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Application of GRAS11 to regulation of synthesis of plant terpenoids and/or development of glandular hairs

A technology of terpenoids and plants, applied in the field of genetic engineering, can solve the problems of unclear regulation and weak regulation, and achieve the effects of improving insect and disease resistance, promoting the development of glandular hairs, and improving nutritional quality

Pending Publication Date: 2021-08-06
TOBACCO RES INST CHIN AGRI SCI ACAD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

The regulation of these transcription factors on the metabolism of terpenoids is relatively weak, and mainly through the regulation of TPS gene expression, the regulation effect on the gene expression of the upstream precursor synthesis pathway (MEP and MVA pathway) is not yet clear, and the use of these transcription factors by genetic engineering technology The application of factors regulating the synthesis and metabolism of plant terpenoids has not been reported yet.
So far, there are no reports on the regulation of plant terpenoid synthesis and glandular hair development by GRAS transcription factors

Method used

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  • Application of GRAS11 to regulation of synthesis of plant terpenoids and/or development of glandular hairs
  • Application of GRAS11 to regulation of synthesis of plant terpenoids and/or development of glandular hairs
  • Application of GRAS11 to regulation of synthesis of plant terpenoids and/or development of glandular hairs

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0043] Example 1: Isolation and sequence analysis of GRAS11 gene fragments

[0044] The tomato leaf material was thoroughly ground in liquid nitrogen, transferred to a 2ml centrifuge tube, mixed with 1ml Trizol (Invitrogen, Cat. 15596-018), and left at room temperature for 5 minutes. Centrifuge at 12000rpm for 10min, and discard the precipitate. Add 0.2ml chloroform to the supernatant, mix well, and centrifuge at 12000rpm for 10min. Take the supernatant and add 0.5ml isopropanol to precipitate RNA. Centrifuge at 12000 rpm for 10 min, wash the precipitate with 75% ethanol, dry in vacuum, and dissolve in 50 μl of water.

[0045] Use RNA PCR system (TaKaRa, Cat.DRR019A) to synthesize cDNA, the reaction solution is: 10×RT buffer: 1 μl; dNTP: 1 μl; MgCl 2 : 2μl; Oligo dT: 0.5μl; RNA: 1μl; RNase Inhibitor: 0.5μl; AMV Reverse Transcriptase: 0.5μl; 2 O: 4 μl. The reaction conditions are: react at 42°C for 30 minutes, and react at 85°C for 5 minutes.

[0046] use FastPfu DNA Po...

Embodiment 2

[0049] Embodiment 2: vector construction

[0050] Using pEASY-GRAS11 as a template, the forward primer VIGS-GRAS11-F-BamHI (5'-GGATCCTTGATGTTGAGAAACTTCGC-3') and the reverse primer VIGS-GRAS11-R-XbaI (5'-TCTAGATGTCGAATCTTTGGAACCAC-3') were used for PCR amplification. Gene amplification, PCR amplification conditions: 95°C for 5min; 95°C for 30s, 55°C for 30s, 72°C for 30s; 30 cycles, recover target gene fragments after agarose gel electrophoresis. The gene fragment and the vector pTRV2 were double-digested with BamHI and XbaI respectively, and the digested products were recovered, mixed according to the molar ratio of the vector and the gene fragment at a ratio of 1:1, and ligated at 16°C for 30 minutes (Takara Company, DNA Ligation Kit Ver.2.1) . The ligation product was transformed into E.coli DH5α, spread on LB solid plates containing 50mg / L Kanamycin (Kan), and cultured in the dark at 37°C for 12-16 hours. Single colonies were picked for PCR clone screening, and positive ...

Embodiment 3

[0052] Example 3: Gene expression detection

[0053] The tomato leaves, roots and other materials were thoroughly ground in liquid nitrogen, transferred to a 2ml centrifuge tube, mixed with 1ml Trizol (Invitrogen, Cat. 15596-018), and left at room temperature for 5 minutes. Centrifuge at 12000rpm for 10min, and discard the precipitate. Add 0.2ml chloroform to the supernatant, mix well, and centrifuge at 12000rpm for 10min. Take the supernatant and add 0.5ml isopropanol to precipitate RNA. Centrifuge at 12000 rpm for 10 min, wash the precipitate with 75% ethanol, dry in vacuum, and dissolve in 50 μl of water.

[0054]Use RNA PCR system (TaKaRa, Cat.DRR019A) to synthesize cDNA, the reaction solution is: 10×RT buffer: 1 μl; dNTP: 1 μl; MgCl 2 : 2μl; Oligo dT: 0.5μl; RNA: 1μl; RNase Inhibitor: 0.5μl; AMV Reverse Transcriptase: 0.5μl; 2 O: 4 μl. The reaction conditions are: 42°C for 30 minutes, 85°C for 5 minutes.

[0055] CFX Connect Real-Time System (Bio-Rad Laboratories, I...

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PUM

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Abstract

The invention relates to the technical field of gene engineering, in particular to an application of GRAS11 to regulation of synthesis of plant terpenoids and / or development of glandular hairs. According to the invention, firstly, amplification is performed in Solanumlycopersicum L. to obtain a transcription factor GRAS11, and the transcription factor GRAS11 is specifically expressed in glandular hairs of leaves and stems. The overexpression of GRAS11 can significantly enhance the expression level of terpene synthase and upstream precursor synthetic pathway genes in tissues such as leaves, thereby improving the yield of terpenoids and increasing the volume of glandular hairs. The invention provides beneficial values for research and application in aspects of improving crop nutritional quality, improving crop insect resistance and disease resistance and the like in genetic engineering.

Description

technical field [0001] The invention relates to the technical field of genetic engineering, in particular to the application of GRAS11 in regulating the synthesis of plant terpenoids and / or the development of glandular hairs. Background technique [0002] Terpenoids are the largest family of plant secondary metabolites with an estimated 80,000 structures. According to the number of five-carbon units on the core skeleton, terpenoids are divided into hemiterpenes, monoterpenes, sesquiterpenes, diterpenes, sesquiterpenes, and triterpenes. Terpenoids exist widely in plants and play an important role in resisting pathogens and herbivorous insects and attracting insect pollination. Some terpenoids are also used as drugs, pesticides, spices, etc., and have important economic value. Enhancing the biosynthetic pathway of endogenous terpenoids in plants and increasing the yield of compounds is of great significance for improving crop quality and improving crop resistance to insects ...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/29C07K14/415C12N15/82A01H5/12A01H5/04A01H6/82
CPCC07K14/415C12N15/8222C12N15/8241C12N15/8243
Inventor 杨长青
Owner TOBACCO RES INST CHIN AGRI SCI ACAD
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