Looking for breakthrough ideas for innovation challenges? Try Patsnap Eureka!

Cold-resist gene PtrERF109 of trifoliate orange and application thereof in cold resistance genetic improvement of plants

A gene and plant technology, applied in the direction of plant gene improvement, application, plant peptides, etc., can solve problems such as unreported ERF transcription factors, achieve the effect of reducing agricultural production costs and achieving environmental friendliness

Active Publication Date: 2018-12-18
HUAZHONG AGRI UNIV
View PDF3 Cites 8 Cited by
  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, there is no report in the prior art that ERF transcription factors can resist cold

Method used

the structure of the environmentally friendly knitted fabric provided by the present invention; figure 2 Flow chart of the yarn wrapping machine for environmentally friendly knitted fabrics and storage devices; image 3 Is the parameter map of the yarn covering machine
View more

Image

Smart Image Click on the blue labels to locate them in the text.
Viewing Examples
Smart Image
  • Cold-resist gene PtrERF109 of trifoliate orange and application thereof in cold resistance genetic improvement of plants
  • Cold-resist gene PtrERF109 of trifoliate orange and application thereof in cold resistance genetic improvement of plants
  • Cold-resist gene PtrERF109 of trifoliate orange and application thereof in cold resistance genetic improvement of plants

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0065] Cloning of full-length cDNA of PtrERF109 gene in Hovenia trifoliate

[0066] Using Hovenia trifoliate cDNA as a template, high-fidelity enzymes were used to amplify. The amplification system is shown in Table 1, and the amplification procedure is shown in Table 2. The sequences of the amplification primers are:

[0067] PtrERF109-F:5'-ATTCCAGAGCCAACACGAAC-3'

[0068] PtrERF109-R:5'-GAACGTGGGATTTCGCCAGC-3';

[0069] AxyPrep-96 DNA Gel Recovery Kit (Axygene, USA) was used to purify and recover the amplified product, and the purified product was compared with 18-T vector (TaKaRa, Japan) was used for ligation, and the ligation system was shown in Table 3-3. After incubation at 16°C for 30 min, it was transformed into Escherichia coli competent Trans5α.

[0070] Table 1 Gene amplification system

[0071] Table 1 Reaction system of gene amplification

[0072]

[0073] Table 2 Gene amplification PCR program

[0074] Table 2 PCR program for gene amplification

[0075]...

Embodiment 2

[0085] qRT-PCR analysis of PtrERF109 gene under different stress conditions

[0086] The expression pattern of the PtrERF109 gene was analyzed by real-time fluorescent quantitative PCR (qRT-PCR), and the quantitative reagent was QuantiNova TM SYBRGreen PC (QIGEN, Germany), the method refers to the instruction manual, and the reaction system is shown in Table 5.

[0087] Table 5 Quantitative PCR reaction system

[0088]

[0089] Actin from Citrus trifoliate was used as an internal reference gene, and the cDNA obtained by reverse transcription was used as a template. Each sample was repeated four times, and the gene primers used in the reaction are listed in Appendix II. Quantitative PCR reaction in Applied Biosystems QuanStudio TM 7 Flex Real-Time PCR system (ABI, USA) was completed, and the reaction program is shown in Table 6. After the reaction, use 2 -ΔΔCtAlgorithms perform calculations on gene expression. The internal reference gene qRT-PCR primers are:

[0090...

Embodiment 3

[0098] Analysis of subcellular localization and transcriptional activation of PtrERF109 gene

[0099] The ORF region of PtrERF109 (without the stop codon) was amplified and constructed on the 101LYFP vector. The YFP protein was located at the 3' end of the gene, and the expression was driven by the CaMV35S promoter. 35S:PtrERF109-YFP and control 35S:YFP were transiently transformed into the leaf epidermal cells of Nicotiana benthamiana respectively, and laser confocal observation showed that the fluorescence of the control filled the entire epidermal cells, including the cytoplasm and nucleus, while the fluorescence of transformed 35S:PtrERF109-YFP Concentrated only in the nucleus, which was further confirmed by DAPI staining. In addition, cotransfection with mCherry fluorescent protein localized in the nucleus also obtained the same results, indicating that PtrERF109 is a nuclear localized protein (see image 3 A in A, B in 3).

[0100] It has been verified that PtrERF109 i...

the structure of the environmentally friendly knitted fabric provided by the present invention; figure 2 Flow chart of the yarn wrapping machine for environmentally friendly knitted fabrics and storage devices; image 3 Is the parameter map of the yarn covering machine
Login to View More

PUM

PropertyMeasurementUnit
Molecular weightaaaaaaaaaa
Login to View More

Abstract

The invention discloses a cold-resist gene PtrERF109 of Poncirus trifoliata and application thereof in cold resistance genetic improvement of plants. The PtrERF109 gene is a transcription factor isolated and cloned from extremely-cold-resist Poncirus trifoliata, and the sequence is as shown in SEQ. ID NO. 1. Overexpression RNAi vectors of the gene are constructed, and the vectors of the PtrERF109gene are respectively introduced into tobacco, lemon and Poncirus trifoliata by Agrobacterium-mediated genetic transformation to obtain transgenic plants. Verification of biological functions of the obtained transgenic plants indicates that the cloned PtrERF109 gene has the function of improving the cold resistance of the plants. The discovery of the gene provides new genetic resources for anti-reverse molecular design and breeding of the plants, and provides new genetic resources for the implementation of green agriculture and water-saving agriculture. The development and utilization of the genetic resources are conducive to reducing agricultural production costs and achieving environmental friendliness.

Description

technical field [0001] The invention belongs to the field of plant genetic engineering. It specifically involves the isolation and cloning of a transcriptional regulator PtrERF109 from Poncirus trifoliata, and also involves the application of this gene in the genetic improvement of plant cold resistance. The gene is overexpressed in plants, and the cold resistance of transgenic plants obtained is significantly improved. . Background technique [0002] Low temperature will destroy the integrity of plant cell membranes, generate reactive oxygen species, reduce enzyme activity, and inhibit the normal function of cells (Chinnusamy et al., 2007). During the long-term evolution process, plants have formed an efficient and complex response mechanism to resist low temperature damage, that is, to make a series of adjustments at the molecular, cellular and physiological levels to reduce the damage caused by low temperature to plants. The various physiological activities related to t...

Claims

the structure of the environmentally friendly knitted fabric provided by the present invention; figure 2 Flow chart of the yarn wrapping machine for environmentally friendly knitted fabrics and storage devices; image 3 Is the parameter map of the yarn covering machine
Login to View More

Application Information

Patent Timeline
no application Login to View More
IPC IPC(8): C07K14/415C12N15/29C12N15/82A01H5/00A01H6/82A01H6/78
CPCC07K14/415C12N15/8273
Inventor 刘继红王敏黄小三
Owner HUAZHONG AGRI UNIV
Who we serve
  • R&D Engineer
  • R&D Manager
  • IP Professional
Why Patsnap Eureka
  • Industry Leading Data Capabilities
  • Powerful AI technology
  • Patent DNA Extraction
Social media
Patsnap Eureka Blog
Learn More
PatSnap group products

Browse by: Latest US Patents, China's latest patents, Technical Efficacy Thesaurus, Application Domain, Technology Topic, Popular Technical Reports.

© 2024 PatSnap. All rights reserved.Legal|Privacy policy|Modern Slavery Act Transparency Statement|Sitemap|About US| Contact US: help@patsnap.com