis pathways, etc.

Inactive Publication Date: 2019-02-15
HUAZHONG AGRI UNIV
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  • Abstract
  • Description
  • Claims
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AI Technical Summary

Problems solved by technology

[0010] In the prior art, in the tomato germplasm with high expression of related genes, these genes were not polymerized by marker-assisted selection, so that the polymerized plants could increase the content of ascorbic acid in tomato fruits and leaves, and at the same time improve the resistance of tomatoes to oxidative stress, which cannot be used for breeding high-quality Tomatoes and Resistance Breeding Provide the Basis
[0011] (2) The ability to increase AsA content by regulating a single structural gene is limited
As AsA is at the center of the plant's primary metabolic network, and its synthesis pathway is complex and easily affected by the external environment, it is difficult to reveal its regulatory mechanism.

Method used

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  • Method for improving vitamin C content of tomato quality component by multi-gene polymerization
  • Method for improving vitamin C content of tomato quality component by multi-gene polymerization
  • Method for improving vitamin C content of tomato quality component by multi-gene polymerization

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Experimental program
Comparison scheme
Effect test

Embodiment 1

[0062] Embodiment 1: Obtaining of the transgenic polymeric material of the present invention

[0063] In the present invention, the aggregated plants are obtained by using the monovalent transgenic lines (SlGMP-OE, SlGME-OE, SlGGP-OE, and SlGPP-OE) preserved in the laboratory by means of conventional hybridization, and tomato conventional lines (Solanum lycopersicumcv. Ailsa Craig (AC) ) as a control to analyze the effect of transgene aggregation on AsA content in tomato. The specific methods for the molecular identification of the hybrid offspring of transgenic materials are as follows:

[0064]1. When the hybridized transgenic aggregated plants and control plants grow to 3-4 true leaves, DNA is extracted by the CTAB method:

[0065] A. Take young leaves (2 to 3 pieces) and place them at the bottom of a 1.5ml centrifuge tube;

[0066] B. Freshly prepare the sample extract and place it at room temperature;

[0067] C. Add 200 μl of sample extract, grind the sample fully wit...

Embodiment 2

[0084] Embodiment 2: Determination of tomato material ascorbic acid of the present invention

[0085] The determination of ascorbic acid includes the determination of reduced ascorbic acid and total ascorbic acid, wherein the total ascorbic acid includes reduced ascorbic acid and oxidized ascorbic acid. The determination method refers to the method of Hu Tixu's doctoral dissertation (2015). The specific steps are as follows: sample preparation. Select the fresh sample of the sample to be tested (the present invention is tomato red ripe fruit), freeze it with liquid nitrogen, and grind it into powder. Weigh about 0.2 g of leaf powder or about 0.4 g of fruit powder in a 2 mL centrifuge tube pre-cooled with liquid nitrogen, and add 1 mL of 6% TCA (trichloroacetic acid, purchased from Sinopharm Chemical Reagent Co., Ltd.) pre-cooled on ice The solution was mixed well, extracted on ice in the dark for 15 minutes, centrifuged at 12000r / min at 4°C for 15 minutes; sample processing ...

Embodiment 3

[0089] Example 3: Analysis of gene expression in leaves and fruits of tomato material of the present invention

[0090] The total RNA of leaves and fruits of related materials was extracted (Trizol method, the kit was purchased from Invitrogen), and the RNA was reverse-transcribed into cDNA using the reverse transcription kit HiScript 1st Strand cDNA Synthesis Kit (purchased from Novizyme Biotechnology Co., Ltd.). Using LightCycler480SYBR Green I Master Kit (Roche, http: / / www.roche.com / ), perform fluorescence quantitative PCR (qRT-PCR) according to the kit steps to detect the relative expression of ascorbic acid anabolic genes. The qRT-PCR primers used in the experiment are shown in Table 1 (the primers were synthesized by Beijing Tianyi Huiyuan Biotechnology Co., Ltd.):

[0091] Table 1 qRT-PCR primers used in experiments

[0092]

[0093] The qRT-PCR reaction system is SYBR-Green Mix 5 μL, forward primer + reverse primer 0.5 μL + 0.5 μL, cDNA 4 μL. The qPCR reacti...

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Abstract

The invention belongs to the field of biotechnology, and discloses a method for improving the vitamin C content of a tomato quality component by multi-gene polymerization. According to the method, keystructural genes S1GMP, SlGME, SlGGP and SlGPP are selected in the pathway of polymerizing D-Man/L-Gal by hetero-binding labeling; the AsA content and AsA excretion content in petioles and stalks ofdifferent polymerized transgenic lines are determined; and the increment on the AsA content, AsA transport capacity and tolerance to oxidative stress of tomatoes through gene polymerization are analyzed. By cross-polymerizing the overexpression transgenic lines of the four key structural genes in the ascorbic acid synthesis pathway, namely D-mannose/L-galactose pathway, so that the ascorbic acid content and transport in tomato fruits and leaves are increased in polymerized plants, the tomato resistance to oxidative stress is improved simultaneously, and a novel method is provided for cultivation of high-quality tomatoes and resistance breeding.

Description

technical field [0001] The invention belongs to the field of biotechnology, and in particular relates to a method for increasing the vitamin C content of tomato quality components through multi-gene polymerization. Background technique [0002] At present, the existing technologies commonly used in the industry are as follows: [0003] As the main quality component of vegetables, L-ascorbic acid (AsA, vitamin C) is a class of abundant hexose lactone compounds synthesized in plants and most animals. As an important antioxidant and enzyme cofactor, AsA plays a key role in various plant physiological processes, including removing reactive oxygen species (ROS), enhancing plant antioxidant capacity, participating in premature aging and programmed cell death (PCD), regulating Plant cell elongation and division, etc. (Gallie 2013). The ROS produced by plants under photorespiration and photosynthesis are mainly concentrated in chloroplasts, while studies have reported that 30% to ...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/82A01H5/00A01H1/02A01H6/82
CPCA01H1/02C12N15/8243C12N15/8271
Inventor 张余洋叶杰叶志彪李汉霞张俊红
Owner HUAZHONG AGRI UNIV
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