Modification of transcriptional repressor binding site in NF-YC4 promoter for increased protein content and resistance to stress

A protein content, NF-YC4 technology, applied to cells modified by introducing foreign genetic material, applications, chemical instruments and methods, etc., can solve problems such as unexplained functions

Active Publication Date: 2018-01-02
IOWA STATE UNIV RES FOUND
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

However, its role in such pe...

Method used

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  • Modification of transcriptional repressor binding site in NF-YC4 promoter for increased protein content and resistance to stress
  • Modification of transcriptional repressor binding site in NF-YC4 promoter for increased protein content and resistance to stress
  • Modification of transcriptional repressor binding site in NF-YC4 promoter for increased protein content and resistance to stress

Examples

Experimental program
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Embodiment 1

[0132] This example describes the preparation and transformation of 35S::QQS and 35S::AtNF-YC4 fusion constructs.

[0133] The 35S::QQS and 35S::AtNF-YC4 fusion constructs were made by cloning the amplified full-length coding sequence into the binary vector pB2GW7, as described above (Li et al. (2015), supra). Briefly, the QQS coding domain sequence (CDS) or the NF-YC4 CDS from Arabidopsis thaliana (AtNF-YC4) was inserted into the vector pB2GW7 (see Figures 1a and 1b for vector maps), which contains the Bar gene ( glufosinate acetyltransferase gene) under the control of Pnos (nos promoter) and Tnos (nos terminator) of nopaline synthase from tobacco (Nicotiana tabacum). The CDS on this vector is controlled by p35S (Cauliflower Mosaic Virus (CaMV) 35S promoter) and terminated by T35S (CaMV 35S). Only the region between LB and RB is introduced into plants to generate transgenic plants.

[0134] The constructs were introduced into Agrobacterium tumefaciens strain GV3101 for tran...

Embodiment 2

[0141] This example describes the analysis of plant composition.

[0142] Determination of leaf composition (starch staining and quantification and protein), in Arabidopsis, in seedling shoots 20 days after planting (DAP) in a growth chamber, and in rice, once in plants from 30 DAP Measured on the fallen leaves (next to the flag leaves) of the branches. 3 (for starch) or 5 (for protein) plants per replicate, 3 replicates from each independent T2 line (Arabidopsis) and T3 line (rice) were analyzed. Leaves were harvested at the end of the light phase. Seeds of Arabidopsis, rice and soybean were harvested from individual plants. For leaf and seed protein quantification, plant material was roasted at 71°C. Dried leaf / seed tissue (0.07 g) was used for each assay. Measure total protein content. Total nitrogen content was determined with LECO CHN-2000 ((LECO, St. Joseph, MI) and converted into protein content (Li et al. (2015), supra).

[0143] The whole aerial part of Arabidop...

Embodiment 3

[0146] This example describes a yeast two-hybrid assay.

[0147] The Matchmaker System 3 (Clontech, Mountain View, CA) was used to identify QQS-interacting proteins using an Arabidopsis (type Columbia) cDNA library constructed from 3-day-old etiolated seedlings (Arabidopsis.org / servlets / TairObject?type=library&id =23). For the interactive yeast two-hybrid assay, QQS and AtNF-YC4 were cloned into pGBKT7 (bait vector) and pGADT7 (trap vector), respectively.

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Abstract

Method of increasing protein content in a eukaryotic cell comprising an NF-YC4 gene comprising modifying the transcriptional repressor binding site; method of producing a plant with increased proteincontent comprising crossing and selecting for increased protein content; method of increasing resistance to a pathogen or a pest in a plant comprising an NF-YC4 gene comprising modifying the transcriptional repressor binding site, alone or in further combination with expressing QQS in the plant; method for producing a plant with increased resistance to a pathogen or a pest comprising crossing andselecting for increased resistance to the pathogen or the pest; a cell, collection of cells, tissue, organ, or organism in which the NF-YC4 gene comprises a promoter comprising a transcriptional repressor binding site that has been modified so that the transcriptional repressor cannot prevent transcription of the NF-YC4; hybrid plants; and seeds.

Description

[0001] Statement Regarding Federally Sponsored Research or Development [0002] The work described herein was supported at least in part by grants from the National Science Foundation, MCB grant numbers 0209789 and 0951170. Accordingly, the US Government has certain rights in this invention. technical field [0003] The present disclosure relates to increasing the protein content of eukaryotic cells, increasing the resistance of plants to stresses, such as abiotic (such as salt, drought and pollution) or biotic (such as pathogens and pests) stresses, with modified transcriptional repressor binding sites Dot promoters so that transcriptional repressors cannot block transcription of genes NF-YC4, QQS, TALENS, CRISPR / Cas9, tissue culture, hybrid and backcross plants, hybrid plants, regenerative cells and seeds. Background of the invention [0004] Protein deficiency is a major health problem in developing countries. Low protein intake causes mental retardation, developmental ...

Claims

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Application Information

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IPC IPC(8): C12N5/10A01H5/00A01H5/10
CPCC07K14/415C12N15/8205C12N15/8243C12N15/8281C12N15/8283C12N15/8286C12N15/8241C12N15/8247Y02A40/146C12N15/8251C12N15/8279C12N15/8216A01H5/00A01H1/02A01H1/122A01H1/021A01H5/10A01H6/4636A01H6/542
Inventor 李灵E·S·维特勒
Owner IOWA STATE UNIV RES FOUND
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