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Plant drought inducible synthetic promoter SP18 and application thereof

An inducible and promoter technology, applied in the fields of biotechnology and genetic engineering, can solve the problem of natural promoters not being able to 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

[0005] The purpose of the present invention is to solve the problem that the natural promoter cannot finely regulate the gene, and provide a plant drought-inducible promoter SP18

Method used

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  • Plant drought inducible synthetic promoter SP18 and application thereof
  • Plant drought inducible synthetic promoter SP18 and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0015] Example 1 Design of plant drought-inducible promoter SP18

[0016] 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, the motif identification software was used to identify the motifs of each cluster, and finally 46 common expression elements of promoters related to stress regulation were obtained. Among them, E2F VARAIANT, RAVI, ABRE, CBF1, Root specific motif, G-box, and Root specific motif were selected as basic cis elements, and each element was repeated twice in series, with a specific sequence interval of 10 bases between the two elements. The above designed fragment was then connected with a 131 bp Root base minipromoter sequence and constructed, named Synthetic Promoter 18, referred to as SP18, and its nucleotide sequence was SEQ ID No.1.

Embodiment 2

[0017] Example 2 Construction of promoter SP18 expression vector

[0018] The sequence of SP18 (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-SP18 vector and the pCAMBIA3301 plasmid were used respectively Sac l and Nco I carried out double enzyme digestion, after reclaiming the digestion products respectively, through connection, transformation and identification, the expression vector pCAMBIA3301-SP18 was obtained ( figure 1 ); the vector pCAMBIA3301-SP18 was transformed into Agrobacterium rhizogenes K599 for infecting soybean cotyledon nodes.

Embodiment 3

[0019] Example 3 Obtaining of Hairy Roots of Transgenic SP18 Soybean

[0020] Using the established soybean hairy root induction system, Agrobacterium rhizogenes K599 containing the pCAMBIA3301-SP18 vector 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 :

[0021] (1) After mixing sodium hypochlorite and concentrated hydrochloric acid 1:1, sterilize soybean seeds with chlorine gas overnight;

[0022] (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;

[0023] (3) Cultivate for about 6 days in an artificial climate chamber at 25°C, under light for 16 hours;

[0024] (4) Agrobacterium rhizogenes K599 containing the recombinant plasmid pCAMBIA3301-SP18 was cultured overnight in an incubator at 28°C;

[0025] (5) S...

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Abstract

The invention discloses a plant drought inducible synthetic promoter SP18, a plant expression vector and application of the promoter SP18 in cultivation of drought-resistant transgenic plants. The nucleotide sequence of the plant drought inducible synthetic promoter SP18 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; the identification of the situation that the promoter SP18 drives the expression of a reporter gene GUS in a soybean hairy root genetic transformation system shows that the promoter SP18 can be induced by drought stress, and the expression intensity is similar to that of a CaMV 35s promoter.

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 SP18 and its application. Background technique [0002] At present, the selection and breeding of crop varieties in my country mainly rely on traditional breeding techniques. Over the years, people have bred a large number of high-yield and high-quality varieties through extensive cross-breeding, making important contributions to the production of agricultural products and national food security. However, with the gradual excavation of important genetic resources, the bottleneck effect of traditional breeding is becoming more and more apparent, and it is becoming more and more difficult to breed new varieties. At present, biotechnology has become one of the main bodies of the new scientific and technological revolution, and the bioindustry is promoting the rapid development of agricultural biotechnology. Transge...

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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