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A Tamarix salt stress-responsive gene tcsbp1 and its miRNA resistance target rtcsbp1 and its application

A technology of salt stress and genetics, applied in the fields of application, genetic engineering, plant genetic improvement, etc.

Active Publication Date: 2022-07-01
NANJING FORESTRY UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

At present, there is no report on the SBP transcription factor of Tamarix chinensis. The cloning and development of the SBP transcription factor of Tamarix chinensis will not only help to elucidate the molecular mechanism of its high salt tolerance, but also provide a basis for screening important salt-tolerant genes and stress-resistant genetic breeding. The theoretical basis and molecular tools are of great value to the utilization of saline-alkali land and the improvement of comprehensive agricultural production capacity

Method used

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  • A Tamarix salt stress-responsive gene tcsbp1 and its miRNA resistance target rtcsbp1 and its application
  • A Tamarix salt stress-responsive gene tcsbp1 and its miRNA resistance target rtcsbp1 and its application
  • A Tamarix salt stress-responsive gene tcsbp1 and its miRNA resistance target rtcsbp1 and its application

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0024] Example 1: Cloning of TcSBP1 gene by RACE technology

[0025] Based on the TcSBP1 transcript of Tamarix RNA-seq data, 5' and 3' RACE primers were designed, and two specific products were amplified by nested PCR, cloned and sequenced by T-vector, and the sequencing results were spliced ​​by overlapping regions, and the complete cDNA was obtained. long. details as follows:

[0026] I. Primer Design

[0027] 3' RACE forward primer (F):

[0028] Outer Primer: 5′-GAATTATTATGTGGCACGAAGGGTAG-3′;

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

[0030] 3' RACE reverse primer (R):

[0031] Outer Primer:

[0032] 5'-CTAATACGACTCACTATAGGGCAAGCAGTGGTATCAACGCAGAGT-3';

[0033]Inner Primer: 5'-CTAATACGACTCACTATAGGGC-3';

[0034] 5'RACE forward primer (F):

[0035] Outer Primer:

[0036] 5'-CTAATACGACTCACTATAGGGCAAGCAGTGGTATCAACGCAGAGT-3';

[0037] Inner Primer: 5'-CTAATACGACTCACTATAGGGC-3';

[0038] 5'RACE reverse primer (R):

[0039] Outer Primer: 5′-AGCAGCTATGA...

Embodiment 2

[0075] Example 2: Analysis of TcSBP1 Gene Expression Pattern by Fluorescence Quantitative PCR

[0076] The response to salt stress of TcSBP1 gene was verified by real-time quantitative PCR technology. The fluorescent quantitative PCR primers were designed based on the CDS region of the TcSBP1 gene, and the internal reference primers were designed based on the TIF gene of Tamarix. The sequences are as follows:

[0077] TcSBP1 forward primer: 5′-ATTTAACCAGCAATGAACCGCAACA-3′;

[0078] TcSBP1 reverse primer: 5′-ACCTGCAGGCTTACGTGTGT-3′;

[0079] TIF forward primer: 5'-ACCACAGGAGTGTCCACCACA-3';

[0080] TIF reverse primer: 5'-TGATGCTTTGCGTGCCAGTG-3'.

[0081] Use saturated dye evagreen (Biotium company) and fluorescence quantitative PCR instrument Viia7 (ABI company) to detect the fluorescence intensity of the PCR process in real time. For the specific PCR system, refer to the evagreen instructions, and calculate and determine by comparing the number of cycles that reach the fluor...

Embodiment 3

[0083] The mega-primer PCR method was used for multi-point mutation to obtain the ORF after synonymous mutation of the miR156 response element of TcSBP1. The specific steps are as follows:

[0084] According to the codon degeneracy, synonymous mutation primers are designed to introduce as many mutation sites as possible, but do not have a poly structure with more than 3 bases, while retaining at least 10nt complementary bases on the flanks. The TcSBP1 mutation primer is 5′- ACAGAATCATCTCATGTAAGCTGTTTCAGTAATCCCATGATC-3', in one round of PCR reaction, a round of PCR product fragments were obtained by amplifying the TcSBP1 mutation primer and the TcSBP1 ORF reverse primer of Example 1. In the second round of PCR reaction, the reaction product of one round was used as the megaprimer, the TcSBP1 ORF forward primer of Example 1, and PCR was performed to obtain the resistance target rTcSBP1 fragment introduced with multiple site mutations. The first and second rounds of PCR were both ...

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Abstract

The invention discloses a tamarisk salt stress response gene TcSBP1 and its miRNA resistance target rTcSBP1 and its application. The nucleotide sequence of the Tamarix salt stress response gene TcSBP1 is shown in SEQ ID No.1, and the amino acid sequence of the expressed protein is shown in SEQ ID No.2. In the present invention, the miR156 response element of the TcSBP1 gene is synonymously mutated by the large primer mutation technology to obtain the miR156 resistance target rTcSBP1, the nucleotide sequence of which is shown in SEQ ID No.3. Under salt stress, TcSBP1 was post-transcriptionally regulated by miR156, while rTcSBP1 was not. The salt stress-responsive gene TcSBP1 of Tamarix and its miR156 resistance target rTcSBP1 are of great value in the research and application of plant salt tolerance or tree resistance breeding.

Description

technical field [0001] The invention belongs to the technical field of plant genetic engineering, and in particular relates to the application of a tamarisk salt stress response gene TcSBP1 and its miRNA resistance target rTcSBP1. Background technique [0002] There is a large area of ​​saline-alkali land that cannot be effectively utilized in my country. Cultivating salt-tolerant germplasm through stress-resistant breeding can increase the area of ​​arable land and increase grain yield. Compared with herbs and gramineous crops, salt-tolerant tree species can grow normally in salinized soils and coastal beaches, and have unique high salt-tolerance characteristics. The salt-tolerance mechanism is the theoretical basis for stress resistance breeding. Resources are of great significance for the cultivation of high salt-tolerant crop varieties. Tamarix chinensis Lour. (Tamarix chinensis Lour.) is the most widely distributed among the tamarisk species in my country. It grows in ...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N15/29C12N15/82C12N15/65C07K14/415A01H5/00
CPCC07K14/415C12N15/8271C12N15/8273C12N15/65
Inventor 徐立安叶友菊王建文陈彩慧吴雅琼辛月
Owner NANJING FORESTRY UNIV
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