Rice seed glutelin glub-5 gene terminator and its application
A gene and transgenic plant technology, applied in application, angiosperm/flowering plants, introduction of foreign genetic material using vectors, etc., can solve the problems of insignificant improvement of related traits, time-consuming and labor-intensive, etc., to increase the added value of science and technology, and achieve large-scale applications. Prospects, effects of increasing expression and accumulation levels
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Embodiment 1
[0038] Embodiment 1, the acquisition of rice seed glutelin GluB-5 gene terminator (tGluB-5)
[0039] According to the cDNA sequence of rice glutelin GluB-5 gene (GenBank number is AK107238), search the genome DNA sequence of glutelin GluB-5 gene from GenBank, the 497bp sequence after the stop codon is the rice seed glutelin GluB-5 of the present invention. 5 gene terminator (tGluB-5), design primers to amplify tGluB-5. To facilitate vector construction, restriction sites (underlined) were added to the primers respectively. The forward primer of tGluB-5 is tGluB-5SacF: 5′-AA GAGCTC ACCCAAGGCATTATACTAA-3'(Sac I), the reverse primer is tGluB-5EcoR:5'-A GAATTC AAACTTGGTGCCACGACACTG-3' (EcoR I).
[0040] 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 and tGluB-5SacF and tGluB-5EcoR as primers, PCR amplified tGluB-5 sequence. The PCR reaction program was...
Embodiment 2
[0041] Example 2, Vector Construction and Transformation of Rice Seed Glutenin GluB-5 Gene Terminator (tGluB-5)
[0042] 1. Construction of tGluB-5 fusion GUS gene plant expression vector
[0043] Digest the pMD-tGluB-5 plasmid with Sac I and EcoR I, recover the 497bp restriction fragment of tGluB-5, and insert the fragment into pGluB-3-nos containing GluB-3 promoter and pGluC of GluC promoter respectively The stable transformation vector pGluB-3-tGluB-5 was constructed between the Sac I and EcoR I double restriction recognition sites of -nos (the schematic diagram of which is shown in image 3 Shown in A) and pGluC-tGluB-5 (the schematic diagram of its structure is shown in image 3 shown in B).
[0044] pGluB-3-nos and pGluC-nos were 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
[0045] GluB-3 Forward Primer 5'-CCC AAGCTT ATTTTACTTGTACTGTTTAACC-3'...
Embodiment 3
[0058] Example 3, Functional Verification of Rice Seed Glutenin GluB-5 Gene Terminator (tGluB-5)
[0059] 1. T 0 Histochemical detection of GUS in transgenic rice
[0060] T of transgenic rice Kitaake / pGluB-3-nos and Kitaake / pGluB-3-tGluB-5 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: the T of transgenic rice Kitaake / pGluB-3-nos and Kitaake / pGluB-3-tGluB-5 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 ferrocyanide, 1.0mM X-Gluc, 0.1% Triton X-100), re...
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