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Rice seed 26kD globulin Glb-1 gene terminator and application thereof

A gene and transgenic plant technology, applied in the field of the 26kD globulin Glb-1 gene terminator of rice seeds, can solve the problems of time-consuming and laborious, and the improvement effect of related traits is not obvious, and achieves great application prospects, increases the added value of science and technology, and improves expression. and cumulative level effects

Active Publication Date: 2011-11-23
INST OF BOTANY CHINESE ACAD OF SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, less research has been done on other terminators that can enhance gene expression
Since some important agronomic traits and plant secondary metabolites are controlled by multiple genes, increasing the expression of a single gene has no obvious effect on the improvement of related traits
Time-consuming and labor-intensive transformation of multiple genes through traditional transformation methods such as repeated transformation or hybridization

Method used

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  • Rice seed 26kD globulin Glb-1 gene terminator and application thereof
  • Rice seed 26kD globulin Glb-1 gene terminator and application thereof
  • Rice seed 26kD globulin Glb-1 gene terminator and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0034] Embodiment 1, the acquisition of rice seed 26kD globulin Glb-1 gene terminator (tGlb-1)

[0035] According to the cDNA sequence of rice 26kD globulin Glb-1 gene (GenBank number is AY427575), search the genome DNA sequence of 26kD globulin Glb-1 gene from GenBank, the sequence of 506bp after the stop codon is rice seed 26kD of the present invention The terminator (tGlb-1) of the globulin Glb-1 gene was designed to amplify tGlb-1. To facilitate vector construction, restriction sites (underlined) were added to the primers respectively. The forward primer of tGlb-1 is tGlb-1 SacF: 5′-AA GAGCTC TAGGCTTAGCTGGCTTGCCT-3'(Sac I), reverse primer is tGlb-1 EcoR: 5'-A GAATTC AAGGACGGATTTGAATTGAG-3' (EcoR I).

[0036] The CTAB method was used to extract a small amount of genomic DNA from the leaves of wild-type rice Kitaake (Qu et al., J.Exp.Bot.2008, 59:2417-2424), using it as a template, and using tGlb-1SacF and tGlb-1EcoR as primers , The PCR reaction program is: 94°C pre-d...

Embodiment 2

[0037] Embodiment 2, the stable expression vector construction of rice seed 26kD globulin Glb-1 gene terminator (tGlb-1)

[0038] 1. Construction of plant stable expression vector fused with GUS gene of tGlb-1

[0039] Digest the pMD-tGlb-1 plasmid with Sac I and EcoR I, recover the 506bp fragment of tGlb-1, and insert the fragment into the SacI and EcoR I double enzymes of pGluB-3-nos containing the GluB-3 promoter A stable transformation vector pGluB-3-tGlb-1 was constructed between the cut recognition sites (the schematic diagram of which is shown in image 3 shown). pGluB-3-nos was constructed according to the method described in the literature Qu et al., J.Exp.Bot.2008, 59: 2417-2424: the genomic DNA of rice Taichung 65 was used as a template, and

[0040] GluB-3 Forward Primer 5'-CCC AAGCTT ATTTTACTTGTACTGTTTAACC-3'(HIndIII) and

[0041] GluB-3 reverse primer 5'AAA CCCGGG AGCTTTCTGTATATGCTAATG-3'(SmaI) is a primer,

[0042] The GluB-3 promoter was amplified by PC...

Embodiment 3

[0051] Example 3, Functional verification of rice seed 26kD globulin Glb-1 gene terminator (tGlb-1)

[0052] 1. T 0 GUS histochemical detection of transgenic rice Kitaake / pGluB-3-nos and Kitaake / pGluB-3-tGlb-1

[0053] T of transgenic rice Kitaake / pGluB-3-nos and Kitaake / pGluB-3-tGlb-1 positive for PCR detection 0The generation plants were subjected to histochemical staining, and the non-transgenic wild-type rice Kitaake plants were used as the control. The specific steps are: the T of transgenic rice Kitaake / pGluB-3-nos and Kitaake / pGluB-3-tGlb-1 0 Part of the leaves, roots, and stem tissues of the generation plant and the non-transgenic wild-type rice Kitaake plant were cut into small pieces; the seeds at the filling stage 17 days after flowering were cut longitudinally from the middle with a scalpel. Soak the treated sample in GUS staining reaction solution (0.1M sodium phosphate buffer (pH 7.0), 10mM Na 2 - EDTA (pH7.0), 5mM potassium ferricyanide, 5mM potassium ferroc...

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Abstract

The invention discloses a rice seed 26kD globulin Glb-1 gene terminator and application thereof. The terminator is a DNA (deoxyribonucleic acid) molecule from the following: 1) a DNA molecule composed of nucleotide sequence disclosed as Sequence 1 in the sequence table; 2) a molecule which is at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% homological with the defined DNA; and 3) a molecule crossbred with the defined DNA sequence. By cooperating with different promoters, the terminator disclosed by the invention can enhance the expression and accumulation level of exogenous gene in the target plant, lays foundation for researching seed quality improvement, molecular medicine farms and the like, and has wide application prospects.

Description

technical field [0001] The invention relates to a rice seed 26kD globulin Glb-1 gene terminator and application thereof. Background technique [0002] The latest development of plant biotechnology not only realizes the improvement of traditional agronomic traits (such as increasing crop yield, enhancing disease resistance, insect resistance, herbicide resistance, improving quality, etc.), but also makes plants become bioreactors for biomedicine and industrial products . Most gramineous crops have the characteristics of high yield, low production cost, storage resistance, easy control of production scale, direct consumption, etc., and they have the ability of post-translational modification in vivo, so they become ideal carriers for the second generation of transgenic products. In recent years, the use of rice seeds to produce exogenous proteins and edible vaccines with health effects has developed rapidly, and has achieved great success. Such as vitamin A, glycinin which c...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/113C12N15/63C12N5/10C12N1/21C12N7/01C12N15/82A01H5/00C12R1/91
Inventor 曲乐庆李文静
Owner INST OF BOTANY CHINESE ACAD OF SCI
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