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Identification and application of plant anther specific-expression promoter pTaASG004

An anther-specific, promoter technology, applied in the field of DNA application and isolated DNA, can solve problems such as influence on plant growth and development, gene silencing with high homology of promoter sequence, and long waiting time.

Active Publication Date: 2015-11-11
BEIJING NEXT GENERATION HYBRID WHEAT BIOTECHNOLOGY CO LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

At present, some constitutive strong promoters are widely used in the field of agricultural biotechnology, such as the CaMV35S promoter and the corn Ubiquitin-1 promoter. , the improvement effect is often not obvious because the time (developmental stage-specific) or space (tissue-organ-specific) of the target gene expression is not well controlled, or the gene expression induced by these constitutive promoters is too high. The growth and development of plants are affected, these are the obstacles encountered in the current use of constitutive strong promoters combined with functional genes to improve crop quality
[0004] In addition, when studying certain metabolic processes or regulatory pathways, it is often necessary to transform two or more genes on the same pathway into the same line, transforming one of the genes to obtain a transgenic plant and then transforming another gene, or It takes a long time to wait for the hybridization after the two genes have been transformed separately. In order to improve the efficiency and shorten the time for transforming multiple genes, it has recently been reported that a new vector can be used to transform multiple genes at the same time, but in multi-gene If the same promoter is used repeatedly during transformation, gene silencing may also occur due to the high homology of the promoter sequence

Method used

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  • Identification and application of plant anther specific-expression promoter pTaASG004
  • Identification and application of plant anther specific-expression promoter pTaASG004
  • Identification and application of plant anther specific-expression promoter pTaASG004

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0042] Example 1. Genome-wide expression profiling analysis of wheat anthers at different developmental stages and acquisition of anther expression contig at later stages of pollen development

[0043] Wheat anthers whose pollen was in meiosis, mononucleate, binucleate and trinucleate were collected, total RNA was extracted with Trizol (Invitrogen), treated with DNaseI (Promega), and then mRNA was purified (Ambion). The purified mRNA was subjected to reverse transcription (Invitrogen), ultrasonic fragmentation (Fisher), library preparation (illumina) and amplification (illumina), and finally a sequencing reaction on an illumina machine.

[0044] The results of high-throughput sequencing of the wheat transcriptome were first assembled by Trinity software, and the resulting spliced ​​sequences were further removed from redundancy and similarity clustering. For the expression change analysis of the spliced ​​transcript contig, the high-throughput sequencing sequence in each sampl...

Embodiment 2

[0046] Example 2.RT-PCR verification of tissue expression specificity of TaASG004 gene

[0047] Wheat is an allohexaploid consisting of three sets of genomes A, B, and D. The average copy number of genes is 2.8, of which nearly half of the genes (46%) have 3-4 copies, and 12% of the genes have 1 -2 copies, 42% of genes had ≥5 copies. Starting from the sequence of comp169138_c0_seq2 (as shown in SEQIDNO: 1), using the sequencing information of common wheat published by CerealsDB and IWGSC (International Wheat Genome Sequencing Consortium), and the wheat ancestor Uraltu wheat (Triticumurartu, A genome donor) published in Nature in 2013 and The sequencing information of Aegilopstauschii (D genome donor) was electronically cloned, and three TaASG004 genes were obtained, which were named TaASG004-1, TaASG004-2 and TaASG004-3. The cDNA sequences of the three TaASG004 genes are shown in SEQ ID NO :2, shown in SEQIDNO:3 and SEQIDNO:4, the homology between the three is about 95%. Des...

Embodiment 3

[0057] Example 3. Obtaining of TaASG004-1, TaASG004-2 and TaASG004-3 Gene Promoter Sequences and Analysis of Cis Elements

[0058] Starting from the cDNA sequences of TaASG004-1, TaASG004-2 and TaASG004-3 genes, using the sequencing information of common wheat published by CerealsDB and IWGSC (International Wheat Genome Sequencing Consortium), and the wheat ancestor Triticum urartu (Triticum urartu, A genome) published in Nature in 2013 donor) and Aegilopstauschii (Aegilopstauschii, D genome donor) were electronically cloned, and the promoters of TaASG004-1, TaASG004-2 and TaASG004-3 genes were obtained, which were named TaASG004-1 promoter, TaASG004 -2 promoter and TaASG004-3 promoter have lengths of 2005bp, 1997bp and 2196bp respectively, and their sequences are shown in SEQ ID NO: 5, SEQ ID NO: 6 and SEQ ID NO: 7 respectively.

[0059] Using PlantCARE database and PLACE database, cis-element analysis was performed on TaASG004-1 promoter, TaASG004-2 promoter and TaASG004-3 p...

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Abstract

The invention belongs to the technical field of plant biology and particularly relates to separation, function identification and application of a plant anther specific-expression promoter. The promoter is specifically expressed in plant anther, so that the promoter is excellent in application prospect in the field of plant transgenosis.

Description

technical field [0001] The present invention belongs to the field of plant biotechnology, in particular, the present invention relates to isolated DNA, which can guide the specific transcription and / or expression of nucleic acid operably linked downstream of it in plant anthers. In addition, the present invention also relates to expression cassettes and plants containing the DNA, and to applications of the DNA. Background technique [0002] Plant gene regulation is mainly carried out at the transcriptional level, which is coordinated by a variety of cis-acting elements and trans-acting factors. The promoter is an important cis-acting element. It is a DNA sequence located in the upstream region of the 5′ end of the structural gene to regulate gene transcription. It can activate RNA polymerase to accurately combine with the template DNA to ensure accurate and efficient transcription. play a key role in transcriptional regulation. According to the different characteristics of...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/113C12N15/82
Inventor 马力耕李健邓兴旺
Owner BEIJING NEXT GENERATION HYBRID WHEAT BIOTECHNOLOGY CO LTD
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