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

A key gene tcarf6 of Tamarix salt stress response and its application

A key gene, salt stress technology, applied in tamarisk salt stress response key gene TcARF6 and its application field

Active Publication Date: 2020-01-31
NANJING FORESTRY UNIV
View PDF3 Cites 0 Cited by
  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0004] At present, there is no report on the ARF family of Tamarix plants. Cloning, development and utilization of these gene resources will not only help to elucidate the molecular regulation mechanism of forest tree salt tolerance, but also promote the process of tree molecular breeding. inestimable value

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
  • A key gene tcarf6 of Tamarix salt stress response and its application
  • A key gene tcarf6 of Tamarix salt stress response and its application

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0020] 1. Cloning of TcARF6 gene by RACE technology

[0021] Based on the existing results of tamarix salt stress transcriptome sequencing research, RACE primers at the 5' and 3' ends were designed to obtain corresponding PCR products, cloned into T-vectors, and sequenced after positive screening of the inserted fragments. The sequence of the sequencing results passed The overlapping regions were spliced ​​to obtain the full-length cNDA. RNA derived from

[0022] TcARF6 RACE primers:

[0023] RACE Adapter contains a uniquely designed Adapter Primer sequence (5'-CTAATACGACTCACTATAGGGCAAGCAGTGGTATCAACGCAGAGT-3') to facilitate primer design.

[0024] 3'RACE Forward Primer:

[0025] Outer Primer: 5'-TACAGCATCCTCAACAGCAAATGGT-3';

[0026] Inner Primer: 5'-TAGACATGGTTGGTACAGACTC-3';

[0027] 3'RACE reverse primer:

[0028] Outer Primer: 5'-CTAATACGACTCACTATAGGGCAAGCAGTGGTATCAACGCAGAGT-3';

[0029] Inner Primer: 5-'CTAATACGACTCACTATAGGGC-3';

[0030] 5'RACE Forward Primer:

...

Embodiment 2

[0076] The response of TcARF6 gene to salt stress was verified by fluorescent quantitative PCR technology. Fluorescent quantitative PCR primers were designed based on the ORF region of TcARF6 gene, and internal reference primers were designed based on the TIFY gene of Tamarix chinensis. The sequence is as follows:

[0077] TcARF6 forward primer: 5'-TCTGGGCATTCGGCGAGCTA-3';

[0078] TcARF6 reverse primer: 5'-GCGGCTATTTGTCGCTGCTG-3';

[0079] TIFY forward primer: 5'-TGGAGTAACTGAACCAGGGAGGAG-3';

[0080] TIFY forward primer: 5'-GGCTGTAGGTGCCTGAACTGG-3'.

[0081] Utilize saturating dye evagreen (Biotium company) and fluorescence quantitative PCR instrument Viia7 (ABI company) to detect the fluorescence intensity of the PCR process in real time. The specific PCR system refers to the instructions of evagreen, and calculates and determines by comparing the cycle number of the TcARF6 gene and the internal reference that reach the fluorescence threshold. Relative expression of TcARF6...

Embodiment 3

[0082] Embodiment 3 TcARF6 gene plant expression vector construction

[0083] The overexpression vector of TcARF6 gene was constructed by gateway technology. Using specific PCR primers (TcARF6ORF primers), cDNA was used as a template to carry out PCR amplification, and the TcARF6 gene ORF was constructed into the entry vector. The entry vector was pCRTM8 / GW / TOPOTM vector (Invitrogen). The reaction system is: Fresh PCR product (purified) 10-20ng; Salt solution 1μL; pCRTM8 / GW / TOPOTM vector 1μL; add sterile ddH 2 O to make up 6 μL. The reaction procedure is: stand at room temperature for 30 min.

[0084] Pick positive clones from the screening culture plate for PCR detection and sequencing verification, the entry vector with TcARF6 gene and the plant expression vector pH35GS for LR reaction. Vector plasmid such as figure 2shown. The reaction system is: linearized dentry clone 100ng; purified destination vector (100ng / μL) 1.5μL; LR Clonase IIenzyme mix 2μL; add TE (pH 8.0) ...

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

No PUM Login to View More

Abstract

The invention discloses a tamarix salt stress response key gene TcARF6 and its application. The nucleotide sequence of the key gene TcARF6 is shown in SEQ ID NO.1. The expression protein of tamarix salt stress response key gene TcARF6, its amino acid sequence is shown in SEQ ID NO.2. The present invention uses tamarix salt stress treated tamarix as material, and clones TcARF6 gene through RACE technology. Through real-time fluorescent quantitative detection technology, the expression pattern of TcARF6 gene was detected after Tamarix tamarix was stressed, and the key of its response to stress was verified. At the same time, the gateway technology was used to construct the overexpression vector pH35GS‑TcARF6 of Tamarix tamarix. Driven by the promoter P35GS, TcARF6 can be highly expressed in the transgene. The relative quantification of TcARF6 in response to salt stress showed that TcARF6 was only specifically and rapidly down-regulated in roots, which verified the key role of this gene in stress response and has important application value in the field of forest tree resistance breeding.

Description

technical field [0001] The invention belongs to the technical field of plant genetic engineering, and in particular relates to a key gene TcARF6 in response to tamarind salt stress and an application thereof. Background technique [0002] There are more than 200,000 square kilometers of saline-alkali land in my country and it is growing continuously. The inability of saline-alkali land to be used in agricultural production has led to an increasingly acute contradiction that my country's food production cannot meet the needs of population growth. There is an urgent need for plant resources to efficiently transform saline-alkali land; Tamarix is ​​a native tree species in my country , which is one of the most salt-tolerant woody plants, plays an important role in maintaining the ecological stability of coastal wetland salinized areas such as the Yellow River Delta, and is also an important resource for the construction of coastal shelterbelts in my country. Although there are m...

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
Patent Type & Authority Patents(China)
IPC IPC(8): C12N15/29C12N15/82C07K14/415A01H5/00A01H6/00
CPCC07K14/415C12N15/8273
Inventor 徐立安王建文陈彩慧王玮胥猛
Owner NANJING FORESTRY 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