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Tamarix chinensis salt stress response key gene TcARF6 and application thereof

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

Active Publication Date: 2017-07-21
NANJING FORESTRY UNIV
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  • 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

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  • Tamarix chinensis salt stress response key gene TcARF6 and application thereof
  • Tamarix chinensis salt stress response key gene TcARF6 and application thereof

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) ...

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Abstract

The invention discloses a tamarix chinensis salt stress response key gene TcARF6 and application thereof. The nucleotide sequence of the key gene TcARF6 is as shown in SEQ ID NO.1. The amino acid sequence of expression protein of the tamarix chinensis salt stress response key gene TcARF6 is as shown in SEQ ID NO.2. The TcARF6 gene is cloned by taking tamarix chinensis subjected to tamarix chinensis salt stress treatment as a material and by an RACE technology. The expression mode of the TcARF6 gene after the tamarix chinensis is stressed is detected by a real-time fluorescent quantitative detection technology, and the criticality of the gene responding to stress is verified. Moreover, a tamarix chinensis overexpression vector pH35GS-TcARF6 is established by a gateway technology, and the TcARF6 can be efficiently expressed in a transgene under the drive of a promoter P35GS. TcARF6 relative quantification in the salt stress response process indicates that the TcARF6 only performs down-regulated expression in a root specifically and rapidly, the key action of the gene in stress response is verified, and an important application value in the field of forest resistance transfer breeding is achieved.

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

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Application Information

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IPC IPC(8): C12N15/29C12N15/82C07K14/415A01H5/00
CPCC07K14/415C12N15/8273
Inventor 徐立安王建文陈彩慧王玮胥猛
Owner NANJING FORESTRY UNIV
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