Cloning and application of tomato ATG8f gene

A tomato gene technology, applied in the field of tomato ATG8f gene cloning, can solve the problems of decreased pollen vigor, affecting yield and quality, falling flowers and fruits, etc., and achieves the effect of rapid detection

Inactive Publication Date: 2016-07-27
ZHEJIANG UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0002] In recent years, my country's protected horticulture industry has developed rapidly, and the production area is still growing at a rate of about 10% per year. However, due to the simple structure of the facilities and backward facilities and equipment, the production of protected vegetables often encounters adversities such as low temperature and low light in winter and

Method used

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  • Cloning and application of tomato ATG8f gene
  • Cloning and application of tomato ATG8f gene
  • Cloning and application of tomato ATG8f gene

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0060] Cloning of Tomato ATG8f Gene

[0061] 1. Tomato Total RNA Extraction

[0062] Adopt PlanttotalRNAextractionkit (TIANGEN) to extract the total RNA of young tomato leaves, and its steps are:

[0063] (1) Take 0.1g of leaves and grind them in liquid nitrogen, add 1mL of lysate, and then process them with a homogenizer;

[0064] (2) Place the homogenized sample at 15-30°C for 5 minutes to completely separate the nucleic acid-protein complex;

[0065] (3) Centrifuge at 12,000 rpm for 5 minutes at 4°C, remove the supernatant, and transfer to a new RNase-free centrifuge tube.

[0066] (4) Add 200 μL of chloroform, cover the tube cap, shake vigorously for 15 seconds, and place at room temperature for 3 minutes;

[0067] (5) Centrifuge at 12,000rpm for 10min at 4°C. The sample will be divided into three layers: a yellow organic phase, an intermediate layer and a colorless aqueous phase. RNA is mainly in the aqueous phase, and the volume of the aqueous phase is about the lysat...

Embodiment 2

[0085] GFP gene clone

[0086] Design specific primers (GFP-F and GFP-R in Table 2) according to the GFP sequence, and add a restriction enzyme site (AscI) to the GFP-F primer, and then amplify the GFP gene to obtain PCR2.

[0087] The PCR reaction system is as follows:

[0088]

[0089] PCR reaction condition setting:

[0090]

[0091] Table 2 GFP gene amplification primer sequence list

[0092]

Embodiment 3

[0094] GFP-ATG8f vector construction

[0095] 1. PCR amplification

[0096] GFP-F and SlATG8f-R were used as primers, and PCR products 1 and 2 were used as templates to amplify, purify, digest, and connect to pFGC1008 vector.

[0097] The PCR reaction system is as follows:

[0098]

[0099] PCR reaction condition setting:

[0100]

[0101] 2. Cut glue recycling

[0102] After the PCR reaction was completed, the target band was recovered and purified according to the DNA Agarose Gel Recovery Kit (TIANGEN), and the specific steps were as follows:

[0103] (1) Column equilibration step: add 500 μL of equilibrium solution BL to the adsorption column CA2 (the adsorption column is placed in the collection tube), centrifuge at 12,000 rpm (~13,400×g) for 1 min, pour off the waste liquid in the collection tube, and the adsorption Column CA2 is put back in the collection tube again;

[0104] (2) Cut out a single target DNA band from the agarose gel (cut off the excess part as...

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Abstract

The invention discloses cloning and application of a tomato ATG8f gene. The gene plays an important role in a tomato autophagosome formation process. The tomato ATG8f gene is cloned, and an agrobacterium tumefaciens engineering bacterium A containing a tomato ATG8f gene over-expression vector is constructed; the agrobacterium tumefaciens engineering bacterium A is used for carrying out mediated transformation on an explant of a target plant to prepare an ATG8f gene transgenic plant; the vegetative growth of the plant is vigorous; the agrobacterium tumefaciens engineering bacterium A is infected with nicotiana benthamiana and a point-shaped autophagosome can be observed. The tomato ATG8f gene disclosed by the invention has important application value in cultivation of good tomato varieties and autophagosome detection.

Description

technical field [0001] The invention belongs to the field of plant genetic engineering, and in particular relates to the cloning and application of tomato ATG8f gene. Background technique [0002] In recent years, my country's protected horticulture industry has developed rapidly, and the production area is still growing at a rate of about 10% per year. However, due to the simple structure of the facilities and backward facilities and equipment, the production of protected vegetables often encounters adversities such as low temperature and low light in winter and spring, and continuous cropping obstacles. The photosynthetic efficiency of tomatoes, cucumbers and other protected vegetables is significantly reduced, the pollen vigor is reduced, and the flower and fruit drop is serious, which in turn affects their yield and quality, and has become a bottleneck problem restricting the development of the protected vegetable industry. [0003] Tomato (Solanum lycopersicum L.) is a t...

Claims

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Application Information

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IPC IPC(8): C12N15/29C12N15/11C12N15/10C12N15/84A01H5/00
CPCC07K14/415C12N15/10C12N15/8205C12N15/8273
Inventor 周杰王玉喻景权
Owner ZHEJIANG UNIV
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