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Method for obtaining tomatoes with high lycopene content and resistance to bacterial wilt through recombining genes

A lycopene and recombinant gene technology, applied in the field of plant transgenic technology, can solve the problems of food safety and environmental safety concerns of genetically modified crops, and achieve the effects of eliminating the safety of genetically modified food, increasing the content of lycopene, and facilitating screening

Inactive Publication Date: 2012-03-21
GUANGZHOU UNIVERSITY
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Their gene products need to be evaluated for safety and environmental impact, which has raised concerns about the food safety and environmental safety of some genetically modified crops

Method used

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  • Method for obtaining tomatoes with high lycopene content and resistance to bacterial wilt through recombining genes
  • Method for obtaining tomatoes with high lycopene content and resistance to bacterial wilt through recombining genes
  • Method for obtaining tomatoes with high lycopene content and resistance to bacterial wilt through recombining genes

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0039] Construction of Intermediate Vector Containing PSY2 Gene

[0040] According to the published tomato PSY2 gene sequence 【1】 For EF534739, the upstream and downstream primers of the gene were designed with the biological software DNAStar, named PSY2F and PSY2R respectively, and BamHI and SacI restriction enzyme sites were introduced respectively. The primer sequences are as follows: PSY2F: 5'AAC GGATCCGTGTATCAAAGGTAAGTAAGGGAAC-3'; PSY2R: 5'ATAGAGCTCACTTGCTAGTGGGGAAGTTG-3'.

[0041] Extract the total RNA of tomato seedlings, and use this as a template to carry out RT-PCR reaction. After the recovered PSY2 gene and pBI101.2 plasmid are digested with BamHI and SacI respectively, the large fragment of pBI101.2 plasmid after digestion is recovered by gel cutting and PSY2 gene fragments were ligated overnight at 16°C with T4 DNA ligase. The ligation product was transformed into E. coli DH5α strain, and Kan-resistant clones were picked for BamHI and SacI double digestion verif...

Embodiment 2

[0044] Construction of intermediate vector containing promoter E8

[0045] According to the fruit-specific E8 promoter sequence of tomato Cherry species reported by Deikman et al., two PCR primers were designed with the biological software DNAStar, respectively E8F: 5'gCAAGCTTA GGAATTTCACGAAATCG3' introduced HindIII restriction site; E8R: 5'CgGGATCCTCTTTTGCACTGTGAATGAT3' introduced BamHI restriction site.

[0046] The total RNA of tomato seedlings was extracted and used as a template for RT-PCR reaction. After the recovered E8 gene was double-digested with HindIII and BamHI, the digested E8 gene fragment was recovered by gel cutting.

[0047] React the pMDT-18 vector with the recovered E8 fragment overnight at 16°C, transform the ligation product into E.coli DH5α, spread it on the LB medium containing Amp, pick a single colony in the LB liquid medium, and culture it overnight at 37°C, a small amount Plasmids were extracted for PCR identification. And named pMDTE8.

Embodiment 3

[0049] Construction of Intermediate Vector Containing Promoter E8 and PSY2 Gene

[0050] After the constructed pMDTE8 and pBIPSY2 plasmids were digested by HindIII and BamHI respectively, the large fragment of pBIPSY2 plasmid and the E8 promoter fragment were recovered by gel cutting, and ligated overnight at 16°C by T4 DNA ligase. The plasmid was transformed into Escherichia coli E.coliDH5α strain, and the Kan-resistant clone was verified by PCR. The recombinant plasmid successfully verified by PCR was sequenced, and the recombinant plasmid with correct connection was named pBEPSY2.

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Abstract

The invention provides a method for obtaining tomatoes with high lycopene content and resistance to bacterial wilt through recombining genes, and belongs to the technical field of biological genes. An intermediate carrier is constructed before PSY2 (phytoene synthase) and E8 (ethylene) promoters are inserted into a GFP (Green Fluorescent Protein) sequence of a basic carrier pX6-GFP, and then genetic transformation is carried out to obtain the tomatoes with high lycopene content and resistance to bacterial wilt. The basic carrier pX6-GFP gene is positioned between a left boundary (LB) sequence and a right boundary (RB) sequence of a carrier T-DNA (Triplex Deoxyribose Nucleic Acid), XVE heterozygous protein and an inducer 17-beta-estradiol contained in the basic carrier pX6-GFP gene are combined, the expression of CRE recombinase can be started so as to remove selected markers, and the problems in food safety and environment safety of genetically modified crops are solved. The genetic transformation of unmarked carriers in the invention not only can eliminate the doubt of people on genetically modified food safety, but also can provide a new idea for obtaining new varieties of tomatoes with the resistance to bacterial wilt.

Description

technical field [0001] The invention relates to the technical field of biological genes, in particular to a plant transgenic technology method. Background technique [0002] Tomatoes are one of the most widely cultivated fruits and vegetables in Guangdong. Tomato varieties suitable for winter and spring cultivation in South China include "Suifeng" and "Jinfeng No. 1". There is a huge demand for tomatoes in Guangdong, and the average daily consumption in Guangzhou alone reaches 10,000-25,000 kilograms. In addition, Guangdong is one of the three major domestic tomato sauce production areas, and its export sales have risen linearly. In 2005, the total output of tomato paste in my country was 1 million tons, of which one-third was produced in Guangdong. With China's rapid economic growth in recent years, people's living standards have gradually improved, and their awareness of tomato products has also gradually increased. It is inevitable that the consumption will increase yea...

Claims

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Application Information

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IPC IPC(8): C12N15/63A01H5/00
Inventor 王小兰刘顺枝
Owner GUANGZHOU UNIVERSITY
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