Photoinduced promoter gene and application thereof

A light-induced and promoter technology, applied in the field of genetic engineering, to achieve the effect of solving food safety problems

Active Publication Date: 2017-07-28
JIANGSU ACADEMY OF AGRICULTURAL SCIENCES
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

This light-inducible promoter derived from rice RbcS gene and intron sequence is beneficial to solve the food safety problem of seeds in transgenic rice without affecting the growth and development of plants

Method used

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  • Photoinduced promoter gene and application thereof
  • Photoinduced promoter gene and application thereof
  • Photoinduced promoter gene and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0037] Example 1 Synthesis and sequence analysis of rice light-inducible promoter p727 fragment

[0038] 1. Promoter cloning

[0039] Unexpectedly, the inventors found that in Nipponbare of ssp.japonica of Oryzasativa (rice), the promoter p727 (SEQ ID NO.1), which contains the OsrbcS gene and intron sequences, contains multiple light-responsive cis-acting elements As well as the essential elements of the promoter, it was found through follow-up experiments that unexpectedly, the promoter p727 also has light induction and leaf tissue specificity, and the exogenous gene can be specifically and moderately expressed in the leaves of transgenic rice. Roots, seeds (embryo and endosperm) not expressed in.

[0040]Hind III and Xba I restriction sites were introduced at both ends, and the p727 fragment was artificially synthesized after the corresponding sequences were amplified from the rice genome. The p727 fragment of the promoter is a 417bp restriction product ( figure 1 ), in t...

Embodiment 2

[0044] Embodiment 2 constructs and transforms the recombinant binary expression plasmid (such as image 3 shown);

[0045] 1. Extract the plasmids of p1300-GUSplusNOS and p-OsRBCS-727 (Axygen plasmid mini-prep kit). p1300-GUSplusNOS was transformed from pCAMBIA1300 binary expression vector (purchased from CAMBIA Company), the GUSplus-NOS gene was added between the Kpn I and EcoR I restriction sites, and there was no promoter sequence upstream.

[0046] 2. Double digestion of p1300-GUSplusNOS plasmid and p-OsRBCS-727 plasmid with restriction endonucleases Hind III and Xba I (purchased from NEB Company).

[0047] 3. Gel recovery (Axygen Gel Recovery Kit) p1300-GUSplusNOS digested plasmid and promoter p727 fragment, ligated at 16°C for 30 minutes (T4 ligase, purchased from NEB Company), and transformed into Trans10 competent produced by Quanshijin Company In the cells, the positive clones were selected by smearing, and the promoter amplification primers were used for sequencing...

Embodiment 3

[0048] Example 3 Agrobacterium-mediated genetic transformation of rice and positive detection of transgenic plants

[0049] The plant expression vector p-OsRBCS-727:GUSplus constructed in Example 2 was transformed into the Agrobacterium tumefaciens strain EHA105 (gifted by Nanjing Agricultural University) by the cold shock method, and then infected with the japonica rice variety Nipponbare (Nipponbare of ssp.japonica of Oryza sativa (rice)) mature embryo-induced callus.

[0050] After co-culture, they were selected with 50 mg / L hygromycin.

[0051] Operations such as rice tissue culture, Agrobacterium-mediated transformation, selection of resistant callus and plant regeneration refer to general methods in the field.

[0052] Positive plants were positively detected by PCR method, and the primers were all internal primers of GUSplus gene (see below). The product size is 400bp.

[0053] GUSplus-F:5'-TAGTTTTTCTCTTCATTTTCT-3'SEQ ID NO2;

[0054] GUSplus-R: 5'-GCTTGTTACGAATGACT...

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Abstract

The invention provides a photoinduced promoter gene which has a nucleotide sequence or a homologous nucleotide sequence thereof. The photoinduced promoter gene comprises a RbcS gene from paddy Nipponbare of ssp.japonica of Oryza sativa (rice) and an intron sequence. The invention further provides a method of induced expression of exogenous gene in plants. The method includes: introducing a vector into a plant callus cell; growing to be a transgenic plant with photoinduced expression. The photoinduced promoter gene is utilized to replace a constitutive promoter to obtain a binary expression vector containing the photoinduced promoter; genetic transformation technology is utilized to guide the binary expression vector into a plant genome, and directional operation of a target gene can be realized to obtain a transgenic plant with photoinduced expression of the target gene.

Description

technical field [0001] The invention belongs to the field of genetic engineering, and in particular relates to a light-inducible promoter gene and its application. Background technique [0002] A promoter is an integral part of a gene that is recognized by RNA polymerase and starts transcription. Constitutive promoters are widely used in plant transgenics, such as CaMV 35S promoter, ubiquitin promoter and actin promoter, which are commonly used in transgenic Bt gene insect-resistant rice. Constitutive expression of the source protein in transgenic plants can also bring about many negative effects. For example, it increases the metabolic burden of plants, affects the growth and development of plants, causes plant growth retardation, short plants, and reduced yield; in addition, the use of constitutive promoters may induce gene silencing, which affects the application of transgenic technology; With the growing concern of plant food safety issues, constitutive promoters canno...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/113C12N15/82A01H5/00
CPCC12N15/113C12N15/8237
Inventor 谢雅晶刘贤金何鑫徐重新张霄
Owner JIANGSU ACADEMY OF AGRICULTURAL SCIENCES
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