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Rice seed glutelin GluD-1 gene terminator and application thereof

A gene and rice technology, applied in the field of rice seed glutenin GluD-1 gene terminator, can solve the problems of time-consuming and labor-intensive improvement of related traits, etc., achieve great application prospects, increase technological added value, and improve seed quality Effect

Inactive Publication Date: 2013-04-03
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 glutelin GluD-1 gene terminator and application thereof
  • Rice seed glutelin GluD-1 gene terminator and application thereof
  • Rice seed glutelin GluD-1 gene terminator and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0034] Embodiment 1, the acquisition of rice seed glutelin GluD-1 gene terminator (tGluD-1)

[0035] According to the cDNA sequence of rice glutelin GluD-1 gene (GenBank number is AY429650), search the genomic DNA sequence of glutelin GluD-1 gene from GenBank, the sequence of 589bp behind the stop codon is the rice seed glutelin GluD of the present invention -1 gene terminator (tGluD-1), design primers to amplify tGluD-1. To facilitate vector construction, restriction sites (underlined) were added to the primers respectively. The forward primer of tGluD-1 is tGluD-1 SacF: 5′-AA GAGCTC AACTTATAGATTATGAGTAC-3′(Sac I), reverse primer is tGluD-1 EcoR: 5′-A GAATTC GTGACGTATCTCCTCTTT-3' (EcoR I).

[0036] A small amount of genomic DNA was extracted from the leaves of wild-type rice Kitaake (Qu et al., J. Exp. Bot. 2008, 59: 2417-2424) by CTAB method. Using it as a template, tGluD-1 SacF and tGluD-1 EcoR As a primer, the tGluD-1 sequence was amplified by PCR. The reaction prog...

Embodiment 2

[0037] Embodiment 2, the stable expression vector construction of rice seed glutelin GluD-1 gene terminator (tGluD-1)

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

[0039] Digest the pMD-GluD-1 plasmid with Sac I and EcoR I, recover the 589bp fragment of tGluD-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-tGluD-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 the GluB-3 forward primer was used

[0040] 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 a...

Embodiment 3

[0049] Example 3, Functional Verification of Rice Seed Glutenin GluD-1 Gene Terminator (tGluD-1)

[0050] 1. T 0 GUS Histochemical Detection of Transgenic Rice Kitaake / pGluB-3-tGluD-1

[0051] T of transgenic rice Kitaake / pGluB-3-nos and Kitaake / pGluB-3-tGluD-1 positive for PCR detection 0 The 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: transgenic rice Kitaake / pGluB-3-nos and Kitaake / pGluB-3-tGluD-1T 0Part 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 ferrocyanide, 1.0mM X-Gluc, 0.1% Trito...

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Abstract

The invention discloses a rice seed glutelin GluD-1 gene terminator and application thereof. The terminator is a DNA (Deoxyribonucleic Acid) molecule selected from (1) or (2) or (3): (1) a DNA molecule consisting of a nucleotide sequence shown as a sequence 1 in a sequence table, (2) a molecule having at least 70 percent, at least 75 percent, at least 80 percent, at least 85 percent, at least 90 percent, at least 95 percent, at least 96 percent, at least 97 percent, at least 98 percent or at least 99 percent of homology with a DNA sequence defined in (1), and (3) a molecule which is hybridized with the DNA sequence defined in (1) or (2) under a severe condition. By matching the terminator with different promoters, the expression and accumulation levels of an exogenous gene in a target plant can be raised, and foundations are laid for the researches on the improvement of seed quality with a biotechnology, molecular medicine farmlands and the like; and the terminator has great application prospect.

Description

technical field [0001] The invention relates to a rice seed glutelin GluD-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 can l...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N15/113C12R1/91
Inventor 曲乐庆李文静
Owner INST OF BOTANY CHINESE ACAD OF SCI