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Plant drought inducible synthetic promoter SP16 and application thereof

An SP16, inducible technology, applied in the field of biotechnology and genetic engineering, can solve the problem that natural promoters cannot finely regulate genes

Active Publication Date: 2019-05-31
JILIN AGRICULTURAL UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0006] The purpose of the present invention is to provide a plant drought-inducible promoter SP16 to solve the problem that the natural promoter cannot finely regulate the gene

Method used

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  • Plant drought inducible synthetic promoter SP16 and application thereof
  • Plant drought inducible synthetic promoter SP16 and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0016] Example 1 Design of plant drought-inducible promoter SP16

[0017] By combining the soybean stress transcriptome sequencing data with the existing soybean stress expression profile data in Genbank, 63 target genes regulated by drought were screened out, and then they were listed for cluster module classification; after obtaining 4 clusters, they were identified using motifs The software identified motifs for each cluster separately, and finally obtained 46 common expression elements of promoters related to stress regulation; among them, ERF1, ABFs, ABRE, ABRE like, and Root specific motifs were selected as basic cis-elements, and 2 One copy of ERF1 element, 2 copies of ABFs element, 1 copy of ABRE element, 2 copies of Root specific motif, 2 copies of ABRE like element, 2 copies of Root specific motif constitute a series unit, each two elements With a specific sequence interval of 10 bases in the middle, the above design fragment was connected with a 131bp Root base mini...

Embodiment 2

[0018] Example 2 Construction of promoter SP16 expression vector

[0019] The sequence of SP16 (SEQ ID No.1) was synthesized into the vector pUC57 by artificial synthesis method, and enzyme cutting sites were added at both ends of the sequence Sac l and Nco I; the pUC57-SP16 vector and the pCAMBIA3301 plasmid were used respectively Sac l and Nco I carried out double enzyme digestion, and after recovering the enzyme digestion products respectively, through connection, transformation and identification, the expression vector pCAMBIA3301-SP16 was obtained ( figure 1 ); the vector pCAMBIA3301-SP16 was transformed into Agrobacterium rhizogenes K599 for infecting soybean cotyledonary nodes.

Embodiment 3

[0020] Example 3 Obtaining of Hairy Roots of Transgenic SP16 Soybean

[0021] Using the established soybean hairy root induction system, Agrobacterium rhizogenes K599 containing the pCAMBIA3301-SP16 carrier was used to infect soybean cotyledon nodes. After induction and identification, transgenic positive hairy roots were obtained; drought stress treatment was carried out; the specific method was as follows :

[0022] (1) After mixing sodium hypochlorite and concentrated hydrochloric acid 1:1, sterilize soybean seeds with chlorine gas overnight;

[0023] (2) Wash the soybeans with sterile water and plant them in high-temperature and high-pressure sterilized vermiculite, and moisturize with 1 / 4 Hoagland's solution;

[0024] (3) Cultivate for about 6 days in an artificial climate chamber at 25°C, under light for 16 hours;

[0025] (4) Agrobacterium rhizogenes K599 containing the recombinant plasmid pCAMBIA3301-SP16 was cultured overnight in an incubator at 28°C;

[0026] (5) ...

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Abstract

The invention discloses a plant drought inducible synthetic promoter SP16, a plant expression vector and application of the promoter SP16 in cultivation of drought-resistant transgenic plants. The nucleotide sequence of the plant drought inducible synthetic promoter SP16 is shown in SEQ ID No.1; the nucleotide sequence shown in SEQ ID No.1 is inserted into a vector plasmid of the plant expressionvector; a soybean hairy root genetic transformation system is used for identifying that the promoter SP16 is induced by drought stress, that is, a GUS reporter gene is driven to be expressed under thedrought stress condition, the GUS gene is not expressed under the unstressed condition, and the expression intensity is similar to that of a CaMV 35s promoter. The artificially designed and constructed plant drought inducible promoter SP16 can be directly applied to crop transgenic drought-resistant breeding.

Description

technical field [0001] The invention belongs to the field of biotechnology and genetic engineering, and in particular relates to a plant drought-inducible promoter SP16 and its application. Background technique [0002] A synthetic promoter is a DNA sequence artificially designed according to the element composition and sequence structure characteristics of a natural synthetic promoter. A well-designed artificial promoter can achieve finer regulation of genes, not only can control the expression intensity of genes, but also can accurately control the expression site and expression time of genes. In the field of plant biotechnology in the future, through the correct design and use of artificial synthetic promoters, the expression of a single gene or multiple genes can be carefully regulated so that they can directly respond to specific environmental, physiological or chemical signals. [0003] In 2012, Koschmann et al. published an article in Plant Physiology, which elaborat...

Claims

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Application Information

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IPC IPC(8): C12N15/113C12N15/82A01H5/00A01H6/54
Inventor 李海燕李晓薇王法微王南刘伟灿周永刚董园园刘秀明姚娜刘欣高延妍
Owner JILIN AGRICULTURAL UNIV
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