A rice stress-inducible promoter p ossalt1 and its application

A promoter and inducible technology, applied in the fields of biotechnology and plant genetic engineering, can solve the problems of ineffective regulation of target genes, unfavorable practical application and promotion, and short plants

Active Publication Date: 2022-03-18
HUNAN UNIV OF SCI & ENG
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, most of the current genetic engineering operations use constitutive promoters to drive the expression of these key stress-resistant genes. Although the obtained transgenic plants can show strong resistance to stress, the constitutive promoters will drive the expression of the key stress-resistant genes. The constant and continuous expression of genes in various plant tissues leads to excessive consumption of materials and energy in cells, and the expression of target genes cannot be effectively regulated in time and space, so sometimes it will bring some negative effects, such as causing short stature of plants , developmental delay and waste of material and energy are not conducive to potential practical application promotion

Method used

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  • A rice stress-inducible promoter p <sub>ossalt1</sub> and its application
  • A rice stress-inducible promoter p <sub>ossalt1</sub> and its application
  • A rice stress-inducible promoter p <sub>ossalt1</sub> and its application

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Experimental program
Comparison scheme
Effect test

Embodiment 1

[0034] P OsSalT1 Cloning of gene promoters

[0035] according to P OsSalT1The promoter sequence of primer6.0 is used to design primers, obtains a pair of primers, and sequence is as shown in SEQ ID NO:2 and SEQ ID NO:3, as shown in table 2, P1 (underline is PstI restriction site) and P2 ( The underline is the BamHI restriction site) and the restriction site was added in front of the primer. The rice genomic DNA was used as a template for PCR amplification. Amplification conditions: 94°C, 3min; 94°C, 30s, 57°C, 30s, 72°C, 2min10s, 30 cycles; 72°C extension 10min. PCR products were separated by 1% agarose gel electrophoresis figure 1 As shown, UV cut glue. The target fragments were recovered using a gel extraction kit, and connected into the cloning vector pEASY-Blunt for sequencing.

[0036] Primers were designed based on the sequence of ATAF1, and the primer pair sequences are shown in SEQ ID NO:4 and SEQ ID NO:5. As shown in Table 2, A1 (the underline is the restrictio...

Embodiment 2

[0040] P OsSalT1 Construction of promoter expression vector and transformation of Arabidopsis

[0041] The promoter of OsSalT1 was cloned into the plant binary vector pCambia1300-221 to drive the expression of GUS gene. The plasmid map of pCambia1300-221 is as follows figure 2 shown. Digest pCambia1300-221-GUS and the pEASY-Blunt cloning vector containing the promoter fragment with PstI and BamHI, and recover the target fragment and vector fragment. Use T4 DNA ligase to ligate overnight at 22°C to obtain pCambia1300-221-P OsSalT1 -GUS plasmid such as image 3 As shown, then transform Escherichia coli XL1-Blue, extract and identify recombinant plasmids such as Figure 4 shown. Wild-type Arabidopsis was transformed by Agrobacterium-mediated genetic transformation of Arabidopsis inflorescences.

[0042] Digest pCambia1300-221-P with BamHI and SacI OsSalT1 -GUS and the pEASY-Blunt cloning vector containing ATAF1, recovering the target fragment and the vector fragment. Use...

Embodiment 3

[0044] Screening of Transgenic Arabidopsis and Treatment of Abiotic Stress

[0045] Hygromycin was used to screen transgenic Arabidopsis seeds, and the homozygous transgenic strains of the T2 generation were identified. Genome PCR verification results showed that P OsSalT1 The promoter and GUS specific primers amplified bands of 2100bp and 280bp respectively, the results are as follows Figure 6 , indicating that the vector was successfully transferred into the recipient. After abiotic stress (drought, low temperature, high salt) treatment and histochemical staining analysis results are as follows Figure 7 shown. Transplant Arabidopsis thaliana cultured in 1 / 2MS solid medium for about 2 weeks into 1 / 2MS liquid medium for 48 hours. Part of the seedlings were treated with 300mmol / L NaCl medium for 4 hours with high salt; part of the seedlings were placed on filter paper , simulated drought treatment for 4h; some seedlings were treated with 100μmol / LSA medium for 4h; some seed...

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Abstract

The invention relates to the technical fields of biotechnology and plant genetic engineering, in particular, the invention relates to a rice stress-inducible promoter P OsSalT1 and its application. The promoter is derived from the rice stress resistance gene OsSalT, and its DNA sequence is shown in SEQ ID NO:1. The downstream of the above-mentioned promoter is connected with a gene to be expressed (such as a stress resistance gene) and a recombinant expression vector is constructed, and the transgenic rice transformed with the recombinant expression vector is induced by abiotic stress conditions (high salt, low temperature, drought) to regulate the gene to be expressed. gene expression. The invention can improve the stress resistance of rice, and plays an important role in ensuring national food security, economic development and social stability.

Description

technical field [0001] The invention relates to the technical fields of biotechnology and plant genetic engineering, in particular, the invention relates to a rice stress-inducible promoter P OsSalT1 and its application. Background technique [0002] Rice is a crop that is extremely sensitive to temperature. The common problem in the world's main rice producing areas is the problem of low temperature and chilling damage. During the planting process of rice, if the budding stage, seedling stage and booting flowering stage are subjected to chilling damage, the growth of rice seedlings will be retarded, the number of tillers will be reduced, and eventually the rice yield will be greatly reduced. In addition, temperature is also an important environmental factor affecting rice grain quality. [0003] Soil salinization has seriously affected the safety of rice planting and has become a major factor affecting the high yield of rice. The impact of salt stress on rice is mainly du...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N15/113C12N15/11C12N15/82C12N15/29A01H5/00A01H5/10A01H6/46A01H6/20
CPCC07K14/415C12N15/8237C12N15/8238C12N15/8273
Inventor 刘永昌何福林向军曾丽亚李英袁志辉张斌刘小文
Owner HUNAN UNIV OF SCI & ENG
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