Novel method for prolonging shelf life of tomato fruit by utilizing genetic engineering technology

A genetic engineering and shelf life technology, applied in genetic engineering, recombinant DNA technology, botanical equipment and methods, etc., can solve the problem of not significantly delaying fruit ripening and softening, affecting fruit nutrients, active ingredients and flavor, unsuitable Production application and other issues, to achieve the effect of large application value and extended shelf life

Inactive Publication Date: 2018-05-08
CHENGDU INST OF BIOLOGY CHINESE ACAD OF S
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

At present, the target manipulation genes of genetic engineering mainly include genes related to ethylene synthesis, ethylene signal transduction and cell wall degradation. Among these genes, some gene modifications do not significantly delay fruit ripening and

Method used

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  • Novel method for prolonging shelf life of tomato fruit by utilizing genetic engineering technology
  • Novel method for prolonging shelf life of tomato fruit by utilizing genetic engineering technology
  • Novel method for prolonging shelf life of tomato fruit by utilizing genetic engineering technology

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0033] Example 1: cDNA cloning and spatiotemporal expression analysis of tomato S1EB1 gene

[0034] 1. SlEB1 gene sequence analysis

[0035] Search the tomato genome database, tomato has two AtEB1 homologous genes, namely SlEB1a (Solyc03g116370) and SlEB1b (Solyc02g092950); the DNA sequence of SlEB1a is shown in SEQ ID NO 1; the DNA sequence of SlEB1b is shown in SEQ ID NO 2.

[0036] 2. Extraction of tomato total RNA

[0037] (1) Take an equal amount of tomato roots, stems, leaves, flowers and fruits at different development stages, divide them into groups, and grind them fully in liquid nitrogen; then add TRIzol Reagent (1mL TRIzol Reagent per 100mg sample), and fully After mixing, place at room temperature for 5 min.

[0038] Wherein, the different developmental stages are: IG1 (Immature Green1, immature green stage I), IG2 (Immature Green1, immature green stage II), IG3 (Immature Green1, immature green stage III), MG (MatureGreen, mature green stage ), Br (Breaker, brea...

Embodiment 2

[0073] Example 2: Construction of the overexpression vector of the SlEB1a gene and the RNAi vector of the SlEB1a / SlEB1b gene

[0074] In order to verify the role of SlEB1a gene, an overexpression vector of SlEB1a gene was constructed; at the same time, considering that SlEB1a and SlEB1b genes may have functional redundancy, an RNAi vector of SlEB1a / SlEB1b gene was constructed to simultaneously silence SlEB1a and SlEB1b genes.

[0075] In the following sequence, the underlined sequence indicates the restriction endonuclease cutting site in the brackets after the sequence. If there are multiple restriction enzymes in the brackets, the restriction endonuclease site represented by the sequence is the same as the restriction endonuclease in the brackets. Enzymes correspond one to one in sequence; The sequence in is the linker sequence.

[0076] 1. Construction of SlEB1a overexpression vector

[0077] In order to facilitate subsequent detection, the GFP gene was connected to the ...

Embodiment 3

[0120] Embodiment three: Agrobacterium-mediated genetic transformation transforms tomato variety AC +

[0121] The overall idea is: transform the overexpression vector pBI121:GFP-SlEB1a of the SlEB1a gene and the RNAi vector pBI121:SlEB1a / SlEB1bRi of the SlEB1a / SlEB1b gene into Agrobacterium EHA105 respectively, and then transform the tomato variety AC + , Tomato AC + Seeds were preserved in our laboratory. Specific steps are as follows:

[0122] 1. Agrobacterium transformation

[0123] On ice, add 10 μL of plasmid to Agrobacterium EHA105 competent cells, mix gently, and place for 40 minutes; liquid nitrogen cold shock for 1 minute; quickly put in a 37°C water bath for 5 minutes; place on ice for 2 minutes; LB culture solution (5g / LYeast extract, 10g / L NaCl, 10g / L tryptone) 600μL, 170rpm, 28℃ shaking culture for 4h; Spread evenly on LB solid medium containing 50mg / L kanamycin and 100mg / L rifampicin; incubate the plate upside down at 28°C for 2 days, and the grown colonies...

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Abstract

The invention belongs to the technical field of biology, and particularly relates to a novel method for prolonging a shelf life of a tomato fruit by utilizing a genetic engineering technology. The shelf life is mainly related to ethylene synthesis, ethylene signal conduction, cell wall degradation and the like, and the existing technology has some researches on related target control genes, but maturing and softening of the fruit are not effectively delayed after the genes are modified or nutrient ingredients, active ingredients and the like of the fruit are also affected although maturing andsoftening of the fruit can be delayed, and therefore, the existing technology is not suitable for production application. The SlEB1 gene of the tomato is tightly related to synthesis of ethylene andcell wall degradation, and the shelf life of the tomato generated by transgenosis is extremely prolonged after the gene is silent, and therefore, the novel method can be practically applied to production and life.

Description

technical field [0001] The invention belongs to the field of biotechnology, and in particular relates to a new method for extending the shelf life of tomato fruit by using genetic engineering technology. Background technique [0002] Tomatoes are representative of fruit vegetables. Tomato fruit is bright in color, thick and juicy, unique in flavor, and rich in vitamins, organic acids, amino acids, minerals, and flavonoids. And many other benefits, so it is deeply loved by consumers. [0003] The global annual tomato net output value exceeds 50 billion US dollars, but due to the short shelf life of tomatoes, more than 25% of the total output per year rots and deteriorates, resulting in huge economic losses. Tomato fruit is a respiratory climacteric fruit, which is characterized in that the respiration is strengthened in the early stage of fruit ripening, a large amount of ethylene is synthesized, and the fruit is rapidly overripe and softened in a short period of time. The...

Claims

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Application Information

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IPC IPC(8): C12N15/82A01H5/00A01H6/82
CPCC12N15/8205
Inventor 汪松虎蒲勤华许春燕黄维藻李辉高兰阳
Owner CHENGDU INST OF BIOLOGY CHINESE ACAD OF S
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