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Application of transcription factor involved in regulating tomato steroid alkaloid synthesis

A technology of tomato steroid organism and steroid alkaloid, applied in the biological field, can solve the problem of unclear metabolic regulation mechanism and other problems

Active Publication Date: 2019-12-03
SICHUAN UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Recently, using multi-omics analysis and molecular biology techniques, the molecular mechanism of steroidal alkaloid synthesis in tomato has been basically determined, from the glycolysis pathway through the mevalonate pathway to the cycloartenol pathway to generate cholesterol. Hydroxylation, oxidation, transamination, cyclization and other reactions generate steroidal alkaloids, but the mechanism of metabolic regulation is still unclear

Method used

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  • Application of transcription factor involved in regulating tomato steroid alkaloid synthesis
  • Application of transcription factor involved in regulating tomato steroid alkaloid synthesis
  • Application of transcription factor involved in regulating tomato steroid alkaloid synthesis

Examples

Experimental program
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Effect test

Embodiment 1

[0023] Embodiment 1, the cloning of tomato SlbHLH114 gene

[0024] (1) Extraction of total RNA from tomato leaves

[0025] Take an appropriate amount of tomato leaves, quick-frozen in liquid nitrogen, and use a rapid grinder to thoroughly pulverize three times at 45Hz, 30s, add lysate, and extract total RNA according to the instructions of Plant RNA Extraction Kit V1.5 (BIOFIT). The RNA quality was detected by agarose gel electrophoresis, and the RNA concentration was detected by a NanoDrop 2000 spectrophotometer.

[0026] (2) Cloning of tomato SlbHLH114 gene

[0027] Using the extracted total RNA as a template, cDNA was synthesized according to the instructions of the Takara reverse transcription kit; gene-specific primers were designed according to the sequence of the SlbHLH114 gene, the specific sequence is as follows:

[0028] SlbHLH114 upstream primer: 5'-atggaacaactcgcggtttc-3' (SEQ ID NO.1);

[0029] SlbHLH114 downstream primer: 5'-atttacaaatcatagctagtaagaa-3' (SEQ I...

Embodiment 2

[0032] Embodiment 2, the construction of the plant expression vector containing SlbHLH114

[0033] To study the effect of SlbHLH114 gene on the content of steroidal alkaloids in tomato, overexpression vectors pBin19-E8-SlbHLH114 and 35S-SlbHLH114-NPTII vectors were constructed respectively. The non-specific arms of the respective vectors were added to the forward and reverse primers, and the primers are shown in Table 1.

[0034] Table 1, pBin19-E8-SlbHLH114 and 35S-SlbHLH114-NPTII vector construction primer sequences

[0035]

[0036]Using the correctly sequenced T4 vector containing the SlbHLH114 gene as a template, using SEQ ID NO.5 and SEQ ID NO.6, and SEQ ID NO.7 and SEQ ID NO.8 as primers respectively, using Takara’s Primerstar Max high-fidelity enzyme Perform PCR amplification. Linked into pBin19-E8-GW and P35S-SlbHLH114-T35S-PnoS-NPTII-TnoS-Ω1 vectors (Ying, Su et al.2019, Sarrion-Perdigones, Vazquez-Vilar et al.2013) through gateway and goldenbraid respectively, ...

Embodiment 3

[0038] Example 3, Agrobacterium tumefaciens-mediated genetic transformation of SlbHLH114 overexpression vector to obtain transgenic tomato plants

[0039] (1) Obtaining of Agrobacterium tumefaciens Engineering Bacteria Containing pBin19-E8-SlbHLH114 and 35S-SlbHLH114-NPTII Expression Vectors

[0040] The pBin19-E8-SlbHLH114 and 35S-SlbHLH114-NPTII expression vectors obtained in Example 2 were transformed into Agrobacterium tumefaciens (such as EHA105) by electroporation, and PCR verification was performed. The results showed that the pBin19-E8-SlbHLH114 and 35S-SlbHLH114-NPTII expression vectors had been successfully constructed into the Agrobacterium tumefaciens strain.

[0041] (2) Transformation of tomato with SlbHLH114 gene mediated by Agrobacterium tumefaciens

[0042] a. Tomato sterile cotyledon preparation and pre-cultivation

[0043] Disinfect the seeds with 10ml, 5% PPM (fungus inhibitor), 3×MS solution for 4 hours; about 30 seeds per bottle, spread on 1 / 2MS (Murash...

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Abstract

The invention discloses application of a transcription factor involved in regulating tomato steroid alkaloid synthesis. The amino acid sequence of the transcription factor SlbHLH114 is as shown in SEQID NO.4, and the nucleotide sequence is as shown in SEQ ID NO.3. Overexpression of the transcription factor has a positive regulating effect on the synthesis of tomato steroid alkaloids, so the steroid alkaloid content of transgenic lines can be improved by overexpressing the transcription factor SlbHLH114 in plants, and a theoretical basis is provided for tomato quality improving and excellent breeding.

Description

technical field [0001] The invention relates to the field of biotechnology, in particular to the application of a transcription factor involved in regulating the synthesis of tomato steroidal alkaloids. Background technique [0002] Steroidal alkaloids (SAs) and their glycosylated forms (steroidal alkaloid glycosides, SGAs) are nitrogen-containing toxic compounds in the plant family, also known as Solanaceous alkaloids, belonging to the Solanaceae (Solanaceae), especially Is a common ingredient in the Solanum family. In plants, steroidal alkaloids often act as antibiotics, providing plants with a pre-existing chemical barrier against various pathogens, such as α-tomatine, which acts by disrupting membranes, leading to leakage of electrolytes and depolarization of membrane potential . Studies have shown that due to the presence of steroidal glycoalkaloids and acetylated steroidal glycoalkaloids in tomato cell membranes, the toxicity of α-tomatine to plant cells is reduced, ...

Claims

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Application Information

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IPC IPC(8): C07K14/415C12N15/29C12N15/82A01H5/00A01H6/82
CPCC07K14/415C12N15/8243
Inventor 张阳李彦周璐付饶
Owner SICHUAN UNIV
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