Tissue specificity expression promoter PD540 and application of the same in rice modification

A promoter, rice technology, applied in the field of genetic engineering, can solve the problems of waste, plant burden, death, etc.

Inactive Publication Date: 2007-08-15
HUAZHONG AGRI UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

This not only causes a burden on the plant body, causes the plant to express extra proteins, causing waste, but also causes the physiological and metabolic disorders of the plant body, causing its death (Karlowski et al., 2003; Ehsani et al., 2003; Miyao et al., 2003); more importantly, exogenous genes are abundantly expressed in fruits or organs eaten by humans, which has caused concerns about the safety of transgenics

Method used

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  • Tissue specificity expression promoter PD540 and application of the same in rice modification
  • Tissue specificity expression promoter PD540 and application of the same in rice modification
  • Tissue specificity expression promoter PD540 and application of the same in rice modification

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0029] Example 1: Isolation of clone P D54O Promoter

[0030] 1. Identification of tissue-specific expression genes in rice using cDNA chip technology

[0031] The present invention utilizes a full growth stage balanced cDNA library of a rice variety Minghui 63 (Oryza sativa ssp. indica), which is widely used in China, for cDNA chip analysis. The library consists of cDNAs from 15 different tissues of rice at different growth stages and different physiological states (Chu et al., 2003). Microarrays were fabricated and analyzed using published methods (Zhou et al., 2002). In order to ensure the reliability of the hybridization results, each cDNA clone has two sites on the cDNA chip, which are located at symmetrical positions on the same grid (Fig. 2).

[0032] The present invention uses total RNA from rice leaves, leaf sheaths, stems, roots, embryos and endosperms to be reverse-transcribed into cDNAs and labeled with radioactive isotopes as probes, respectively hybridized wit...

Embodiment 2

[0041] Example 2: P D54O Functional verification of promoters

[0042] 1. Construction of genetic transformation vector

[0043] The pCAMBIA1381 vector used ( FIG. 6 ) is a series vector of the commonly used Agrobacterium planta-mediated genetic transformation vector pCAMBIA1301 (Sun et al., 2004). This vector carries a promoter-less reporter gene GUS, which was donated by CAMBIA Laboratory (Center for the Application of Molecular Biology to International Agriculture) in Australia.

[0044] According to the sequence analysis results, restriction endonucleases SmaI and SphI were selected to digest the subcloning of the 6.5kb fragment of 119H12 to obtain a promoter fragment of about 2.1kb, which included the region from -2134 to +41 (relative to The translation start codon ATG is +1), named P D54O . The end of the obtained fragment was smoothed with T4 DNA polymerase; at the same time, the genetic transformation vector pCAMBIA1381 was digested with a blunt-ended restriction ...

Embodiment 3

[0047] Example 3: P D54O Promoter application

[0048] 1.P D54O Construction and Transformation of Fusion Vector with Anti-insect Gene Bt

[0049] In order to test P D54OThe application value of the promoter in rice insect resistance, the present invention replaces the reporter gene GUS in the aforementioned pCAMBIA1381 vector with the Bt insect resistance gene Cry1Ac (Cheng et al., 1998), adopts the method described in Example 2 to P D54O The promoter was ligated with the pCAMBIA1381 vector carrying the Cry1Ac gene. The obtained recombinant plasmid was named P D54O -Cry1Ac (Figure 9). Will P D54O - Electrotransformation of the Cry1Ac plasmid (Sambrook and Russell, 2001) into Agrobacterium strain EHA105 (Sun et al., 2004).

[0050] The P D54O The -Cry1Ac plasmid was introduced into the rice variety Mudanjiang 8, and 20 positive independent transformed plants were obtained. Utilize the BT-Cry1Ab / 1Ac gold-labeled immunological rapid detection test strip (purchased from ...

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Abstract

The invention discloses a DNA fragment separating colony and functional checking of rice tissue special expressing promoter PD54O in plant gene engineering technical domain, which is characterized by the following: the PD54O promoter expresses in rice leaf and leaf sheath and does not express in germ and endosperm; transferring genetic rice plant can resist the invasion of borer with the expression of PD54O regulate and control insect-resisting gene; it does not express Bt protein in seeds of food.

Description

technical field [0001] The invention relates to the technical field of genetic engineering. It specifically relates to the separation and cloning, functional verification and application of a DNA fragment. The DNA fragment contains a promoter of gene D54O encoding rice photosystem II 10 kD protein, which can be expressed in green tissue of rice, but not expressed in embryo and endosperm. After linking the fragment with the reporter gene GUS, it is directly transferred into the plant body, and the reporter gene in the transgenic plants is only expressed in green tissues such as leaves and leaf sheaths. After the fragment is fused with the insect-resistant Bt gene, it is transformed into rice, and the genetically transformed plants show resistance to insect pests under the condition of naturally developing insects in the field. Background technique [0002] Rice is one of the main food crops in the world. More than 2 billion people in the world rely on rice as a staple food,...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/29C12N15/82
Inventor 王石平蔡萌魏君
Owner HUAZHONG AGRI UNIV
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