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Gene silencing

A silencing and genetic technology, applied in genetic engineering, organic chemistry, biochemical equipment and methods, etc., can solve problems such as inducible chromatin modification

Inactive Publication Date: 2015-10-21
THE ROCKEFELLER UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

In most cases in rice, IRRNA targeting promoter regions can trigger DNA methylation of homologous sequences, but it cannot induce chromatin modification and TGS (Okano et al., 2008)

Method used

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Experimental program
Comparison scheme
Effect test

Embodiment 1

[0087] vector construction

[0088] A 1731 bp Arabidopsis thaliana wild-type (WT) genomic fragment (position -274 to -2004; transcription start site is +1) from the AT2G17690 (SDC) promoter region was cloned using standard PCR and cloning techniques into the Gateway entry vector to generate pENTR / PSDC. A 475 bp genomic DNA fragment (-9 to -483; transcription start site is +1) from the promoter region of AT1G80080 (TMM) was cloned into the Gateway entry vector to generate pENTR / PTMM-S. The primers used to clone the two genomic DNA fragments contained restriction enzyme sites, AvrII in the forward primer and SpeI and AscI in the reverse primer. pENTR / PSDC was cut into linearized pENTR / PSDC by SpeI and AscI double enzymes. pENTR / PTMM-S was digested with AvrII and AscI to release the PTMM-S DNA fragment. Because the digested ends of AvrII and SpeI are compatible, the digested PTMM-SDNA fragment can be ligated into linear pENTR / PSDC to generate pENTR / PSDC-PTMM-S. pENTR / PTMM-S a...

Embodiment 2

[0132] Plant Material and Transformation

[0133] Arabidopsis thaliana WT (Col-0) and Nicotiana benthamiana were used in this study. Stable transformation of Arabidopsis was performed by the flower dip method described by Zhang et al. (2006). Transformed plants were selected on selection medium (1x MS salts + 1% sucrose + 0.5 g / L MES + 100 mg / L carbenicillin + 10 mg / L basta + 8% agar pH 5.7). Clustered stomatal phenotypes were assessed microscopically using 2-week-old T1 and T2 seedlings. T3 homozygous seedlings were used for DNA methylation and chromatin modification assays. Nicotiana N. benthamiana leaf Agro-infiltration (Voinnet et al., 2003) was used to measure the levels of small RNAs produced by the different constructs.

Embodiment 3

[0135] stomatal phenotype observation

[0136]The stomatal phenotype was assessed microscopically using cotyledons of 2-week-old T1 and T2 seedlings. In wild type (WT), stomata appear on the lower surface of the cotyledon, which are separated by at least one intervening epidermal cell. Seedlings were assessed as exhibiting a tmm mutant phenotype (clustered stomatal phenotype) if two or more stomata were located together. For each transformation event of the construct, 32-64 T1 independent lines and 24 T2 independent lines were evaluated to determine penetrance of the tmm phenotype.

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Abstract

The present invention relates to transcriptional gene silencing (TGS) of endogenes in plants, plant tissue and plant cells. More specifically, the present invention relates to nucleic acid constructs that are capable of more effectively silencing genes of interest, such as endogenes, in plants, plant tissue and plant cells by TGS. The present invention further relates to methods of more effectively reducing endogenous gene expression in plants, plant tissues or plant cells by TGS using the nucleic acid constructs of the invention.

Description

[0001] Cross References to Related Applications [0002] This application claims priority to US Provisional Patent Application 61 / 738,651, filed December 18, 2012. This application is hereby incorporated by reference. [0003] Sequence submission [0004] A sequence listing in electronic form is filed with this application. The title of the sequence listing is 2312128PCTSequenceListing.txt, created on December 5, 2013, and the size is 27kb. The information in the sequence listing in electronic form is incorporated by reference in its entirety. Background of the invention [0005] The present invention relates to transcriptional gene silencing (TGS) of endogenous genes (endogenes) in plants, plant tissues and plant cells. More particularly, the present invention relates to nucleic acid constructs capable of more efficiently silencing a gene of interest, such as an endogenous gene, in plants, plant tissues and plant cells by TGS. The present invention further relates to a m...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): A01H5/10C07H21/04C12N15/82
CPCC12N15/8216C12N15/8218C12N15/113
Inventor N-H·蔡Q·牛S·邓
Owner THE ROCKEFELLER UNIV