A plant drought-inducible artificially synthesized promoter sp2 and its application

A promoter, inducible technology, applied in the field of genetic engineering, can solve the problems of low specificity and low expression activity

Inactive Publication Date: 2020-07-24
JILIN AGRICULTURAL UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, the activities of the natural promoters currently isolated and identified are limited, such as low expression activity and low specificity, etc.

Method used

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  • A plant drought-inducible artificially synthesized promoter sp2 and its application
  • A plant drought-inducible artificially synthesized promoter sp2 and its application
  • A plant drought-inducible artificially synthesized promoter sp2 and its application

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0018] Example 1. Design of plant stress-inducible promoter SP2

[0019] 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. Select G-box, ABRE, ABF, SORLIP1, CCA1, and E2F as the basic cis-elements, each element is repeated 4 times in series, and the two elements are separated by a random sequence of 7-9 bases. Synthetic Promoter 2, referred to as SP2, is constructed by connecting with 35S core promoter Core CaMV 35S, and its nucleotide sequence is SEQ ID No.1.

Embodiment 2

[0020] Example 2. Construction of promoter SP2 expression vector

[0021] The sequence of SP2 (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 Bam H l and Hind III. The pUC57-SP2 vector and pBI121 plasmid were used respectively Bam H l and Hind III carried out double enzyme digestion, and after recovering the enzyme digestion products respectively, after connection, transformation and identification, the expression vector pBI121-SP2 (such as figure 1 ). The vector pBI121-SP2 was transformed into Agrobacterium EHA105 for infecting tobacco.

Embodiment 3

[0022] Example 3. Obtaining of Transgenic SP2 Tobacco

[0023] The leaf disc transformation method (Horsch et al. 1988) was followed. The Agrobacterium solution is diluted with sterilized water to 106-107 cells / mL (generally about 30 times diluted). Take sterile tobacco leaves, remove the main veins, cut the leaves into square shapes, then immerse them in the diluted bacterial solution, and infect for 8-10 minutes.

[0024] 1) Co-culture

[0025] After taking out the leaf explants, blot the bacteria solution on sterile filter paper, place the leaf surface down on the surface of the MS medium, and culture in the dark at 24°C-25°C for 2 days.

[0026] 2) Selective culture

[0027] Transfer the co-cultured leaves to the selective differentiation medium (containing 250 mg / L cephalosporin; 100 mg / L kanamycin), at 28°C, with 16 hours of light per day, and change the medium every 15-20 days.

[0028] 3) Rooting culture

[0029] When the differentiated resistant shoot grows to ab...

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Abstract

The invention discloses a soybean-induced promoter SP2, which belongs to the construction of an artificially synthesized promoter. It uses G-box, ABRE, ABF, SORLIP1, CCA1, and E2F as basic cis elements, and each element repeats four times in series. The elements are separated by a random sequence of 7-9 bases, and the above designed fragments are then connected with the 35S core promoter Core CaMV 35S to construct, named SP2. The identification of the expression of the reporter gene GUS driven by the SP2 promoter in transgenic tobacco showed that the SP2 promoter can be induced by drought, salt, saline-alkali, ABA and low temperature stress, and is extremely sensitive to drought stress. It can be directly applied to crop transgenic stress resistance breeding.

Description

technical field [0001] The invention belongs to the field of genetic engineering, in particular to a plant drought-inducible promoter SP2 and its application in transgenic plants. [0002] technical background [0003] A promoter regulates the expression of a gene by binding to one or more transcription factors. Each promoter has its specific sequence structure and binding site. The main structure of the promoter includes the core promoter sequence and upstream promoter elements, which can guide the transcription of nearby DNA segments. Promoters play a very important role in the initiation and regulation of gene transcription, and are important building blocks in genetic engineering. In recent years, with the development of synthetic biology, artificially synthesized functional elements have shown great application potential in the field of metabolic engineering. The metabolic flux of cells is mainly regulated by the transcriptional level, and the promoter is an important ...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N15/113C12N15/82C12N15/62A01H5/00A01H6/54A01H6/82
CPCC07K14/415C12N15/8273
Inventor 李海燕李晓薇王法微刘伟灿王南董园园刘秀明姚娜刘欣肖洪庆戢舒涵
Owner JILIN AGRICULTURAL UNIV
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