Identification and application of promoter pTaASG019 featuring plant anther-specific expression

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

Active Publication Date: 2016-02-10
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 promoter pTaASG019 featuring plant anther-specific expression
  • Identification and application of promoter pTaASG019 featuring plant anther-specific expression
  • Identification and application of promoter pTaASG019 featuring plant anther-specific expression

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] Embodiment 2.RT-PCR verifies the tissue expression specificity of TaASG019 gene

[0047] Since 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 comp106333_c0_seq1 (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 sequence information of Aegilopstauschii (Aegilopstauschii, D genome donor) was electronically cloned, and three TaASG019 genes were obtained, named TaASG019-1, TaASG019-2 and TaASG019-3, among which comp106333_c0_seq1 corresponds to TaASG019-1. The cDNA sequences of the TaASG019 gene are respectively shown in SEQ ID NO: 2, SEQ ID NO...

Embodiment 3

[0057] Example 3. Obtaining of TaASG019-2 and TaASG019-3 Gene Promoter Sequences and Analysis of Cis Elements

[0058]Starting from the cDNA sequences of TaASG019-2 and TaASG019-3 genes, using the sequencing information of common wheat published by CerealsDB and IWGSC (International Wheat Genome Sequencing Consortium), and the wheat ancestor Triticumurartu (Triticum urartu, A genome donor) published in Nature in 2013 and The sequence information of Aegilopstauschii (D genome donor) was electronically cloned, and the promoters of TaASG019-2 and TaASG019-3 genes were obtained, which were named TaASG019-2 promoter and TaASG019-3 promoter respectively, and their lengths were respectively They are 1918bp and 2064bp, and their nucleotide sequences are shown in SEQ ID NO:5 and SEQ ID NO:6 respectively.

[0059] Using the PlantCARE database and the PLACE database, the TaASG019-2 promoter and the TaASG019-3 promoter were analyzed in cis elements. Such as image 3 As shown, the transl...

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Abstract

The invention belongs to the technical field of plant biology and particularly relates to isolation, functional identification and application of a promoter pTaASG019 featuring plant anther-specific expression. The promoter featuring plant anther-specific expression has promising 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): C12N15/113C12N15/82A01H5/00
Inventor 李健马力耕邓兴旺
Owner BEIJING NEXT GENERATION HYBRID WHEAT BIOTECHNOLOGY CO LTD
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