Authentication and uses of adversity specificly induced two-directional expression activity rice promotor CPIP

A technique for inducing expression and promoters, which is applied in the field of plant genetic engineering and can solve problems such as the lack of bidirectional strong inducible expression activity

Inactive Publication Date: 2006-07-26
HUAZHONG AGRI UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

But so far there is no report on the identification of a promoter with bidirectional strong inducible expression activity and its use in genetic transformation

Method used

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  • Authentication and uses of adversity specificly induced two-directional expression activity rice promotor CPIP
  • Authentication and uses of adversity specificly induced two-directional expression activity rice promotor CPIP
  • Authentication and uses of adversity specificly induced two-directional expression activity rice promotor CPIP

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0022] Example 1, promoter CPIP isolation and identification

[0023] Through the analysis of the drought-induced gene expression profile of the rice variety "Zhonghan 5" (a commercial variety provided by the Shanghai Academy of Agricultural Sciences), a cDNA that was strongly induced by drought (the expression level increased by more than 12 times in the late stage of drought stress) was found. Sequence analysis confirmed that the cDNA was a complete full-length cDNA encoding a chymotrypsin inhibitor gene, and the accession number in the GenBank database was AY878695; for the convenience of analysis, the gene was named OsCPI1. Using the DNA sequence of OsCPI1 to search the rice genome sequence, there is a homologous gene (named OsCPI2) with a sequence similarity of 82% at a position about 1118 base pairs away from the gene, and its transcription direction is opposite to that of OsCPI1. In the KOME database (http: / / cdna01.dna.affrc.go.jp / cDNA / ), a full-length cDNA with a 100% ...

Embodiment 2

[0025] Example 2, detection of induced expression of rice endogenous genes OsCPI1 and OsCPI2

[0026] The rice variety "Zhenshan 97" (a rice variety widely cultivated in China) was used as the material, and were subjected to drought, high-salt stress and abscisic acid (ABA) treatments at the 3-leaf stage. Drought treatment is to absorb the moisture of rice seedling roots and expose to the air at 0h, 30min, 2h, 4h, 6h sampling, high-salt stress is to irrigate the seedlings planted in the soil in 200mM NaCl solution and dry them at 0h, 3h, 1h , sampled after 3d. For ABA treatment, the roots of seedlings were soaked in 100 μM ABA solution and samples were taken after 0h, 30min, 3h, 6h, 24h and 72h. After extracting the total RNA (Trizol reagent, Invitrogen) of the leaves, RNA transfer was carried out (with reference to J. Sambrook et al., Molecular Cloning Experiment Guide, the third edition, Jin Dongyan et al. (translation), Science Press, 2002, Beijing), and Northern hybridiz...

Embodiment 3

[0027] Example 3, identification of promoter CPIP drought-induced activity

[0028] The embodiment of the present invention is to construct the GUS gene expression vector of the promoter CPIP and transform it into the rice variety "Zhonghua 11" (commercial variety from the Crop Research Institute of the Chinese Academy of Agricultural Sciences) to quantitatively detect the drought-induced expression activity of the promoter CPIP. The specific operation is as follows:

[0029] First, the PCR product of the promoter CPIP isolated in Example 1 was ligated into pGEM-T Easy vector (purchased from Promega), transformed into Escherichia coli DH10B (purchased from Promega), and positive clones were screened by blue and white spots. Because the BamHI restriction site was added when designing the primers, the product was connected to the multiple cloning site of the expression vector pCAMBIA1391Z (from the carrier publicly used by CAMBIA, and the carrier contains the GUS reporter gene) ...

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Abstract

The invention discloses a promotor CPIP from Oryza Sativa L in the plant genetic engineering domain, which is characterized by the following: the promotor CPIP possesses SEQ ID NO: 1 with nucleic acid sequence, which controls two alfapsin inhibition sub-genes simultaneously; the alfapsin inhibition sub-gene is controlled by promotor CPIP in backward-forward direction, which makes answering reaction with arid, salt-forced and abscisic acid(ABA); the promotor region contains a plurality of hostile environment answering cis-form appliance elements; the promotor CPIP controls GUS report gene in backward-forward direction to converse rice; the GUS gene is induced and expressed strongly in the condition of arid and salt-forced.

Description

technical field [0001] The invention belongs to the technical field of plant genetic engineering. Specifically, it relates to the isolation, identification and application of a plant stress-specific inducible promoter. The promoter is named CPIP, which is induced by drought or salt and has bidirectional inducible expression activity. It is applied to plant stress resistance genes, especially for the genetic transformation of important crop stress resistance genes, to achieve the purpose of improving plant stress resistance. Background technique [0002] Rice is one of the most important food crops, and my country is the first rice country in the world. Abiotic stress (drought, chilling injury, soil salinization, etc.) seriously threatens rice yield and quality. In the face of adversity stress, many signal transduction systems of plants are turned on, and a group of response genes are induced, including functional protein genes that directly play...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/29C12N15/82
Inventor 熊立仲黄越敏
Owner HUAZHONG AGRI UNIV
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