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A key gene tcsbp5 regulating the salt tolerance of Tamarix and its application

A key gene, salt tolerance technology, applied in the fields of application, genetic engineering, plant genetic improvement, etc.

Active Publication Date: 2022-04-19
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 factors of Tamarix plants. The cloning and development of Tamarix SBP transcription factors will not only help to elucidate the molecular regulation mechanism of high salt tolerance in halophytes, but also provide important information for the screening of important salt tolerance genes and stress resistance. Genetic breeding provides a theoretical basis and molecular tools, and has important application value for the utilization of saline-alkali land and the improvement of comprehensive agricultural production capacity

Method used

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  • A key gene tcsbp5 regulating the salt tolerance of Tamarix and its application
  • A key gene tcsbp5 regulating the salt tolerance of Tamarix and its application
  • A key gene tcsbp5 regulating the salt tolerance of Tamarix and its application

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0021] Example 1: Cloning of the TcSBP5 gene by RACE technology

[0022] Based on tamarix RNA-seq data, TcsBP5 sequences were designed for 5′ and 3′ RACE primers, two specific products were amplified by nested PCR, and sequenced by T-vector cloning, and the sequencing results were spliced by overlapping regions to obtain the full length of cNDA.

[0023] The details are as follows:

[0024] I. Primer design

[0025] The forward primers for 3′RACE are:

[0026] Outer PrimerF：

[0027] Inner PrimerF：

[0028] The 3′RACE reverse primers are:

[0029] Outer PrimerR：

[0030]

[0031] Inner PrimerR：

[0032] The 5′ RACE forward primer is

[0033] Outer PrimerF：

[0034]

[0035] Inner PrimerF：

[0036] The 5′RACE reverse primers are:

[0037] Outer PrimerR：

[0038] Inner PrimerR：

[0039] II.3′RACE reaction process:

[0040] (1) Reverse transcription, adding the following components to a small centrifuge tube placed on ice: 1 μg Total RNA (tamarind leaf for plant material), ...

Embodiment 2

[0074] Example 2: TcSBP5 gene plant expression vector construction

[0075] The hyperexpression vector of TcSBP5 gene was constructed by channel cloning technology. Using a specific PCR primer (TcSBP5 ORF primer in Example 1), using cDNA as a template, PCR amplification, the TcSBP5 gene ORF is constructed into an entry vector. The entry vector is pCRTM8 / GW / TOPOTM vector (Invitrogen). The reaction system was: Fresh PCR product (purified) 10-20ng, Salt solution 1μL, pCRTM8 / GW / TOPOTM vector 1μL, and sterile ddH2O supplemented with 6μL. The reaction procedure is: let stand at room temperature for 30 min.

[0076] Positive clones were selected from screening plates for sequencing validation, and positive entry vectors performed LR reactions with plant expression vector PBI121GW. Carrier plasmids such as Figure 1as shown. The reaction system is: 100 ng of entry vector, PBI121GW vector (100 ng / μL) 1.5 μL, LR Clonase II enzyme mix 2 μL, and 10 μL of TE (pH 8.0) plus TE. Reaction conditi...

Embodiment 3

[0077] Example 3: genetic transformation of TcSBP5 gene

[0078] The constructed PBI121GW-TcSBP5 overexpression vector was transferred to Agrobacterium strain EHA105 by liquid nitrogen freeze-thaw method, and TcSBP5 gene was transferred to Arabidopsis thaliana by agrobacterium tidbit infestation. The resulting positive Arabidopsis seeds were measured for germination rate and plant phenotypic observation on MS solid medium containing 0.3% NaCl (51 mM). Figure 2 PCR detection of TcSBP5 inserts for 10 successfully transformed transgenic Arabidopsis thaliana; Figure 3 Transgenic Arabidopsis thaliana, which is hyperexpressed with TcSBP5, was compared with the overall morphology of wild-type thaliana, and the growth matrix was MS solid medium containing 0.3% NaCl (51 mM). 1 was 28d growth of 0.3% NaCl medium for transgenic Arabidopsis thaliana, 2 was 14 days for 14 days after growth of transgenic Arabidopsis thaliana 0.3% NaCl medium was transferred to NACl-free MS medium for 14 days, 3...

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Abstract

The invention discloses a key gene TcSBP5 regulating tamarix salt tolerance, the nucleotide sequence of which is shown in SEQ ID No.1. The protein product expressed by TcSBP5 is Tamarix SBP transcription factor, and its amino acid sequence is shown in SEQ ID No.2. The invention obtains Arabidopsis thaliana overexpressing the TcSBP5 gene by transforming Arabidopsis thaliana, the salt-tolerant germination rate of its seeds is significantly reduced, the salt-tolerant physiological index of its plants is significantly reduced, and the typical salt-sensitive phenotype of growth inhibition is generally shown , indicating that the gene is an important key factor regulating plant salt tolerance, and has important application value in the field of forest tree salt tolerance breeding.

Description

Technical field [0001] The present invention belongs to the field of plant genetic engineering technology, specifically relates to a key gene TcSBP5 regulating the salt tolerance of Tamarix and its applications. Background [0002] According to the statistics of the Food and Agriculture Organization of the United Nations, 6.5% of the total land area is unculculable saline-alkali land, China's saline-alkali land area of about 200,000 square kilometers, saline alkali land produces high salinity stress, directly affect crop yield, through stress-resistant breeding means to make full use of saline-alkali land, to increase crop yield, solve the food crisis is of great significance. Although some general salt tolerance mechanisms have been elucidated in model species such as Arabidopsis thaliana and rice, many studies of salt tolerance genes have shown that the current effect of salt tolerance improvement in transgenic plants using these genes is not ideal, cannot meet the needs in ac...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N15/29C07K14/415C12N15/82A01H5/00A01H5/10A01H6/20
CPCC07K14/415C12N15/8273
Inventor 胥猛王建文叶友菊陈彩慧樊航
Owner NANJING FORESTRY UNIV
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