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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  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

Their gene products need to be evaluated for safety and environmental impact, which has rais

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

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Experimental program
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Example Embodiment

[0038] Example 1

[0039] Construction of an intermediate vector containing PSY2 gene

[0040] According to the published tomato PSY2 gene sequence 【1】 EF534739, used the biological software DNAStar to design the upstream and downstream primers of the gene, named PSY2F, PSY2R, respectively, introduced BamHI and SacI restriction enzyme sites, the primer sequence is as follows: PSY2F: 5'AAC GGATCCGTGTATCAAAGGTAGTAAGGGAAC-3'; PSY2R: 5'ATAGAGCTCACTTGCTAGTGGGGAAGTTG-3'.

[0041] The total RNA of tomato seedlings was extracted and used as a template for RT-PCR reaction. The recovered PSY2 gene and pBI101.2 plasmid were digested with BamHI and SacI respectively, and the gel was cut to recover the large fragment of pBI101.2 plasmid after digestion And PSY2 gene fragment, ligated overnight at 16°C by T4 DNA ligase. The ligation product was transformed into E. coli DH5α strain, and Kan-resistant clones were selected for BamHI and SacI double enzyme digestion verification. The successfully v...

Example Embodiment

[0043] Example 2

[0044] Construction of intermediate vector containing promoter E8

[0045] According to the fruit-specific E8 promoter sequence of Tomato Cherry reported by Deikman et al., two PCR primers were designed with the biological software DNAStar, namely E8F: 5'gCAAGCTTA GGAATTTCACGAAATCG3' to introduce HindIII restriction site; E8R: 5'CgGGATCCTCTTTTGCACTGTGAATGAT3' to introduce 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 digested with HindIII and BamHI, the gel was cut to recover the digested E8 gene fragment.

[0047] The pMDT-18 vector was reacted with the recovered E8 fragment at 16°C overnight, and the ligation product was transformed into E.coli DH5α, spread on LB medium containing Amp, and single colonies were selected in LB liquid medium and cultured at 37°C overnight. The plasmid is extracted and used for PCR identification. And named pMDTE8.

Example Embodiment

[0048] Example 3

[0049] Construction of an intermediate vector containing promoter E8 and PSY2 genes

[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 from the gel, and they were ligated overnight at 16°C by T4 DNA ligase. The plasmid was transformed into E. coliDH5α strain, the Kan resistant clone was verified by PCR, the recombinant plasmid successfully verified by PCR was sequenced, and the correctly connected recombinant plasmid 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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