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A high temperature-inducible promoter for plant green tissue-specific expression and its application

A green tissue and plant expression vector technology, applied in high temperature inducible promoters and application fields, can solve the problems of short plants, energy consumption, etc.

Active Publication Date: 2021-09-28
ZHEJIANG UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0005] The purpose of this application is to provide an inducible and tissue-specific promoter to solve the defects of energy consumption and plant shortness caused by the overexpression of constitutive promoters in plants under non-essential conditions. Under high temperature conditions, a large number of expression of target genes can be driven in plant green tissues, which can be applied to plant stress resistance genetic engineering

Method used

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  • A high temperature-inducible promoter for plant green tissue-specific expression and its application
  • A high temperature-inducible promoter for plant green tissue-specific expression and its application
  • A high temperature-inducible promoter for plant green tissue-specific expression and its application

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0026] Example 1: Real-time fluorescent quantitative PCR analysis of the expression characteristics of the RCA1 gene

[0027] 1. Use the RNAprep Pure Polysaccharide Polyphenol Plant Total RNA Extraction Kit (Tiangen Biochemical Technology Co., Ltd.) to extract the samples to be tested (young leaves, mature leaves, stems, and petals after three-year-old cutting seedlings of Rhododendron hainan treated at 37°C for 3 hours at high temperature , pistil, stamen) each sample RNA, agarose electrophoresis to detect the integrity, Nanodrop to determine the concentration.

[0028] 2. Take 1 μg RNA and use PrimeScript TM RT reagent Kit with gDNA Eraser (PerfectReal Time) kit (TaKaRa Company) was used to generate cDNA by reverse transcription, which was used as a template for quantitative PCR after dilution. Fluorescent quantitative PCR was carried out using the SYBR dye method, the instrument used was Roche LightCycler 480II, and 18S rRNA was used as an internal reference, according to...

Embodiment 2

[0034] Example 2: pRCA1 promotes the stable expression of the GUS gene

[0035] 1. Cloning of pRCA1

[0036] 1. Using the CTAB method to extract the genomic DNA of Rhododendron hainanensis.

[0037] 2. Using the Rhododendron hainanensis genomic DNA as a template, using the high-fidelity enzyme PrimeSTAR Max, the primer pair consisting of primers pRCA1-F1: 5'-ACTCGGCACAGCTACTACC-3' and pRCA1-R1: 5'-CAAAGGTGGAAACGGCAG-3' was used for PCR amplification , to obtain the PCR product.

[0038] The reaction system was 25 μL: 1 μL of upper and lower primers, 12.5 μL of Max mix, 1 μL of genomic DNA, 9.5 μL of ddH2O; the PCR reaction program was pre-denaturation at 98°C for 60 s; followed by denaturation at 98°C for 10 s, annealing at 57°C for 15 s, extension at 72°C for 60 s, and 30 cycle; 72°C extension for 10 min.

[0039]3. Carry out agarose gel electrophoresis to the PCR amplification product, reclaim the band similar to the size of the target promoter fragment with the DNA gel r...

Embodiment 3

[0060] Example 3: pRCA1 initiates transient expression of LUC gene

[0061] 1. Construction of recombinant plasmid pRCA1::LUC.

[0062] The schematic diagram of the recombinant plasmid pRCA1::LUC is shown in image 3 .

[0063] (1) Construction of plant expression vectors by seamless cloning method. The plant expression vector pGreenⅡ0800-LUC was single digested with Pst I, and the linearized vector fragment was recovered after agarose gel electrophoresis.

[0064] (2) Design the specific primer pRCA1-F5 containing the vector sequence according to the sequence at the gap after single restriction digestion of pGreenⅡ0800-LUC:

[0065] 5'-GCTTGATATCGAATTC CTGCAG CTACTACCAAGCACCTCCGC-3'

[0066] and pRCA1-R5:

[0067] 5'-GGATCCCCCGGG CTGCAG AGAAATCAAGGGTCTGTTTGGGA-3' (the underline is the recognition site of the restriction endonuclease PstI),

[0068] The promoter with vector sequences at both ends was amplified by the high-fidelity enzyme PrimeSTAR Max.

[0069] (3) ...

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Abstract

The invention discloses a high temperature inducible promoter specifically expressed in green tissue of plants and its application. The high-temperature-inducible promoter is named pRCA1, and its sequence is shown in SEQ ID No.1. The promoter pRCA1 of this application has both high temperature inducible and plant green tissue-specific expression characteristics. By constructing the expression vector of pRCA1::target gene, and then introducing the expression vector into the target plant through transgenesis, the target gene can be expressed in the target plant under high temperature conditions. Expressed in green tissue, to achieve the purpose of increasing the expression of the target gene in leaves and other parts under high temperature stress. Under the trend of global warming, it has important application prospects for plant stress resistance genetic engineering.

Description

technical field [0001] The invention relates to the technical field of plant genetic engineering, in particular to a high-temperature-inducible promoter specifically expressed in green tissue of plants and its application. Background technique [0002] A plant promoter is a DNA sequence that can specifically bind to RNA polymerase and its transcription factors and determine the initiation of gene transcription. The promoter is the regulatory center of gene transcription. It not only has basic action elements such as CAAT-box and TATA-box, but also has some cis-acting elements that respond to adversity stress. Transcription under different conditions. Plant promoters can be divided into constitutive, inducible and tissue-specific promoters according to their transcription modes. Constitutive promoters can continuously and efficiently drive gene transcription in different types of cells and at different stages of cell development, and the transcriptional activity is relative...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N15/113C12N15/82A01H5/00A01H5/10A01H6/20A01H6/82
CPCC07K14/415C12N15/8223C12N15/8237C12N15/8273
Inventor 王秀云李铮刘冰周泓夏宜平
Owner ZHEJIANG UNIV
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